Transferrin receptor antigen-binding domains and uses therefor

Abstract

The present disclosure is generally directed to antigen-binding domains that specifically bind to human transferrin receptor (TfR) and their use in transport across the blood brain barrier (BBB).

Description

TRANSFERRIN RECEPTOR ANTIGEN-BINDING DOMAINS AND USES THEREFORCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application Nos. 63 / 819,227 (filed June 6, 2025), 63 / 743,994 (filed January 10, 2025), 63 / 681,554 (filed August 9, 2024), and 63 / 660,907 (filed June 17, 2024), 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_031PC04_SL.xml; Size: 255,956 bytes; and Date of Creation: June 10, 2025) is herein incorporated by reference in its entirety.FIELD OF THE PRESENT DISCLOSURE

[0003] The present disclosure relates to antigen-binding domains that specifically bind to human transferrin receptor (TfR). These antigen-binding domains can cross the blood brain barrier and can transport other agents (e.g., therapeutically active agents) associated with the antigen-binding domain across the blood brain barrier.BACKGROUND

[0004] 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. Alterations of the BBB are an important component of pathology and progression of different neurological diseases. 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 some therapeutics to the CNS has relied on injection of the therapeutic directly into the CNS. However, such injections are invasive procedures that have efficacy that is limited bythe 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 a high dose 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. Accordingly, improved products and methods for delivering therapeutics across the BBB are needed.SUMMARY OF THE PRESENT DISCLOSURE

[0005] Provided herein are antigen-binding domains, complexes, fusion proteins, antibodies, and multispecific proteins that specifically bind to human transferrin receptor (TfR), and methods of making and using the same.

[0006] In some aspects, provided herein is an antigen-binding domain that specifically binds to human transferrin receptor (TfR), wherein the 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, VL CDR2, and VL CDR3 sequences comprising the amino acid sequences of: SEQ ID NOs:22, 23, 24, 34, 32, and 33, respectively; SEQ ID NOs:22, 23, 26, 34, 32, and 33, respectively; SEQ ID NOs:22, 23, 25, 35, 32, and 33, respectively; SEQ ID NOs:22, 23, 25, 36, 32, and 33, respectively; SEQ ID NOs:22, 23, 25, 37, 32, and 33, respectively; SEQ ID NOs:22, 23, 25, 38, 32, and 33, respectively; SEQ ID NOs: 22, 23, 167, 34, 32, and 33, respectively; SEQ ID NOs:27, 30, 29, 42, 40, and 41, respectively; SEQ ID NOs:27, 28, 29, 43, 40, and 41, respectively; SEQ ID NOs:27, 28, 29, 44, 40, and 41, respectively; or SEQ ID NOs:27, 30, 29, 45, 40, and 41, respectively.

[0007] In some aspects, provided herein is an antigen-binding domain that specifically binds to human transferrin receptor (TfR), wherein the antigen-binding domain comprises (i) VH CDR1, VH CDR2, and VH CDR3 sequences and (ii) VL CDR1, VL CDR2, and VL CDR3 sequences as set forth in: SEQ ID NOs:46 and 48, respectively; SEQ ID NOs:50 and 48, respectively; SEQ ID NOs:49 and 51, respectively; SEQ ID NOs:49 and 52, respectively; SEQ ID NOs:49 and 53, respectively; SEQ ID NOs:49 and 54, respectively; SEQ ID NOs:55 and 48, respectively; SEQ ID NOs:58 and 59, respectively; SEQ ID NOs:56 and 60, respectively; SEQ ID NOs:56 and 61, respectively; SEQ ID NOs:58 and 62, respectively; or SEQ ID NOs:56 and 57, respectively.

[0008] In some aspects, the 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 least95%, or at least 99% identical to the amino acid sequences of: SEQ ID NOs:46 and 48, respectively; SEQ ID NOs:50 and 48, respectively; SEQ ID NOs:49 and 51, respectively; SEQ ID NOs:49 and 52, respectively; SEQ ID NOs:49 and 53, respectively; SEQ ID NOs:49 and 54, respectively; SEQ ID NOs:55 and 48, respectively; SEQ ID NOs:58 and 59, respectively; SEQ ID NOs:56 and 60, respectively; SEQ ID NOs:56 and 61, respectively; SEQ ID NOs:58 and 62, respectively; SEQ ID NOs:85 and 87, respectively; SEQ ID NOs:89 and 87, respectively; SEQ ID NOs:88 and 90, respectively; SEQ ID NOs:88 and 91, respectively; SEQ ID NOs:88 and 92, respectively; SEQ ID NOs:88 and 93, respectively; SEQ ID NOs:94 and 87, respectively; SEQ ID NOs:97 and 98, respectively; SEQ ID NOs:95 and 99, respectively; SEQ ID NOs:95 and 100, respectively; SEQ ID NOs:97 and 101, respectively; SEQ ID NOs: 102 and 104, respectively; SEQ ID NOs: 106 and 104, respectively; SEQ ID NOs: 105 and 107, respectively; SEQ ID NOs: 105 and 108, respectively; SEQ ID NOs: 105 and 109, respectively; SEQ ID NOs:105 and 110, respectively; SEQ ID NOs: 111 and 104, respectively; SEQ ID NOs: 114 and 115, respectively; or SEQ ID NOs:56 and 57, respectively.

[0009] In some aspects, provided herein is an antigen-binding domain that specifically binds to human TfR, wherein the antigen-binding domain comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO:46, 49, 50, 55, 56, 58, 85, 88, 89, 94, 95, 97, 102, 105, 106, 111, or 114.

[0010] In some aspects, provided herein is an antigen-binding domain that specifically binds to human TfR, wherein the antigen-binding domain comprises a VH and a VL, wherein the VL comprises the amino acid sequence of SEQ ID NO:48, 51, 52, 53, 54, 59, 60, 61, 62, 104, 107, 108, 109, 110, or 115.

[0011] In some aspects, the antigen-binding domain comprises a VH and VL comprising the amino acid sequences of: SEQ ID NOs:46 and 48, respectively; SEQ ID NOs:50 and 48, respectively; SEQ ID NOs:49 and 51, respectively; SEQ ID NOs:49 and 52, respectively; SEQ ID NOs:49 and 53, respectively; SEQ ID NOs:49 and 54, respectively; SEQ ID NOs: 55 and 48, respectively; SEQ ID NOs:58 and 59, respectively; SEQ ID NOs:56 and 60, respectively; SEQ ID NOs:56 and 61, respectively; SEQ ID NOs:58 and 62, respectively; SEQ ID NOs:85 and 87, respectively; SEQ ID NOs:89 and 87, respectively; SEQ ID NOs:88 and 90, respectively; SEQ ID NOs:88 and 91, respectively; SEQ ID NOs:88 and 92, respectively; SEQ ID NOs:88 and 93, respectively; SEQ ID NOs: 94 and 87, respectively; SEQ ID NOs: 97 and 98, respectively; SEQ ID NOs:95 and 99, respectively; SEQ ID NOs:95 and 100, respectively; SEQ ID NOs:97 and 101, respectively; SEQ ID NOs: 102 and 104, respectively; SEQ ID NOs: 106 and 104,respectively; SEQ ID NOs: 105 and 107, respectively; SEQ ID NOs: 105 and 108, respectively; SEQ ID NOs: 105 and 109, respectively; SEQ ID NOs: 105 and 110, respectively; SEQ ID NOs: 111 and 104, respectively; or SEQ ID NOs: 114 and 115, respectively.

[0012] In some aspects, the antigen-binding domain binds to an epitope comprising residue D356 of SEQ ID NO: 1. In some aspects, the antigen-binding domain is capable of crossing the blood brain barrier (BBB). In some aspects, the antigen-binding domain binds to cynomolgus monkey TfR. In some aspects, the antigen-binding domain binds human TfR with an affinity of 0.01 nM to 50 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity of 51 nM to 750 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity of 751 nM to 10,000 nM. In some aspects, the antigen-binding domain is internalized in blood-brain barrier epithelial cells, optionally wherein the antigen-binding domain is internalized in blood-brain barrier epithelial cells greater than 5-fold or greater than 40-fold as compared to internalization by an isotype control, further optionally wherein the blood-brain barrier epithelial cells are HCMEC / D3 cells. In some aspects, the antigen-binding domain does not significantly reduce TfR levels in a mouse brain following administration of the antigenbinding domain as measured in a whole brain lysate. In some aspects, the antigen-binding domain accumulates at least 4-fold or at least 5-fold more than an isotype control in vessel- depleted mouse brain.

[0013] In some aspects, the antigen-binding domain comprises a VH and a VL on a single polypeptide chain. In some aspects, the antigen-binding domain comprises a single-chain fragment variable (scFv). In some aspects, the antigen-binding domain comprises a VH and a VL and further comprises a disulfide staple comprising a disulfide bond: (i) between a cysteine residue in the VH and a cysteine residue in the VL of the antigen-binding domain, (ii) between a cysteine residue in the VH and a cysteine residue outside the VH, or (iii) between a cysteine residue in the VL and a cysteine residue outside the VL. In some aspects, (i) the disulfide staple is formed in part by the substitution of a residue with cysteine in the VH domain; (ii) the disulfide staple is formed in part by the substitution of a residue with cysteine in the VL domain; (iii) the VH and VL are linked by a linker, and the disulfide bond is formed in part by a cysteine residue in the linker; and / or (iv) the disulfide staple is formed by the substitution of a residue with cysteine in the VH domain and the substitution of a residue with cysteine in the VL domain. In some aspects, (i) the substitution of the residue with cysteine is at position 44 of the VH, with numbering according to Kabat; (ii) the substitution of the residue with cysteine is at position 100 of the VL, with numbering according to Kabat; or (iii) the substitutions of residues with cysteinesare at position 44 of the VH and position 100 of the VL, with numbering according to Kabat. In some aspects, the antigen-binding domain comprises a disulfide-stapled single-chain fragment variable (dsFv) comprising an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 132-142. In some aspects, the scFv is in the orientation VH-linker-VL. In some aspects, the scFv is in the orientation VL-linker-VH. In some aspects, the linker (i) is about 5 to about 25 amino acids, is about 5 to about 20 amino acids, is about 10 to about 25 amino acids, or is about 10 to about 20 amino acids and / or (ii) comprises the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO:5) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:6).

[0014] In some aspects, the antigen-binding domain comprises a VH on a first polypeptide and a VL on a second polypeptide.

[0015] In some aspects, provided herein is an antigen-binding domain that specifically binds to human TfR, wherein the antigen-binding domain is a VHH comprising (i) the VH CDR1, VH CDR2, and VH CDR3 as set forth in any one of SEQ ID NOs:48, 51, 52, 53, 54, 59, 60, 61, 62, 87, 90, 91, 92, 93, 98, 99, 100, 101, 104, 107, 108, 109, 110, or 115 or (ii) the VH of any one of SEQ ID NOs:48, 51, 52, 53, 54, 59, 60, 61, 62, 87, 90, 91, 92, 93, 98, 99, 100, 101, 104, 107, 108, 109, 110, or 115, optionally wherein the VHH is capable of crossing the blood brain barrier (BBB).

[0016] In some aspects, provided herein is an antibody comprising the antigen-binding domain provided herein.

[0017] In some aspects, provided herein is complex or fusion protein comprising an antigenbinding domain provided herein and a heterologous protein or peptide. In some aspects, the antigen-binding domain and heterologous protein or peptide are linked by an amino acid linker. In some aspects, the linker is (GGGGS)x3 (SEQ ID NO:7), (GGSGG)x3 (SEQ ID NO:8), or GGSGG (SEQ ID NOV). In some aspects, the heterologous protein or peptide (a) comprises the amino acid sequence of P-glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), clusterin (APOJ), Reelin, very low density lipoprotein receptor (VLDLR), apolipoprotein E receptor 2 (APOER2, low-density lipoprotein receptor-related protein 8 (LRP8)), Tripeptidyl Peptidase 1 (CLN2 / TPP1), or Alpha-L-Iduronidase (IDUA), Iduronate 2- Sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosaminide N- acetyltransferase (HGSNAT), and N-acetyl-alpha-glucosaminidase (NAGLU), N- sulfoglucosamine sulfohydrolase (SGSH), ubiquitin protein ligase E3 A (UBE3 A), or a variant orportion thereof or (b) specifically binds to beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, Glycoprotein nonmetastatic protein B (GPNMB), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6A (MS4A6A), or Transmembrane Protein 106B (TMEM106b). In some aspects, the complex comprises a first polypeptide comprising from N’ to C’ terminal, a VH of the antigen-binding domain, an Fc domain, and the heterologous protein or peptide; and a second polypeptide comprising a VL of the antigen-binding domain. In some aspects, the complex comprises from N’ to C’ terminal a first polypeptide comprising the heterologous protein or peptide and a first Fc domain; a second polypeptide comprising from N’ to C’ terminal a VH of the antigen-binding domain and a second Fc domain; and a third polypeptide comprising a VL of the antigen-binding domain. In some aspects, the first Fc domain comprises a knob mutation and the second Fc domain comprises a hole mutation. In some aspects, the first Fc domain comprises a hole mutation and the second Fc domain comprises a knob mutation.

[0018] In some aspects, provided herein is a multi-specific protein comprising a first antigenbinding domain that is an antigen-binding domain provided herein linked to an antibody or second antigen-binding domain. In some aspects, the antibody or second antigen-binding domain specifically binds to (i) a CNS antigen or (ii) a cancer antigen. In some aspects, the antibody or second antigen-binding domain comprises a heavy chain constant region. In some aspects, the first antigen-binding domain is linked, optionally via an amino acid linker, to the C-terminus of the heavy chain constant region. In some aspects, the first antigen-binding domain is linked to the second antigen-binding domain or antibody by an amino acid linker wherein the linker is (GGGGS)x3 (SEQ ID NO:7), (GGSGG)x3 (SEQ ID NO:8), or GGSGG (SEQ ID NOV). In some aspects, the first antigen-binding domain is linked directly to the antibody or second antigen-binding domain. In some aspects, the multi-specific protein comprises a truncation at a terminus of the first antigen-binding domain, a truncation at a terminus of the antibody or second antigen-binding domain, or truncations at termini of both the first antigen-binding domain andthe antibody or second antigen-binding domain. In some aspects, the antibody or second antigenbinding domain comprises a C-terminal truncation of one, two, or three residues. In some aspects, the antibody or second antigen-binding domain comprises an N-terminal truncation of one, two, or three residues. In some aspects, the antibody or second antigen-binding domain comprises an IgGl antibody or antigen-binding fragment thereof and comprises a truncation of residues PGK. In some aspects, the first antigen-binding domain is linked to the C-terminus of the antibody.

[0019] In some aspects, the antibody or second antigen-binding domain specifically binds to a cancer antigen, and the antibody or second antigen-binding domain is selected from the group consisting of rituximab, cetuximab, trastuzumab, pertuzumab, bevacizumab, nivolumab, pembrolizumab, atezolizumab, avelumab, and durvalumab. In some aspects, the antibody or second antigen-binding domain specifically binds to a cancer antigen, and the antibody or second antigen-binding domain is selected from the group consisting of a version of rituximab comprising reduced effector function, a version of cetuximab comprising reduced effector function, a version of trastuzumab comprising reduced effector function, a version of pertuzumab comprising reduced effector function, a version of bevacizumab comprising reduced effector function, a version of nivolumab comprising reduced effector function, a version of pembrolizumab comprising reduced effector function, a version of atezolizumab comprising reduced effector function, a version of avelumab comprising reduced effector function, and a version of durvalumab comprising reduced effector function. In some aspects, the reduced effector function comprises a mutation of LALA-P331S and / or LALA-P329S. In some aspects, the antibody or second antigen-binding domain that binds to a cancer antigen is a version of rituximab comprising reduced effector function. In some aspects, the multi-specific protein comprises the amino acid sequences of SEQ ID NOs: 80-82.

[0020] In some aspects, the antibody or second antigen-binding domain specifically binds to matrix metalloproteinase 9 (MMP-9). In some aspects, the multi-specific protein comprises the amino acid sequences of SEQ ID NOs: 152 and 153. In some aspects, the antibody or second antigen-binding domain is an IgG4 antibody or antigen-binding domain. In some aspects, the multi-specific protein comprises the amino acid sequences of: (i) SEQ ID NOs: 154, 155, and 156, (ii) SEQ ID NOs:157, 158, and 156, (iii) SEQ ID NOs: 159, 160, and 156, (iv) SEQ ID NOs: 161, 155, and 156, (v) SEQ ID NOs: 162, 158, and 156, (vi) SEQ ID NOs:163, 160, and 156, (vii) SEQ ID NOs: 164, 155, and 156, (viii) SEQ ID NOs: 165, 158, and 156, or (ix) SEQ IDNOs: 166, 160, and 156. In some aspects, the antibody or second antigen-binding domain is andecaliximab.

[0021] In some aspects, the multi-specific protein is bispecific. In some aspects, the multispecific protein is bivalent, trivalent, or tetravalent. In some aspects, the multi-specific protein is bivalent. In some aspects, the multi-specific protein is trivalent, optionally wherein the trivalent protein comprises the antigen-binding domain that binds to human TfR and two antigen-binding domains that bind to a CNS antigen.

[0022] In some aspects, the multi-specific protein is tetravalent, optionally wherein the tetravalent protein comprises two of the antigen-binding domains that bind to human TfR and two antigen-binding domains that bind to a CNS antigen. In some aspects, the antibody or second antigen-binding fragment thereof comprises a constant region comprising a knob mutation and a constant region comprising a hole mutation. In some aspects, the antigen-binding domain is linked, optionally via an amino acid linker, to the constant region comprising a hole mutation. In some aspects, the antigen-binding domain is linked, optionally via an amino acid linker, to the constant region comprising a knob mutation. In some aspects, the CNS antigen is a brain antigen. In some aspects, the CNS antigen is not TfR.

[0023] In some aspects, the antibody or second antigen-binding domain comprises a mutation that reduces effector function, optionally wherein the mutation that reduces effector function comprises (i) L234A, L235A, and / or P331S and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S. In some aspects, the antibody or second antigen-binding domain comprises a constant region comprising a knob mutation and a mutation that reduces effector function, optionally wherein the mutation that reduces effector function comprises (i) L234A, L235A, and / or P331 S and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S. In some aspects, the antibody or second antigen-binding domain comprises a constant region comprising a hole mutation and a mutation that reduces effector function, optionally wherein the mutation that reduces effector function comprises (i) L234A, L235A, and / or P331 S and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S. In some aspects, the antibody or second antigen-binding domain is an IgG antibody or antigen-binding fragment thereof. In some aspects, the IgG antibody or antigenbinding fragment thereof is an IgGl antibody or antigen-binding fragment thereof or an IgG4 antibody or antigen-binding fragment thereof.

[0024] In some aspects, the antibody or second antigen-binding domain specifically binds to a CNS antigen that is beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE),apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, Glycoprotein nonmetastatic protein B (GPNMB), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6A (MS4A6A), or Transmembrane Protein 106B (TMEM106b).

[0025] In some aspects, the multi-specific protein is capable of binding FcRn.

[0026] In some aspects, the antibody or second antigen-binding domain, complex or fusion protein, or multi-specific protein is linked to an imaging agent.

[0027] In some aspects, provided herein is a composition comprising a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode the multi-specific protein provided herein, wherein the first polynucleotide encodes a first heavy chain, the second polynucleotide encodes a second heavy chain and the antigen-binding domain that specifically binds to human TfR, and the third polynucleotide encodes a light chain.

[0028] In some aspects, provided herein is a composition comprising a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode the multi-specific protein provided herein, wherein the first polynucleotide encodes a first heavy chain and a first copy of the antigen-binding domain that specifically binds to human TfR, the second polynucleotide encodes a second heavy chain and a second copy of the antigen-binding domain that specifically binds to human TfR, and the third polynucleotide encodes a light chain, optionally wherein the first and second copies of the antigen-binding domains that bind to human TfR comprise the same amino acid sequence. In some aspects, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation. In some aspects, the ratio of the first, second, and third polynucleotides is about 1 :3:6. In some aspects, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

[0029] In some aspects, provided herein is a composition comprising a first polynucleotide and a second polynucleotide, wherein the first and second polynucleotides encode the multi-specific protein provided herein, wherein the first polynucleotide encodes a heavy chain and the antigen-binding domain that bind to human TfR, and wherein the second polynucleotide encodes a light chain.

[0030] In some aspects, provided herein is a complex comprising an antigen-binding domain provided herein and a polynucleic acid. In some aspects, the polynucleic acid is an ASO, an siRNA, or an RNAi agent. In some aspects, the polynucleic acid is an siRNA. In some aspects, the siRNA suppresses the product of a gene related to a neurological disease or disorder, optionally wherein the neurological disease or disorder is selected from Alzheimer's disease (AD), Huntington’s disease, dystonia, ataxia, Bell’s palsy, stroke, dementia, Lewy body 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, encephalitis, traumatic brain injury, and limbic-predominant age- related TDP-43 encephalopathy (LATE). In some aspects, the siRNA targets tau mRNA, alpha- synuclein mRNA, or NLRP3 mRNA. In some aspects, the antigen-binding domain comprises a VH and VL, wherein the VH or VL comprise an amino acid substitution of a residue to cysteine, wherein the cysteine is the site of attachment of the siRNA of the complex, and wherein the VH and VL comprise amino acid sequences at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 98% identical to the amino acid sequences of: SEQ ID NOs:46 and 47, respectively; SEQ ID NOs:46 and 48, respectively; SEQ ID NOs:50 and 48, respectively; SEQ ID NOs:49 and 48, respectively; SEQ ID NOs:49 and 51, respectively;SEQ ID NOs:49 and 52, respectively; SEQ ID NOs:49 and 53, respectively; SEQ ID NOs:49 and 54, respectively; SEQ ID NOs:55 and 48, respectively; SEQ ID NOs:56 and 57; respectively;SEQ ID NOs:58 and 59, respectively; SEQ ID NOs:56 and 60, respectively; SEQ ID NOs:56 and 61, respectively; SEQ ID NOs:58 and 62, respectively; SEQ ID NOs:85 and 86, respectively;SEQ ID NOs:85 and 87, respectively; SEQ ID NOs:88 and 87, respectively; SEQ ID NOs:89 and 87, respectively; SEQ ID NOs:88 and 90, respectively; SEQ ID NOs:88 and 91, respectively;SEQ ID NOs:88 and 92, respectively; SEQ ID NOs:88 and 93, respectively; SEQ ID NOs:94 and 87, respectively; SEQ ID NOs:95 and 96, respectively; SEQ ID NOs:97 and 98, respectively; SEQ ID NOs:95 and 99, respectively; SEQ ID NOs:95 and 100, respectively; SEQ ID NOs:97 and 101, respectively; SEQ ID NOs: 102 and 103, respectively; SEQ ID NOs: 102 and 104, respectively; SEQ ID NOs: 105 and 104, respectively; SEQ ID NOs: 106 and 104, respectively; SEQ ID NOs: 105 and 107, respectively; SEQ ID NOs: 105 and 108, respectively; SEQ ID NOs: 105 and 109, respectively; SEQ ID NOs: 105 and 110, respectively; SEQ ID NOs:l l l and104, respectively; SEQ ID NOs: 112 and 113, respectively; or SEQ ID NOs: 114 and 115, respectively.

[0031] In some aspects, provided herein is a host cell comprising a composition provided herein.

[0032] In some aspects, provided herein is an isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain of an antigen-binding domain provided herein.

[0033] In some aspects, provided herein is an isolated polynucleotide comprising a nucleic acid molecule encoding the light chain variable region of an antigen-binding domain provided herein.

[0034] In some aspects, provided herein is an isolated vector comprising a polynucleotide provided herein.

[0035] In some aspects, provided herein is an isolated vector comprising a nucleic acid molecule encoding the heavy chain variable region of an antigen-binding domain provided herein and a nucleic acid molecule encoding the light chain variable region of the antigen-binding domain.

[0036] In some aspects, provided herein is a host cell comprising a polynucleotide or a vector provided herein. In some aspects, the host cell is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, Rl. l, B-W, L-M, COS 1, COS 7, BSC1, BSC40, BMT10 cell, plant cell, insect cell, and human cell in tissue culture.

[0037] In some aspects, provided herein is a method of producing an antigen-binding domain or multi-specific protein comprising culturing a host cell provided herein so that the antigenbinding domain or multi-specific protein is produced, optionally wherein the method further comprises isolating the antigen-binding domain or multi-specific protein from the culture. In some aspects, provided herein is an isolated antigen-binding domain or multi-specific protein thereof produced by the method.

[0038] In some aspects, an antibody provided herein, a complex or fusion protein provided herein, or a multi-specific protein provided herein further comprises a cytotoxic drug. In some aspects, the cytotoxic drug is a microtubule disrupting agent, optionally wherein the microtubule disrupting agent is monomethyl auri statin E (MMAE).

[0039] In some aspects, provided herein is an antibody drug conjugate (ADC) comprising a drug and (i) an antigen-binding domain provided herein, (ii) an antibody provided here, (iii) a complex or fusion protein provided herein, or (iv) a multi-specific protein provided herein. In some aspects, the ADC binds to tissue factor (TF), human epidermal growth factor receptor(HER2), B7-H4, or Nectin-4. In some aspects, the ADC comprises a microtubule disrupting agent, optionally wherein the microtubule disrupting agent is monomethyl auristatin E (MMAE). In some aspects, the ADC comprises a version of tisotumab vedotin, disitamab vedotin, felmetatug vedotin, enfortumab vedotin, or trastuzumab deruxtecan with reduced effector function.

