Transferrin receptor (TFR)-binding variable domains of a heavy chain only antibody (VHH), conjugates and uses thereof

Abstract

Provided herein are TfR binding VHH polypeptides which bind to human and non-human primate TfR, conjugates thereof, and pharmaceutical compositions comprising these. Provided are also methods of use comprising administering TfR binding VHH polypeptides, conjugates thereof or pharmaceutical compositions thereof to deliver agents, including therapeutic agents, to treat a disease or disorder.

Description

TRANSFERRIN RECEPTOR (TFR)-BINDING VARIABLE DOMAINS OF A HEAVY CHAIN ONLY ANTIBODY (VHH), CONJUGATES AND USES THEREOFREFERENCE TO A SEQUENCE LISTING

[0001] This application contains a sequence listing which has been submitted electronically in XML format. The Sequence Listing XML is incorporated herein by reference. Said XML file is named 31083_WO.xml created May 2, 2025, 166 kilobytes in size.BACKGROUND

[0002] TfR is a type II homodimeric transmembrane glycoprotein consisting of two identical 90 kDa subunits linked by two disulfide bridges (Jing SQ, Trowbridge IS (1987) Identification of the intermolecular disulfide bonds of the human transferrin receptor and its lipid-attachment site. EMBO J 6:327-331; McClelland, A., Kuhn, L. C, & Ruddle, F. H. (1984). The human transferrin receptor gene: Genomic organization, and the complete primary structure of the receptor deduced from a cDNA sequence. Cell 39, 267-274). Each monomer has a short cytoplasmic N-terminal domain of 61 amino acids containing a YTRF (Tyrosine-Threonine- Arginine-Phenylalanine) internalization motif, a single hydrophobic transmembrane segment of 27 amino acids, and a large C-terminal extracellular domain of 670 amino acids, containing a trypsin cleavage site and a transferrin binding site (Aisen, P. (2004), Transferrin receptor 1, Int. J. Biochem. Cell Biol., 36 (2004), pp. 2137-2143). Each subunit is capable of binding a transferrin polypeptide. The extracellular domain has one O-glycosylation site and three N-glycosylation sites, the latter being particularly important for the proper folding and transport of the receptor to the cell surface (Hayes et al., Identification of the O-linked glycosylation site of the human transferrin receptor;Glycobiology, 2 (1992), pp. 355-359). There are also palmitylation sites in the intramembranous domain, that presumably anchor the receptor and allow its endocytosis (Alvarez et al., 1990, A point mutation in the cytoplasmic domain of the transferrin receptor inhibits endocytosis. Biochem. I 267, 31-35; Omary and Trowbridge, 1981 Biosynthesis of the human transferrin receptor in cultured cells; Volume 256, Issue 24, 25 December 1981, Pages 12888-12892). In addition, an intracellular phosphorylation site is present, whose functions are uncertain, and which plays no role in endocytosis.

[0003] The TfR is involved in the incorporation of iron, transported by its transferrin ligand, and in the regulation of cell growth (Neckers and Trepel 1986, Transferrin receptor expression and the control of cell growth, Cancer Invest 1986;4(5):461-70; Ponka and Lok 1999, The transferrin receptor: role in health and disease, Int. J. Biochem. Cell Biol., 31 (10) (1999), pp. 1111-1137,). There are two types of transferrin receptors: the TfRJ receptor and a homologous receptor, TfR2, expressed primarily in the liver. In the context of the invention, the term TfR. is used to designate the TfRl homologue.

[0004] The TfR receptor is expressed at high level by highly proliferating cells, whether healthy or neoplastic (Gatter et al., 1983; Transferrin receptors in human tissues: their distribution and possible clinical relevance, J Clin Pathol. 1983 May;36(5):539-45). Many studies have shown high levels of TfR expression in cancer cells compared to healthy cells. Targeting agents to TfR may thus be suitable for disease treatment, including highly proliferative cells diseases such as cancer.

[0005] The blood brain barrier (BBB) is a selective semipermeable border of capillary endothelial cells that prevents solutes, including pathogens, from passing into the central nervous system (CNS). The BBB allows the passage of some small polypeptides by passive diffusion and the cells of BBB actively transport metabolic products crucial to neural function such as glucose and amino acids across the barrier using specific transport proteins. The BBB has neuroprotective function by tightly controlling access to the brain; but it also impedes access of therapeutic agents to CNS.

[0006] BBB shuttles for improving passage of the therapeutic agents across the blood brain barrier and into the CNS have been described. However, attempts at using anti-TfR antibodies to shuttle therapeutic agents across the BBB have proven challenging. To date, there are no approved TfR binding shuttles or conjugates for the treatment of CNS diseases.

[0007] This transmembrane glycoprotein is found on the surface of many normal cell types. TfR is expressed on various tissues, including skeletal muscle, the heart, and adipose tissues. As such TfR is an attractive target for developing targeted shuttles for the transport of agents across cellular membranes.

[0008] Therefore, there remains a need for improved TfR binding VHH polypeptides and conjugates for pharmaceutical uses that can deliver agents across cell membranes, includingwithout limitation the BBB into the CNS, into skeletal muscle, including the heart, into adipose tissue, and other TfR expressing tissues for the treatment of various diseases.SUMMARY

[0009] The present invention provides TfR-binding variable domains of heavy chain only antibody (VHH) (TfR-binding VHH or TfR-binding VHH polypeptides). TfR-binding VHH polypeptides of the invention are recombinant polypeptides engineered with certain advantageous properties. In certain embodiments, TfR-binding VHH polypeptides bind human TfR and nonhuman (cynomolgus) TfR. In certain embodiments, TfR-binding VHH polypeptides bind human TfR, rodent TfR and non-human (cyno) TfR. In certain embodiments, TfR-binding VHH polypeptides do not comprise any N- or O- linked glycosylation sites. In certain embodiments, TfR-binding VHH polypeptides bind protein A. In certain embodiments the binding to TfR from different species is balanced.

[0010] In certain embodiments TfR-binding VHH polypeptides are conjugated (TfR-binding VHH conjugates) to at least one agent, including without limitation scaffolds, half-life extenders, therapeutic, imaging and / or diagnostic agents In certain embodiments TfR-binding VHH polypeptides are conjugated to one, two or three different agents. In certain embodiments TfR- binding VHH polypeptides are conjugated to one or two or more different agents. In certain embodiments, the TfR-binding VHH conjugates transport agents, including without limitation therapeutic, imaging and / or diagnostic agents into any cell expressing a TfR receptor. In some embodiments, the TfR-binding VHH conjugates transport agents across the BBB. In some embodiments, the TfR-binding VHH conjugates transport agents into muscle cells.