[0040] In some aspects, provided herein is a pharmaceutical composition comprising (i) an antibody provided herein or an antigen-binding fragment thereof, a complex or fusion protein provided herein, or a multi-specific protein provided herein, and (ii) a pharmaceutically acceptable carrier. In some aspects, the antibody or an antigen-binding fragment thereof, complex, fusion protein, or multi-specific protein is increased in the brain following administration to a subject as compared to an isotype control.

[0041] In some aspects, provided herein is a method of treating a neurological disease or disorder in a subject comprising administering an antibody provided herein or an antigen-binding fragment thereof, a complex or fusion protein provided herein, a multi-specific protein provided herein, or a pharmaceutical composition provided herein to the subject. In some aspects, administration increases delivery of the antibody or antigen-binding fragment thereof, complex or fusion protein, multi-specific protein, or pharmaceutical composition into the brain by at least 50%, at least 100%, at least 200%, at least 500% or at least 1000% as compared to an isotype control. In some aspects, administration increases delivery of the antibody or antigen-binding fragment thereof, complex or fusion protein, multi-specific protein, or pharmaceutical composition into the frontal cortex, the entorhinal cortex and / or the hippocampus. In some aspects, the neurological disease or disorder is selected from 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, the neurological disease or disorder is selected from Alzheimer's disease (AD), Huntington’s disease, dystonia, ataxia, Bell’s palsy, stroke, dementia, Lewy body 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, encephalitis, traumatic brain injury, and limbic- predominant age-related TDP-43 encephalopathy (LATE). In some aspects, the dementia is frontotemporal dementia (FTD). In some aspects, the neurological disease or disorder is Alzheimer’s disease. In some aspects, the Alzheimer's disease is early onset Alzheimer’s disease, prodromal Alzheimer’s disease, mild Alzheimer’s disease, or late onset Alzheimer’s disease. Insome 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.

[0042] In some aspects, provided herein is a method of treating a lysosomal storage disease in a subject comprising administering an antibody provided herein or an antigen-binding fragment thereof, a complex or fusion protein provided herein, a multi-specific protein provided herein, or a pharmaceutical composition provided herein to the subject. 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.

[0043] In some aspects, provided herein a method of transporting a fusion protein, antibody or an antigen-binding fragment thereof, or multi-specific protein across the BBB of a subject, comprising administering to the subject an antibody provided herein or an antigen-binding fragment thereof, a complex or fusion protein provided herein, a multi-specific protein provided herein, or a pharmaceutical composition provided herein. In some aspects, the concentration of the antibody or antigen-binding fragment thereof, complex or fusion protein, multi-specific protein, or pharmaceutical composition is increased in the brain following administration as compared to an isotype control. In some aspects, administration of antibody or antigen-binding fragment thereof, complex or fusion protein, multi-specific protein, or pharmaceutical composition does not result in reticulocyte count reduction in the subject, as compared to an isotype control.

[0044] In some aspects, provided herein is a method of imaging a CNS antigen within a subject, comprising administering to the subject an antibody provided herein or antigen-binding fragment thereof linked to an imaging agent, a complex or fusion protein provided herein linked to an imaging agent, or a multi-specific protein provided herein linked to an imaging agent and locating the imaging agent within the subject.

[0045] In some aspects, provided herein is a method of detecting a CNS antigen in vitro, comprising contacting an in vitro sample with an antibody provided herein or antigen-binding fragment thereof linked to an imaging agent, a complex or fusion protein provided herein linked to an imaging agent, or a multi-specific protein provided herein linked to an imaging agent and locating the imaging agent within the sample.

[0046] In some aspects, provided herein is a use of an antibody provided herein or the antigenbinding fragment thereof, a complex or fusion protein provided herein, a multi-specific proteinprovided herein, or a pharmaceutical composition provided herein, in a method of treating provided herein. In some aspects, provided herein is an antibody provided herein or an antigenbinding fragment thereof, a complex or fusion protein provided herein, a multi-specific protein provided herein, or a pharmaceutical composition provided herein for use in a method of treating provided herein.

[0047] It is to be understood that one, some, or all of the properties of the various aspects described herein can be combined to form other aspects of the present disclosure. These and other aspects of the disclosure will be immediately apparent to one of skill in the art. These and other aspects of the disclosure are further described by the detailed description that follows.BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG. 1 shows exemplary formats for complexes disclosed herein comprising an anti- TfR antigen-binding domain and a heterologous polypeptide, such as an enzyme. FIG. 1 includes examples of 1+1 formats ((i)-(iv)), 2+1 formats ((v)-(vi)), and 2+2 formats ((vii)-(viii)). The 1+1 format in FIG. 1 (i) shows an enzyme-Fc domain - anti-TfR scFv fusion protein. The 1+1 format in FIG. 1 (ii) shows an anti-TfR antigen-binding domain (Fv) - Fc domain - enzyme complex. The 1+1 format in FIG. 1 (iii) shows an anti-TfR antigen-binding domain (Fv) - Fc region (i.e. a Fc domain in a hole format and a second Fc domain in a knob format) - enzyme complex. The 1+1 format in FIG. 1 (iv) shows a complex comprising an enzyme linked to the N-terminal of a Fc domain in a knob format, and an anti TfR antigen-binding domain (Fv) linked to the N- terminal of a Fc domain in a hole format. The 2+1 format in FIG. 1 (v) shows a complex comprising an anti-TfR antigen-binding domain (Fv) linked to the N-terminal of a Fc domain in a knob format, an enzyme linked to the C-terminal of the Fc domain in the knob format, and an enzyme linked to the C-terminal of a Fc domain in a hole format. The 2+1 format in FIG. 1 (vi) shows a complex comprising an enzyme linked to the N-terminal of a Fc domain in a knob format, an enzyme linked to the N-terminal of a Fc domain in a hole format, and an anti-TfR scFv linked to the C-terminal of the Fc domain in the knob format. The 2+2 format in FIG. 1 (vii) shows a complex comprising an anti-TfR antibody and an enzyme linked to the C-terminal of both Fc domains of the anti-TfR antibody. The 2+2 format in FIG. 1 (viii) shows a complex comprising an enzyme linked to the N-terminal of a Fc domain in a knob format, an anti-TfR scFv linked to the C-terminal of the Fc domain in the knob format, an enzyme linked to the N-terminal of a Fc domain in a hole format, and an anti-TfR scFv linked to the C-terminal of the Fc domain in the hole format.

[0049] FIG. 2A shows exemplary formats for bispecific complexes disclosed herein. FIG. 2A (i) shows an exemplary 2+1 bispecific format; FIG. 2A (ii) shows an exemplary 1+1 format; and FIG. 2A (iii) shows another exemplary 1+1 format with two different VHH domains.

[0050] FIG. 2B shows a mvFc-scFv schematic for a bivalent and bispecific antibody. This mvFc-scFv antibody example contains one scFv that targets TfR and one heavy chain and light chain pair that bind to a different target. A mvFc-scFv could also contain one heavy chain and light chain pair that targets TfR an and scFv that binds to a different target.

[0051] FIG. 2C shows an example of a 2+1 bispecific antibody.

[0052] FIG. 2D shows an example of a 2+2 bispecific antibody.

[0053] FIG. 3A shows 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 different concentrations of antibody.

[0054] FIG. 3B shows nonspecific binding (BVP) of anti-TfR antibodies (42Q, 24 A, L-21, L- 6, 39.38, and L-19), negative control antibody, and positive control antibody at different concentrations of antibody.

[0055] FIG. 4A shows 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 10 pg / mL.

[0056] FIG. 4B shows 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.

[0057] FIG. 5 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.

[0058] FIGs. 6A and 6B 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. 6A) and fold-change over isotype control (FIG. 6B).

[0059] FIG. 7 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.

[0060] FIG. 8 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.

[0061] FIG. 9 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).

[0062] FIG. 10 shows GCase activity in a rescue assay in SH-SY5Y GBA knockout cells with various GCase-BBB-binding complexes (N-term monozyme WCAC hTfR15.WH8.1.42Q, N- term monozyme WCAC hTfR.9.1B.39.27.L35, and N-term monozyme WCAC hTfR15.WH8.L24A) and mFc_PD-WCAC.

[0063] FIG. 11 shows confocal microscopy images of SH-SY-5Y GBA knockout cells showing GCase-TfR (upper right), early endosomal marker (EEA-1; bottom left), late endosomal / lysosomal marker (LAMP-1; bottom right), and overlay of all markers (upper left).

[0064] FIG. 12 shows exemplary formats of bispecifics comprising (i) an antigen-binding domain that specifically binds to human TfR and (ii) rituximab with LALA-P331S mutation.

[0065] FIG. 13 shows the assessment of huTfR-huPGRN variants for binding to human sortilin using an ELISA.

[0066] FIG. 14 shows the evaluation of hematological parameters in huTfR-KI mice 24 hrs after administration of huTfR-huPGRN fusion proteins at various doses.

[0067] FIG. 15 shows the assessment of surface huTfR levels on blood reticulocytes in huTfR- KI mice 24 hrs after dosing with huTfR-huPGRN variants or isotype-control-huPGRN.

[0068] FIG. 16 shows the brain levels of huTfR-huPGRN or isotype-control-huPGRN detected in vessel-depleted brain lysates from huTfR-KI mice dosed once at various dose levels and collected 6 hrs or 24 hrs post-dose.

[0069] FIG. 17 shows TfR levels in whole brain lysates of mice dosed with huTfR-huPGRN fusion proteins. Tissues were collected at 6 hours or 24 hours post-dosing and analyzed by quantitative immunoblotting.

[0070] FIGs. 18A-18C show the serum pharmacokinetic profiles of human TfR-knock-in mice treated with GCase-TfR constructs for treatment groups Gl, G2, and G4 (FIG. 18A), groups G3 and G5 (FIG. 18B), and groups G1-G5 (FIG. 18C).

[0071] FIG. 19 shows vessel-depleted brain concentrations of GCase-TfR constructs in human-TfR knock-in mice for the indicated treatment groups.

[0072] FIGs. 20A-20B show absolute reticulocyte counts of human TfR knock-in mice in treatment groups G1-G6 at 24 hours (FIG. 20A) or 72 hours (FIG. 20B) post-injection. Asterisk (*) indicates statistically significant difference between G3 and G6.

[0073] FIG. 21 shows exemplary formats of complexes comprising an anti-TfR antigenbinding domain and a free cysteine as a conjugation site for a polynucleic acid. The approximate location of the free cysteine is indicated by a star.

[0074] FIG. 22 shows a human TfR apical domain sequence (SEQ ID NO:84) with selected residues identified for mutagenesis.

[0075] FIG. 23 shows the results of binding of anti-TfR antibodies L7-2, 24A, 39.38, and trontinemab to TfR apical domain variants normalized to the TfR apical domain parent (par) sequence.

[0076] FIG. 24 shows the structure of a human TfR complex with the approximate positions of binding epitopes of various anti-TfR antibodies indicated.

[0077] FIG. 25 shows the effect of anti-TfR antibodies L7-2, 39.38, and LIO-16 on levels of circulating reticulocytes and TfR expression in huTfR KI mice. Mice were administered 3 mg / kg, 10 mg / kg, or 30 mg / kg of Iso-TfR antibody variants (L7-2 hlgGl WT, LIO-16 hlgGl WT,39.38-hIgGl WT); levels of circulating reticulocytes and TfR expression levels were measured 1 day (DI) and 7 days (D7) post-dose. Levels of circulating reticulocytes are shown as percent reticulocytes.

[0078] FIG. 26 shows serum antibody levels in huTfR KI mice after administration of 3 mg / kg, 10 mg / kg, or 30 mg / kg of Iso-TfR antibody variants (L7-2 hlgGl WT, LIO-16 hlgGl WT, 39.38-hIgGl WT). Serum PK was measured 1 day (DI) and 7 days (D7) post-dose.

[0079] FIG. 27 shows serum antibody levels in huTfR KI mice administered 3 mg / kg of Remtemetug and TfR-Remternetug antibody variants (L7-2 hlgGl WT, LI 0-16 hlgGl WT,39.38-hIgGl WT). Mice were dosed twice, 14 days apart. Serum PK was measured at day 1 (DI), day 7 (D7), and day 14 (DI 4) after the first dose and 24h after the second dose (day 15; D15).

[0080] FIG. 28 shows brain uptake of antibodies in huTfR KI mice dosed with 3 mg / kg of Remtemetug and TfR-Remternetug antibody variants (L7-2 hlgGl WT, LI 0-16 hlgGl WT,39.38-hIgGl WT). Antibody concentration (ng / mg total protein) in vessel-depleted brain after 24 hours was measured.DETAILED DESCRIPTION OF THE PRESENT DISCLOSURE

[0081] The present disclosure relates to antigen-binding domains that specifically bind to human transferrin receptor (TfR), and complexes, fusion proteins, antibodies and antigen-binding fragments thereof comprising such antigen-binding domains, methods of making and using such antigen-binding domains, complexes, fusion protein, antibodies, and antigen-binding fragments thereof; pharmaceutical compositions comprising such antigen-binding domains, complexes, fusion proteins, antibodies, and antigen-binding fragments thereof; nucleic acids encoding such antigen-binding domains, antibodies, and antigen-binding fragments thereof; and host cells comprising nucleic acids encoding such antigen-binding domains, complexes, fusion proteins, antibodies, and antigen-binding fragments thereof.

[0082] Exemplary anti-TfR antigen-binding domains and fusion proteins, antibodies, and antigen-binding fragments thereof comprising such antigen-binding domains, methods of making the same, methods of measuring the affinity and activity of the same, and methods of using the same are provided in International Application Publication No. WO2024 / 026472, which is herein incorporated by reference.

[0083] 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, bi- specific format comprising (i) one antigen-binding domain that binds to human TfR and (ii) one antigen-binding domain that binds to a CNS antigen that is not TfR. Such a “1+1” format can also comprise an Fc domain or Fc region.

[0084] In some aspects, a complex of the present disclosure has a “1+1 format” comprises (i) an antigen-binding domain that binds to human TfR; and (ii) a protein or polypeptide useful for protein replacement therapy or an enzyme or fragment thereof useful for enzyme replacement therapy. Such a “1+1” format can also comprise an Fc domain or Fc region.

[0085] In some aspects, a multi-specific binding protein of the present disclosure with a “2+1 multi-specific binding protein format” comprises a trivalent, bi-specific format comprising (i) an antigen-binding domain that binds to human TfR, and (ii) an antibody comprising two antigenbinding domains that bind to a CNS antigen that is not TfR, wherein the antibody comprises two heavy chains and two light chains; wherein the antigen-binding domain that binds to human TfR is linked to the C-terminus of one of the two antibody heavy chains.

[0086] In some aspects, a multi-specific binding protein of the present disclosure with a “2+1 multi-specific binding protein format” comprises a trivalent, bi-specific format comprising (i) anantigen-binding domain that bind to a CNS antigen that is not TfR, and (ii) an antibody comprising two antigen-binding domains that binds to TfR, wherein the antibody comprises two heavy chains and two light chains; wherein the antigen-binding domain that binds to the CNS antigen that is not human TfR is linked to the C-terminus of one of the two antibody heavy chains.

[0087] In some aspects, a complex of the present disclosure has a “2+1 format” comprising (i) an antigen-binding domain that binds to human TfR, (ii) two copies of a protein or polypeptide useful for protein replacement therapy or an enzyme or fragment thereof useful for enzyme replacement therapy. Such a “2+1” format can also comprise an Fc domain or Fc region.

[0088] In some aspects, a multi-specific binding protein of the present disclosure with a “2+2 multi-specific binding protein format” comprises a tetravalent, bi-specific format comprising (i) two antigen-binding domains that bind to human TfR and (ii) an antibody comprising two antigen-binding domains that bind to a CNS antigen that is not TfR, 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 antigen-binding domain that binds to human TfR is linked to the C-terminus of the other of the two antibody heavy chains.

[0089] In some aspects, a multi-specific binding protein of the present disclosure with a “2+2 multi-specific binding protein format” comprises a tetravalent, bi-specific format comprising (i) two antigen-binding domains that bind to a CNS antigen that is not human TfR and (ii) an antibody comprising two antigen-binding domains that bind to human TfR, wherein the antibody comprises two heavy chains and two light chains; wherein one antigen-binding domain that binds to a CNS antigen that is not human TfR is linked to the C-terminus of one of the two antibody heavy chains, and the other antigen-binding domain that binds to a CNS antigen that is not human TfR is linked to the C-terminus of the other of the two antibody heavy chains.

[0090] In some aspects, a complex of the present disclosure has a “2+2” format comprising (i) two antigen-binding domains that bind to human TfR and (ii) two copies of a protein useful for protein replacement therapy or an enzyme or fragment thereof useful for enzyme replacement therapy. Such a “2+2” format can also comprise an Fc domain or Fc region.

[0091] In some aspects, a fusion protein or complex provided herein comprises (i) an antigenbinding domain that specifically binds to human TfR, (ii) a protein useful for protein replacement therapy or an enzyme or fragment thereof useful for enzyme replacement therapy, and (iii) an Fc portion. In some aspects, the antigen-binding domain that specifically binds to human TfR andthe protein / enzyme useful for replacement therapy are linked to the N-terminus of the Fc portion of the fusion protein or complex. In some aspects, the antigen-binding domain that specifically binds to human TfR and the protein / enzyme useful for replacement therapy are both linked to the C-terminus of the Fc portion of the fusion protein or complex. In other aspects, the antigenbinding domain that specifically binds to human TfR is linked to the N-terminus of the Fc portion and the protein / enzyme useful for replacement therapy is linked to the C-terminus of the Fc portion of the fusion protein or complex. In other aspects, the protein / enzyme useful for replacement therapy is linked to the N-terminus of the Fc portion of the fusion protein or complex and the antigen-binding domain that specifically binds to human TfR is linked to the C- terminus of the Fc portion.

[0092] In some aspects, disclosed herein is a fusion protein or complex comprising (i) a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human TfR, (ii) a Fc domain, and (iii) a single copy of a protein useful for protein replacement therapy or an enzyme or fragment thereof useful for enzyme replacement therapy, wherein the single scFv, VHH, or Fab antigen-binding domain that binds to human TfR is linked to the N-terminus of the Fc domain and the protein / enzyme useful for replacement therapy is linked to C-terminus of the Fc domain. In some aspects, the Fc is a single chain, engineered monovalent Fc. An example of this 1+1 format of a fusion protein or complex is shown as (ii) in Fig. 1. Such monovalent formats can have improved properties for purification and manufacturing.

[0093] In some aspects, disclosed herein is a fusion protein or complex comprising (i) a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human TfR, (ii) a Fc domain, and (iii) a single copy of a protein useful for protein replacement therapy or an enzyme or fragment thereof useful for enzyme replacement therapy, wherein the single scFv, VHH, or Fab antigen-binding domain that specifically binds to human TfR and the protein / enzyme useful for replacement therapy are both linked to the N-terminus of the Fc domain. An example of this 1+1 format of a fusion protein or complex is shown as (iv) in Fig. 1. Such N-terminal monozyme formats can result in improved serum PK (longer half-life).

[0094] In some aspects, disclosed herein is a complex comprising (i) an antigen-binding domain that specifically binds to human TfR and (ii) an polynucleic acid. In some aspects, disclosed herein is a complex comprising (i) an antigen-binding domain that specifically binds to human TfR and (ii) an RNAi agent. In some aspects, disclosed herein is a complex comprising (i) an antigen-binding domain that specifically binds to human TfR and (ii) an antisense oligonucleotide (ASO). In some aspects, disclosed herein is a complex comprising (i) an antigen-binding domain that specifically binds to human TfR and (ii) an siRNA. Exemplary formats are depicted in FIG. 21. The format shown on the left side of FIG. 21 (Format #1) is an anti-TfR antigen-binding domain (Fv) - Fc region (i.e., a Fc domain in a knob format and a second Fc domain in a hole format) with a free cysteine on the hole Fc domain for use as a conjugation site for the polynucleic acid (e.g., siRNA, ASO, or RNAi agent) of the complex. The format shown in the middle of FIG. 21 (Format #2) is a “2+1” format with the anti-TfR antigen-binding domain as an scFv on the C-terminus of a constant domain of a knob-in-hole IgG with inert Fabs and a free cysteine on the hole chain for use as a conjugation site for the polynucleic acid (e.g., siRNA, ASO, or RNAi agent). The format shown on the right of FIG. 21 (Format #3) is a “Fab-only” format comprising a free cysteine engineered on a variable domain of the anti-TfR antigenbinding domain without an Fc.

[0095] 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

[0096] The terms "central nervous system" and "CNS" refer to the complex of nerve tissues that control bodily function and includes the brain, spinal cord.

[0097] The terms "blood brain barrier" and "BBB" refer to a network of brain capillary endothelial cells that are closely sealed by tight junctions.

[0098] 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). 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.

[0099] A "central nervous system antigen" or "CNS antigen" is an antigen expressed in the CNS, including the brain, which can be targeted with an antibody or small molecule. Examples of such antigens include, without limitation: beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2),tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, [3-glucocerebrosidase (Gcase or GBA), progranulin (PGRN), Prosaposin (PSAP), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), interleukin 6 receptor (IL6R), TNF receptor 1 (TNFR1), interleukin 1 beta (IL1 |3), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), glycoprotein nonmetastatic melanoma protein B (GPNMB), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6A (MS4A6A), ubiquitin protein ligase E3A (UBE3A), or Transmembrane Protein 106B (TMEM106b).

[0100] A "brain antigen" is a CNS antigen expressed in the brain.

[0101] The terms "Transferrin receptor " "TfR," “TfR polypeptide f and “Z / R 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. In some aspects, the TfR is human TfR. As used herein, the term “ human TfR' refers to a polypeptide with the amino acid sequence of SEQ ID NO: 1.MMDQARS AF SNLFGGEPLS YTRF SLARQ VDGDNSHVEMKL AVDEEENADNNTKANVT KPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPA ARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFK LSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHA NFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELS FFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSD WKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPG AAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSL HLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVE KLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEV AGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFR ATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF (SEQ ID NO:1)

[0102] 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.

[0103] 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 Tf with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting TfR. In one aspect, the extent of binding of an anti-TfR antibody to an unrelated, non-TfR polypeptide is less than about 10% of the binding of the antibody to TfR as measured, e.g., by a radioimmunoassay (RIA). In certain aspects, 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). In certain aspects, an anti-TfR antibody binds to an epitope of TfR that is conserved among TfR from different species. In certain aspects, an anti-TfR antibody binds to an epitope of TfR comprising residue D356 of SEQ ID NO: 1.

[0104] 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, linearantibodies, 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 be monospecific 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.

[0105] 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.

[0106] 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 for targeting TfR 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-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 aspects, 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., 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- TfR antigen-binding domain binds to an epitope of TfR that is conserved among TfR from different species. In certain aspects, an anti-TfR antigen-binding domain binds to an epitope of TfR comprising residue D356 of SEQ ID NO: 1.

[0107] 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 an Fc 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 acidencoding 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.

[0108] “Native IgG antibodies’" are usually 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.

[0109] Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab"" fragments, and a residual “Fc"" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire light chain along with the variable region domain of the heavy chain (VH), and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, z.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the 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. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0110] The Fc fragment comprises the carboxy-terminal portions of both heavy chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells. [OHl] “Fv"" is the minimum antibody fragment which comprises a complete antigenrecognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanates six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0112] “Single-chain Fv ” also abbreviated as “sFv ” or “scFv ” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some aspects, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.

[0113] The term "diabodies" refers to small antibody fragments prepared by constructing scFv fragments (see preceding paragraph) 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, z.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.

[0114] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. The variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, 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, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in a sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with antigen. In some aspects, the variable region is a human variable region. In some aspects, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In some aspects, the variable region is a primate (e.g., non-human primate) variable region. In some aspects, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs). 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-bindingfragment thereof. In certain aspects, CDRs can be determined according 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 35A 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). In some aspects, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme. Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDRH1 loop, when numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35 A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). 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. In some aspects, the CDRs can be “contact” CDRs. The “contact” CDRs are based on an analysis of the available complex crystal structures. The residues from each of these CDRs are noted below.LOOD 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 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

[0115] 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.

[0116] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0117] 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.

[0118] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0119] 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 the constant regions. Light chain amino acid sequences are well known in the art. In some aspects, the light chain is a human light chain.

[0120] 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).

[0121] 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.

[0122] 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 antibodyfragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins or complexes ccomprising 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.

[0123] 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.

[0124] 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 humanizedantibodies 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.

[0125] 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.

[0126] “Framework' or “FR' residues are those variable-domain residues other than the CDR residues as herein defined.

[0127] 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. In some aspects, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0128] 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.

[0129] 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 residuemeans 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.

[0130] 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.

[0131] As used herein a “Fc region” or “fragment crystallizable 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 domains. 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, a Fc region may not contain any K447 residues, may contain at least one polypeptide containing 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 compliment system. 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] “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.

[0136] 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), 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 a protein useful for protein replacement therapy or an enzyme useful for enzyme replacement therapy, an antigen-bindingdomain, antibody or antigen-binding fragment thereof that binds to a CNS antigen, an antigenbinding domain than specifically binds to TfR, an Fc region, and / or an Fc domain. Accordingly, non-limiting examples of a “complex” comprising an antigen-binding domain and a protein or enzyme useful for replacement therapy include (a) a fusion protein comprising the antigenbinding domain and the protein or enzyme useful for replacement therapy in a single polypeptide chain (e.g., as shown in Figure l(i)), and (b) three proteins connected via noncovalent proteinprotein interactions, wherein the first protein contains the protein or enzyme useful for replacement therapy and an Fc domain, the second protein contains a VH of the antigen-binding domain, and the third protein contains a VL of the antigen-binding domain (e.g., as shown in Figure l(iv)). Formats of other exemplary complexes are provided in Figures 1 and 2.

[0137] "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 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 kon / koff. 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), stoppedflow analysis, and colorimetric or fluorescent protein melting analyses. (See, e.g., Estep et al, (2013) AL4Z> 5(2):270-8.)

[0138] 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 a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of amolecule 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 to" 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.

[0139] 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:2). 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, wherein N is any amino acid (SEQ ID NO:3). A glycine-serine linker is one which contains both glycine and serine in any proportion, e.g., GGGS (SEQ ID NO:4). 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.

[0140] 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 conservative substitutions 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 orMEGALIGN™ (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.

[0141] The term "epitope" includes any determinant capable of being bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets that antigen, and when the antigen is a polypeptide, includes specific amino acids that directly contact the antibody. Most often, epitopes reside on polypeptides, but in some instances, can reside on other kinds of molecules, such as nucleic acids. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics, and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of polypeptides and / or macromolecules.

[0142] An antibody that "binds to the same epitope" as a reference antibody refers to an antibody that contacts the same amino acid residues on the antigen as the reference antibody. The ability of an antibody to bind to the same epitope as a reference antibody can be determined using peptide scanning mutagenesis or high throughput alanine scanning mutagenesis. In the latter methodology, a comprehensive mutation library of antigen, or a portion thereof (e.g., the extracellular domain), can be generated by mutating each individual amino acid residue to alanine (or if the amino acid residue is alanine, then to another residue such as serine) and testing each mutant for binding to a target antibody or antigen-binding fragment thereof.

[0143] An antibody is said to "competitively inhibit" binding of a reference antibody to a given epitope if it preferentially binds to that epitope or an overlapping epitope such that it blocks, to some degree, binding of the reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays. An antibody can be said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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 techniques are 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.

[0148] “Polynucleotide,” “polynucleic acid,” 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.

[0149] 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 invention. In some aspects, the host cell is an isolated host cell.

[0150] “ 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.

[0151] As used herein, the term ’’treatmen ' 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.

[0152] 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.

[0153] 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 effect of 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.

[0154] 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.

[0155] 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.

[0156] 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 or microbial infection, inflammation, ischemia, neurodegenerative disease, seizure, behavioral disorders, and a lysosomal storage disease.

[0157] A “Lysosomal storage disorder" or (LSD) as used herein refers to an 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.

[0158] “Protein replacement therapy” or “PRT” refers to a medical treatment that supplements or replaces a protein in a patient in whom that particular protein is deficient or absent.

[0159] An "enzyme replacement therapy enzyme" or "ERT enzyme" refers to an enzyme that is deficient in a lysosomal storage disorder. An "ERT enzyme variant" refers to a functional variant, including allelic and splice variants, of a wild-type ERT enzyme or a fragment thereof, where the ERT enzyme variant has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding wild-type ERT enzyme or fragment thereof, e.g., when assayed under identical conditions. A "catalytically active fragment" of an ERT enzyme refers to a portion of a full-length ERT enzyme or a variant thereof, where the catalytically active fragment has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full-length ERT enzyme or variant thereof, e.g., when assayed under identical conditions.

[0160] 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.

[0161] 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.

[0162] As used herein, a molecule that "targets" another molecule refers to a molecule capable of specifically interacting with the other molecule or with a polynucleic acid encoding the other molecule. Unless indicated otherwise, the specific interaction can occur under physiological conditions. For example, an antibody or antigen-binding fragment thereof that "targets" a molecule, e.g., a protein, is capable of specifically binding to that molecule (e.g., protein). A polynucleic acid (e.g., siRNA) that "targets" another polynucleotide (e.g., mRNA or miRNA) can hybridize to that polynucleotide or a complementary sequence thereof. A polynucleotide (e.g., siRNA) that "targets" a protein can hybridize to a polynucleotide encoding that protein or a complementary sequence thereof.

[0163] 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 presence of 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.Anti-TfR Antigen-Binding Domains

[0164] Provided herein are antigen-binding domains that specifically bind to human TfR.

[0165] 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 BBB. Accordingly, in some aspects, provided herein are antigen-binding domains that specifically bind to human TfR that are capable of being internalized in BBB epithelial cells such as HCMEC / D3 cells.

[0166] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the six CDRs of an antibody listed in Tables 4 and 5 (i.e., the three VH CDRs of the antibody listed in Table 4 and the three VL CDRs of the same antibody listed in Table 5).

[0167] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the six CDRs of an antibody listed in Table 4, 5, and / or 6. In some aspects, the CDRs of such an antigen-binding domain can be determined according to the Chothia numbering scheme, which refers to the location of immunoglobulin 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). Typically, when using the Kabat numbering convention, the Chothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56, and the Chothia CDR- H3 loop is present at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop is present at light chain amino acids 24 to 34, the Chothia CDR-L2 loop is present at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97. The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).

[0168] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the six Chothia CDRs of an antibody listed in Table 4, 5, and / or 6. In some aspects, such as an antigen-binding domain that specifically binds to human TfR comprises one or more CDRs, in which the Chothia and Kabat CDRs have the same amino acid sequence. In some aspects, provided herein are antigen-binding domains that specifically binds to human TfR and comprise combinations of Kabat CDRs and Chothia CDRs.

[0169] 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 Table 4, 5, and / or 6 as determined by the method in MacCallum RM et al.

[0170] 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 Chothia structural 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 Table 4, 5, and / or 6 as determined by the AbM numbering scheme.

[0171] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the six IMGT CDRs of an antibody listed in Table 4, 5, and / or 6 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.

[0172] 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 aspecific 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.

[0173] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the VH of an antibody listed in Table 6.

[0174] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the VL of antibody listed in Table 6.

[0175] 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 6 (i.e., the VH of the antibody listed in Table 6 and the VL of the same antibody listed in Table 6).

[0176] 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 in Table 6 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 6. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 4 and 5 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0177] 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 6 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 6. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 4 and 5 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0178] 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 6 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 6. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 4 and 5 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0179] 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 VHamino acid sequence of an antibody in Table 6 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 6. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 4 and 5 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0180] 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 6 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 6. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 4 and 5 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0181] 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 6 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 6. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 4 and 5 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0182] 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 6 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 6. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 4 and 5 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0183] 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 6 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 6. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 4 and 5 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0184] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is identical to a VH amino acid sequence of an antibody in Table 6 and (ii) a VL comprising an amino acid sequence that is identical to the VL amino acid sequence of the same antibody in Table 6. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 4 and 5 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0185] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is identical to a VH amino acid sequence of an antibody in Table 6, except for a single cysteine substitution and (ii) a VL comprising an amino acid sequence that is identical to the VL amino acid sequence of the same antibody in Table 6, except for a single cysteine substitution. In some aspects, the antigenbinding domain that specifically binds to human TfR comprises a VH and VL comprising an amino acid sequence from Table 6 with no more than 2 amino acid substitutition, no more than 5 amino acid substitutions, or no more than 10 amino acid substitutions. In some aspects, the substitutions would not be in the CDR sequences.

[0186] In some aspects, provided herein is an antigen-binding domain that binds to the same TfR epitope as an antibody comprising a VH amino acid sequence of an antibody in Table 6 and a VL amino acid sequence of the same antibody in Table 6. In some aspects, the epitope comprises residue D356 of SEQ ID NO: 1.

[0187] 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 6 and a VL amino acid sequence of the same antibody in Table 6.

[0188] 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 6). 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 can comprisethe amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO:5). Such a linker can comprise the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:6).

[0189] 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).

[0190] 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).

[0191] 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

[0192] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR also binds to cynomolgus monkey TfR.

[0193] In certain aspects, an antibody that binds to TfR has a dissociation constant (KD) of about 0.01 nM to about 50 nM, about 51 nM to about 750 nM, about 751 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 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).

[0194] 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: 27, 28, 29, 39, 40, and 41, respectively.

[0195] In some aspects, the anti-TfR antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 56 and 57, respectively.

[0196] 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: 22, 23, 25, 35, 32, and 33, respectively.

[0197] In some aspects, the anti-TfR antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 49 and 51, respectively.

[0198] 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: 22, 23, 24, 31, 32, and 33, respectively.

[0199] In some aspects, the anti-TfR antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 46 and 47, respectively.

[0200] 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: 22, 23, 25, 38, 32, and 33, respectively.

[0201] In some aspects, the anti-TfR antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 49 and 54, respectively.

[0202] In some aspects, an antigen-binding domain provided herein specifically binds to human TfR 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) of about 51 nM to about 750 nM (e.g., about 100 nM to about 750 nM or about 100 nM to about 700 nM), or about 751 nM to about 10,000 nM (e.g., about 1000 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.

[0203] 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 (e.g., 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 (e.g., about 100 nM to about 1250 nM or about 150 nM to about 1250 nM), or about 1251 nM to about 10,000 nM (e.g., 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.

[0204] 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 (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) and cynomolgus TfR with an affinity of about0.01 nM to about 50 nM (e.g., 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 (e.g., 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 (e.g., 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 (e.g., about 1000 nM to about 5000 nM) and cynomolgus TfR with an affinity of about 1251 nM to about 10,000 nM (e.g., 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.

[0205] In some aspects, an antigen-binding domain provided herein specifically binds to human TfR with an affinity from about 0.01 nM to about 50 nM, such as about 0.01 nM to about 1 nM, about 0.01 nM to about 5 nM, about 0.01 nM to about 25 nM, about 1 nM to about 5 nM, about 1 nM to about 25 nM, about 1 nM to about 50 nM, about 5 nM to about 25 nM, about 5 nM to about 50 nM, about 10 nM to about 25 nM, about 10 nM to about 50 nM, about 25 nM to about 50 nM, and values and ranges there between.

[0206] In some aspects, an antigen-binding domain provided herein specifically binds to human TfR with an affinity from about 51 nM to about 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 there between.

[0207] In some aspects, an antigen-binding domain provided herein specifically binds to human TfR with an affinity from 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.

[0208] In some aspects, an antigen-binding domain provided herein that binds to human TfR with moderate to high affinity (e.g., 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.

[0209] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR does not significantly reduce TfR as measured in whole brain lysates (e.g., as measured according to the assay in Example 8).

[0210] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR accumulates at least about 4-fold or at least about 5-fold more than an isotype control in vessel-depleted human TfR knock-in mouse brain after peripheral injection (e.g., as measured using the assay in Example 6).

[0211] Also provided herein are antigen-binding domains that bind to the same epitope of TfR as a TfR antigen-binding domain provided herein. In some aspects, the epitope of TfR comprises residue D356 of SEQ ID NO: 1. Also provided herein are antigen-binding domains that competitively inhibit binding to TfR of TfR antigen-binding domain provided herein.Agents Comprising Anti-TfR Antigen-Binding Domains

[0212] Provided herein are agents (e.g., fusion proteins, complexes, multi-specific (e.g., bispecific) proteins, antibodies, antigen-binding fragments thereof, etc.) comprising an antigenbinding domain that specifically binds to human TfR.

[0213] The present antigen-binding domains may be used, in various aspects, to form complexes (such as fusion proteins, multi-specific proteins, and conjugates) with cargo molecules, including enzymes for enzyme replacement therapy, antibodies or antibody fragments thereof to form multi-specific proteins, neurotrophic factors, proteins or peptides, gene therapy vectors such as adeno-associated virus vectors, lentiviral vectors, adenoviral vectors, or non-viral vectors, small molecule drugs, polynucleotides, or nanoparticles or liposomes loaded with a therapeutic agent.Agents Comprising Anti-TfR Antigen-Binding Domains and Polynucleic Acids

[0214] In some aspects, the agent comprises a complex comprising an antigen-binding domain that specifically binds to human TfR and a polynucleic acid. In some aspects, the polynucleic acid comprises RNA and / or DNA nucleic acid molecules. In some aspects, the polynucleic acid comprises chemically modified nucleic acid molecules (RNA and / or DNA). In some aspects, the polynucleic acid comprises unmodified nucleic acid molecules (RNA and / or DNA). In some aspects, the polynucleic acid can be an antisense oligonucleotide (ASO), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA, a DNA / RNA hybrid, or one or more CRISPR components. In some aspects, the polynucleic acid is single stranded (e.g., ASO). In some aspects, the polynucleic acid is double stranded (e.g., siRNA). In some aspects, the polynucleic acid is 14-30 nucleotides in length, 20-24 nucleotides in length, or 19-21 nucleotides in length.

[0215] In some aspects, the complex comprises an antigen-binding domain that specifically binds to human TfR and a polynucleic acid, such as an siRNA. The TfR-targeting antigenbinding domain can then be used to deliver the polynucleic acid, such as an siRNA, across the blood-brain barrier wherein the polynucleic acid, such as an siRNA, can be released into, for example, brain cells. In some aspects, when the polynucleic acid is an siRNA, release into cells will achieve targeted gene silencing.

[0216] The polynucleic acid, such as an siRNA, can be conjugated to the anti-TfR antigenbinding domain through direct or indirect methods using any suitable techniques known in the art. These methods include, but are not limited to, bi-functional cross-linking, such as chemical cross-linkers or click chemistry, thiol-mal eimide chemistry, disulfide bond formation, enzyme- mediated conjugation, streptavidin-biotin interaction, genetic fusion, such as inclusion of a sequence which can hybridize with a sequence on the siRNA, or any other suitable means. In some aspects, any of the anti-TfR antigen-binding domains or other sequences fused to or complexed with the anti-TfR antigen-binding domain disclosed herein are modified to comprise a free cysteine suitable for use as a conjugation site for a polynucleic acid (such as an siRNA), allowing for the efficient attachment and functional integration of the polynucleic acid (such as an siRNA) with the antigen-binding domain. In some aspects, modifications to the anti-TfR antigen-binding domain can be made to enhance the stability, efficacy, or specificity of the conjugation or to improve the pharmacokinetic properties of the conjugated molecule.Furthermore, in some aspects, the complex may comprise a spacer molecule or linkers with varying lengths and chemistries between the antigen-binding domain and the polynucleic acid (such as an siRNA) molecule, which can be optimized to maintain the functional activity of both the polynucleic acid (such as an siRNA) and the antigen-binding domain.

[0217] Exemplary formats for a complex comprising (i) an antigen-binding domain that specifically binds to human TfR and (ii) a polynucleic acid are illustrated in FIG. 21. In some aspects, the polynucleic acid comprises an siRNA. Format #1 of FIG. 21 shows a format comprising a knob-in-hole with a single anti-TfR Fab and a free cysteine on the hole chain for use as a conjugation site for the polynucleic acid. Format #2 of FIG. 21 shows a “2+1” format with the TfR antigen-binding domain as an scFv on the C-terminus of a constant domain of a knob-in-hole IgG with inert Fabs, and a free cysteine on the hole chain for use as a conjugation site for the polynucleic acid. Format #3 of FIG. 21 shows a “Fab-only” format comprising a free cysteine engineered directly on a variable chain of a TfR antigen-binding domain without Fc. The polynucleic acid is conjugated at the free cysteine of the Fab. Any other suitable formatscomprising a complex comprising any of the anti-TfR antigen-binding domains disclosed herein and a polynucleic acid, such as an siRNA, are contemplated, including any suitable means of conjugating the polynucleic acid, directly or indirectly, to the anti-TfR antigen-binding domain.

[0218] In any of the aspects disclosed herein, wherein a complex comprising (i) an antigenbinding domain that specifically binds to human TfR and (ii) a polynucleic acid, such as an siRNA, is provided, the antigen-binding domain that specifically binds to human TfR may be any anti-TfR domain disclosed herein comprising an amino acid substitution of a residue with a cysteine, wherein the cysteine is capable of functioning as a conjugation site for the polynucleic acid, such as an siRNA. In some aspects, the anti-TfR antigen-binding domain is associated with a constant domain comprising an amino acid substitution of a residue with a cysteine, wherein the cysteine is capable of functioning as a conjugation site for the polynucleic acid, such as an siRNA. Exemplary conjugation strategies are depicted in the formats of FIG. 21.

[0219] In some aspects, a complex provided herein comprises (i) an antigen-binding domain that specifically binds to human TfR and (ii) a polynucleic acid. In some aspects, a complex provided herein comprises (i) an antigen-binding domain that specifically binds to human TfR and (ii) a small interfering RNA (siRNA). In some aspects, a complex provided herein comprises (i) an antigen-binding domain that specifically binds to human TfR and (ii) an siRNA that suppresses the product of a gene related to a neurological disease or disorder. In some aspects, the neurological disease or disorder is selected from Alzheimer's disease (AD), Huntington’s disease, dystonia, ataxia, Bell’s palsy, stroke, dementia, Lewy body 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, encephalitis, traumatic brain injury, and limbic-predominant age-related TDP-43 encephalopathy (LATE).

[0220] In some aspects, a complex provided herein comprises (i) an antigen-binding domain that specifically binds to human TfR and (ii) an siRNA that suppresses the product of a gene related to amyotrophic lateral sclerosis, such as superoxide dismutate 1 (SOD1).

[0221] In some aspects, a complex provided herein comprises (i) an antigen-binding domain that specifically binds to human TfR and (ii) an siRNA that suppresses the product of a gene related to duplication syndrome, such as methyl CpG binding protein 2 (MECP2).

[0222] In some aspects, a complex provided herein comprises (i) an antigen-binding domain that specifically binds to human TfR and (ii) an siRNA that suppresses the product of a gene related to Alzheimer’s disease, such as beta-secretase-antisense transcript (BACE1-AS), cyclindependent kinase 5 (Cdk5), amyloid precursor protein (APP), tau, voltage-dependent anionselective channel 1 (VDAC-1), beta-secretase 1 (BACE1), presenilin-1 (PS-1), Rho-associated protein kinase II (ROCK-II), a mutant form of presenilinl (such as L392V PS-1), inhibitor 2 of protein phosphatase 2A (12 PP-2A), acetyl-CoA acetyltransferase 1 (ACAT-1), or Nogo receptor.

[0223] In some aspects, a complex provided herein comprises (i) an antigen-binding domain that specifically binds to human TfR and (ii) an siRNA that suppresses the product of a gene related to Parkinson’s disease, such as alpha-synuclein.

[0224] In some aspects, a complex provided herein comprises (i) an antigen-binding domain that specifically binds to human TfR and (ii) an siRNA that suppresses the product of a gene related to Huntington’s disease, such as huntingtin protein (Htt gene product).

[0225] In some aspects, a complex provided herein comprises (i) an antigen-binding domain that specifically binds to human TfR and (ii) an siRNA that suppresses the product of a gene associated with the genetic aspect of spinal cord injury, such as glial fibrillary acid protein (GFAP), vimentin, ephrin B (EphB3), inducible nitric oxide synthase (iNOS), nischarin, or RhoA.

[0226] In some aspects, a complex provided herein comprises (i) an antigen-binding domain that specifically binds to human TfR and (ii) an siRNA that suppresses the product of a gene related to multiple sclerosis, such as T-box transcription factor (T-bet), Notch homolog 1 translocation-associated (Notchl), LINGO-1, nuclear receptor subfamily 4 group A member 2 (NR4A2), TIR-domain-containing adapter-inducing interferon-P (TRIF), caspase-2, or calcium / calmodulin dependent protein kinase II (CaMKII).

[0227] In some aspects, a complex provided herein comprises (i) an antigen-binding domain that specifically binds to human TfR and (ii) an siRNA that targets a miR33. In some aspects, a complex provided herein comprises (i) an antigen-binding domain that specifically binds to human TfR and (ii) an siRNA that targets USP30. In some aspects, a complex provided herein comprises (i) an antigen-binding domain that specifically binds to human TfR and (ii) an siRNA that targets Bini. In some aspects, a complex provided herein comprises (i) an antigen-binding domain that specifically binds to human TfR and (ii) an siRNA that targets TORC1.

[0228] In some aspects, a complex provided herein comprises (i) an antigen-binding domain that specifically binds to human TfR and (ii) an siRNA that targets NLR family pyrin domain containing 3 (NLRP3).

[0229] In some aspects, a complex provided herein comprises (i) an antigen-binding domain that specifically binds to human TfR and (ii) an siRNA that targets tau (such as an siRNA that targets tau mRNA).Fusion Proteins

[0230] In some aspects, a complex or fusion protein provided herein comprises: an antigenbinding domain that specifically binds to human TfR and a heterologous protein or polypeptide. In some aspects, the heterologous protein is a protein or polypeptide or fragment thereof useful in protein replacement therapy (PRT). In some aspects, the heterologous polypeptide is an enzyme (e.g., an enzyme for use in enzyme replacement therapy (ERT)) or a catalytically active fragment thereof. In some aspects, the heterologous polypeptide is an ERT enzyme or an ERT enzyme variant, or a catalytically active fragment thereof.

[0231] In some aspects, provided herein is a multi-specific protein comprising the amino acid sequences of SEQ ID NOs:65, 66, and 67. In some aspects, provided herein is a multi-specific protein comprising the amino acid sequences of SEQ ID NOs:65, 68, and 69. In some aspects, provided herein is a multi-specific protein comprising the amino acid sequences of SEQ ID NOs:65, 68, and 70. In some aspects, provided herein is a multi-specific protein comprising the amino acid sequences of SEQ ID NOs:65, 68, and 71. In some aspects, provided herein is a multi-specific protein comprising the amino acid sequences of SEQ ID NOs:65, 72, and 73.

[0232] In some aspects, the heterologous polypeptide in a complex or fusion protein provided herein is a growth factor. In some aspects, the heterologous polypeptide in complex or a fusion protein provided herein is a decoy receptor. In some aspects, the heterologous polypeptide in a complex or fusion protein provided herein is P-glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), clusterin (APOJ), Reelin, Tripeptidyl Peptidase 1 (CLN2 / TPP1), Alpha-L-Iduronidase (IDUA), Iduronate 2-Sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), N-acetyl-alpha- glucosaminidase (NAGLU), N-sulfoglucosamine sulfohydrolase (SGSH), ubiquitin protein ligase E3 A (UBE3 A), or a variant thereof, or a catalytically active fragment thereof.

[0233] In some aspects, the heterologous polypeptide in a complex or fusion protein provided herein is very low density lipoprotein receptor (VLDLR) or apolipoprotein E receptor 2 (APOER2; also known as low-density lipoprotein receptor-related protein 8 (LRP8)), or a variant thereof, or a catalytically active fragment thereof.

[0234] In some aspects, the heterologous protein in the fusion protein is N-terminal to the antigen-binding domain that specifically binds to human TfR. In some aspects, the heterologous protein or polypeptide in the fusion protein is C-terminal to the antigen-binding domain that specifically binds to human TfR. In some aspects, the heterologous protein or polypeptide and the antigen-binding domain that specifically binds to human TfR are directly connected via a peptide bond. In some aspects, the heterologous fusion protein and the antigen-binding domain that specifically binds to human TfR are connected via a linker, e.g., a peptide linker. In some aspects, the fusion protein comprises an antigen-binding domain and a heterologous protein or polypeptide and an Fc portion. In some aspects, the antigen-binding domain and the heterologous protein or polypeptide are linked to the N-terminus of the Fc portion of the fusion protein. In other aspects, the antigen-binding domain is linked to the N-terminus of the Fc portion and the heterologous protein or polypeptide is linked to the C-terminus of the Fc portion of the fusion protein. In other aspects, the antigen-binding domain is linked to the C-terminus of the Fc portion and the heterologous protein or polypeptide is linked to the N-terminus of the Fc portion of the fusion protein.Bispecific and Multi-specific Proteins

[0235] 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 and an antigen-binding domain that specifically binds to a CNS antigen or a brain antigen. In some aspects, the CNS antigen or brain antigen is not TfR. 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 antigenbinding domain provided herein.

[0236] In some aspects, a multi-specific protein provided herein comprises a first antigenbinding domain that binds to human TfR and a second antigen-binding domain. The first antigen-binding domain that binds to human TfR can be any antigen-binding domain that binds to human TfR provided herein (such as an antibody, Fab, scFv, or VHH). The second antigenbinding domain can be an antigen-binding domain that specifically binds to a CNS antigen or a brain antigen. In some aspects, the CNS antigen or brain antigen is not TfR.

[0237] In some aspects, a multi-specific protein provided herein comprises an antigen-binding domain that binds to human TfR linked to an antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof can bind a CNS antigen or brain antigen. In some aspects, the CNS antigen or brain antigen is not TfR. In some aspects, such a multi-specific protein can be in a 2+1 multi-specific binding format or a 2+2 multi-specific binding format.

[0238] In some aspects, a multi-specific protein provided herein comprises a TfR antigenbinding domain that is an scFv linked to an antibody that binds to a CNS antigen, 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.

[0239] In some aspects, a multi-specific protein provided herein comprises an anti-TfR antigen-binding domain that is a Fab linked to an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains. In some aspects, the Fab is linked to the C-terminus of one of the two antibody heavy chains, e.g., via a protein linker.