[0011] In non-limiting embodiments the agent is: a polypeptide, including without limitation an antigen binding VHH domain or polypeptide; a peptide; a nucleic acid molecule, including without limitation double stranded (ds)RNA such as siRNA, saRNA, antisense oligonucleotide (ASO), and a guide RNA; a dye; a small molecule, nanoparticles (NPs); lipid NPs; a liposome, an exosome; an antibody, an antibody Fc portion or antigen binding fragments thereof; a viral particle, or any combination thereof. In some embodiments, one of the agents is a polypeptide which extends the half-life (“half-life extender”) of the TfR-binding VHH polypeptides. In some embodiments, the half-life extender is a VHH polypeptide which extend the half-life of the TfR-binding VHHpolypeptide and the second agent is a nucleic acid. In some embodiments, the half-life extending VHH polypeptide is an albumin binding VHH polypeptide. In some embodiments, a therapeutic agent includes for example a nucleic acid molecule, including without limitation double stranded (ds)RNA such as siRNA, saRNA, antisense oligonucleotide (ASO), and a guide RNA; a small molecule; nanoparticles (NPs); lipid NPs; a liposome; an exosome; an antibody, an antibody Fc portion or antigen binding fragments thereof; a viral particle, or any combination thereof.

[0012] In some aspects, provided is a compound comprising an amino acid sequence which is at least 90% identical to SEQ ID NO: 1, 6, 8, 13, 18, 20, 23, 28, 32, 35, 38, 41, 44, 47, 49, 52, 64, or 66. A compound comprising an amino acid sequence which is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% identical to SEQ ID NO: 1, 6, 8, 13, 18, 20, 23, 28, 32, 35, 38, 41, 44, 47, 49, 52, 64, or 66. In some embodiments the compound further comprises an agent conjugated to an amino acid sequence which is at least 90% identical to SEQ ID NO: 1, 6, 8, 13, 18, 20, 23, 28, 32, 35, 38, 41, 44, 47, 49, 52, 64, or 66. In some embodiments the compound further comprises an agent conjugated to an amino acid sequence which is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% identical to SEQ ID NO: 1, 6, 8, 13, 18, 20, 23, 28, 32, 35, 38, 41, 44, 47, 49, 52, 64, or 66. In some embodiments, the compound binds to human and non-human primate (NHP) TfR. In some embodiments, the compound binds to mouse TfR.

[0013] In some aspects, provided is a Transferrin Receptor 1 (TfR) binding variable domain of a heavy chain only antibody (VHH) (TfR binding VHH) comprising three complementarity determining regions (CDR)l, CDR2 and CDR3 in any one of the following sequences: SEQ ID NO: 1; SEQ ID NO: 8; SEQ ID NO: 13; SEQ ID NO: 20; SEQ ID NO: 23; SEQ ID NO: 28; SEQ ID NO: 32; SEQ ID NO: 35; SEQ ID NO: 38; SEQ ID NO: 41; SEQ ID NO: 44; SEQ ID NO: 47; SEQ ID NO: 49; SEQ ID NO: 52; SEQ ID NO: 64; or SEQ ID NO: 66.

[0014] In some embodiments of the TfR binding VHH, CDR1, CDR2 and CDR3 are grafted in a human or humanized framework. In some embodiments, the framework is VH3-23.

[0015] In some embodiments provided are (TfR) binding variable domain of a heavy chain only antibody (VHH) (TfR binding VHH) comprising CDR1, CDR2 and CDR3, wherein:a. CDR1 comprises SEQ ID NO: 2, CDR 2 comprises SEQ ID NO: 3 and CDR3 comprises SEQ ID NO: 4; b. CDR1 comprises SEQ ID NO: 9, CDR 2 comprises SEQ ID NO: 10 and CDR3 comprises SEQ ID NO: 11; c. CDR1 comprises SEQ ID NO: 14, CDR 2 comprises SEQ ID NO: 15 and CDR3 comprises SEQ ID NO: 16; d. CDR1 comprises SEQ ID NO: 14, CDR 2 comprises SEQ ID NO: 21 and CDR3 comprises SEQ ID NO: 16; e. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 26; f. CDR1 comprises SEQ ID NO: 29, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 30; g. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33; h. CDR1 comprises SEQ ID NO: 36, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33; i. CDR1 comprises SEQ ID NO: 39, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33; j. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 42 and CDR3 comprises SEQ ID NO: 33; k. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 45 and CDR3 comprises SEQ ID NO: 33; l. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 21 and CDR3 comprises SEQ ID NO: 33; m. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 50 and CDR3 comprises SEQ ID NO: 33; n. CDR1 comprises SEQ ID NO: 53, CDR 2 comprises SEQ ID NO: 54 and CDR3 comprises SEQ ID NO: 33, wherein CDR1 and CDR2 comprise any combination of amino acids XI and X2 in CDR1, wherein Xl= Q or T and X2= A or S, and X3, X4, and X5 in CDR2, wherein X3= N or S, X4= A, G or T, and X5= R or Y;o. CDR1 comprises SEQ ID NO: 53, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33, wherein CDR1 comprises any combination of amino acids XI and X2, wherein Xl= Q or T and X2= A or S; or p. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 54 and CDR3 comprises SEQ ID NO: 33, wherein CDR2 comprises any combination of amino acids X3, X4, and X5, wherein X3= N or S, X4= A, G or T, and X5= R or Y.