[0240] In some aspects, the multi-specific protein comprises 1) an antigen-binding domain that binds TfR, 2) a second antigen-binding domain that binds a different CNS or brain antigen, 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 protein. In other aspects, the multispecific protein comprises 1) an antigen-binding domain that comprises a heavy chain variable region and binds TfR, 2) a second antigen binding domain that comprises a heavy-chain variable region and binds a different CNS or brain antigen, and 3) an Fc region, wherein the TfR antigenbinding domain and the second antigen-binding domain are connected or linked to the Fc region of the multi-specific protein. In some aspects, the multi-specific protein comprises an antigenbinding domain that binds TfR, a second antigen-binding domain that binds a different CNS or brain antigen, and an Fc region. In some aspects, the TfR antigen-binding domain and the second antigen-binding domain are connected or linked to the N-terminus of the Fc portion of the multispecific protein. In other aspects, the TfR antigen-binding domain is connected or linked to the N-terminus of an Fc portion of the multi-specific 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 TfR antigen-binding domain 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.

[0241] In some aspects, a multi-specific protein provided herein comprises two copies of a TfR antigen-binding domain that is an scFv and an antibody that binds to a CNS antigen, wherein theantibody comprises two heavy chains and two light chains, wherein one of the two copies of the antigen-binding domain is linked to the C-terminus of one of the antibody heavy chains, and wherein the other copy of the antigen-binding domain is linked to the C-terminus of the other antibody heavy chain. In some aspects, the scFvs are linked to the heavy chains via a protein linker.

[0242] In some aspects, a multi-specific protein provided herein comprises two copies of a TfR antigen-binding domain that is a Fab and an antibody that binds to a CNS antigen, wherein the antibody comprises two heavy chains and two light chains, wherein one of the two copies of the antigen-binding domain is linked to the C-terminus of one of the antibody heavy chains, and wherein the other copy of the antigen-binding domain is linked to the C-terminus of the other antibody heavy chain. In some aspects, the Fabs are linked to the heavy chains via a protein linker.

[0243] As provided herein, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific 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 invention is not limited to any particular bispecific format or method of producing it. Accordingly, bispecific proteins 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, e.g., that binds to a CNS antigen or a brain antigen.

[0244] Examples of bispecific molecules that can be used in the present disclosure include, e.g., (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 trivalentbispecific 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 WO20 19 / 246288, which is incorporated by reference.

[0245] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof or multi-specific protein provided herein is multivalent (e.g., bivalent). In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein is trivalent (e.g., in the 2+1 format or a 2+1 multi-specific binding format). In some aspects, a trivalent format comprises a single TfR antigen-binding domain provided herein and two antigen-binding domains that bind to a CNS antigen or a brain antigen. 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.

[0246] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein is tetravalent (e.g., in the 2+2 format or the 2+2 multi-specific binding format). In some aspects, a tetravalent format comprises two TfR antigenbinding 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 antigenbinding domains comprise the same amino acid sequence. In some aspects, one or both of the TfR antigen-binding domains is an scFv. In some aspects, one or both of the anti-TfR antigenbinding domains is a Fab. 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.

[0247] A complex, fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein can comprise a linker, e.g., linking an anti-TfR antigen-binding domain to a heterologous protein, antibody or antigen-binding fragment thereof, or other antigenbinding domain. 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:7). The linker can comprise the amino acid sequence (GGSGG)x3 (SEQ ID NO:8). The linker can comprise the amino acid sequence GGSGG (no repeats) (SEQ ID NO:9). The linker can be 1 to 20 amino acids in length.

[0248] Engagement of TfR with antibodies has been associated previously with acute clinical signals and a decrease in circulating reticulocyte count. Fc engineering, such as reducing the Fc effector function, and tuning of the anti-TfR affinity, to efficiently transport cargo across the blood-brain barrier while minimizing off-target effects, can ameliorate these liabilities. Effectorless Fc domains and TfR antigen-binding domains spanning various affinities are disclosed herein accordingly. In addition, without being bound to theory, when the TfR antigenbinding domain is part of a complex, multi-specific protein, or fusion protein, the spatial distance and rigidity of the link between the TfR antigen-binding domain and the protein that it is fused to (such as a heterologous protein, an antibody or antigen-binding fragment thereof, or another antigen-binding domain) may be tuned to further reduce the potential for inducing deleterious side effects, such as engagement of Fc gamma receptors, which may lead to ADCC and / or CDC, such as when the complex is engaged to TfR on reticulocytes. Increasing rigidity of the molecule may prevent simultaneous binding to TfR (such as TfR on reticulocytes) and Fc gamma receptors, which may be further promoted by the particular binding epitope of the anti-TfR antigen-binding domain. See Examples 16-17 herein.

[0249] In some aspects, in a complex, such as a multi-specific protein or fusion protein, comprising an antigen-binding domain that specifically targets TfR, the link between an anti-TfR antigen-binding domain and the protein that it is fused to, such as a heterologous protein, an antibody or antigen-binding fragment thereof, or another antigen-binding domain, may be a short amino acid linker, such as less than 10 amino acids, less than 9 amino acids, less than 8 amino acids, less than 7 amino acids, less than 6 amino acids, less than 5 amino acids, less than 4 amino acids, from 2-9 amino acids, from 2-8 amino acids, from 2-7 amino acids, from 2-6 amino acids, or from 2-5 amino acids. In some aspects, the short linker is GGSGG (SEQ ID NO:9).

[0250] In some aspects, to further increase stability and / or rigidity of the complex, such as a fusion protein or multi-speicfic protein, the anti-TfR antigen-binding domain is directly linked to the heterologous protein, antibody or antigen-binding fragment thereof, or another antigenbinding domain. For example, in some aspects, the N-terminus of the VH of an anti-TfR antigenbinding domain disclosed herein is fused directly to the C-terminus of a heterologous protein, antibody or antigen-binding fragment thereof, or another antigen-binding domain. In some aspects, the N-terminus of the VH of an anti-TfR antigen-binding domain disclosed herein is fused directly to the C-terminus of an Fc domain. In some aspects, the C-terminus of the VH of an anti-TfR antigen-binding domain disclosed herein is fused directly to the N-terminus of a heterologous protein, antibody or antigen-binding fragment thereof, or another antigen-bindingdomain. In some aspects, the C-terminus of the VH of an anti-TfR antigen-binding domain disclosed herein is fused directly to the N-terminus of an Fc domain.

[0251] In some aspects, to further increase stability and / or rigidity of the complex, such as a multi-specific protein or fusion protein, the termini of one or both of the proteins in the complex protein may be truncated, which may be considered a “negative linker” or “single- or dualtruncation” format. For example, in some aspects, one, two, or three of the terminal residues of a VH or VL of one of the TfR antigen-binding domains may be truncated. In some aspects, the N- terminal residues QV are truncated from the N-terminus of the VH domain of any of the TfR antigen-binding domains disclosed herein (see, for example, the VH and VL domains of Table 6A compared to those listed in Table 6). In some aspects, one, two, three, four, or five of the terminal residues of the heterologous protein, antibody or antigen-binding fragment thereof, or other antigen-binding domain may be truncated. For example, in a multi-specific protein, such as one comprising an antibody-VH-linker-VL, wherein the VH and VL are one of the anti-TfR antibodies disclosed herein, the conserved C-terminal residues PGK of the Fc of the antibody may be truncated. In another example, in a multi-specific protein, such as one comprising an antibody-VH-linker-VL, wherein the VH and VL are one of the anti-TfR antibodies disclosed herein, the C-terminal residues PGK from the C-terminus of the antibody may be truncated and the N-terminal residues QV from the N-terminus of the VH may be truncated (e.g., a “-5 linker” format). Table 6A sets forth exemplary N-terminal truncation versions of the VH domains of the anti-TfR antibodies disclosed herein, which may be used in these “negative linker” formats. In some aspects, the multi-specific protein, such as one comprising an antibody-VH-linker-VL, comprises the VH and VL of a pair set forth in Table 6A. In some aspects, the multi-specific protein, such as one comprising an antibody-VH-linker-VL, comprises the VH and VL of a VH and VL pair set forth in Table 6A, and the C-terminus of the antibody is truncated.

[0252] A complex, fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein comprising an anti-TfR antigen-binding domain may comprise a “disulfide staple”. A disulfide staple refers to the engineering of one or more disulfide bonds to stabilize a molecule’s structure, enhance its stability, and / or improve any functional properties compared to the structure without the engineered disulfide staple. See, e.g., Reiter et al., 1994, Biochemistry, 33:5451-5459 and Wetherill et al., 2012, Protein Engineering, Design & Selection, 25:321-329. A disulfide staple is in addition to any disulfide bonds naturally present in the molecule, such as intra-chain disulfide bonds in the VH and / or VL. scFvs comprise two variable domains (a VH and a VL) that are linked by a flexible linker. Typically, there is a weakinteraction between the VH and VL domains, which can lead to aggregation and / or reduced stability. To ameliorate these liabilities, the complexes, fusion proteins, antibodies or antigenbinding fragments thereof, or multi-specific proteins disclosed herein may comprise a disulfide staple, such as in an anti-TfR scFv antigen-binding domain. In some aspects, the disulfide staple is formed in part by the substitution of a residue with cysteine in a variable heavy domain disclosed in Table 6. In some aspects, the disulfide staple is formed in part by the substitution of a residue with cysteine in a variable light domain disclosed in Table 6. In some aspects, the disulfide staple is formed by the substitution of a residue with cysteine in a variable heavy domain disclosed in Table 6 and the substitution of a residue with cysteine in a variable light domain disclosed in Table 6.

[0253] In some aspects, the substitution of a residue with cysteine is located in a framework region of the variable light and / or variable heavy domains disclosed in Table 6. In some aspects, the substitution of a residue with cysteine is not located in any of the CDR regions of a variable light and / or variable heavy domain disclosed in Table 6, such as the CDRs disclosed in Table 4 and Table 5. In some aspects, the disulfide staple is formed in part by the substitution of position 44 with cysteine in a VH domain disclosed in Table 6, with numbering according to Kabat. In some aspects, the disulfide staple is formed in part by the substitution of position 100 with cysteine in a VL domain disclosed in Table 6, with numbering according to Kabat. Any suitable positions in the VH and VL domains disclosed in Table 6 may be substituted with cysteine to form the disulfide staple, provided that the domains retain specific binding to TfR. In some aspects, the disulfide staple is according to the sequence of the VH and VL pairs disclosed in Table 6B. In some aspects, the VH and VL of the anti-TfR antigen-binding domain are in the scFv format and comprise a linker in the orientation VH-linker-VL or VL-linker-VH. In some aspects, the one or more residues of the linker may comprise an amino acid substitution of a residue with cysteine, wherein the one or more cysteine residues form the disulfide staple with a complementary cysteine in the VH and / or VL domains of the anti-TfR antigen-binding domain. Thus, in some aspects, the antigen-binding domain is in the orientation VH-linker-VL or VL- linker-VH, and the linker comprises a cysteine residue and the VH or VL domain comprise a complementary cysteine residue which forms at least part of the disulfide staple. In some aspects, the antigen-binding domain is in the orientation VH-linker-VL or VL-linker-VH, and the linker comprises a first cysteine residue and a second cysteine residue, wherein the first cysteine residue forms a disulfide bond with a complementary cysteine residue in the VH domain, and the secondcysteine residue forms a disulfide bond with a complementary cysteine residue in the VL domain.

[0254] In some aspects, the introduction of a disulfide staple to the anti-TfR antigen-binding domains disclosed herein improves stability of the antigen-binding domain. While introduction of a disulfide staple may improve the stability or pharmacokinetic properties of the antigen-binding domain, or a complex comprising said antigen-binding domain, it is contemplated that such engineered disulfide-stapled anti-TfR antigen-binding domains may exhibit lower affinity for TfR compared to an antigen-binding domain lacking a disulfide staple while still retaining specific binding to TfR and ability to transport cargo molecules across the blood-brain barrier. Thus, in some aspects, the introduction of a disulfide staple to an anti-TfR antigen-binding domain disclosed herein reduces the affinity of the anti-TfR antigen-binding domain for TfR.

[0255] A complex, fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein can comprise a constant region. In some aspects, a TfR antigenbinding 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.

[0256] In some aspects a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific 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. Withrespect 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.

[0257] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific 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, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific 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.

[0258] Non-limiting examples of human constant region sequences have been described in e.g., U.S. Patent No. 5,693,780 and Kabat EA etal., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242).

[0259] 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, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein provided herein can comprise a constant region comprising a knob mutation and a constant region comprising a hole mutation.Bispecific or Multi-specific Proteins that Bind to TfR and to Cancer Antigens

[0260] In some aspects, a bispecific or multispecific protein provided herein comprises an antigen-binding domain that specifically binds to human TfR and an antigen-binding domain that specifically binds to a cancer antigen. Such bispecific or multi-specific proteins can comprise, for example, an antibody or antigen-binding fragment thereof that binds to a target expressed on the surface of a cell (e.g., a tumor cell) in the CNS and an anti-TfR antigen-binding domain. The anti-TfR antigen-binding domain can facilitate crossing the BBB, and the antibody or antigenbinding fragment thereof can target and induce cell death of the tumor cells. The bi specific ormulti-specific protein can be a bispecific or multi-specific protein with reduced effector function. Exemplary formats of a multispecific protein comprising an antigen-binding domain that specifically binds to human TfR and to a cancer antigen are shown in Figure 12.

[0261] In some aspects, the bispecific or multi-specific proteins provided herein comprise an antigen-binding domain that binds to TfR and an antibody or antigen-binding fragment thereof that binds to CD20. In some aspects, the antibody or antigen-binding fragment thereof that binds to CD20 is rituximab. In some aspects, the antibody or antigen-binding fragment thereof that binds to CD20 is a version of rituximab with reduced effector function. In some aspects, the antibody or antigen-binding fragment that binds to CD20 (e.g., rituximab) with reduced effector function contains the LALA-P331 S and / or LALA-P329S mutation. Such bispecific and multispecific proteins can be used for treating chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma, which has manifested in the CNS and / or brain.

[0262] In some aspects, the bispecific or multi-specific proteins provided herein comprise an antigen-binding domain that binds to TfR and an antibody or antigen-binding fragment thereof that binds to epidermal growth factor receptor (EGFR). In some aspects, the antibody or antigenbinding fragment thereof that binds to EGFR is cetuximab. In some aspects, the antibody or antigen-binding fragment thereof that binds to EGFR is a version of cetuximab with reduced effector function. In some aspects, the antibody or antigen-binding fragment that binds to EGFR with reduced effector function (e.g., cetuximab) contains the LALA-P331 S and / or LALA-P329S mutation. Such bispecific and multi-specific proteins can be used for treating head and neck cancer and colorectal cancer, which has metastasized to the CNS and / or brain.

[0263] In some aspects, the bispecific or multi-specific proteins provided herein comprise an antigen-binding domain that binds to TfR and an antibody or antigen-binding fragment thereof that binds to HER2. In some aspects, the antibody or antigen-binding fragment thereof that binds to HER2 is trastuzumab. In some aspects, the antibody or antigen-binding fragment thereof that binds to HER2 is a version of trastuzumab with reduced effector function. Such bispecific and multi-specific proteins can be used for treating breast cancer and gastric cancer, which has metastasized to the CNS and / or brain. In some aspects, the antibody or antigen-binding fragment thereof that binds to HER2 is pertuzumab. In some aspects, the antibody or antigen-binding fragment thereof that binds to HER2 is a version of pertuzumab with reduced effector function. In some aspects, the antibody or antigen-binding fragment that binds to HER2 with reduced effector function (e.g., pertuzumab) contains the LALA-P331 S and / or LALA-P329S mutation.Such bispecific and multi-specific proteins can be used for treating breast cancer, which has metastasized to the CNS and / or brain.

[0264] In some aspects, the bispecific or multi-specific proteins provided herein comprise an antigen-binding domain that binds to TfR and an antibody or antigen-binding fragment thereof that binds to vascular endothelial growth factor (VEGF). In some aspects, the antibody or antigen-binding fragment thereof that binds to VEGF is bevacizumab. In some aspects, the antibody or antigen-binding fragment thereof that binds to VEGF is a version of bevacizumab with reduced effector function. In some aspects, the antibody or antigen-binding fragment that binds to VEGF with reduced effector function (e.g., bevacizumab) contains the LALA-P331S and / or LALA-P329S mutation. Such bispecific and multi-specific proteins can be used for treating colorectal cancer, lung cancer, breast cancer, kidney cancer, ovarian cancer, cervical cancer, and brain cancer, which has manifested or metastasized to the CNS and / or brain.

[0265] In some aspects, the bispecific or multi-specific proteins provided herein comprise an antigen-binding domain that binds to TfR and an antibody or antigen-binding fragment thereof that binds to programmed cell death protein 1 (PD-1). In some aspects, the antibody or antigenbinding fragment thereof that binds to PD-1 is nivolumab. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-1 is a version of nivolumab with reduced effector function. In some aspects, the antibody or antigen-binding fragment that binds to PD-1 with reduced effector function (e.g., nivolumab) contains the LALA-P331 S and / or LALA-P329S mutation. Such bispecific and multi-specific proteins can be used for treating non-small cell lung cancer (NSCLC), renal cell carcinoma, gastric cancer, gastroesophageal junction cancer, esophageal cancer, melanoma, urothelial carcinoma, pleural mesothelioma, head and neck cancer, hepatocellular carcinoma, colorectal cancer, and Hodgkin lymphoma, which has manifested or metastasized to the CNS and / or brain. In some aspects, the antibody or antigenbinding fragment thereof that binds to PD-1 is pembrolizumab. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-1 is a version of pembrolizumab with reduced effector function. Such bispecific and multi-specific proteins can be used for treating melanoma, NSCLC, head and neck cancer, Hodgkin lymphoma, B-cell lymphoma, urothelial cancer, bladder cancer, colorectal cancer, gastric cancer, gastroesophageal junction cancer, cervical cancer, biliary tract cancer, Merkel cell carcinoma, renal cell carcinoma, endometrial carcinoma, squamous cell carcinoma, and breast cancer, which has manifested or metastasized to the CNS and / or brain which can metastasize to the CNS and / or brain.

[0266] In some aspects, the bispecific or multi-specific proteins provided herein comprise an antigen-binding domain that binds to TfR and an antibody or antigen-binding fragment thereof that binds to programmed death-ligand 1 (PD-L1). In some aspects, the antibody or antigenbinding fragment thereof that binds to PD-L1 is atezolizumab. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-L1 is a version of atezolizumab with reduced effector function. In some aspects, the antibody or antigen-binding fragment that binds to PD-L1 with reduced effector function (e.g., atezolizumab) contains the LALA-P331 S and / or LALA- P329S mutation. Such bispecific and multi-specific proteins can be used for treating non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), hepatocellular carcinoma, melanoma, and alveolar soft part sarcoma (ALPS), which has metastasized to the CNS and / or brain. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-L1 is avelumab. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-L1 is a version of avelumab with reduced effector function. In some aspects, the antibody or antigen-binding fragment that binds to PD-L1 with reduced effector function (e.g., avelumab) contains the LALA-P331S and / or LALA-P329S mutation. Such bispecific and multi-specific proteins can be used for treating urothelial cancer, Merkel cell carcinoma, and renal cell carcinoma, which has metastasized to the CNS and / or brain. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-L1 is durvalumab. In some aspects, the antibody or antigen-binding fragment thereof that binds to PD-L1 is a version of durvalumab with reduced effector function. In some aspects, the antibody or antigen-binding fragment that binds to PD-L1 with reduced effector function (e.g., durvalumab) contains the LALA-P331 S and / or LALA-P329S mutation. Such bispecific and multi-specific proteins can be used for treating non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), biliary tract cancer, and hepatocellular carcinoma, which has metastasized to the CNS and / or brain.Bispecific or Multi-specific Proteins that Bind to TfR and to Other CNS Antigens

[0267] In some aspects, the bispecific or multi-specific proteins provided herein comprise an antigen-binding domain that binds to TfR and an antibody or antigen-binding fragment thereof that binds to matrix metalloproteinase-9 (MMP-9). Matrix metalloproteinase-9 (MMP-9), also known as 92 kDa type IV collagenase, 92 kDa gelatinase or gelatinase B (GELB), is a matrixin, a class of enzymes that belong to the zinc-metalloproteinases family involved in the degradation of the extracellular matrix. MMP-9 specifically breaks down type IV and V collagens, gelatin, and elastin. It plays a critical role in various physiological processes including tissue remodeling, wound healing, embryonic development, and angiogenesis. MMP-9 plays a critical role in thebreakdown of the BBB, cerebral edema, and hemorrhagic transformation following ischemic stroke. MMP-9 upregulation correlates with infarct size and neurological deterioration. Inhibition of MMP-9 in rodent models leads to reduced infarct volumes and improved outcomes. However, systemic MMP-9 inhibition carries risks due to MMPs’ roles in tissue remodeling.Fc Domains and Regions

[0268] A complex, fusion protein, antibody or antigen-binding fragment thereof, or multispecific protein 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.

[0269] 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, the Fc domain or region or fragment thereof is a wild-type IgG2.

[0270] In some aspects provided herein, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided 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 region of the same isotype). In some aspects, the modified Fc domain or region or fragment thereof is a modified IgG2 Fc comprising one or more amino acid substitutions (e.g., relative to a wild-type IgG2 Fc). In some aspects, the modified Fc domain or region or fragment thereof is a modified IgG2 Fc comprising one or more modifications relative to a wild-type IgG2 Fc. In some aspects, the modified Fc domain or region or fragment thereof is a modified IgG4 Fc relative to a wild-type IgG4 Fc. In some aspects, the modified Fc domain or region or fragment thereof is a modified IgG4 Fc domain comprising one or more modifications relative to a wild-type IgG4 Fc. 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 some aspects, the one or more amino acidsubstitutions in the modified IgG4 Fc are selected from IgG4-S228P and / or IgG4-S228P / L235E, where the amino acid position is according to the EU numbering convention. In some aspects, the one or more amino acid substitutions are selected from N297A (Bolt S et al. (1993) 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), 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 domain or region or fragment thereof comprises the amino acid substitutions L234A, L235A, and P331S (LALAPS) according to EU numbering. In some aspects, the Fc domain or region or fragment thereof comprises the amino acid substitutions L234A, L235A, and P329S (LALAPS) according to EU numbering. In some aspects of any of the modified IgG Fc, the Fc domain or region or fragment thereof comprises N325S and L328F mutations according to EU numbering. In some aspects, the Fc domain or region or fragment thereof comprises P329G or P329S according to EU numbering.

[0271] In some aspects, the Fc domain or region or fragment thereof comprises S228P according to EU numbering. In some aspects, the Fc domain or region or fragment thereof is a modified IgG4 Fc and comprises S228P according to EU numbering. In some aspects, the Fc domain or region or fragment thereof comprises L235E according to EU numbering. In some aspects, the Fc domain or region or fragment thereof comprises S228P and L235E according to EU numbering. In some aspects, the modified Fc domain or region or fragment thereof is a modified IgG4 Fc and comprises S228P and L235E according to EU numbering.

[0272] In some aspects, the modified Fc domain or region or fragment thereof is a modified IgG2 Fc domain comprising one or more amino acid substitutions (e.g., relative to a wild-type IgG2 Fc). In some aspects, the modified Fc domain or region or fragment thereof is a modified IgG2 Fc domain comprising one or more modifications relative to a wild-type IgG2 Fc. In some aspects, the modified Fc domain or region or fragment thereof is a modified IgG4 Fc domain relative to a wild-type IgG4 Fc. In some aspects, the modified Fc domain or region or fragment thereof is a modified IgG4 Fc domain comprising one or more modifications relative to a wildtype IgG4 Fc. 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 some aspects, the one or more aminoacid substitutions in the modified IgG4 Fc are selected from IgG4-S228P and / or IgG4- S228P / L235E, where the amino acid position is according to the EU numbering convention.

[0273] Any suitable Fc domain or Fc region or fragment thereof is contemplated in the complexes, fusion proteins, antibodies, or antigen-binding fragments thereof disclosed herein, and exemplary Fc domains are provided in Table 1, below.Table 1: Exemplary Fc Domains

[0274] In some aspects provided herein, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein is a bi-specific fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein. Bi-specific 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 complex, fusion protein, 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.

[0275] In some aspects provided herein, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein comprises a mutation to promote heterodimerization of Fc regions. In some aspects, a dimerized Fc region of a bispecific provided herein is formed by Fc regions 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 Fc region 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

[0276] 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;W01996 / 027011; WO 1998 / 050431; W02006 / 028936; W02009 / 089004; WO2011 / 143545; WO2014 / 067011; WO2012 / 058768; WO2018 / 027025; US2014 / 0363426; US2015 / 0307628; US2018 / 0016354; US2015 / 0239991; US2017 / 0058054; USPN5731168; USPN7183076; USPN9701759; USPN9605084; USPN9650446; USPN8216805; USPN8765412; and USPN8258268.

[0277] 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.

[0278] Additionally, complementary Fc polypeptides of an Fc heterodimer include “knob-into- hole” configurations to promote heterodimerization of two Fc polypeptides. “Knob-into-hole” technology is described in e.g., 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 smallerones (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. In some aspects, a knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two subunits of the Fc domain. In some aspects, the subunit of the Fc domain comprising the knob modification additionally comprises the amino acid substitution S354C, and the subunit of the Fc domain comprising the hole modification 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 domain, 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.

[0279] Exemplary paired amino acid modifications of complementary Fc polypeptides of an Fc heterodimeric configuration are set forth below in the table below (EU numbering).Table 2: Exemplary paired Fc modifications for heterodimeric Fc domains

[0280] Some agents provided herein comprise antigen-binding fragments of antibodies. 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 scFvfragments, see, e.g., WO 93 / 16185; and U.S. Patent Nos. 5571894 and 5587458. For discussion 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. 5869046.

[0281] 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. Some aspects, a single-domain antibody is a human single-domain antibody (see, e.g., U.S. Patent No. 6248516).

[0282] 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.

[0283] As provided herein, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein can be chimeric. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4816567. 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.

[0284] As provided herein, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein 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. 5530101, 5693761; 5693762; and 5585089. 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. 5766886 and 5869619. 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 atleast 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.

[0285] 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 US Patent Nos. 5821337, 7527791, 6982321, 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)).

[0286] As provided herein, a complex fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein 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).

[0287] 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. 5545807, EP 546073, and EP 546073. See also, for example, U.S. Patent Nos. 6075181 and 6150584describing XENOMOUSE™ technology; U.S. Patent No. 5770429 describing HUMAB® technology; U.S. Patent No. 7041870 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.

[0288] 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. 7189826 (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.

[0289] In some aspects provided herein, 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-affinityantibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al. EMBO J. 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers 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: US Patent No. 5750373, and US Patent Publication Nos. 2007 / 0292936 and 2009 / 0002360. Antibodies isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0290] As provided herein, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein can comprise an antigen-binding domain that binds to a CNS antigen or a brain antigen. In some aspects, the CNS antigen or brain antigen can be beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6 A (MS4A6A), Transmembrane Protein 106B (TMEM106b), or matrix metalloproteinase-9 (MMP-9). In some aspects, the CNS antigen or brain antigen can be beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33, sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), sialic acid binding Ig-like lectin 11 (Siglecl 1), glycoprotein nonmetastatic melanoma protein B (GPNMB), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A(MS4A4A), Membrane Spanning 4-Domains A 6 A (MS4A6A), MSA4A4E, Transmembrane Protein 106B (TMEM106b), CR1, ABCA1, ABCA7, HLA-DR1, HLA-DR5, IL1RAP, TREML2, IL-34, SORL1, ADAMI, or matrix metalloproteinase-9 (MMP-9).

[0291] In some aspects, the CNS antigen or brain antigen can be reelin, very low density lipoprotein receptor (VLDLR), or apolipoprotein E receptor 2 (APOER2; also known as low- density lipoprotein receptor-related protein 8 (LRP8)).

[0292] In some aspects, the CNS or brain antigen is on a cancer cell within the central nervous system. In some aspects, the CNS or brain antigen is a cell surface target on a hematological cancer cell selected from B7H3, BCMA, CD125, CD166, CD19, CD20, CD205, CD22, CD25, CD30, CD37, CD39, CD73, and CD79b. In some aspects, the CNS or brain antigen is a tumor cell target selected from siglec-3 or CD33, siglec-5, siglec-7, siglec-9, siglec 14, PILRA, IL18- BP, MerTK, ACKR1, ALK, AXL, CD25, CD44v6, CD46, CD56 (NCAM), CDH6 (cadherin 6), CEACAM 5 (CD66E), EGFR, EGFR viii, ETBR, FGFR (1-4), Folate Receptor alpha, GAL-3BP (galectin binding protein), GD2, GD3, GloboH (globohexasylceramide), gplOO, gpNMB, HER2, HER3, HER4, IGFR1, KIT, LIV1A, LRRC15 (leucine rich repeat containing 15), MET, NaPi2B, PDL1, PMEL17, PRAME, PSMA, PTK7 (CCK4; colon carcinoma kinase), RON, ROR1, TF (tissue factor), and TROP2.

[0293] As provided herein, a complex fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein can comprise an antigen-binding domain that binds to a CNS antigen or a brain antigen. As provided herein, a multi-specific protein provided herein can comprise an antigen-binding domain that binds to a CNS antigen or a brain antigen. The antigen-binding domain that binds to a CNS antigen or a brain antigen can comprise a VH and a VL. Exemplary CNS antigen-binding VH and VL sequences are provided below. Additional VH and VL and antigen-binding domain sequences are found in US2017 / 0224702, US 2018 / 0002433, US 2021 / 0236634, and US 2021 / 0238265, each of which is herein incorporated by reference in its entirety.Table 3: Exemplary CNS Antigen-Binding VH and VL Sequences

[0294] As provided herein, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein can be capable of crossing the BBB as a resultof the fact that the anti-TfR antigen-binding domain in the fusion protein, antibody or antigenbinding fragment thereof, or multi-specific protein is capable of crossing the BBB.

[0295] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein is internalized in blood-brain barrier epithelial cells greater than 10-fold as compared to internalization by an isotype control. The blood-brain barrier endothelial cells can be, e.g., HCMEC / D3 cells.

[0296] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein does not significantly reduce TfR as measured in whole brain lysates (e.g., as measured according to the assay in Example 8).

[0297] In some aspects, a fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein accumulates at least 4-fold more than an isotype control in vessel-depleted mouse brain. In some aspects, a fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein accumulates at least 5 -fold more than an isotype control in vessel-depleted mouse brain.

[0298] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein binds the apical domain of human TfR with an affinity of about 5 to about 50 nM (e.g., as measured by Biacore using the assay provided in Example 2).

[0299] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein binds the apical domain of cynomolgus monkey TfR with an affinity of about 5 to about 50 nM (e.g., as measured by Biacore using the assay provided in Example 2).

[0300] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein binds the apical domain of human TfR with an affinity of about 5 to about 50 nM and binds the apical domain of cynomolgus monkey TfR with an affinity of about 5 to about 50 nM (e.g., as measured by Biacore using the assay provided in Example 2).

[0301] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein binds the apical domain of human TfR with an affinity of about 51 to about 1000 nM (e.g., as measured by Biacore using the assay provided in Example 2).

[0302] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein binds the apical domain of cynomolgus monkeyTfR with an affinity of about 51 to about 1000 nM (e.g., as measured by Biacore using the assay provided in Example 2).

[0303] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein binds the apical domain of human TfR with an affinity of about 51 to about 1000 nM and binds the apical domain of cynomolgus monkey TfR with an affinity of about 51 to about 1000 nM (e.g., as measured by Biacore using the assay provided in Example 2).

[0304] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein binds the apical domain of human TfR with an affinity of about 1001 to about 5000 nM (e.g., as measured by Biacore using the assay provided in Example 2).

[0305] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein binds the apical domain of cynomolgus monkey TfR with an affinity of about 1001 to about 5000 nM (e.g., as measured by Biacore using the assay provided in Example 2).

[0306] In some aspects, a complex, fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein provided herein binds the apical domain of human TfR with an affinity of about 1001 to about 5000 nM and binds the apical domain of cynomolgus monkey TfR with an affinity of about 1001 to about 5000 nM (e.g., as measured by Biacore using the assay provided in Example 2).Antibody Drug Conjugates Comprising Anti-TfR Antigen-Binding Domains

[0307] In some aspects, provided herein are antibody drug conjugates (ADCs) comprising an anti-TfR antigen-binding domain. Such ADCs can comprise, for example, an antibody or antigen-binding fragment thereof that binds to a target expressed on the surface of a cell (e.g., a tumor cell) in the CNS, a cytotoxic drug, and an anti-TfR antigen-binding domain. In some aspects, such an ADC further comprises a linker. For example, the linker can conjugate the cytotoxic drug to the antibody or antigen-binding fragment thereof that binds to a target expressed on the surface of a cell (e.g., a tumor cell) in the CNS. The anti-TfR antigen-binding domain can facilitate crossing the BBB; the antibody or antigen-binding fragment thereof can target the cytotoxic drug to cells that express the target (e.g., tumor cells); and the cytotoxic drug can kill the tumor cells. In some aspects, such ADCs have reduced effector function. In some aspects, the ADCs comprise an antibody or antigen-binding fragment with LALA-P331 S and / orLALA-P329S mutation. Such ADCs can be useful, e.g., for the treatment of CNS cancers, glioblastoma, and brain metastatis of a solid cancer.

[0308] In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, an antibody or antigen-binding fragment thereof that binds to tissue factor (TF), and a cytotoxic drug. In some aspects, an ADC provided herein comprises an anti- TfR antigen-binding domain provided herein, tisotumab or antigen-binding fragment thereof, and a cytotoxic drug, e.g., monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, tisotumab or antigen-binding fragment thereof with reduced effector function, and a cytotoxic drug, e.g., monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, an antibody or antigen-binding fragment thereof that binds to to a target expressed on the surface of a cell (e.g., a tumor cell) in the CNS, and monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, tisotumab or antigen-binding fragment thereof, and monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain and tisotumab vedotin. Such ADCs can be useful, e.g., for the treatment of glioblastoma.

[0309] In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, an antibody or antigen-binding fragment thereof that binds to human epidermal growth factor receptor (HER2), and a cytotoxic drug, e.g., monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, trastuzumab or antigen-binding fragment thereof, and a cytotoxic drug. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, trastuzumab or antigen-binding fragment thereof, and monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, trastuzumab or antigen-binding fragment thereof with reduced effector function, and monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain and disitamab vedotin. Such ADCs can be useful, e.g., for the treatment of brain metastasis of breast cancer or other solid tumors.

[0310] In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, an antibody or antigen-binding fragment thereof that binds to human B7-H4, and a cytotoxic drug e.g., monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, brentuximab or antigenbinding fragment thereof, and a cytotoxic drug. In some aspects, an ADC provided hereincomprises an anti-TfR antigen-binding domain provided herein, brentuximab or antigen-binding fragment thereof, and monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, brentuximab or antigen-binding fragment thereof with reduced effector function, and monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain and felmetatug vedotin. Such ADCs can be useful, e.g., for the treatment of brain metastasis.

[0311] In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, an antibody or antigen-binding fragment thereof that binds to human nectin-4, and a cytotoxic drug e.g., monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, enfortumab or antigenbinding fragment thereof, and a cytotoxic drug. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, enfortumab or antigen-binding fragment thereof, and monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, enfortumab or antigen-binding fragment thereof with reduced effector function, and monomethyl auristatin E. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain and enfortumab vedotin. Such ADCs can be useful, e.g., for the treatment of brain metastasis.

[0312] In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, an antibody or antigen-binding fragment thereof that binds to human HER2, and a cytotoxic drug e.g., deruxtecan. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, trastuzumab or antigen-binding fragment thereof, and a cytotoxic drug. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, trastuzumab or antigen-binding fragment thereof, and deruxtecan. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain provided herein, trastuzumab or antigen-binding fragment thereof with reduced effector function, and deruxtecan. In some aspects, an ADC provided herein comprises an anti-TfR antigen-binding domain and trastuzumab deruxtecan. Such ADCs can be useful, e.g., for the treatment of brain metastasis of breast cancer or other solid tumors.Polynucleotides and Methods of Making Anti-TfR Antigen-Binding Domains and Agents Comprising the Same

[0313] In some aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding an antigen-binding domain that specifically bind to human TfR, complex, fusionprotein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein or a domain thereof described herein, and vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells).

[0314] 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 bind to human TfR provided herein and a nucleic acid molecule encoding the light chain of an antigen-binding domain that specifically bind to human TfR provided herein.

[0315] 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 an 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.

[0316] 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 holemutation. In some aspects, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

[0317] 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.

[0318] Also provided herein are polynucleotides comprising a nucleotide sequence encoding an antigen-binding domain that specifically bind to human TfR, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein 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.

[0319] A polynucleotide comprising a nucleotide sequence encoding an antigen-binding domain that specifically bind to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein 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 to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein.

[0320] Polynucleotides provided herein can be, e.g., 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.

[0321] 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 complex or fusion protein 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 -methylinosine, 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.

[0322] In some aspects, provided herein are polynucleotides encoding a complex, fusion protein comprising an antigen-binding protein provided herein and a heterologous polypeptide. In some aspects, the heterologous polypeptide comprises an antigen binding domain that binds to beta-secretase 1 (BACE1), Amyloid beta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, Glycoprotein nonmetastatic protein B (GPNMB), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6A (MS4A6A), Transmembrane Protein 106B (TMEM106b), or matrix metalloproteinase-9 (MMP-9). In some aspects, provided herein are polynucleotides encoding a complex or fusion protein comprising an antigen-binding domain provided herein and a heterologous polypeptide. In some aspects, the heterologous polypetide comprises the amino acid sequence of P-glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), clusterin (APOJ), Reelin, Tripeptidyl Peptidase 1 (CLN2 / TPP1), or Alpha-L-Iduronidase (IDUA), Iduronate 2-Sulfatase (IDS), glucosamine (N- acetyl)-6-sulfatase (GNS), heparan-alpha-glucosaminide N-acetyltransf erase (HGSNAT), and N- acetyl-alpha-glucosaminidase (NAGLU), N-sulfoglucosamine sulfohydrolase (SGSH), ubiquitin protein ligase E3 A (UBE3 A), or a variant or portion thereof. In some aspects, the heterologous polypetide comprises the amino acid sequence of very low density lipoprotein receptor (VLDLR) or apolipoprotein E receptor 2 (APOER2; also known as low-density lipoprotein receptor-related protein 8 (LRP8)). In some aspects, the polynucleotide is mRNA (e.g., synthetic mRNA). In some aspects, disclosed herein are polynucleotides encoding a complex or fusion protein disclosed herein comprising a heterologous polypeptide. In some aspects, the heterologous polypeptide is an ERT enzyme or an ERT enzyme variant, or a catalytically active fragment thereof. In some aspects, the heterologous polypeptide comprises P-glucocerebrosidase (GCaseor GBA), progranulin (PGRN), Prosaposin (PSAP), or a catalytically active fragment thereof. In some aspects, the heterologous polypeptide in a complex or fusion protein provided herein is a growth factor. In some aspects, the heterologous polypeptide in a complex or fusion protein provided herein is a decoy receptor. In some aspects, the heterologous polypeptide in a complex or fusion protein provided herein is progranulin (PGRN), prosaposin (PSAP), or survival motor neuron protein (SMN). In some aspects, the heterologous protein is an enzyme selected from a-L Iduronidase (IDUA), Iduronate-2-sulphatase (IDS), N-acetylgalactoslamine-6-sulphatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulphatase (arylsulphatase B; ARSB), acid sphingomyelinase (ASM), P-glucocerebrosidase (GCase or GBA), galactosylceramide beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, betahexosaminidase B, aryl sulphatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme replacement thereof, such as larodinase, idursulphase, elosulphase alpha or galsuphase, or a variant thereof, or a catalytically active fragment thereof. In some aspects, the heterologous protein is an enzyme selected from clusterin (APOJ), Reelin, ubiquitin protein ligase E3 A (UBE3 A), Tripeptidyl Peptidase 1 (CLN2 / TPP1), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosaminide N- acetyltransferase (HGSNAT), and N-acetyl-alpha-glucosaminidase (NAGLU), a-L Iduronidase (IDUA), Iduronate-2-sulphatase (IDS), N-acetylgalactoslamine-6-sulphatase (GALNS), N- sulfoglucosamine sulfohydrolase (SGSH), N-acetylgalactosamine-4-sulphatase (aryl sulphatase B; ARSB), acid sphingomyelinase (ASM), P-glucocerebrosidase (GCase or GBA), galactosylceramide beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, betahexosaminidase B, aryl sulphatase A, beta-galactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme replacement thereof, such as larodinase, idursulphase, elosulphase alpha or galsuphase, or a variant thereof, or a catalytically active fragment thereofa-L Iduronidase (IDUA), Iduronate-2-sulphatase (IDS), N- acetylgalactoslamine-6-sulphatase (GALNS), N-sulfoglucosamine sulfohydrolase (SGSH), N- acetylgalactosamine-4-sulphatase (aryl sulphatase B; ARSB), acid sphingomyelinase (ASM), P- glucocerebrosidase (GCase or GBA), galactosylceramide beta-galactosidase, glucosylceramidase, beta-hexosaminidase A, beta-hexosaminidase B, aryl sulphatase A, betagalactosidase, acid ceramidase, alpha-glucosidase, lysosomal acid lipase, lysosomal protease, a synthetic enzyme replacement thereof, such as larodinase, idursulphase, elosulphase alpha or galsuphase, or a variant thereof, or a catalytically active fragment thereof. In some aspects, the polynucleotide is mRNA (e.g., synthetic mRNA).

[0323] In some aspects, the heterologous protein is an enzyme selected from very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (APOER2; also known as low- density lipoprotein receptor-related protein 8 (LRP8)), or a variant thereof, or a catalytically active fragment thereof. In some aspects, the polynucleotide is mRNA (e.g., synthetic mRNA).

[0324] 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, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein, for recombinant expression in a host cell, e.g., in a mammalian host cell. A vector for the production of the antigen-binding domain that specifically bind to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein 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 an antigen-binding domain that specifically bind to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein 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).

[0325] In certain aspects, provided herein are expression systems comprising polynucleotides comprising nucleotide sequences encoding an antigen-binding domain that specifically bind to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein described herein, or a domain thereof described herein. 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.

[0326] 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, complex, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein described herein, or a domain thereof described herein. In some aspects, for the expression of double-chained 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 an antigenbinding 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.

[0327] In some aspects, provided herein are methods for producing an antigen-binding domain that specifically binds to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein in a host cell. In some aspects, provided herein are methods for producing a single chain antigenbinding domain that specifically binds to human TfR, an Fc domain, and a heterologous protein or polypeptide as described herein in a host cell. In some aspects, provided herein are methods for producing a single chain antigen-binding domain that specifically binds to human TfR, an Fc domain, and a second antigen-binding domain, as described herein in a host cell. 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 an antigen-binding domain that specifically binds to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein.

[0328] A variety of host-expression vector systems can be utilized to express an antigenbinding domain that specifically bind to human TfR, complex, fusion protein, antibody, antigenbinding fragment thereof, or multi-specific protein 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 an antigen-binding domain that specifically bind to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specificprotein 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 an antigen-binding domain that specifically bind to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein 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 an antigen-binding domain that specifically bind to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein 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 an antigen-binding domain that specifically bind to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein 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, an antigen-binding domain that specifically bind to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein is produced by CHO cells or NSO cells.

[0329] 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.

[0330] Once an antigen-binding domain that specifically bind to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein 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 affinity for 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 antigen-binding domain that specifically bind to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof described herein can be fused to heterologous polypeptide sequences to facilitate purification.

[0331] In some aspects, once an antigen-binding domain that specifically binds to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein is isolated or purified. Generally, an isolated or purified antigen-binding domain that specifically bind to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein is one that is substantially free of other proteins. For example, in some aspects, a preparation of an antigen-binding domain that specifically binds to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein is substantially free of cellular material and / or chemical precursors.Pharmaceutical Compositions

[0332] Provided herein are compositions comprising an antigen-binding domain that specifically binds to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein described herein. In some aspects, the antigen-binding domain that specifically bind to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein having the desired degree of purity is present in a formulation comprising, e.g., a physiologically acceptable carrier, excipient or stabilizer (Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). 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-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0333] In some aspects, a pharmaceutical composition comprises an antigen-binding domain that specifically bind to human TfR, complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein 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)). 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.

[0334] Also provided herein are pharmaceutical compositions comprising a polynucleotide encoding an antigen-binding domain that specifically binds to human TfR, a complex, a fusion protein, an antibody or antigen-binding fragment thereof, or multi-specific protein described herein. In some aspects, the polynucleotide is RNA. In some aspects, the polynucleotide is synthetic mRNA. In some aspects, the pharmaceutical composition comprising a polynucleotide further comprises a lipid-based transfection reagent.

[0335] 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.

[0336] 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.

[0337] 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.Methods of Using Anti-TfR Antigen-Binding Domains and Agents Comprising the Same

[0338] Antigen-binding domains that specifically bind to human TfR, complexes, fusion proteins, antibodies, antigen-binding fragments thereof, and multi-specific proteins comprising such antigen-binding domains as provided herein can advantageously be transported across a blood brain barrier. Accordingly, provided herein are methods of administering or transporting an antigen-binding protein that specifically binds to human TfR or a complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein comprising an antigenbinding protein that specifically binds to human TfR, across the blood brain barrier of a subject comprising administering to the subject an antigen-binding protein that specifically binds to human TfR or a complex, fusion protein, antibody, antigen-binding fragment thereof, or multispecific protein comprising an antigen-binding protein that specifically binds to human TfR.

[0339] In view of the ability of antigen-binding domains that specifically bind to human TfR and complexes, fusion proteins, antibodies, antigen-binding fragments thereof, and multi-specific proteins comprising such antigen-binding domains as provided herein to be transported across a blood brain barrier, they 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 an antigen-binding protein that specifically binds to human TfR or a complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein comprising an antigen-binding protein that specifically binds to human TfR. The neurological disease or disorder can be, for example, a neuropathy disorder, a neurodegenerative disease, cancer, anocular 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, motor neuron 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.

[0340] In some aspects, provided herein is a method of treating a lysosomal storage disease with a complex or fusion protein 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 ),

[0341] Antigen-binding domains that specifically bind to human TfR and complexes, fusion proteins, antibodies, antigen-binding fragments thereof, and multi-specific proteins comprising such antigen-binding domains as provided herein can be used to detect an antigen (e.g., a CNS antigen or a brain antigen). Antigen-binding domains that specifically bind to human TfR and complexes, fusion proteins, antibodies, antigen-binding fragments thereof, and multi-specificproteins comprising such antigen-binding domains for such purposes can be labeled. Exemplary labels include, for example, radioisotopes (e.g.,64CU) and fluorescent labels. Accordingly, methods of detecting an antigen using an antigen-binding protein that specifically binds to human TfR or a complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein comprising an antigen-binding protein that specifically binds to human TfR described herein are provided. In some aspects, a method of detecting an antigen in the CNS (e.g., brain) of a subject comprises administering an antigen-binding protein that specifically binds to human TfR or a copmplex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein comprising an antigen-binding protein that specifically binds to human TfR to the antigen 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 fusion protein, antibody or antigen-binding fragment thereof, or multi-specific protein disclosed herein and locating the imaging agent within the sample.

[0342] Antigen-binding domains that specifically bind to human TfR and complexes, fusion proteins, antibodies, antigen-binding fragments thereof, and multi-specific proteins comprising such antigen-binding domains as 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 an antigen-binding protein that specifically binds to human TfR or a complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein comprising an antigen-binding protein that specifically binds to human TfR for use as a diagnostic. In some aspects, the antigen-binding protein that specifically binds to human TfR or a complex, fusion protein, antibody, antigen-binding fragment thereof, or multi-specific protein comprising an antigen-binding protein that specifically binds to human TfR comprises a detectable label.

[0343] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0344] 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: Generation of Affinity-Tuned Anti-TfR Antibodies

[0345] To generate affinity-tuned TfR antibodies, humanized anti-TfR antibodies TfR.9.1B.39.38, TfR.15.WH8.1.24A, and TfR.15.WH8.1.42Q were selected for further affinity tuning. The generation of humanized antibodies TfR.9.1B.39.38, TfR.15. WH8.1.24 A and TfR.15.WH8.1.42Q was described in International Publication No. WO2024 / 026472. Amino acid substitutions at selected positions in the CDRs were performed to generate affinity variants. The variants were expressed in Expi293 culture, and either the supernatant or purified protein was used for screening.

[0346] The antibody sequences of the TfR.9. IB.39.38, TfR.15.WH8.1.24A, and TfR.15.WH8.1.42Q antibodies and the affinity-tuned anti-TfR antibodies (or "affinity variants") are provided in Tables 4-6 below.Table 4. Heavy Chain CDR Sequences of Anti-TfR AntibodiesTable 5. Light Chain CDR Sequences of Anti-TfR AntibodiesTable 6. VH and VL Sequences of Anti-TfR AntibodiesTable 6A. Truncated VH and VL Sequences of Anti-TfR AntibodiesTable 6B. Disulfide-Stapled VH and VL Sequences of Anti-TfR AntibodiesExample 2: Assessment of Affinity of Anti-TfR Binding Domains

[0347] To assess the affinity of anti-TfR antibodies, a Biacore T200 instrument was used to evaluate the binding kinetics (Global Life Sciences Solutions USA LLC, Marlborough, MA). The anti-TfR antibodies in this example were formatted as Fabs with a monovalent Fc. Anti-TfR antibodies were prepared by dilution in running buffer HBS-EP+ (Teknova) with BSA (MP Biomedicals) and captured by an anti-Fab antibody (Cat. 28958325, Cytiva) immobilized on a CM4 Sensor S Series sensor chip surface (Cytiva) that was prepared according to the manufacturer’s recommendations. A fixed concentration of antibody was captured on an individual flow cell followed by a concentration series of human or cyno apical TfR analyte injected over the active and reference flow cells. Blank injections were used for doublereferencing. All surfaces were regenerated with 10 mM glycine pH 2.1 buffer (Cytiva) at the end of each injection cycle. Data were processed and analyzed using Biacore T200 BiaEvaluation software (Cytiva), which fits the association and disassociation phases to a 1 : 1 binding model to generate kinetic rate constants konand koir. The equilibrium dissociation constant KD (nM) for the interaction between the antibody and protein was then calculated from the kinetic rate constants using the equation KD = k0ff / k0n. Results for the various anti-TfR binding domains are shown in Table 7.