[0016] In some embodiments of the TfR binding VHH: a. CDR1 consists essentially of SEQ ID NO: 2, CDR 2 consists essentially of SEQ ID NO: 3 and CDR3 consists essentially of SEQ ID NO: 4; b. CDR1 consists essentially of SEQ ID NO: 9, CDR 2 consists essentially of SEQ ID NO: 10 and CDR3 consists essentially of SEQ ID NO: 11; c. CDR1 consists essentially of SEQ ID NO: 14, CDR 2 consists essentially of SEQ ID NO: 15 and CDR3 consists essentially of SEQ ID NO: 16; d. CDR1 consists essentially of SEQ ID NO: 14, CDR 2 consists essentially of SEQ ID NO: 21 and CDR3 consists essentially of SEQ ID NO: 16; e. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 25 and CDR3 consists essentially of SEQ ID NO: 26; f. CDR1 consists essentially of SEQ ID NO: 29, CDR 2 consists essentially of SEQ ID NO: 25 and CDR3 consists essentially of SEQ ID NO: 30; g. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 25 and CDR3 consists essentially of SEQ ID NO: 33; h. CDR1 consists essentially of SEQ ID NO: 36, CDR 2 consists essentially of SEQ ID NO: 25 and CDR3 consists essentially of SEQ ID NO: 33; i. CDR1 consists essentially of SEQ ID NO: 39, CDR 2 consists essentially of SEQ ID NO: 25 and CDR3 consists essentially of SEQ ID NO: 33; j. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 42 and CDR3 consists essentially of SEQ ID NO: 33; k. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 45 and CDR3 consists essentially of SEQ ID NO: 33;l. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 21 and CDR3 consists essentially of SEQ ID NO: 33; m. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 50 and CDR3 consists essentially of SEQ ID NO: 33; or n. CDR1 consists essentially of SEQ ID NO: 53, CDR 2 consists essentially of SEQ ID NO: 54 and CDR3 consists essentially of SEQ ID NO: 33, wherein CDR1 and CDR2 comprise any combination of amino acids XI and X2 in CDR1, wherein Xl= Q or T and X2= A or S, and X3, X4, and X5 in CDR2, wherein X3= N or S, X4= A, G or T, and X5= R or Y; o. CDR1 comprises SEQ ID NO: 53, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33, wherein CDR1 comprises any combination of amino acids XI and X2, wherein XI = Q or T and X2= A or S; or p. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 54 and CDR3 comprises SEQ ID NO: 33, wherein CDR2 comprises any combination of amino acids X3, X4, and X5, wherein X3= N or S, X4= A, G or T, and X5= R or Y.

[0017] In some embodiments of the TfR binding VHH: a. CDR1 consists of SEQ ID NO: 2, CDR 2 consists of SEQ ID NO: 3 and CDR3 consists of SEQ ID NO: 4; b. CDR1 consists of SEQ ID NO: 9, CDR 2 consists of SEQ ID NO: 10 and CDR3 consists of SEQ ID NO: 11; c. CDR1 consists of SEQ ID NO: 14, CDR 2 consists of SEQ ID NO: 15 and CDR3 consists of SEQ ID NO: 16; d. CDR1 consists of SEQ ID NO: 14, CDR 2 consists of SEQ ID NO: 21 and CDR3 consists of SEQ ID NO: 16; e. CDR1 consists of SEQ ID NO: 24, CDR 2 consists of SEQ ID NO: 25 and CDR3 consists of SEQ ID NO: 26; f. CDR1 consists of SEQ ID NO: 29, CDR 2 consists of SEQ ID NO: 25 and CDR3 consists of SEQ ID NO: 30; g. CDR1 consists of SEQ ID NO: 24, CDR 2 consists of SEQ ID NO: 25 and CDR3 consists of SEQ ID NO: 33;h. CDR1 consists of SEQ ID NO: 36, CDR 2 consists of SEQ ID NO: 25 and CDR3 consists of SEQ ID NO: 33; i. CDR1 consists of SEQ ID NO: 39, CDR 2 consists of SEQ ID NO: 25 and CDR3 consists of SEQ ID NO: 33; j . CDR1 consists of SEQ ID NO: 24, CDR 2 consists of SEQ ID NO: 42 and CDR3 consists of SEQ ID NO: 33; k. CDR1 consists of SEQ ID NO: 24, CDR 2 consists of SEQ ID NO: 45 and CDR3 consists of SEQ ID NO: 33; l. CDR1 consists of SEQ ID NO: 24, CDR 2 consists of SEQ ID NO: 21 and CDR3 consists of SEQ ID NO: 33; m. CDR1 consists of SEQ ID NO: 24, CDR 2 consists of SEQ ID NO: 50 and CDR3 consists of SEQ ID NO: 33; or n. CDR1 consists of SEQ ID NO: 53, CDR 2 consists of SEQ ID NO: 54 and CDR3 consists of SEQ ID NO: 33, wherein CDR1 and CDR2 comprise any combination of amino acids XI and X2 in CDR1, wherein Xl= Q or T and X2= A or S, and X3, X4, and X5 in CDR2, wherein X3= N or S, X4= A, G or T, and X5= R or Y; o. CDR1 consists of SEQ ID NO: 53, CDR 2 consists of SEQ ID NO: 25 and CDR3 consists of SEQ ID NO: 33, wherein CDR1 comprises any combination of amino acids XI and X2, wherein Xl= Q or T and X2= A or S; or p. CDR1 consists of SEQ ID NO: 24, CDR 2 consists of SEQ ID NO: 54 and CDR3 consists of SEQ ID NO: 33, wherein CDR2 consists of any combination of amino acids X3, X4, and X5, wherein X3= N or S, X4= A, G or T, and X5= R or Y.

[0018] In some embodiments, the TfR binding VHH comprises an amino acid sequence which is at least 90% identical to SEQ ID NO: 1, 6, 8, 13, 18, 20, 23, 28, 32, 35, 38, 41, 44, 47, 49, 52, 64, 66, 75, 80, or 81. In some embodiments, the TfR binding VHH comprises an amino acid sequence which is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% identical to SEQ ID NO: 1, 6, 8, 13, 18, 20, 23, 28, 32, 35, 38, 41, 44, 47, 49, 52, 64, 66, 75, 80, or 81. In some embodiments, the TfR binding VHH comprises an amino acid sequence of SEQ ID NO: 1, 6, 8, 13, 18, 20, 23, 28, 32, 35, 38, 41, 44, 47, 49, 52, 64, 66, 75, 80, or 81. In some embodiments, the TfR binding VHH consists essentially of an amino acid sequence of SEQ ID NO: 1, 6, 8, 13, 18, 20, 23, 28, 32, 35, 38, 41, 44, 47, 49, 52, 64, 66, 75, 80, or 81.

[0019] In some embodiments, the TfR binding VHH binds to human and non-human primate (NHP) TfR. In some embodiments, the TfR binding VHH binds optionally to mouse TfR.