[0348] Additional measurements are performed with the anti-TfR antibodies reformatted as an scFv in a 2+1 antibody format. The affinity of anti-TfR antibodies for the full-length ECDs ofboth human and cynomolgus TfR are also assessed. Assessments are also performed in the reverse orientation by immobilizing the apical domain of TfR or the ECD of TfR and evaluating binding of the anti-TfR antibody. Binding affinities are further calculated both by a kinetic model, as above, but also via a steady-state equilibrium model. Assessments are also performed using bio-layer interferometry in addition to surface plasmon resonance.Table 7. Affinities of anti-TfR antibodies in (Fab format)Example 3: Nonspecific Binding Assay for Anti-TfR Antibodies

[0349] To assess nonspecific binding of the anti-TfR antibodies with varying affinities ("affinity variants"), an ELISA assay was performed to assess binding to baculovirus particles (BVP) and double stranded DNA (dsDNA). BVP particles derived from insect cells contain phospholipid, carbohydrate, glycoproteins, extracellular matrix, nucleic acids, viral capsid,allowing for detection of electrostatic and hydrophobic interactions (Hotzel et al., mAbs, 2012). dsDNA is one of the agents used in ELISA assay to study polyreactivity in natural antibody repertoires during B-cell maturation. A majority of the antibodies expressed from early immature B cells showed ELISA binding to dsDNA, ssDNA, insulin, and LPS (Wardemann et al., 2003).

[0350] An ELISA plate was coated with either BVP particles or dsDNA, washed and incubated with blocking buffer, washed and incubated with the anti-TfR antibodies tested, followed by detection with anti-human IgG HRP antibody. A BVP score was calculated from the OD450 value of the sample wells compared to the background wells, which did not have any antibody added.

[0351] Anti-TfR affinity variants in Table 8 were tested for nonspecific binding to BVP and dsDNA as described above. In the BVP binding assay, the following concentrations of each antibody were tested: 1 pM, 0.33 pM, 0.11 pM or 0.037 pM. Control antibodies (isotype, negative and positive control) were also tested at these concentrations. In the dsDNA binding assay, antibodies were tested at 10 pg / mL.Table 8. Anti-TfR Antibodies Tested for Non-specific Binding to BVP and dsDNA

[0352] As shown in FIGs. 3A and 3B, all anti-TfR affinity variants showed low BVP scores.As shown in FIGs. 4A and 4B, all anti-TfR affinity variants showed low dsDNA binding.Example 4: Assessment of Characteristics for Manufacturing of Anti-TfR Affinity Variants

[0353] The physiochemical and biochemical properties of a protein can impact protein stability and can pose potential risks during manufacturing. In this study, an assessment was conducted by testing selected anti-TfR antibodies in IgG format, in order to determine and identify these characteristics. Several analytical techniques were utilized as shown in Table 9 below, demonstrating that the properties of those selected anti-TfR antibodies met the criteria for further development.Table 9: Overview of Characteristics for Selected Anti-TfR Affinity VariantsExample 5: In-vitro Cell Uptake of Anti-TfR Affinity Variants

[0354] Anti-human TfR affinity variant antibodies were assessed for extent of cell uptake using hCMEC / D3 cells in an acute 2-hour incubation assay. Anti-TfR affinity variants were fused to an N-terminus of a monovalent Fc (mvFc), with an isotype control scFv fused to the C-terminus of the mvFc ("anti-TfR Fab-mvFc-Iso-scFv") and were assessed by a 2-point titration at 500 nM and 100 nM concentrations. Anti-TfR antibodies H6-4, H3-7, 24A, L7-2, LIO-16, 24A.42Q, L10- 1, L-35, LI 0-8, and 42Q and an isotype control were tested. FIG. 5 shows the detection of the anti-TfR antibodies at 500 nM concentration. Cell uptake was observed for anti-TfR antibodies but not for the format-matched isotype control antibody. Generally, the extent of cell uptake correlated with the affinity of the antibodies (see FIG. 5). The cell uptake was proportionally weaker when incubated at the lower dose of 100 nM (data not shown).Example 6: Brain Uptake of Anti-TfR Affinity Variants

[0355] In vivo studies were conducted to determine the brain penetration of a panel of anti-TfR antibodies with varying affinities to human transferrin receptor (TfR) apical domain ranging from double digit nM to single digit uM (see Table 7). huTfR knockin (KI) mice were injected with a single dose of antibody at 5 mg / kg in the Fab-mvFc monovalent format to determine the brain uptake at the 24-hour time point. Brain uptake was determined from both vessel-depleted and whole brain fractions to assess whether the higher affinity anti-TfR antibodies were retained in the brain vasculature.

[0356] To produce vessel depleted brain fractions, brain tissues were minced and homogenized in HBSS buffer (MilliporeSigma #550370) containing 10 mM HEPES (#15630130, Gibco) using a hand-operated tissue grinder. Subsequently, samples were centrifuged to separate the vessel portion from the parenchymal portion, lysed in RIPA with protease inhibitors, and total protein concentration in the lysates was determined by BCA Assay (Pierce #23225). Lysates were subsequently frozen on dry ice and stored at -80°C until analysis.

[0357] Antibody concentrations in the brain were measured using an MSD (Meso Scale Discovery) assay. Antibody concentrations were normalized to the total protein concentration in the lysate and then graphed as either antibody concentration (ng antibody / mg total protein) or fold-change over the format matched isotype control antibody.

[0358] At 24-hour post i.v. injection, an increase in brain uptake in vessel depleted brain was observed for all anti-TfR antibodies as compared to the format-matched isotype control (FIGs. 6A and 6B). FIG. 6A shows brain uptake of various anti-TfR affinity variants (administered at 5 mg / kg) after 24 hrs in vessel-depleted brain. FIG. 6B shows the fold change over the matched isotype control in vessel-depleted brain.

[0359] In addition, whole brain antibody concentration was determined, and a ratio of vessel depleted to whole brain antibody concentration was calculated (FIG. 7). The data indicates that some fraction of the higher affinity anti-TfR antibodies remained in the vessels at the 24 hour timepoint, while the lower affinity TfR-antibodies (such as H3-7) had equivalent antibody levels in vessel depleted or whole brain, indicating that there was not a significant retention in the blood vessels. A similar trend was also observed in another in vivo study with other anti-TfR affinity variants (data not shown).

[0360] Overall, this study showed that the anti-TfR antibodies can drive significant brain uptake even at a low dose of 5 mg / kg.Example 7: Analysis of Blood Reticulocyte Levels

[0361] To assess the safety of higher affinity TfR antibodies, the population of reticulocytes in whole blood samples at 24 hours after administration was assessed. Whole blood samples from animals dosed with anti-TfR affinity variants or isotype control antibodies at 5 mg / kg were drawn from the animals prior to perfusion and collected into K2EDTA tubes. A hematological analysis was performed (Idexx Laboratories (Fremont CA)). A fully automated diagnostic instrument SYSMEX XT-V was used for routine hematology testing. The complete blood count (CBC) and reticulocyte count were measured by flow cytometry.

[0362] As shown in FIG. 8, no significant effect on blood reticulocyte levels was observed among groups in this experiment at the 24-hour timepoint. Although some variability was observed, there were no substantial decreases in reticulocyte levels or associated acute clinical symptoms.Example 8: Assessment of TfR Degradation by Anti-TfR Affinity Variants

[0363] High-affinity TfR binders have been reported to drive TfR to the lysosome and induce TfR degradation (Bien-Ly, N. et al., J. Exp. Med. 2014 Feb 10;211(2):233-44.), which is undesirable as it may impact the capacity of TfR to transport its natural ligand and any cargo attached to the anti-TfR antibody. To determine whether the anti-TfR affinity variants led to TfR degradation, the whole brain lysate from the in vivo study described above in Example 6 were prepared in RIPA buffer with protease and phosphatase inhibitors. Total protein concentration in the lysates were determined by BCA assay (Pierce #23225), and 40 pg of each sample were separated by SDS-PAGE. The gels were transferred to nitrocellulose membranes for immunoblot. An anti-TfR antibody detecting both human and mouse TfR (Thermo #13-6800) and mouse anti-GAPDH (Millipore MAB374) were incubated overnight. Images were captured using a Biorad ChemiDoc system and analyzed with Image Lab 6.1 software. Band intensities for TfR were normalized to levels of GAPDH for each sample and the isotype control mean.

[0364] FIG. 9 shows that TfR levels in animals treated with anti-TfR antibodies L-21, L-6, and L-35 were reduced in comparison to the isotype controls; however, this difference was not statistically significant. The TfR levels in anti-TfR antibodies 42Q, 24A, and 39.38 also did not decrease significantly. Overall, these results suggest that the anti-TfR antibodies tested did not significantly degrade TfR, with a subset of the anti-TfR antibodies slightly reducing TfR levels at the 24-hour timepoint and 5 mg / kg dose.Example 9: Engineering of GCase - Anti-TfR Affinity Variant Molecules

[0365] Further experiments were conducted to test the ability of an anti-TfR affinity variant described herein to transport a protein cargo across the blood-brain barrier. Complexes were engineered that combined a P-glucocerebrosidase (GCase) enzyme (wild-type or mutant GCase) with one or more of the affinity-tuned anti-TfR antibodies described herein. Table 10 shows the sequences of wild-type (WT) Human GCase and a GCase mutant used in subsequent examples.Table 10. Human GCase and GCase Mutant Sequences

[0366] Table 11 shows example sequences for various complexes of a GCase mutant protein and an anti-TfR affinity variant antibody (referred to herein as “GCase-TfR”).

[0367] The engineered complexes of the GCase mutant and an anti-TfR affinity variant antibody were expressed in a format called “N-term monozyme,” which is shown in FIG. l(iv).Table 11. Sequences for Complex of GCase and Anti-TfR Affinity Variant AntibodyExample 10: Complex of GCase and Anti-TfR Affinity Variant Traffics to the Lysosome and Rescues GBA Deficiency in Human Neuroblastoma Cells

[0368] To determine the localization and activity of a GCase-TfR complex, GBA deficient SH- SY5Y cells were generated using GBA targeting sgRNA (Synthego) and rCas9 (IDT) technology. Single clones were isolated by limiting dilution and maintained in culture in DMEM:F12 media supplemented with 10% FBS. Thirty thousand cells were treated with different concentrations of recombinant GCase-TfR for 2h at 37°C in 96-U bottom untreated plates. Untreated wild-type SH-SY5Y cells were used as controls. After uptake, cells were pelleted and the culture media was removed. The cells were resuspended with 150pM PFB- FDGlu (MedChemExpress) and incubated at 37°C for Ih. The cells were then pelleted, washed once in cold FACS Staining buffer (BD) and resuspended in FACS Staining Buffer with DAPI. Data analysis and calculation of median fluorescence intensity (MFI) values was performed with FlowJo (TreeStar) software version 10.0.8.

[0369] The following GCase-TfR complexes were tested: N-term monozyme WCAC hTfR15.WH8.1.42Q (with an affinity to TfR apical domain of 19 nM), N-term monozyme WCAC hTfR_01B_3927.L35 (274 nM), N-term monozyme WCAC hTfR15.WH8.1.24A (390 nM) and a control molecule called “mvFc_PD-WCAC” (a monovalent Fc connected by a linker “PD” to a GCase WCAC mutant) with no TfR binding.

[0370] As shown in FIG. 10, treatment with a recombinant GCase mutant without an anti-TfR antibody (“mvFc-PD-WCAC”) did not rescue GBA deficient SH-SY5Y cells to the level of wild type cells. Combining the recombinant GCase mutant (WCAC) to an anti-TfR affinity variant antibody resulted in a full rescue of GCase activity in GBA deficient SH-SY5Y cells below 5nM, which is an achievable concentration in vivo. By testing different anti-TfR affinity variants, itwas determined that there was a similar relationship in human cells as previously observed in mouse cells (data not shown). Thus, the data in FIG. 10 demonstrates that GCase-TfR complexes can be taken up through a TfR-dependent mechanism and fully rescue GCase loss of function in human neuroblastoma cells in vitro.

[0371] As shown in FIG. 11, GCase-TfR complexes co-localized with the early endosomal marker EEA-1 and the late endosomal / lysosomal marker LAMP-1, consistent with trafficking from the extracellular space to the lysosome.Example 11: Engineering of an anti-CD20-TfR Bispecific Protein

[0372] A bispecific antibody comprising an anti-TfR antigen-binding domain, anti-CD20 Fab binding domains from rituximab, and a modified IgGl Fc mutations to reduce effector function (LALA-P331S) was created in silico. The bispecific antibody comprising an anti-TfR antigenbinding domain and rituximab could be designed in one of two exemplary formats as shown in FIG. 12. Table 12 shows the sequences for a bispecific antibody comprising an anti-TfR antigenbinding domain and rituximab with a LALA-P331S sequence.Table 12. Sequences to be used in a Bispecific Antibody Comprising an Anti-TfR Antigen-Binding Domain and IAIA-P331S Rituximab

[0373] Table 13 provides the sequences to create a bispecific antibody comprising an anti-TfR antigen-binding domain and a LALA-P331S rituximab. In some aspects, the constant domain of the heavy chain contains LALA-P329S, which is a different set of mutations to reduce effector function. Figure 12 provides exemplary formats for this bispecific antibody with the sequences in Table 13 forming the bispecific antibody in a 2+1 format. The sequences of Table 13 combine to form Format A of Figure 12.Table 13. Sequences of a Bispecific Antibody Comprising an Anti-TfR Antigen-BindingDomain and LALA-P331S Rituximab

[0374] The bispecific antibody comprising an anti-TfR antigen-binding domain and a LALA- P331S rituximab is produced in cells. The produced bispecific protein will be administered in in vitro and in vivo models to demonstrate their anti-cancer effect on CD20-expressing cancers.Example 12: Binding affinities of anti-human HR- hum an progranuiin fusion proteins for human sortiiin

[0375] Anti-TfR human progranuiin fusion constructs were created by pairing anti-TfR antibodies L10-16, L10-8, 24A, and 39.38 with a human progranuiin on IgGl-LALAPS Fc, as shown in FIG. l(iii). This Example shows that these huTfR-huPGRN constructs (L10- 16.hIgGlLALAPS.hPGRN, L10-8.hIgGlLALAPS.hPGRN, 24A.hIgGlLALAPS.hPGRN, 39.38hIgGlLALAPS.hPGRN) maintain the ability to efficiently bind human sortiiin (a target of hPGRN) similar to isotype-control-hlgGlLALAPS.hPGRN, as shown by ELISAs (FIG. 13). A pre-blocked streptavidin-coated plate (Thermo, 15125) was coated with 1 pg / mL of biotinylated recombinant human sortiiin (Acrobiosy stems, SON-H82E9) in PBS for 24 hours at 4 °C. The fusion proteins were diluted three-fold from a highest concentration of 300 nM to a lowest concentration of 0.0017 nM and incubated for 1 hour at room temperature. Then, the plate was washed four times with PBST. HRP-conjugated anti-human Fc secondary antibodies were added to the plate for 30 minutes at room temperature, followed by washing. The plate was developed using TMB (Surmodics, TMBS-1000-01) and quenched using 2 N H2SO4. Optical density at 450 nm was measured using a microplate reader.

[0376] Binding affinities were determined based on EC50, with L10-8-huPGRN, L10-16- huPGRN, and isotype-control-huPGRN showing similar affinities for human sortiiin ranging from about 0.4 to about 1.2 nM (FIG. 13, left panel). In a separate experiment, 39.38-huPGRNdisplayed an affinity of about 4 nM for human sortilin (FIG. 13, right panel). Additionally, no differences were detected in human sortilin binding for all huTfR-huPGRN variants.

[0377] Sequence of hPGRN:TRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSS CCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGS WGCCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPD ARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLP AHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQG PHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQH CCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWAC CQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHD NQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQ LL (SEQ ID NO:83).Example 13: Brain uptake of huTfR-huPGRN anti-huTfR affinity variants in huTfR KI mice after a single dose

[0378] An in vivo study was performed in human TfR knock-in (hu TfR KI) mice to determine the extent of brain uptake of human PGRN constructs with various human TfR binding sequences displaying different affinities for human TfR.

[0379] Seven- to nine-week-old female homozygous huTfR KI mice, on a C57B1 / 6 background, were used for all studies (Taconic). On the day of treatment, the mice were weighed and dosed via intravenous injection in the tail vein. Mice were dosed with anti-huTfR-huPGRN variants of known differing anti-huTfR affinity: L10-16-huPGRN (3, 10, or 30 mg / kg), 10 mg / kg L10-8-huPGRN, 10 mg / kg 39.38-huPGRN, or 10 mg / kg isotype-control-huPGRN (six mice per group). Three mice from each group were sacrificed 6 hours post-dose and the three remaining mice from each group were sacrificed 24 hours post-dose. During tissue collection, blood was collected via cardiac puncture and all mice then underwent transcardial perfusion with PBS. After perfusion, brains were collected and snap frozen on dry ice. Whole blood samples from animals administered huTfR-huPGRN and isotype-control-huPGRN were obtained from the animals prior to perfusion for hematology analysis (performed at Idexx Laboratories, Fremont CA) using a fully automated diagnostic instrument SYSMEX XT-V. The complete blood count (CBC, data not shown) and reticulocyte count were measured by flow cytometry.

[0380] Mice were dosed with Fab-huFc-huPGRN fusion proteins generated on a human IgGl backbone, with the anti-human TfR or isotype control moieties within the Fab region and the Fcregion mutated to LALAPS to attenuate Fey receptor binding and thus effector functions. The N- terminus of mature mouse progranulin was fused to the C-terminus of the human IgGl backbone to attenuate potential interference with progranulin binding to sortilin via its C-terminus. The intactness of the fusion protein C-termini was evaluated by mass spectrometry.

[0381] As shown in FIG. 14, no major changes in red blood cell count (RBC, a key hematological parameter) were observed in mice administered huTfR-huPGRN or isotype- control-huPGRN fusion proteins. In addition, blood reticulocyte counts did not differ between treatment groups. RBC and blood reticulocyte counts were measured by flow cytometry. Surface TfR levels on blood reticulocytes were analyzed by FACS of whole blood (FIG. 15). No significant differences were observed between the treatment groups.

[0382] Brain fusion protein levels were analyzed using a modified MSD ECLA (FIG. 16). Analyses were performed using vessel-depleted brain lysates to measure brain fusion protein levels in the brain parenchyma. Vessel-depleted brain lysates were generated and the total protein concentrations were measured. Briefly, a 384- or 96-well streptavidin-coated plate (MSD, L21SA-1 or L15SA-1) was incubated with 0.1 pg / mL of biotinylated goat anti-human IgG (SouthemBiotech, 2049-08) in TAB (0.1% BSA and 0.05% Tween-20 in Tris-buffered saline) for 1 hour at room temperature, shaking at 600 rpm or 350 rpm (for 384- and 96-well plates, respectively). After washing with PBST, standards, quality controls (QCs), and samples were added to the plate and incubated for 2 hours at room temperature with shaking. During this time, the detection antibody was prepared by incubating 0.25 pg / mL of ruthenylated goat anti-human IgG with 0.1% mouse serum in TAB for 1 hour at room temperature while rotating. After washing the plate with PBST, the detection antibody was added and incubated for 1 hour at room temperature with shaking. After washing with PBST, 2X MSD Read Buffer T with surfactant (MSD, R92TC) was added to the plate and it was read immediately using an MSD Sector S600 plate reader. For all brain uptake assays, each fusion protein was included as a standard, and sample and QC concentrations were interpolated based on the respective fusion protein standard curve. Antibody concentrations were normalized to the total protein concentration in the lysate for each sample.

[0383] FIG. 16 shows brain levels of anti -huTfR-huPGRN fusion proteins 6 hours and 24 hours after a single dose in huTfR-KI mice. While isotype-control-huPGRN levels were relatively low, mice dosed with L10-16-huPGRN showed a dose-dependent increase in brain levels. At 6 hours post-dose, 3 mg / kg L10-8-huPGRN group reached brain levels that weresimilar to those of the 10 mg / kg L10-16-huPGRN group. Overall, higher huTfR affinity correlated with increased brain fusion protein levels at 6 hours and 24 hours post-dose.

[0384] TfR levels in whole brain lysates were assessed to determine whether the blood-brain barrier (BBB) target showed an affinity-dependent decrease. Cleared whole brain lysates were generated from PBS-perfused brain tissue using N-PER lysis buffer (Thermo, 87792) with Halt protease and phosphatase inhibitors (Thermo, 78466), Qiagen TissueLyser II with 5 mm stainless steel beads (Qiagen), and centrifugation. Brain TfR levels were determined by quantitative immunoblotting. No differences were observed in brain TfR levels as a function of treatment group at 6 hours or 24 hours post-dose (FIG. 17).Example 14: Effect of TfR Affinity on Brain Uptake and Reticulocyte Depletion in Human TfR-knockin Mice Treated with GCase-TfR

[0385] Additional experiments were performed with the GCase-TfR molecules described in Example 9.

[0386] To establish the in vivo relationship between TfR affinity and serum, brain pharmacokinetic (PK), and reticulocyte depletion of GCase-TfR constructs, 8-10 week-old male human TfR-KI mice were injected intravenously with 10 mg / kg or 50 mg / kg GCase-TfR, and their serum and brain PK was monitored over 72 hours. WCAC GCase was used in the GCase- TfR constructs and combined with anti-TfR antibodies 42Q, L7-2, or 24A, as described in Example 9, in the N-terminal monozyme format (as shown in FIG. 1 (iv). See, also, Table 11, above, for sequences of GCase-TfR constructs.

[0387] As a control, a monovalent dGCase3 as disclosed in Pokorna et al. was fused to an Fc (C4-GC3). See Pokorna et al., Design of a stable human acid-P-glucosidase: towards improved Gaucher disease therapy and mutation classification; FEBS J. 2023 Jul; 290(13):3383-3399. dGCase3 has the sequence: ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPAPGTFSRYESTRSGKRMERSMGPIQANW TGTGLLLTLHPEQKFQKIKGFGGAMTDAAAINILKLSPPAQEELLKSYFSEEGIEYNIIRVP MGSCDFSTRTYTYADTPNDFQLKNFSLPEEDTKLKIPLIKRAQALSQRPVSLLAXPWTAP TWLKTNGAMNGKGTLKGQPGDKYHQTWANYFVKFLDAYAKHGLTFWAVTAENEPSA GLIPGYPFQCLGFTPEHQRDFIARNLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLS DPEAAKYVHGIAVHWYLDFLAPAKDTLGETHRLFPDKFLFASEACTGSHFWEQSVRLGS WDRGEQYAHSIIEDLNYWVVGWTDWNLALDPEGGPNWVKNFVDSPIIVDATKDEFYKQPMFYHMGHFSKFIPEGAQRVGLSASQKNDLDAVALMNPDGSAVVVVLNRSDKDVPL TIWDPDVGFIETISPGYSIHTYLWRRQ (SEQ ID NO: 116).

[0388] The treatment groups and sample collection schedule stagger (each stagger N=3) are summarized in Tables 14 and 15, respectively, below. Hpi = hours post infection.Table 14. Treatment GroupsTable 15. Sample collection schedule for each stagger of N=3

[0389] Serum and brain concentrations of GCase-TfR were measured using an anti-Fc sandwich assay on the MesoScale Discovery (MSD) platform. Biotinylated goat anti-human IgG (SouthemBiotech Cat. No. 2049-08) was captured on streptavidin-coated plates (MesoScale Discoveries Cat. No. R32AJ-5) for 1 hour at room temperature. After washing, samples were added at an appropriate dilution and incubated with shaking for 2 hours at room temperature. After a further wash, a ruthenilated goat anti-human IgG antibody (SouthemBiotech Cat. No.2049-01) was added as a detection reagent for 1 hour at room temperature and read on the MSD platform.

[0390] As shown in FIG. 18A, serum PK profiles of the three 10 mg / kg groups were very similar, with a non-significant trend towards faster distribution as the TfR affinity increased (data normalized to 5 -minute or 30-minute timepoints depending on stagger group). As summarized in Table 16, below, the area under the curve (AUC) computed from the raw PK concentrations confirmed the lack of difference between G2 and G4. The AUC of G1 was about 20% lower because of faster distrubtion in the first 30 minutes. This faster distribution was observed in the 50 mg / kg groups, as well. However, serum concentrations of G2 and G4 varied by less than 10% in the first 30 minutes.Table 16. Area under the curve of serum PK

[0391] As shown in FIG. 18B, there was no difference in PK profiles of the two 50 mg / kg groups. The Cmax of G3 and G5 was 7-8 times higher than Gl, G2, and G4 as shown in FIG. 18C. These data suggest that the range of TfR affinities tested had minimal impact on serum PK at 10 mg / kg, and no impact at 50 mg / kg.

[0392] Vessel-depleted brain concentration was measured at the terminal time points 24-hours- or 72-hours-post-inj ection. As shown in FIG. 19, the brain concentrations of Gl, G2, and G4 were similar. The brain concentrations of G3 and G5 were also similar at both time points, and about 2.4-fold higher than the 10 mg / kg groups at 24-hours post injection. These data suggest that at the dose and TfR affinity range tested, brain uptake was not affected by TfR affinity and did not saturate at 10 mg / kg.