[0020] In some embodiments, the TfR binding VHH is conjugated to at least one agent. In some embodiments, the TfR binding VHH is conjugated to one agent. In some embodiments, the TfR binding VHH is conjugated to two agents. In some embodiments, the agent is a polypeptide; a peptide; a nucleic acid molecule; a dye; a small molecule; nanoparticles (NPs); lipid NPs; a liposome; an exosome; an antibody, an antibody Fc portion or antigen binding fragments thereof; a viral particle; or any combination thereof. In some embodiments, the polypeptide agent includes without limitation an antigen binding VHH domain or polypeptide. In some embodiments, the nucleic acid molecule includes without limitation double stranded (ds)RNA such as siRNA, saRNA, antisense oligonucleotide (ASO), and / or a guide RNA.

[0021] In some embodiments, the agent is conjugated to the TfR binding VHH directly. In some embodiments, the agent is conjugated to the TfR binding VHH via a linker.

[0022] In some embodiments, wherein the agent is an siRNA conjugated to the TfR binding VHH, the siRNA agent comprises a sense strand and an antisense strand. In some embodiments the agent is an siRNA conjugated to a cysteine residue in the TfR binding VHH. In some embodiments, the linker is a SMCC linker. In some embodiments, the linker is a SMCC linker conjugated to the antisense strand of the siRNA agent. In some embodiments, the linker is a SMCC linker conjugated to the sense strand of the siRNA agent. In some embodiments, the agent is a half-life extender. In some embodiments, the half-life extender is an albumin (ALB) binding polypeptide. In some embodiments, the ALB binding polypeptide binds human serum albumin. In some embodiments, the albumin binding polypeptide is an albumin (ALB) binding VHH. In some embodiments, the ALB binding VHH binds to human serum albumin.

[0023] In some embodiments, the ALB binding VHH comprises three complementarity determining regions, CDR1, CDR2, and CDR3, wherein CDR1 comprises SEQ ID NO: 61; CDR2 comprises SEQ ID NO:62; and CDR3 comprises SEQ ID NO:63 or SEQ ID NO: 71. In some embodiments, the ALB binding VHH comprises SEQ ID NO: 60 or SEQ ID NO: 70, and optionally comprises a cysteine at the C-terminal end. In some embodiments, the ALB bindingVHH comprises SEQ ID NO: 60 or SEQ ID NO: 70, without the two C-terminal [PP] amino acids, in some embodiments, the ALB binding VHH comprises a cysteine at the C-terminal end.

[0024] In some embodiments, the C-terminal residue of the half-life extender is conjugated either directly or via a linker to the N-terminal residue of the TfR binding VHH. In some embodiments, the C-terminal residue of the TfR binding VHH is conjugated either directly or via a linker to the N-terminal residue of the half-life extender. In some embodiments, the linker is a glycine rich linker. In some embodiments, the glycine rich linker comprises an amino acid sequence of (G4Q)n(SEQ ID NO: 59), wherein n= 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the glycine rich linker comprises the amino acid sequence of (G4Q)n SEQ ID NO:59, wherein n=l, 2, 3, 4 or 5.

[0025] In some embodiments where the TfR binding VHH is conjugated to two agents, wherein the first agent is an ALB binding VHH, and wherein the second agent is an siRNA, which siRNA comprises a sense strand and an antisense strand. In some embodiments, the ALB binding VHH is conjugated via a glycine rich linker to the TfR binding VHH and the siRNA, which comprises a sense strand and an antisense strand, is conjugated via a chemical linker to (1) a cysteine residue in the ALB binding VHH or (2) a cysteine residue in the TfR binding VHH. In some embodiments, the ALB binding VHH is conjugated via a glycine rich linker to the TfR binding VHH, wherein the glycine rich linker comprises an amino acid sequence of (G4Q)n(SEQ ID NO: 59), wherein n= 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and the siRNA which comprises a sense strand and an antisense strand, wherein the sense strand of the siRNA is conjugated via a chemical linker to (1) a cysteine residue in the ALB binding VHH or (2) a cysteine residue in the TfR binding VHH. In some embodiments, the ALB binding VHH is conjugated via a glycine rich linker to the TfR binding VHH, wherein the glycine rich linker comprises an amino acid sequence of (G4Q)n(SEQ ID NO: 59), wherein n= 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and the siRNA which comprises a sense strand and an antisense strand, wherein the antisense strand of the siRNA is conjugated via a chemical linker to (1) a cysteine residue in the ALB binding VHH or (2) a cysteine residue in the TfR binding VHH. In some embodiments, the ALB binding VHH is conjugated via a glycine rich linker to the TfR binding VHH and the siRNA, which comprises a sense strand and an antisense strand, is conjugated via a chemical linker to (1) a C terminal cysteine residue in the ALB binding VHH or (2) a C terminal cysteine residue in the TfR binding VHH. In some embodiments, the ALB binding VHH is conjugated via a glycine rich linker to the TfR binding VHH, wherein the glycine rich linkercomprises an amino acid sequence of (G4Q)n(SEQ ID NO: 59), wherein n= 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and the siRNA which comprises a sense strand and an antisense strand, wherein the sense strand of the siRNA is conjugated via a chemical linker to (1) a C terminal cysteine residue in the ALB binding VHH or (2) a C terminal cysteine residue in the TfR binding VHH. In some embodiments, the ALB binding VHH is conjugated via a glycine rich linker to the TfR binding VHH, wherein the glycine rich linker comprises an amino acid sequence of (G4Q)n (SEQ ID NO: 59), wherein n= 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and the siRNA which comprises a sense strand and an antisense strand, wherein the antisense strand of the siRNA is conjugated via a chemical linker to (1) a C terminal cysteine residue in the ALB binding VHH or (2) a C terminal cysteine residue in the TfR binding VHH. Non-limiting embodiments of a chemical linker are listed in Table 3. In one embodiment, the chemical linker is a SMCC.

[0026] In certain aspects provided are conjugates of the following Formula I:(I) [[first agent]-linker l](n)-[TfR VHH]-[linker 2-[second agent]] (m), wherein the [TfR VHH] comprises the TfR binding VHH of any one of the preceding embodiments; the linker 1 and linker 2 are optional; and a [first agent] and a [second agent], wherein m and n are independent, n=0, 1, 2 or 3, m=0, 1, 2 or 3. In some embodiments, the [first agent], the [second agent] and the [TfR VHH] are conjugated in any order.