[0393] Reticulocyte counts were measured by IDEXX BioAnalytics from whole blood samples collected at the terminal time points in blood collection (K2 EDTA) tubes. As shown in FIG.20A, a trend for lower reticulocytes was observed with higher TfR affinity. For G2 and G3, there was no statistically significant dose-dependent trend. Only the reduction observed in G3 was statistically different from the no TfR-moiety control group, as indicated by the asterisk on FIG. 20A (G6; p=0.048 one-way ANOVA with Dunnett’s multiple comparison test). 72 hours after injection, there was no statistically significant difference in reticulocyte counts between the treated and control groups (FIG. 20B), suggesting a quick recovery of reticulocyte count aftertreatment. This is consistent with the short half-life observed in the PK dataset. These data suggest that the GCase-TfR constructs do not significantly reduce circulating reticulocytes at 10 mg / kg dose, regardless of affinity, likely due to a combination of engineering of the Fc, short half-life, characteristics of the Fab, and the TfR antibody epitope. At a higher dose, there was only a limited and transient reduction in reticulocyte counts observed which resolved by 72-hours post injection.Example 15: Effect of TfR Affinity and Construct Format on Brain Uptake and Target Knockdown of siRNA-conjugated TfRs

[0394] To assess the ability of the present TfR-targeting antigen-binding domains to deliver nucleic acid cargos to the brain, a study is conducted utilizing a representative siRNA targeting SOD1 (superoxide dismutase 1) conjugated to TfR-binding moieties with a range of affinities and in various formats (FIG. 21).

[0395] The formats include “Format #1” in FIG. 21 encompassing a knob-in-hole Fc with a single anti-TfR Fab (constructs #1, #2, #3, and #4) with a single engineered free cysteine; a “2+1” (“Format #2” in FIG. 21) format wherein the TfR-binding domain is an scFv on the C- terminus of the constant domain of a knob-in-hole IgG with a single engineered free cysteine with inert Fabs (constructs #5 and #6); and a “Fab-only” format (“Format #3” in FIG. 21) wherein the siRNA is directly conjugated to an anti-TfR Fab without Fc (constructs #7 and #8). The star in FIG. 21 indicates the approximate position of the free cysteine on the constructs, which is the conjugation site for the siRNA.

[0396] In an exemplary experiment, cells are co-transfected with three expression plasmids encoding the heavy-chain knob, heavy-chain hole, and light-chain in a transient engineered CHO cell line at a ratio of 2: 1 :2 for 7 days. For the “Fab-only” format, only heavy and light chains are used at a 1: 1 ratio. The components are purified from conditioned media by loading supernatant over a protein A column (GE Mab SelectSuRe). The columns are washed with PBS at pH 7.4 and are eluted with 50 mM sodium citrate at pH 3.0 containing 150 mM NaCl and immediately neutralized with IM TRIS at pH 8.0. The components are further purified by cation exchange chromatography (GE Poros XS) with 25 mM sodium acetate at pH 5.0 with a 1 M NaCl gradient. The components are then formulated into PBS at pH 7.4. The purified components may be confirmed by intact mass LC / MS and checked for a purity of >95% by analytical HPLC-SEC.

[0397] The resulting antibodies are functionalized with free cysteines (constructs #1-6 with S239C and constructs #7-8 with K145C) to allow for conjugation through cysteine-maleimidechemistry. Briefly, antibodies are reduced using TCEP tris(2-carboxyethyl)phosphine to liberate free cysteine residues and then reoxidized using DHAA dehydroascorbic acid to re-bridge the interchain disulfides and leave the engineered cysteine(s) available for maleimide-based conjugation. See Cochran et al., J. Med. Chem. 2024 Sep 12;67(17): 14852-14867. SMCC- functionalized siRNA targeting the SOD1 gene are then conjugated to the reduced engineered cysteine(s) of the antibodies to form the siRNA-TfR conjugates. Free SH groups are capped with N-ethylmal eimide (NEM) and the conjugated antibodies are purified to isolate the DAR1- conjugate (DARI meaning a single siRNA conjugated per antibody).

[0398] Anti-SODl siRNA-TfR constructs are assayed for brain uptake and knockdown of SOD1 gene expression in a multi-dose PK / PD study in human TfR knock-in (huTfR-KI) mice. huTfR-KI mice are dosed with 4 weekly intravenous injections of siRNA-TfR or naked siRNA as a control. An additional cohort of mice are dosed with a single ICV injection of naked siRNA as an additional control. Serum samples are collected routinely, such as at 1 hour, 4 hours, 24 hours, and 72 hours post-dosing for plasma PK analysis. Animals are taken down at 72-hours post-final- dosing (day 24) and plasma, brain, and peripheral tissues are collected. Tissues are processed to remove blood vessels and lysed, and tissue lysate and plasma samples are quantified for test article levels. Additionally, SOD1 mRNA levels are quantified by QuantiGene™ Singleplex assay and normalized to vehicle treated control in order to determine the level of knockdown in peripheral and brain tissues. Exemplary anti-SODl siRNA sequences to be tested in the siRNA- TfR constructs are summarized in Table 17 and exemplary constructs #l-#8 are summarized in Tables 18 and 18B, below.Table 17: Anti-SODl siRNA test sequencesSiRNA Nomenclature:• (C16-N): 2'-O-hexadecyl ribonucleotide• (MC-Val-Cit-PABC): Valine-citrulline dipeptide crosslinker (cathepsin-mediated cleavage)• (NHC6): Aminohexyl linker• (SMCC): Succinimidyl-trans-4-(N-maleimidylmethyl)cyclohexane-l -carboxylate• (vinu): 5'-vinylphosphonate-2'-0-methyl ribonucleotide• Nf: 2'-F residues• n: 2'-0Me residues• s: phosphorothioate backbone modificationTable 18A. TfR Sequences for Conjugation with siRNATable 18B. siRNA ConstructsExample 16: Determination of Anti-TfR Antibody Binding Epitope

[0399] A study was designed to determine the binding epitopes of anti-TfR antibodies disclosed herein. A Carterra LSA-XT instrument (Carterra, Salt Lake City, UT) was used to evaluate binding of the antibodies to a series of TfR apical domain mutants that eachincorporated an alanine substitution of a selected residue. The TfR apical domain mutants were derived from the following engineered human TfR apical domain sequence: SSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNDSAQN SVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIV RAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELS (SEQ ID NO:84).

[0400] Amino acid residues with surface-accessible side chains were identified using Molecular Operating Environment (MOE) software (Chemical Computing Group, Montreal, Canada) based on published crystal structures of a TfR apical domain construct (PDB IDs: 6Y76 and 6W3H).

[0401] A subset of these surface-accessible residues in human TfR were selected based on their difference in the murine TfR sequence (murine sequence from NCBI Reference Sequence NP 035768.1 versus human sequences). The anti-TfR antibodies disclosed herein display selective binding to human TfR over murine TfR. See Example 5 and Example 6 of WO 2024 / 026472. These results suggest that the epitopes include at least one of these non-conserved residues found in the human TfR sequence but not in the murine TfR sequence. FIG. 22 shows the sequence of the apical domain construct with these selected residues indicated in bold underlined text. All expressed proteins contained an N-terminal His / Avi tag for capture.

[0402] The TfR apical domain variants were produced by transient expression in HEK293 cells using standard methods. Culture supernatants containing the expressed variants were spotted in an array onto an anti-Avi tag or anti-His tag lawn on a sensor chip that had been prepared according to the manufacturer’s recommended protocol. Briefly, the anti-Avi tag antibody (MAB 10546, R&D Systems) or anti-His tag antibody (GenScript catalog number A00186) was immobilized on an HC30M or HC200M sensor chip (catalog number 4279 or 4827, Carterra, Salt Lake City, UT) by amine coupling using a running buffer without BSA (HBS-EP+, catalog number 8020, Teknova, Hollister, CA). The supernatants containing TfR apical domain variants and control wild-type construct were diluted 10-fold into Running Buffer (HBS-EP+ with 0.5mg / mL BSA, Roche catalog number 3117332001). Purified TfR apical domains were diluted to 10 ug / mL as positive controls. For each antibody tested, TfR apical domains and control proteins were spotted onto the sensor chip for 3-10 minutes using the LSA-XT multi-channel spotting function. After three injections of Running Buffer, antibodies diluted to 2-8 uM in Running Buffer were injected for 3 minutes, followed by 3 minutes of dissociation in Running Buffer. After injection of each test antibody, the chip surface was regenerated with two x 30- second injections of 10 mM Glycine at pH 1.5 (catalog number 3639, Carterra, Salt Lake City,UT) to remove captured and bound proteins, leaving the capture surface free to capture fresh analytes. Data were processed using Kinetics Analysis Software (Carterra, Salt Lake City, UT). Sensorgrams and binding levels were evaluated to identify residues that were required for binding by the antibodies and thus represent a part of the binding epitope.

[0403] FIG. 23 shows the results of the binding study, which reveal that the antibodies bind to different regions of the TfR apical domain. For the antibody in the TfR.9 lineage (TfR.9.1B.39.38), binding was comparable to the parental TfR apical domain construct for all variants except Vari 7, indicating that Y72, by numbering in FIG. 22, is part of its epitope. While its binding to Varl3 (D65A, by numbering of FIG. 22) is reduced, all of the antibodies tested had reduced binding to Varl3, suggesting that Varl3 may have been poorly expressed or folded, resulting in lower binding overall, such that the mutated position in Vari 3 cannot be conclusively evaluated. For both antibodies from the TfR.15 lineage (TfR.15.WH8.1.42Q.L7-2 and TfR.15.WH8.1.24A), binding was comparable to the parental TfR apical domain construct for all variants except Var03, indicating that D31, by numbering in FIG. 22 (D356 by numbering of SEQ ID NO: 1), is part of their epitope. For the commercial antibody trontinemab (Cat. No. HY- P9999, MedChemExpress, Monmouth Junction, NJ), which was included as a control, binding was comparable to the parental TfR apical domain construct for all variants except Vari 6 and Vari 7, suggesting that both residues V71 and Y72 in FIG. 22 are included in its epitope. These findings are summarized in Table 19, below, together with published epitope residues for additional anti-TfR antibodies.Table 19. Variants and Residues Included in Antibody Epitopes* Epitope residue numbering is for mouse TfR homolog.

[0404] The locations of the epitopes for these antibodies, as well as the epitopes for additional published anti-TfR antibodies, are listed in Table 19, above, and highlighted in the structure shown in FIG. 24. The epitope regions are indicated by the space-filling sidechains on the backbone of the ECD dimer structure from the ECD / Transferrin complex molecular structure in PDB ID: 1SUV. These results show that antibodies from the TFR.15.WH8 lineage bind to an epitope on a region of the TfR ECD that is distinct from the epitopes of the other tested or known antibodies. Antibodies from the TfR.9 lineage bind to an epitope that overlaps with those of other published antibodies, trontinemab and TV35, and the parallel location on the mouse homolog by antibody 8D3. A third epitope region (outside of the TfR apical domain) is reported for antibody h26D3.Example 17: Anti-TfR Antibodies that Occlude / Prevent TfR-dependent ADCC and CDC

[0405] Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are key potential safety risks for anti-TfR antibodies, particularly those containing active Fc (such as IgGl) capable of engaging activating Fc gamma receptors (such as FcyRIIIa) on effector cells in vivo. See Couch et al., Addressing safety liabilities of TfR bispecific antibodies that cross the blood-brain barrier, Sci. Transl. Med. 2013 May l;5(183): 183ra57, 1-12. The ability of anti-TfR 2+1 antibodies to drive these unwanted effector functions may be related to the epitope on the transferrin receptor being bound. The TfR.15 lineage family of antibodies binds to a unique epitope on the transferrin receptor that is different from the TfR.9 lineage as well as many other known antibodies, as shown in the preceding Example, and which may protect against ADCC and CDC through the orientation of antigen bound antibody.

[0406] The ability of certain anti-TfR 2+1 antibodies disclosed herein to drive antibodydependent cellular cytotoxicity (ADCC) is evaluated using an ADCC Reporter Bioassay system from Promega (catalog number G7010). This system relies on an engineered Jurkat T cell line stably expressing the FcyRIIIa receptor (VI 58 variant) and an NF AT response element driving expression of firefly luciferase. A variety of TfR-expressing target cells can be used in this assay including Ramos, hCMEC / D3 or even purified reticulocytes to mimic the ability of anti-TfR antibodies to drive ADCC on different cell types in vivo.

[0407] Briefly, antibodies to be tested are aliquoted at 3x their final concentration in assay buffer (RPMI 1640 media + 4% low IgG Serum), in a 3-fold dilution series. Target cells are diluted in assay buffer at a concentration of 1.2xl06per mL, and 25 pL of cells (30,000 per well) are aliquoted to inner wells of a 96-well white assay plate. The outer wells of the plate are filled with 75 pL of assay buffer (no cells or antibody) to minimize edge effects. 25 pL of 3x antibody is added to wells containing cells. Following addition of antibody to target cells, the provided effector cells (frozen at 2xl07cells per mL) are thawed at 37 °C, and 630 pL are added to 3.6mL of warmed (37 °C) assay buffer with gentle mixing. 25 pL of the diluted effector cells (75,000 per well, for an E:T ratio of 2.5: 1) are immediately added to the wells containing target cells and antibody. The plate is then incubated at 37 °C with 5% CO2 for 6 hours to allow for receptor cell activation and luciferase expression. After 6 hours, the plate is equilibrated to room temperature (about 15 minutes), while the Bio-Gio Luciferase Assay Reagent is thawed and mixed. 75 pL of the Luciferase Assay Reagent is subsequently added to each well, and the plate is incubated for 10 minutes on a plate shaker, and luminescence is measured on a BioTek plate reader with data analysis conducted in PRISM.

[0408] Similarly, the ability of anti-TfR 2+1 antibodies to induce CDC may be assayed using a custom CDC assay utilizing complement-active human serum. Briefly, antibodies to be tested are aliquoted at 4x their final concentration in RPMI 1640 media in a 3 -fold dilution series. Target cells are detached, washed once in PBS, and diluted to 2xl06cells / mL in RPMI 1640 media.50 pL of target cells are aliquoted per well (IxlO5cells per well) in round-bottom 96-well plates. 25 pL of the 4x antibody media is added to the washed and diluted target cells. Cells and antibody are incubated at 37 °C for 15 minutes, and then 25 pL of pooled complement human serum (Innovative Research, IPLA-CSER) are added per well as a complement source. The plates are incubated for a further 30-120 minutes at 37 °C. Afterwards, the cells are washed twice with FACS buffer (PBS + 2% FBS + 1 mM EDTA), and 100 pL of 1 :200 diluted anti-C3b-APC antibody (Biolegend catalog number 846106) is added per well and incubated on ice for30 minutes. The cells are then washed twice with FACS buffer and resuspended in 80 pL / well of FACS buffer + 0.25 pl / well of propidium iodide prior to analysis on a flow cytometer.Complement activation is measured as either cell killing (ratio of PI+ cells in each well) or, if the response was too weak to see cell killing, by assessing C3b deposition in the APC channel.Example 18: Disulfide Stapling of Anti-TfR Antibodies

[0409] Single-chain fragment variable (scFv) antibody formats contain two variable domains (VH and VL) that are linked by a flexible linker. Due to weak interaction between the VH and VL domains, some scFvs may exhibit increased risk of aggregation and / or may display reduced stability. To overcome this, two mutations are introduced into the VH and VL framework (VH44 and VL100) to introduce a disulfide bond between the VH and VL domains (see Reiter et al., 1994, Biochemistry, 33:5451-5459 and Wetherill et al., 2012, Protein Engineering, Design & Selection, 25:321-329). Sequences of several TfR scFvs displaying diverse affinities with disulfide stapled mutations in VH-VL orientation are listed below in Table 20 (Table 4 and Table 5 provide CDR sequences of these constructs). The engineered cysteines in Table 20 are bolded and underlined. Several properties of the disulfide stapled TfR dsFvs are assessed, including affinity to human and cyno TfR apical domain, manufacturing characteristics (e.g., assessment of hydrophobic interaction, freeze / thaw stability, heat stress, thermostability, concentratability), in vitro characteristics (hCMEC brain endothelial cell uptake), and in vivo characteristics (e.g., brain uptake, effect on reticulocyte count, serum clearance, etc.).Table 20. Disulfide-stapled scFv (dsFv) SequencesExample 19: Linker Engineering

[0410] A multi-specific 2+1 antibody may contain a flexible linker between the C-terminus of one of the antibody heavy chains and the anti-TfR antigen-binding domain. To increase rigidity of the molecule and reduce the potential for T...

Claims

WHAT IS CLAIMED IS:

1. An antigen-binding domain that specifically binds to human transferrin receptor (TfR), wherein the 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, VL CDR2, and VL CDR3 sequences comprising the amino acid sequences of(i) SEQ ID NOs:22, 23, 24, 34, 32, and 33, respectively;(ii) SEQ ID NOs:22, 23, 26, 34, 32, and 33, respectively;(iii) SEQ ID NOs:22, 23, 25, 35, 32, and 33, respectively;(iv) SEQ ID NOs:22, 23, 25, 36, 32, and 33, respectively;(v) SEQ ID NOs:22, 23, 25, 37, 32, and 33, respectively;(vi) SEQ ID NOs:22, 23, 25, 38, 32, and 33, respectively;(vii) SEQ ID NOs: 22, 23, 167, 34, 32, and 33, respectively;(viii) SEQ ID NOs:27, 30, 29, 42, 40, and 41, respectively;(ix) SEQ ID NOs:27, 28, 29, 43, 40, and 41, respectively;(x) SEQ ID NOs:27, 28, 29, 44, 40, and 41, respectively; or(xi) SEQ ID NOs:27, 30, 29, 45, 40, and 41, respectively.

2. An antigen-binding domain that specifically binds to human transferrin receptor (TfR), wherein the antigen-binding domain comprises (i) VH CDR1, VH CDR2, and VH CDR3 sequences and (ii) VL CDR1, VL CDR2, and VL CDR3 sequences as set forth in:(i) SEQ ID NOs:46 and 48, respectively;(ii) SEQ ID NOs:50 and 48, respectively;(iii) SEQ ID NOs:49 and 51, respectively;(iv) SEQ ID NOs:49 and 52, respectively;(v) SEQ ID NOs:49 and 53, respectively;(vi) SEQ ID NOs:49 and 54, respectively;(vii) SEQ ID NOs: 55 and 48, respectively;(viii) SEQ ID NOs:58 and 59, respectively;(ix) SEQ ID NOs:56 and 60, respectively;(x) SEQ ID NOs:56 and 61, respectively;(xi) SEQ ID NOs: 58 and 62, respectively; or(xii) SEQ ID NOs:56 and 57, respectively.

3. The antigen-binding domain of claim 1 or claim 2, wherein the 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%, or at least 99% identical to the amino acid sequences of:(i) SEQ ID NOs:46 and 48, respectively;(ii) SEQ ID NOs:50 and 48, respectively;(iii) SEQ ID NOs:49 and 51, respectively;(iv) SEQ ID NOs:49 and 52, respectively;(v) SEQ ID NOs:49 and 53, respectively;(vi) SEQ ID NOs:49 and 54, respectively;(vii) SEQ ID NOs:55 and 48, respectively;(viii) SEQ ID NOs:58 and 59, respectively;(ix) SEQ ID NOs:56 and 60, respectively;(x) SEQ ID NOs:56 and 61, respectively;(xi) SEQ ID NOs:58 and 62, respectively;(xii) SEQ ID NOs:85 and 87, respectively;(xiii) SEQ ID NOs:89 and 87, respectively;(xiv) SEQ ID NOs:88 and 90, respectively;(xv) SEQ ID NOs:88 and 91, respectively;(xvi) SEQ ID NOs:88 and 92, respectively;(xvii) SEQ ID NOs:88 and 93, respectively;(xviii) SEQ ID NOs:94 and 87, respectively;(xix) SEQ ID NOs:97 and 98, respectively;(xx) SEQ ID NOs:95 and 99, respectively;(xxi) SEQ ID NOs:95 and 100, respectively;(xxii) SEQ ID NOs:97 and 101, respectively;(xxiii) SEQ ID NOs: 102 and 104, respectively;(xxiv) SEQ ID NOs: 106 and 104, respectively;(xxv) SEQ ID NOs: 105 and 107, respectively;(xxvi) SEQ ID NOs: 105 and 108, respectively;(xxvii) SEQ ID NOs: 105 and 109, respectively;(xxviii) SEQ ID NOs: 105 and 110, respectively;(xxix) SEQ ID NOs: 111 and 104, respectively;(xxx) SEQ ID NOs: 114 and 115, respectively; or(xxxi) SEQ ID NOs:56 and 57, respectively.

4. An antigen-binding domain that specifically binds to human TfR, wherein the antigenbinding domain comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO:46, 49, 50, 55, 56, 58, 85, 88, 89, 94, 95, 97, 102, 105, 106, 111, or 114.

5. An antigen-binding domain that specifically binds to human TfR, wherein the antigenbinding domain comprises a VH and a VL, wherein the VL comprises the amino acid sequence of SEQ ID NO:48, 51, 52, 53, 54, 59, 60, 61, 62, 104, 107, 108, 109, 110, or 115.

6. The antigen-binding domain of any one of claims 1-5, wherein the antigen-binding domain comprises a VH and VL comprising the amino acid sequences of:(i) SEQ ID NOs:46 and 48, respectively;(ii) SEQ ID NOs:50 and 48, respectively;(iii) SEQ ID NOs:49 and 51, respectively;(iv) SEQ ID NOs:49 and 52, respectively;(v) SEQ ID NOs:49 and 53, respectively;(vi) SEQ ID NOs:49 and 54, respectively;(vii) SEQ ID NOs: 55 and 48, respectively;(viii) SEQ ID NOs:58 and 59, respectively;(ix) SEQ ID NOs:56 and 60, respectively;(x) SEQ ID NOs:56 and 61, respectively;(xi) SEQ ID NOs: 58 and 62, respectively;(xii) SEQ ID NOs:85 and 87, respectively;(xiii) SEQ ID NOs: 89 and 87, respectively;(xiv) SEQ ID NOs:88 and 90, respectively;(xv) SEQ ID NOs:88 and 91, respectively;(xvi) SEQ ID NOs:88 and 92, respectively;(xvii) SEQ ID NOs:88 and 93, respectively;(xviii) SEQ ID NOs: 94 and 87, respectively;(xix) SEQ ID NOs:97 and 98, respectively;(xx) SEQ ID NOs:95 and 99, respectively;(xxi) SEQ ID NOs:95 and 100, respectively;(xxii) SEQ ID NOs:97 and 101, respectively;(xxiii) SEQ ID NOs: 102 and 104, respectively;(xxiv) SEQ ID NOs: 106 and 104, respectively;(xxv) SEQ ID NOs: 105 and 107, respectively;(xxvi) SEQ ID NOs: 105 and 108, respectively;(xxvii) SEQ ID NOs: 105 and 109, respectively;(xxviii) SEQ ID NOs: 105 and 110, respectively;(xxix) SEQ ID NOs: 111 and 104, respectively; or(xxx) SEQ ID NOs: 114 and 115, respectively.

7. The antigen-binding domain of any one of claims 1-6, wherein the antigen-binding domain binds to an epitope comprising residue D356 of SEQ ID NO: 1.

8. The antigen-binding domain of any one of claims 1-7, wherein the antigen-binding domain is capable of crossing the blood brain barrier (BBB).

9. The antigen-binding domain of any one of claims 1-8, wherein the antigen-binding domain binds to cynomolgus monkey TfR.

10. The antigen binding domain of any one of claims 1-9, wherein the antigen-binding domain binds human TfR with an affinity of 0.01 nM to 50 nM.

11. The antigen binding domain of any one of claims 1-9, wherein the antigen-binding domain binds human TfR with an affinity of 51 nM to 750 nM.

12. The antigen binding domain of any one of claims 1-9, wherein the antigen-binding domain binds human TfR with an affinity of 751 nM to 10,000 nM.

13. The antigen-binding domain of any one of claims 1-12, wherein the antigen-binding domain is internalized in blood-brain barrier epithelial cells, optionally wherein the antigen-binding domain is internalized in blood-brain barrier epithelial cells greater than5-fold or greater than 40-fold as compared to internalization by an isotype control, further optionally wherein the blood-brain barrier epithelial cells are HCMEC / D3 cells.

14. The antigen-binding domain of any one of claims 1-13, wherein the antigen-binding domain does not significantly reduce TfR levels in a mouse brain following administration of the antigen-binding domain as measured in a whole brain lysate.

15. The antigen-binding domain of any one of claims 1-14, wherein the antigen-binding domain accumulates at least 4-fold or at least 5-fold more than an isotype control in vessel-depleted mouse brain.

16. The antigen-binding domain of any one of claims 1-15, wherein the antigen-binding domain comprises a VH and a VL on a single polypeptide chain.

17. The antigen-binding domain of any one of claims 1-16, wherein the antigen-binding domain comprises a single-chain fragment variable (scFv).

18. The antigen-binding domain of any one of claims 1-17, wherein the antigen-binding domain comprises a VH and a VL and further comprises a disulfide staple comprising a disulfide bond: (i) between a cysteine residue in the VH and a cysteine residue in the VL of the antigen-binding domain, (ii) between a cysteine residue in the VH and a cysteine residue outside the VH, or (iii) between a cysteine residue in the VL and a cysteine residue outside the VL.