[0027] In some embodiments of Formula (I), the first and / or the second agent is a polypeptide; a peptide; a nucleic acid molecule; a dye; a small molecule; nanoparticles (NPs); lipid NPs; a liposome; an exosome; an antibody, an antibody Fc portion or antigen binding fragments thereof; viral particle; or any combination thereof. In some embodiments of Formula (I), the first agent is conjugated via the linker 1 and / or the second agent is conjugated via the linker 2 to the TfR VHH. In some embodiments, wherein n=0 and m=l, wherein when m=l the second agent is a nucleic acid molecule. In some embodiments, wherein n=0 and m=2, wherein one [second agent] is a halflife extender and the other [second agent] is a nucleic acid molecule. In some embodiments of Formula (I), wherein when n=l, the [first agent] is a nucleic acid molecule, and m=2, wherein one [second agent] is a half-life extender and the other [second agent] is a nucleic acid molecule. In some embodiments of Formula (I), at least one of the first or the second agent is a therapeutic agent.

[0028] In some embodiments of Formula (I), wherein the one [first agent] or the other [second agent] is a nucleic acid molecule, the nucleic acid molecule is an siRNA conjugated to the TfR binding VHH, and wherein the siRNA comprises a sense strand and an antisense strand. In some embodiments, the siRNA is conjugated to a cysteine residue in the TfR binding VHH, and wherein the siRNA comprises a sense strand and an antisense strand.

[0029] In some embodiments of Formula (I), the C-terminal residue of the TfR binding VHH is conjugated either directly or via the linker 2 to the N-terminal residue of the half-life extender.

[0030] In some embodiments of Formula (I), wherein n=l, the [first agent] is a nucleic acid molecule, and m=l, wherein one [second agent] is a half-life extender further comprising a nucleic acid molecule, e.g. an siRNA, wherein the nucleic acid molecule is conjugated to a cysteine residue of the half-life extender, and wherein the nucleic acid molecule is conjugated via any one of the linkers in Table 3.

[0031] In some embodiments of Formula (I), one [second agent] is half-life extender which is an albumin (ALB) binding polypeptide. In some embodiments of Formula (I), the ALB binding polypeptide binds human serum albumin. In some embodiments of Formula (I), the ALB binding polypeptide is an ALB binding VHH comprising three complementarity determining regions, CDR1, CDR2, and CDR3, wherein CDR1 comprises SEQ ID NO: 61; CDR2 comprises SEQ ID NO:62; and CDR3 comprising SEQ ID NO:63 or SEQ ID NO: 71. In some embodiments of Formula (I), the ALB binding VHH comprises SEQ ID NO: 60 or SEQ ID NO: 70. In some embodiments of Formula (I), the ALB binding VHH comprises SEQ ID NO: 60 which also comprises a cysteine at the C-terminal end or SEQ ID NO: 70 which also comprises a cysteine at the C-terminal end. In some embodiments of Formula (I), the ALB binding VHH comprises SEQ ID NO: 60 without the two C-terminal [PP] amino acids or SEQ ID NO: 70 without the two C- terminal [PP] amino acids, and each sequence also comprising a cysteine at the C-terminal end.

[0032] In some embodiments of Formula (I), the linker 1 or the linker 2 is a glycine rich linker. In some embodiments of Formula (I), the linker 1 or the linker 2 comprises an amino acid sequence of (G4Q)n (SEQ ID NO:59), wherein n= 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments of Formula (I), wherein the linker 1 or the linker 2 comprises the amino acid sequence of (G4Q)n(SEQ ID NO:59), wherein n=l, 2, 3, 4 or 5.

[0033] In some embodiments of Formula (I), the linker 1 or the linker 2 is any one of the linkers from Table 3. In some embodiments of Formula (I), the linker 1 or the linker 2 is a SMCC linker. In some embodiments of Formula (I), the linker 1 or the linker 2 is a SMCC linker conjugated to the antisense strand of the siRNA agent. In some embodiments of Formula (I), the linker 1 or the linker 2 is a SMCC linker conjugated to the sense strand of the siRNA agent. In some embodiments of Formula (I), when the linker 1 is an SMCC linker, the linker 2 comprises an amino acid sequence of (G4Q)n(SEQ ID NO:59), wherein n= 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0034] A conjugate of the following formulae:11(a) [second agent]-linker 1-[TIR VHH]-linker 2-[ALB VHH] or11(b) [TfR VHH]-linker 1-[ALB VHH]-linker 2-[second agent],

[0035] wherein the [TfR VHH] comprises the TfR binding VHH of any one of the preceding embodiments and [ALB VHH] comprises albumin binding VHH; the linker 1 or the linker 2 is optional. In some embodiments of Formula 11(a) or Formula 11(b), the [second agent], the [TfR VHH] and the [ALB VHH] are conjugated in any order. In some embodiments of Formula 11(a) or Formula 11(b), the second agent is a polypeptide, including without limitation an antigen binding VHH domain or polypeptide; a peptide; a nucleic acid molecule, including without limitation double stranded (ds)RNA such as siRNA, saRNA, antisense oligonucleotide (ASO), and a guide RNA; a dye; a small molecule; nanoparticles (NPs); lipid NPs; a liposome; an exosome; an antibody, an antibody Fc portion or antigen binding fragments thereof; a viral particle; or any combination thereof. In some embodiments of Formula 11(a) or Formula 11(b), the second agent is a therapeutic agent. In some embodiments, the second agent is a nucleic acid molecule, wherein the nucleic acid molecule is double stranded (ds)RNA such as an siRNA, saRNA, antisense oligonucleotide (ASO), or a guide RNA.

[0036] In some embodiments of Formula 11(a) or Formula 11(b), the second agent is conjugated via the linker 1 or the linker 2 to the ALB binding VHH. In some embodiments of Formula 11(a) or Formula 11(b), the second agent is a nucleic acid molecule. In some embodiments of Formula 11(a) or Formula 11(b), the second agent is an siRNA, wherein the siRNA comprises a sense strand and an antisense strand.

[0037] In some embodiments of Formula 11(a) or Formula 11(b), the nucleic acid molecule is an siRNA conjugated via linker 1 to the TfR binding VHH, wherein the siRNA comprises a sense strand and an antisense strand. In some embodiments of Formula 11(a) or Formula 11(b), the nucleic acid molecule is an siRNA conjugated via the linker 1 to a cysteine residue in the TfR binding VHH, wherein the siRNA comprises a sense strand and an antisense strand. In some embodiments of Formula 11(a) or Formula 11(b), the nucleic acid molecule is an siRNA conjugated via the linker 2 to the ALB binding VHH, wherein the siRNA comprises a sense strand and an antisense strand. In some embodiments of Formula 11(a) or Formula 11(b), the nucleic acid molecule is an siRNA conjugated via the linker 2 to a cysteine residue in the ALB binding VHH, wherein the siRNA agent comprises a sense strand and an antisense strand. In some embodiments of Formula 11(a) or Formula 11(b), the ALB binding VHH comprising three complementarity determining regions, CDR1, CDR2, and CDR3, wherein CDR1 comprises SEQ ID NO: 61; CDR2 comprises SEQ ID NO:62; and CDR3 comprising SEQ ID NO:63 or SEQ ID NO: 70. In some embodiments of Formula 11(a) or Formula 11(b), the ALB binding VHH comprises SEQ ID NO: 60 or SEQ ID NO: 70. In some embodiments of Formula 11(a) or Formula 11(b), the ALB binding VHH comprises SEQ ID NO: 60 and / or SEQ ID NO: 70 further comprises a cysteine at the C- terminal end. In some embodiments of Formula 11(a) or Formula II(b)the ALB binding VHH comprises SEQ ID NO: 60 or SEQ ID NO: 70 without the two C-terminal [PP] amino acids, and optionally comprises a cysteine at the C-terminal end.

[0038] In some embodiments of Formula 11(a) or Formula 11(b), the second agent is conjugated to any one of a polypeptide comprising SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 or SEQ ID NO: 58. In some embodiments of Formula 11(a) or Formula 11(b), the second agent is an siRNA which is conjugated to any of the polypeptides comprising SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID N: 57 or SEQ ID NO: 58.

[0039] In some embodiments of Formula II (a), the linker 1 is a SMCC linker. In some embodiments of Formula II (a), the linker 1 is a SMCC linker conjugated to an antisense strand of an siRNA agent and the linker 2 is a glycine rich linker. In some embodiments of Formula 11(a), the linker l is a SMCC linker conjugated to a sense strand of an siRNA agent and the linker 2 is a glycine rich linker.

[0040] In some embodiments of Formula II (b), the linker 1 is glycine rich linker and the linker 2 is a SMCC linker conjugated to an antisense strand of an siRNA agent. In some embodiments of Formula II (b), the linker 1 is glycine rich linker and the linker 2 is a SMCC linker conjugated to a sense strand of an siRNA agent.

[0041] In some embodiments of Formula II (a) or Formula II (b), the glycine rich linker comprises an amino acid sequence of (G4Q)n (SEQ ID NO:59), wherein n=l, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0042] In some embodiments of Formula II (a) or Formula II (b), the linker 1 or the linker 2 comprises the amino acid sequence of (G4Q)n (SEQ ID NO:59), wherein n=l, 2, 3, 4 or 5.

[0043] In certain aspects provided are pharmaceutical compositions comprising the TfR binding VHH of any one of the embodiments disclosed herein or the conjugate of any one of the embodiments disclosed herein and a pharmaceutically acceptable carrier or excipient.

[0044] In certain aspects provided are methods of treating a disease or disorder comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of the TfR binding VHH of any one of the embodiments disclosed herein, a composition comprising a therapeutically effective amount of the conjugate of any one of the embodiments disclosed herein or a therapeutically effective amount of the pharmaceutical composition of any of the embodiments disclosed herein. In certain aspects provided are methods for delivering an agent to a subject comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of the TfR binding VHH of any one of the embodiments disclosed herein, a composition comprising a therapeutically effective amount of the conjugate of any one of the embodiments disclosed herein or a therapeutically effective amount of the pharmaceutical composition of any of the embodiments disclosed herein. In certain embodiments, the composition is administered systemically; subcutaneously or intravenously. In certain aspects provided are compositions for use in therapy.

[0045] In certain aspects provided is use of the TfR binding VHH of any one of the embodiments disclosed herein or the conjugate of any one of the embodiments disclosed herein in the manufacture of a medicament for the treatment of a disease or disorder, or for delivering a therapeutic agent to a subject. In certain aspects provided is use of the TfR binding VHH of anyone of the embodiments disclosed herein or the conjugate of any one of the embodiments disclosed herein in the treatment of a disease or disorder, or for delivering a therapeutic agent to a subject.

[0046] In certain aspects provided is a recombinant nucleic acid molecule comprising a first nucleic acid sequence encoding the TfR binding VHH of anyone of the embodiments disclosed herein. In certain embodiments, the recombinant nucleic acid molecules further comprise a second nucleic acid sequence encoding any one of: a protein agent, an antibody or antigen binding fragment thereof, an antigen binding VHH, or a combination thereof, wherein the second nucleic acid sequence is operably linked, e.g. have common regulatory elements that control expression of the first and second nucleic acid sequence, to the fist nucleic acid sequence. In some embodiments of the recombinant nucleic acid molecule, the second nucleic acid sequence encodes an ALB binding VHH.

[0047] In certain aspects provided is an expression vector comprising the recombinant nucleic acid molecule of any of the embodiments described herein.

[0048] In certain aspects provided is a host cell comprising the recombinant nucleic acid molecule of any of the embodiments described herein. In certain aspects provided is a host cell comprising the expression vector of any of the embodiments described herein. In certain embodiments, the host cell is a mammalian cell, bacterial cell or a yeast cell.DETAILED DESCRIPTION

[0049] In certain aspects, the invention provides a Transferrin Receptor (TfR) binding variable domain of a heavy chain only antibody (VHH) polypeptide (TfR binding VHH polypeptide) comprising three complementarity determining regions CDR1, CDR2 and CDR3, and conjugates thereof. In certain embodiments, the TfR -binding VHH polypeptides and conjugates can be used as shuttles to transport agents, such as therapeutic, imaging and / or diagnostic agents, into cells, including without limitation muscle cells and across the BBB.TfR binding VHH

[0050] VHH domain is the variable region of heavy chain only camelid antibodies that are naturally devoid of light chains. VHH domains are around 15 kDa and are a single polypeptidechain molecule that can bind its cognate antigen as a single domain. The antigen-binding surfaces of VHHs are sometimes more convex (or protruding) than those of conventional antibodies, which are usually flat or concave.

[0051] Llamas can be immunized with human and / or mouse TfRl proteins to generate VHH molecules having desired characteristics. Following isolation and analysis of numerous llama VHH sequences for TfRl binding and TfRl species specificity, selected VHH sequences with desired binding specificity and cross species reactivity can be further engineered to improve and / or optimize their properties, including without limitation antigen binding, developability properties that affect recombinant expression, purification and CMC characteristics, and the ability to develop these VHH polypeptides as therapeutic molecules, e.g. engineering to reduce immunogenicity in human subjects.