19. The antigen-binding domain of claim 18, wherein:(i) the disulfide staple is formed in part by the substitution of a residue with cysteine in the VH domain;(ii) the disulfide staple is formed in part by the substitution of a residue with cysteine in the VL domain;(iii) the VH and VL are linked by a linker, and the disulfide bond is formed in part by a cysteine residue in the linker; and / or(iv) the disulfide staple is formed by the substitution of a residue with cysteine in the VH domain and the substitution of a residue with cysteine in the VL domain.

20. The antigen-binding domain of claim 19, wherein:(i) the substitution of the residue with cysteine is at position 44 of the VH, with numbering according to Kabat;(ii) the substitution of the residue with cysteine is at position 100 of the VL, with numbering according to Kabat; or(iii) the substitutions of residues with cysteines are at position 44 of the VH and position 100 of the VL, with numbering according to Kabat.

21. The antigen-binding domain of any one of claims 1-20, wherein the antigen-binding domain comprises a disulfide-stapled single-chain fragment variable (dsFv) comprising an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 132-142.

22. The antigen-binding domain of any one of claims 17-21, wherein the scFv is in the orientation VH-linker-VL.

23. The antigen-binding domain of any one of claims 17-21, wherein the scFv is in the orientation VL-linker-VH.

24. The antigen-binding domain of claim 22 or 23, wherein the linker (i) is about 5 to about 25 amino acids, is about 5 to about 20 amino acids, is about 10 to about 25 amino acids, or is about 10 to about 20 amino acids and / or (ii) comprises the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 5) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:6).

25. The antigen-binding domain of any one of claims 1-16, wherein the antigen-binding domain comprises a VH on a first polypeptide and a VL on a second polypeptide.

26. An antigen-binding domain that specifically binds to human TfR, wherein the antigenbinding domain is a VHH comprising (i) the VH CDR1, VH CDR2, and VH CDR3 as set forth in any one of SEQ ID NOs:48, 51, 52, 53, 54, 59, 60, 61, 62, 87, 90, 91, 92, 93, 98, 99, 100, 101, 104, 107, 108, 109, 110, or 115 or (ii) the VH of any one of SEQ ID NOs:48, 51, 52, 53, 54, 59, 60, 61, 62, 87, 90, 91, 92, 93, 98, 99, 100, 101, 104, 107, 108,109, 110, or 115, optionally wherein the VHH is capable of crossing the blood brain barrier (BBB).

27. An antibody comprising the antigen-binding domain of any one of claims 1-26.

28. A complex or fusion protein comprising the antigen-binding domain of any one of claims 1-26 and a heterologous protein or peptide.

29. The complex or fusion protein of claim 26, wherein the antigen-binding domain and heterologous protein or peptide are linked by an amino acid linker.

30. The complex or fusion protein of claim 29, wherein the linker is (GGGGS)x3 (SEQ ID NO:7), (GGSGG)x3 (SEQ ID NO:8), or GGSGG (SEQ ID NO:9).

31. The complex or fusion protein of any one of claims 28-30, wherein the heterologous protein or peptide (a) comprises the amino acid sequence of P-glucocerebrosidase (GCase or GBA), progranulin (PGRN), Prosaposin (PSAP), clusterin (APO J), Reelin, very low density lipoprotein receptor (VLDLR), apolipoprotein E receptor 2 (APOER2, low-density lipoprotein receptor-related protein 8 (LRP8)), Tripeptidyl Peptidase 1 (CLN2 / TPP1), or Alpha-L-Iduronidase (IDUA), Iduronate 2-Sulfatase (IDS), glucosamine (N-acetyl)-6-sulfatase (GNS), heparan-alpha-glucosaminide N- acetyltransferase (HGSNAT), and N-acetyl-alpha-glucosaminidase (NAGLU), N- sulfoglucosamine sulfohydrolase (SGSH), ubiquitin protein ligase E3 A (UBE3 A), or a variant or portion thereof or (b) specifically binds to beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha- synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, Glycoprotein nonmetastatic protein B (GPNMB), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6 A (MS4A6A), or Transmembrane Protein 106B (TMEM106b).

32. The complex of any one of claims 28-31, wherein the complex comprises a first polypeptide comprising from N’ to C’ terminal, a VH of the antigen-binding domain, an Fc domain, and the heterologous protein or peptide; and a second polypeptide comprising a VL of the antigen-binding domain.

33. The complex of any one of claims 28-31, wherein the complex comprises from N’ to C’ terminal a first polypeptide comprising the heterologous protein or peptide and a first Fc domain; a second polypeptide comprising from N’ to C’ terminal a VH of the antigenbinding domain and a second Fc domain; and a third polypeptide comprising a VL of the antigen-binding domain.

34. The complex of claim 33, wherein the first Fc domain comprises a knob mutation and the second Fc domain comprises a hole mutation.

35. The complex of claim 33, wherein the first Fc domain comprises a hole mutation and the second Fc domain comprises a knob mutation.

36. A multi-specific protein comprising a first antigen-binding domain that is the antigenbinding domain of any one of claims 1-26 linked to an antibody or second antigenbinding domain.

37. The multi-specific protein of claim 36, wherein the antibody or second antigen-binding domain specifically binds to (i) a CNS antigen or (ii) a cancer antigen.

38. The multi-specific protein of claim 36 or claim 37, wherein the antibody or second antigen-binding domain comprises a heavy chain constant region.

39. The multi-specific protein of claim 38, wherein the first antigen-binding domain is linked, optionally via an amino acid linker, to the C-terminus of the heavy chain constant region.

40. The multi-specific protein of any one of claims 36-39, wherein the first antigen-binding domain is linked to the second antigen-binding domain or antibody by an amino acid linker wherein the linker is (GGGGS)x3 (SEQ ID NO:7), (GGSGG)x3 (SEQ ID NO:8), or GGSGG (SEQ ID NOV).

41. The multi-specific protein of any one of claims 36-39, wherein the first antigen-binding domain is linked directly to the antibody or second antigen-binding domain.

42. The multi-specific protein of any one of claims 36-41, comprising a truncation at a terminus of the first antigen-binding domain, a truncation at a terminus of the antibody or second antigen-binding domain, or truncations at termini of both the first antigen-binding domain and the antibody or second antigen-binding domain.

43. The multi-specific protein of any one of claims 36-42, wherein the antibody or second antigen-binding domain comprises a C-terminal truncation of one, two, or three residues.

44. The multi-specific protein of any one of claims 36-43, wherein the antibody or second antigen-binding domain comprises an N-terminal truncation of one, two, or three residues.

45. The multi-specific protein of claim 44, wherein the antibody or second antigen-binding domain comprises an IgGl antibody or antigen-binding fragment thereof and comprises a truncation of residues PGK.

46. The multi-specific protein of claim 45, wherein the first antigen-binding domain is linked to the C-terminus of the antibody.

47. The multi-specific protein of claim 37, wherein the antibody or second antigen-binding domain specifically binds to a cancer antigen, and the antibody or second antigen-binding domain is selected from the group consisting of rituximab, cetuximab, trastuzumab, pertuzumab, bevacizumab, nivolumab, pembrolizumab, atezolizumab, avelumab, and durvalumab.

48. The multi-specific protein of claim 37, wherein the antibody or second antigen-binding domain specifically binds to a cancer antigen, and the antibody or second antigen-binding domain is selected from the group consisting of a version of rituximab comprising reduced effector function, a version of cetuximab comprising reduced effector function, a version of trastuzumab comprising reduced effector function, a version of pertuzumab comprising reduced effector function, a version of bevacizumab comprising reduced effector function, a version of nivolumab comprising reduced effector function, a version of pembrolizumab comprising reduced effector function, a version of atezolizumabcomprising reduced effector function, a version of avelumab comprising reduced effector function, and a version of durvalumab comprising reduced effector function.

49. The multi-specific protein of claim 48, wherein the reduced effector function comprises a mutation of LALA-P331S and / or L AL A-P329 S.

50. The multi-specific protein of claim 48 or 49, wherein the antibody or second antigenbinding domain that binds to a cancer antigen is a version of rituximab comprising reduced effector function.

51. The mult-specific protein of claim 48, wherein the multi-specific protein comprises the amino acid sequences of SEQ ID NOs: 80-82.

52. The multi-specific protein of claim 36, wherein the antibody or second antigen-binding domain specifically binds to matrix metalloproteinase 9 (MMP-9).

53. The multi-specific protein of claim 52, wherein the amino acid sequences of SEQ ID NOs: 152 and 153.

54. The mutli-specific protein of claim 52 or claim 53, wherein the antibody or second antigen-binding domain is an IgG4 antibody or antigen-binding domain.

55. The multi-specific protein of claim 52 or claim 53, wherein the multi-specific protein comprises the amino acid sequences of:(i) SEQ ID NOs: 154, 155, and 156,(ii) SEQ ID NOs: 157, 158, and 156,(iii) SEQ ID NOs: 159, 160, and 156,(iv) SEQ ID NOs:161, 155, and 156,(v) SEQ ID NOs: 162, 158, and 156,(vi) SEQ ID NOs:163, 160, and 156,(vii) SEQ ID NOs:164, 155, and 156,(viii) SEQ ID NOs: 165, 158, and 156, or(ix) SEQ ID NOs:166, 160, and 156.

56. The multi-specific protein of claim 52 or claim 53, wherein the antibody or second antigen-binding domain is andecaliximab.

57. The multi-specific protein of any one of claims 36-56, wherein the multi-specific protein is bispecific.

58. The multi-specific protein of any one of claims 36-50 and 57, wherein the multi-specific protein is bivalent, trivalent, or tetravalent.

59. The multi-specific protein of claim 58, wherein the multi-specific protein is bivalent.

60. The multi-specific protein of claim 58, wherein the multi-specific protein is trivalent, optionally wherein the trivalent protein comprises the antigen-binding domain that binds to human TfR and two antigen-binding domains that bind to a CNS antigen.

61. The multi-specific protein of claim 58, wherein the multi-specific protein is tetravalent, optionally wherein the tetravalent protein comprises two of the antigen-binding domains that bind to human TfR and two antigen-binding domains that bind to a CNS antigen.

62. The multi-specific protein of any one of claims 36-61, wherein the antibody or second antigen-binding fragment thereof comprises a constant region comprising a knob mutation and a constant region comprising a hole mutation.

63. The multi-specific protein of claim 62, wherein the antigen-binding domain is linked, optionally via an amino acid linker, to the constant region comprising a hole mutation.

64. The multi-specific protein of claim 62, wherein the antigen-binding domain is linked, optionally via an amino acid linker, to the constant region comprising a knob mutation.

65. The multi-specific protein of any one of claims 37-46 and 57-64, wherein the CNS antigen is a brain antigen.

66. The multi-specific protein of any one of claims 37-46 and 57-65, wherein the CNS antigen is not TfR.

67. The multi-specific protein of any one of claims 36-66, wherein the antibody or second antigen-binding domain comprises a mutation that reduces effector function, optionally wherein the mutation that reduces effector function comprises (i) L234A, L235A, and / or P331S and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S.

68. The multi-specific protein of any one of claims 36-67, wherein the antibody or second antigen-binding domain comprises a constant region comprising a knob mutation and a mutation that reduces effector function, optionally wherein the mutation that reduces effector function comprises (i) L234A, L235A, and / or P331 S and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S.

69. The multi-specific protein of any one of claims 36-68, wherein the antibody or second antigen-binding domain comprises a constant region comprising a hole mutation and a mutation that reduces effector function, optionally wherein the mutation that reduces effector function comprises (i) L234A, L235A, and / or P331 S and / or (ii) N325S and / or L328F, and / or (iii) P329G or P329S.

70. The multi-specific protein of any one of claims 36-69, wherein the antibody or second antigen-binding domain is an IgG antibody or antigen-binding fragment thereof.

71. The multi-specific protein of claim 70, wherein the IgG antibody or antigen-binding fragment thereof is an IgGl antibody or antigen-binding fragment thereof or an IgG4 antibody or antigen-binding fragment thereof.

72. The multi-specific protein of any one of claims 37-46 and 57-71, wherein the antibody or second antigen-binding domain specifically binds to a CNS antigen that is beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein, apolipoprotein E (ApoE), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, Glycoprotein nonmetastatic protein B (GPNMB), gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, sortilin (SORT), triggering receptor expressed on myeloid cells 2 (TREM2), CD33 or sialic acid binding Ig-like lectin 3 (Siglec3), sialic acid binding Ig-like lectin 5 (Siglec5), sialic acid binding Ig-like lectin 7 (Siglec7), sialic acid binding Ig-like lectin 9 (Siglec9), Paired immunoglobin like type 2 receptor alpha (PILRA), Membrane Spanning 4-Domains A4A (MS4A4A), Membrane Spanning 4-Domains A 6 A (MS4A6A), or Transmembrane Protein 106B (TMEM106b).

73. The multi-specific protein of any one of claims 36-72, wherein the multi-specific protein is capable of binding FcRn.

74. The antibody of claim 27, complex or fusion protein of any one of claims 28-35, or the multi-specific protein of any one of claims 36-73, wherein the antibody or second antigen-binding domain, complex or fusion protein, or multi-specific protein is linked to an imaging agent.

75. A composition comprising a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode the multispecific protein of any one of claims 36-50 and 57-73, wherein the first polynucleotide encodes a first heavy chain, the second polynucleotide encodes a second heavy chain and the antigen-binding domain that specifically binds to human TfR, and the third polynucleotide encodes a light chain.

76. A composition comprising a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode the multispecific protein of any one of claims 36-50 and 57-73, wherein the first polynucleotide encodes a first heavy chain and a first copy of the antigen-binding domain that specifically binds to human TfR, the second polynucleotide encodes a second heavy chain and a second copy of the antigen-binding domain that specifically binds to human TfR, and the third polynucleotide encodes a light chain, optionally wherein the first and second copies of the antigen-binding domains that bind to human TfR comprise the same amino acid sequence.

77. The composition of claim 75 or 76, wherein the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation.

78. The composition of any one of claims 75-77, wherein the ratio of the first, second, and third polynucleotides is about 1 :3:6.

79. The composition of claim 75 or 76, wherein the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

80. A composition comprising a first polynucleotide and a second polynucleotide, wherein the first and second polynucleotides encode the multi-specific protein of any one of claims 36-50 and 57-73, wherein the first polynucleotide encodes a heavy chain and theantigen-binding domain that bind to human TfR, and wherein the second polynucleotide encodes a light chain.

81. A complex comprising the antigen-binding domain of any one of claims 1-26 and a polynucleic acid.

82. The complex of claim 81, wherein the polynucleic acid is an ASO, an siRNA, or an RNAi agent.

83. The complex of claim 82, wherein the polynucleic acid is an siRNA.

84. The complex of claim 83, wherein the siRNA suppresses the product of a gene related to a neurological disease or disorder, optionally wherein the neurological disease or disorder is selected from Alzheimer's disease (AD), Huntington’s disease, dystonia, ataxia, Bell’s palsy, stroke, dementia, Lewy body 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, encephalitis, traumatic brain injury, and limbic-predominant age-related TDP-43 encephalopathy (LATE).

85. The complex of claim 83 or claim 84, wherein the siRNA targets tau mRNA, alpha- synuclein mRNA, or NLRP3 mRNA.

86. The complex of any one of claims 83-85, wherein the antigen-binding domain comprises a VH and VL, wherein the VH or VL comprise an amino acid substitution of a residue to cysteine, wherein the cysteine is the site of attachment of the siRNA of the complex, and wherein the VH and VL comprise amino acid sequences at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 98% identical to the amino acid sequences of:(i) SEQ ID NOs:46 and 47, respectively;(ii) SEQ ID NOs:46 and 48, respectively;(iii) SEQ ID NOs:50 and 48, respectively;(iv) SEQ ID NOs:49 and 48, respectively;(v) SEQ ID NOs:49 and 51, respectively;(vi) SEQ ID NOs:49 and 52, respectively;(vii) SEQ ID NOs:49 and 53, respectively;(viii) SEQ ID NOs:49 and 54, respectively;(ix) SEQ ID NOs:55 and 48, respectively;(x) SEQ ID NOs:56 and 57; respectively;(xi) SEQ ID NOs:58 and 59, respectively;(xii) SEQ ID NOs:56 and 60, respectively;(xiii) SEQ ID NOs:56 and 61, respectively;(xiv) SEQ ID NOs:58 and 62, respectively;(xv) SEQ ID NOs:85 and 86, respectively;(xvi) SEQ ID NOs:85 and 87, respectively;(xvii) SEQ ID NOs:88 and 87, respectively;(xviii) SEQ ID NOs:89 and 87, respectively;(xix) SEQ ID NOs:88 and 90, respectively;(xx) SEQ ID NOs:88 and 91, respectively;(xxi) SEQ ID NOs:88 and 92, respectively;(xxii) SEQ ID NOs:88 and 93, respectively;(xxiii) SEQ ID NOs:94 and 87, respectively;(xxiv) SEQ ID NOs:95 and 96, respectively;(xxv) SEQ ID NOs:97 and 98, respectively;(xxvi) SEQ ID NOs:95 and 99, respectively;(xxvii) SEQ ID NOs:95 and 100, respectively;(xxviii) SEQ ID NOs:97 and 101, respectively;(xxix) SEQ ID NOs: 102 and 103, respectively;(xxx) SEQ ID NOs: 102 and 104, respectively;(xxxi) SEQ ID NOs: 105 and 104, respectively;(xxxii) SEQ ID NOs: 106 and 104, respectively;(xxxiii) SEQ ID NOs: 105 and 107, respectively;(xxxiv) SEQ ID NOs: 105 and 108, respectively;(xxxv) SEQ ID NOs: 105 and 109, respectively;(xxxvi) SEQ ID NOs: 105 and 110, respectively;(xxxvii) SEQ ID NOs: 111 and 104, respectively;(xxxviii) SEQ ID NOs: 112 and 113, respectively; or(xxxix) SEQ ID NOs: 114 and 115, respectively.

87. A host cell comprising the composition of any one of claims 75-80.

88. An isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain of the antigen-binding domain of any one of claims 1-26.

89. An isolated polynucleotide comprising a nucleic acid molecule encoding the light chain variable region of the antigen-binding domain of any one of claims 1-26.

90. An isolated vector comprising the polynucleotide of claim 88 and / or the polynucleotide of claim 89.

91. An isolated vector comprising a nucleic acid molecule encoding the heavy chain variable region of the antigen-binding domain of any one of claims 1-25 and a nucleic acid molecule encoding the light chain variable region of the antigen-binding domain.

92. A host cell comprising the polynucleotide of claim 88 or 89 or the vector of claim 90 or 91.

93. The host cell of claim 87 or 92, wherein the host cell is selected from the group consisting of E. coli. Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NSO, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, Rl. l, B-W, L-M, COS 1, COS 7, BSC1, BSC40, BMT10 cell, plant cell, insect cell, and human cell in tissue culture.

94. A method of producing an antigen-binding domain or multi-specific protein comprising culturing the host cell of any one of claims 87, 92, and 93 so that the antigen-binding domain or multi-specific protein is produced, optionally wherein the method further comprises isolating the antigen-binding domain or multi-specific protein from the culture.

95. An isolated antigen-binding domain or multi-specific protein thereof produced by the method of claim 94.

96. The antigen-binding domain of any one of claims 1-25, the antibody of claim 27, the complex or fusion protein of any one of claims 28-35, or the multi-specific protein of any one of claims 36-73 further comprising a cytotoxic drug.

97. The antigen-binding domain, the antibody, the complex or fusion protein, or the multispecific protein of claim 96, wherein the cytotoxic drug is a microtubule disruptingagent, optionally wherein the microtubule disrupting agent is monomethyl auristatin E (MMAE).

98. An antibody drug conjugate (ADC) comprising a drug and (i) the antigen-binding domain of any one of claims 1-26, (ii) the antibody of claim 27, (iii) the complex or fusion protein of any one of claims 28-35, or (iv) the multi-specific protein of any one of claims 36-73.

99. An antibody drug conjugate (ADC) comprising a drug and (i) the antigen-binding domain of any one of claims 1-26, (ii) the antibody of claim 27, (iii) the complex or fusion protein of any one of claims 28-35, or (iv) the multi-specific protein of any one of claims 36-73, or (v) an antigen-binding domain comprising 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: 27, 28, 29, 39, 40, and 41, respectively.

100. The ADC of claim 99, wherein the ADC binds to tissue factor (TF), human epidermal growth factor receptor (HER2), B7-H4, or Nectin-4.

101. The ADC of claim 99, wherein the ADC comprises a microtubule disrupting agent, optionally wherein the microtubule disrupting agent is monomethyl auristatin E (MMAE).

102. The ADC of any one of claims 99-101, wherein the ADC comprises a version of tisotumab vedotin, disitamab vedotin, felmetatug vedotin, enfortumab vedotin, or trastuzumab deruxtecan with reduced effector function.

103. A pharmaceutical composition comprising (i) the antibody of claim 27 or an antigenbinding fragment thereof, the complex or fusion protein of any one of claims 28-35, or the multi-specific protein of any one of claims 36-73, and (ii) a pharmaceutically acceptable carrier.

104. The pharmaceutical composition of claim 103, wherein the antibody or an antigenbinding fragment thereof, complex, fusion protein, or multi-specific protein is increased in the brain following administration to a subject as compared to an isotype control.

105. A method of treating a neurological disease or disorder in a subject comprising administering the antibody of claim 27 or an antigen-binding fragment thereof, the complex or fusion protein of any one of claims 28-35, the multi-specific protein of any one of claims 36-46 and 57-73, or pharmaceutical composition of claim 103 or 104 to the subject.

106. The method of claim 105, wherein administration increases delivery of the antibody or antigen-binding fragment thereof, complex or fusion protein, multi-specific protein, or pharmaceutical composition into the brain by at least 50%, at least 100%, at least 200%, at least 500% or at least 1000% as compared to an isotype control.

107. The method of claim 105 or 106, wherein administration increases delivery of the antibody or antigen-binding fragment thereof, complex or fusion protein, multi-specific protein, or pharmaceutical composition into the frontal cortex, the entorhinal cortex and / or the hippocampus.

108. The method of any one of claims 105-107, wherein the neurological disease or disorder is selected from 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.

109. The method of claim 108, wherein the neurological disease or disorder is selected from Alzheimer's disease (AD), Huntington’s disease, dystonia, ataxia, Bell’s palsy, stroke, dementia, Lewy body 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, encephalitis, traumatic brain injury, and limbic-predominant age-related TDP-43 encephalopathy (LATE).

110. The method of claim 109, wherein the dementia is frontotemporal dementia (FTD).

111. The method of claim 109, wherein the neurological disease or disorder is Alzheimer’s disease.

112. The method of claim 111, wherein the Alzheimer's disease is early onset Alzheimer’s disease, prodromal Alzheimer’s disease, mild Alzheimer’s disease, or late onset Alzheimer’s disease.

113. The method of claim 109, wherein the neurological disease or disorder is Parkinson’s disease.

114. The method of any one of claims 105-107, wherein the neurological disease or disorder is frontal temporal epilepsy.

115. The method of any one of claims 105-107, wherein the neurological disease or disorder is autism.

116. The method of any one of claims 105-107, wherein the neurological disease or disorder is lissencephaly.

117. A method of treating a lysosomal storage disease in a subject comprising administering the antibody of claim 25 or an antigen-binding fragment thereof, the complex or fusion protein of any one of claims 28-35, the multi-specific protein of any one of claims 36-46 and 57-73, or pharmaceutical composition of claim 103 or 104 to the subject.

118. The method of claim 117, wherein the lysosomal storage disease is selected from Gaucher disease, Ceroid lipofuscinosis (Batten disease), Mucopolysaccharidosis (MPS) Type I, MPS Type II and MPS Type III.

119. A method of transporting a fusion protein, antibody or an antigen-binding fragment thereof, or multi-specific protein across the BBB of a subject, comprising administering to the subject the antibody of claim 25 or an antigen-binding fragment thereof, the complex or fusion protein of any one of claims 28-35, the multi-specific protein of any one of claims 36-73, or pharmaceutical composition of claim 103 or 104.

120. The method of claim 119, wherein the concentration of the antibody or antigen-binding fragment thereof, complex or fusion protein, multi-specific protein, or pharmaceutical composition is increased in the brain following administration as compared to an isotype control.

121. The method of any one of claims 105-120, wherein administration of antibody or antigenbinding fragment thereof, complex or fusion protein, multi-specific protein, or pharmaceutical composition does not result in reticulocyte count reduction in the subject, as compared to an isotype control.

122. A method of imaging a CNS antigen within a subject, comprising administering to the subject the antibody or antigen-binding fragment thereof, complex or fusion protein, or multi-specific protein of claim 74 and locating the imaging agent within the subject.

123. A method of detecting a CNS antigen in vitro, comprising contacting an in vitro sample with the antibody or antigen-binding fragment thereof, complex or fusion protein, or multi-specific protein of claim 74 and locating the imaging agent within the sample.

124. Use of the antibody of claim 27 or an antigen-binding fragment thereof, the complex or fusion protein of any one of claims 28-35, the multi-specific protein of any one of claims 36-46 and 57-73, or pharmaceutical composition of claim 103 or 104, in the method of any one of claims 105-118.

125. The antibody of claim 27 or an antigen-binding fragment thereof, the complex or fusion protein of any one of claims 28-35, the multi-specific protein of any one of claims 36-46 and 57-73, or pharmaceutical composition of claim 103 or 104 for use in the method of any one of claims 105-118.

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