[0052] The TfR binding VHH polypeptides of the invention typically comprise or consist essentially of three regions of hyper-variability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). The CDRs are important regions for antigen binding specificity. Each VHH polypeptide comprises three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Typically, the CDRs contain most of the residues that form specific interactions with the antigen.

[0053] In some aspects, provided is a compound comprising an amino acid sequence which is at least 90% identical to SEQ ID NO: 1, 6, 8, 13, 18, 20, 23, 28, 32, 35, 38, 41, 44, 47, 49, 52, 64, 66, 75, 80, or 81. A compound comprising an amino acid sequence which is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% identical to SEQ ID NO: 1, 6, 8, 13, 18, 20, 23, 28, 32, 35, 38, 41, 44, 47, 49, 52, 64, 66, 75, 80, or 81. In some embodiments the compound further comprises an agent conjugated to an amino acid sequence which is at least 90% identical to SEQ ID NO: 1, 6, 8, 13, 18, 20, 23, 28, 32, 35, 38, 41, 44, 47, 49, 52, 64, 66, 75, 80, or 81. In some embodiments the compound further comprises an agent conjugated to an amino acid sequence which is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% identical to SEQ ID NO: 1, 6, 8, 13, 18, 20, 23, 28, 32, 35, 38, 41, 44, 47, 49, 52, 64, 66, 75, 80, or 81. In some embodiments, the compound binds to human and non-human primate (NHP) TfR. In some embodiments, the compound binds to mouse TfR.

[0054] In some aspects, provided is a Transferrin Receptor 1 (TfR) binding variable domain of a heavy chain only antibody (VHH) (TfR binding VHH) comprising three complementarity determining regions (CDR)1, CDR2 and CDR3 in any one of the following sequences: SEQ ID NO: 1; SEQ ID NO: 8; SEQ ID NO: 13; SEQ ID NO: 20; SEQ ID NO: 23; SEQ ID NO: 28; SEQ ID NO: 32; SEQ ID NO: 35; SEQ ID NO: 38; SEQ ID NO: 41; SEQ ID NO: 44; SEQ ID NO: 47; SEQ ID NO: 49; SEQ ID NO: 52; SEQ ID NO: 64; or SEQ ID NO: 66.

[0055] In some embodiments of the TfR binding VHH: a. CDR1 comprises SEQ ID NO: 2, CDR 2 comprises SEQ ID NO: 3 and CDR3 comprises SEQ ID NO: 4; b. CDR1 comprises SEQ ID NO: 9, CDR 2 comprises SEQ ID NO: 10 and CDR3 comprises SEQ ID NO: 11; c. CDR1 comprises SEQ ID NO: 14, CDR 2 comprises SEQ ID NO: 15 and CDR3 comprises SEQ ID NO: 16; d. CDR1 comprises SEQ ID NO: 14, CDR 2 comprises SEQ ID NO: 21 and CDR3 comprises SEQ ID NO: 16; e. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 26; f. CDR1 comprises SEQ ID NO: 29, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 30; g. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33; h. CDR1 comprises SEQ ID NO: 36, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33; i. CDR1 comprises SEQ ID NO: 39, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33; j. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 42 and CDR3 comprises SEQ ID NO: 33; k. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 45 and CDR3 comprises SEQ ID NO: 33;l. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 21 and CDR3 comprises SEQ ID NO: 33; m. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 50 and CDR3 comprises SEQ ID NO: 33; n. CDR1 comprises SEQ ID NO: 53, CDR 2 comprises SEQ ID NO: 54 and CDR3 comprises SEQ ID NO: 33, wherein CDR1 and CDR2 comprise any combination of amino acids XI and X2 in CDR1, wherein Xl= Q or T and X2= A or S, and X3, X4, and X5 in CDR2, wherein X3= N or S, X4= A, G or T, and X5= R or Y; o. CDR1 comprises SEQ ID NO: 53, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33, wherein CDR1 comprises any combination of amino acids XI and X2, wherein Xl= Q or T and X2= A or S; or p. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 54 and CDR3 comprises SEQ ID NO: 33, wherein CDR2 comprises any combination of amino acids X3, X4, and X5, wherein X3= N or S, X4= A, G or T, and X5= R or Y.

[0056] In some embodiments of the TfR binding VHH: a. CDR1 consists essentially of SEQ ID NO: 2, CDR 2 consists essentially of SEQ ID NO: 3 and CDR3 consists essentially of SEQ ID NO: 4; b. CDR1 consists essentially of SEQ ID NO: 9, CDR 2 consists essentially of SEQ ID NO: 10 and CDR3 consists essentially of SEQ ID NO: 11; c. CDR1 consists essentially of SEQ ID NO: 14, CDR 2 consists essentially of SEQ ID NO: 15 and CDR3 consists essentially of SEQ ID NO: 16; d. CDR1 consists essentially of SEQ ID NO: 14, CDR 2 consists essentially of SEQ ID NO: 21 and CDR3 consists essentially of SEQ ID NO: 16; e. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 25 and CDR3 consists essentially of SEQ ID NO: 26; f CDR1 consists essentially of SEQ ID NO: 29, CDR 2 consists essentially of SEQ ID NO: 25 and CDR3 consists essentially of SEQ ID NO: 30; g. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 25 and CDR3 consists essentially of SEQ ID NO: 33;h. CDR1 consists essentially of SEQ ID NO: 36, CDR 2 consists essentially of SEQ ID NO: 25 and CDR3 consists essentially of SEQ ID NO: 33; i. CDR1 consists essentially of SEQ ID NO: 39, CDR 2 consists essentially of SEQ ID NO: 25 and CDR3 consists essentially of SEQ ID NO: 33; j. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 42 and CDR3 consists essentially of SEQ ID NO: 33; k. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 45 and CDR3 consists essentially of SEQ ID NO: 33; l. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 21 and CDR3 consists essentially of SEQ ID NO: 33; m. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 50 and CDR3 consists essentially of SEQ ID NO: 33; or n. CDR1 consists essentially of SEQ ID NO: 53, CDR 2 consists essentially of SEQ ID NO: 54 and CDR3 consists essentially of SEQ ID NO: 33, wherein CDR1 and CDR2 comprise any combination of amino acids XI and X2 in CDR1, wherein Xl= Q or T and X2= A or S, and X3, X4, and X5 in CDR2, wherein X3= N or S, X4= A, G or T, and X5= R or Y; o. CDR1 comprises SEQ ID NO: 53, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33, wherein CDR1 comprises any combination of amino acids XI and X2, wherein XI = Q or T and X2= A or S; or p. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 54 and CDR3 comprises SEQ ID NO: 33, wherein CDR2 comprises any combination of amino acids X3, X4, and X5, wherein X3= N or S, X4= A, G or T, and X5= R or Y.

[0057] In some embodiments of the TfR binding VHH, CDR1, CDR2 and CDR3 are grafted in a human or humanized framework. In some embodiments, the framework is VH3-23.

[0058] In some embodiments, the TfR binding VHH comprises an amino acid sequence which is 90% identical to SEQ ID NO: 1, 6, 8, 13, 18, 20, 23, 28, 32, 35, 38, 41, 44, 47, 49, 52, 64, 66, 75, 80, or 81. In some embodiments, the TfR binding VHH comprises an amino acid sequence of SEQ ID NO: 1, 6, 8, 13, 18, 20, 23, 28, 32, 35, 38, 41, 44, 47, 49, 52, 64, 66, 75, 80, or 81. In someembodiments, the TfR binding VHH consists essentially of an amino acid sequence of SEQ ID NO: 1, 6, 8, 13, 18, 20, 23, 28, 32, 35, 38, 41, 44, 47, 49, 52, 64, 66, 75, 80, or 81.

[0059] In some embodiments, the TfR. binding VHH binds to human and non-human primate (NHP) TfR.. In some embodiments, the TfR. binding VHH binds optionally to mouse TfR..

[0060] In some embodiments, the TfR. binding VHH is conjugated to at least one agent. In some embodiments, the TfR. binding VHH is conjugated to one agent. In some embodiments, the TfR. binding VHH is conjugated to two agents. In some embodiments, the agent is a polypeptide; a peptide; a nucleic acid molecule; a dye; a small molecule; nanoparticles (NPs); lipid NPs; a liposome; an exosome; an antibody, an antibody Fc portion or antigen binding fragments thereof; a viral particle; or any combination thereof. In some embodiments, the polypeptide agent includes without limitation an antigen binding VHH domain or polypeptide. In some embodiments, the nucleic acid molecule includes without limitation double stranded (ds)RNA such as siRNA, saRNA, antisense oligonucleotide (ASO), and / or a guide RNA.

[0061] Assignment of amino acid residues to the CDRs may be done according to the well-known schemes, including those described in Kabat (Kabat et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al ., “Canonical structures for the hypervariable regions of immunoglobulins”, Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics database available at IMGT webpage; see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212).

[0062] T fR binding VHH comprising CDRs as described herein, e.g. Table 1, are grafted between human framework regions FR1, FR2, FR3 and FR4 from human VH3 genes, while maintaining select llama amino acids positions 37, 44, 45, 47, and / or 49 in framework 2 and position 84 in framework 3 (framework numbering according Kabat). In some embodiments, the VH3 gene is VH3-23. In some embodiments, the VH3 gene is VH3-7, 3-9, 3-30, 3-33, 3-48, 3-66, 3-74. In some embodiments the framework regions are from any other VH3 or other human VH gene(s) that frame the CDRs as TfR binding VHH polypeptideTable 1. Exemplary amino acid sequences of TfR binding VHH polypeptides and listing of CDRs (CDR numbering according to North). Included are also non-limiting embodiments of nucleic acid sequences which encode the corresponding polypeptide.

[0063] In some embodiments, the polypeptides described in Table 1 without a C-terminal Cysteine further comprise a C-terminal Cysteine. In some embodiments, the polypeptides described in Table 1 further comprise an N-terminal Cysteine.

[0064] As shown in the Examples, TfR binding VHH polypeptides described herein bind TfR in vitro and bi vivo, with binding ECso values as shown in Example 3. Furthermore, all of these TfR binding VHH polypeptides bind both human and NHP (cynomolgus) TfR. Some TfR binding VHH polypeptides which bind human and cynomolgus TfR also bind mouse TfR. Moreover, binding of the TfR binding VHHs to the TfR receptor does not compete with binding of transferrin, the endogenous TfR ligand, and does not affect regular functions of the ligand. In some embodiments, conjugates produced with such TfR binding VHH polypeptides bind TfR in vitro and in vivo, and are transported across the cell membrane in vivo, showing transcytosis. Such TfR binding VHH polypeptides and their conjugates are suitable shuttles for drug delivery or targeting of therapeutic agents.

[0065] In some embodiments, the TfR-binding VHH polypeptides of the invention are VHH polypeptides which competitively inhibit binding of a TfR-binding VHH polypeptides as described in Table 1 to a human and a non-human TfR. In competitive inhibition assay, a test VHH polypeptide can reduce or inhibit or displace the binding of a reference TfR binding VHH polypeptide, e.g. any one of the TfR binding VHH polypeptides described herein, in vitro or in vivo. Competition assays can be performed using standard techniques such as, for instance, competitive ELISA or other binding assays. Typically, a competitive binding assay involves a recombinant cell or membrane preparation expressing a TfR, optionally bound to a solid substrate, an unlabeled test VHH polypeptide (or a phage expressing the same) and a labeled reference TfR binding VHH polypeptide (or a phage expressing the same). Competitive inhibition is measured by determining the amount of labeled TfR binding VHH polypeptide bound in the presence of the test VHH Usually the test VHH is present in excess, such as about 5 to 500 times the amount of reference VHH. Typically, for ELISA, the test VHH is in 100-fold excess. When a test VHH present in excess inhibits or displaces at least 70% of the binding of the reference TfR binding VHH polypeptides to TfR, it is considered as competitively inhibiting the reference TfR binding VHH polypeptides. Typically a competing VHH polypeptide(s) bind epitopes that share common amino acid residues.

[0066] The TfR binding polypeptides as described herein can be recombinantly produced in a host cell, for example, using an expression vector. For example, an expression vector may include a sequence that encodes one or more signal peptides that facilitate secretion of the polypeptide(s) from a host cell. Expression vectors containing a polynucleotide of interest (e.g., a polynucleotide encoding the TfR binding VHH polypeptides) may be transferred into a host cell by well-known methods. Additionally, expression vectors may contain one or more selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to aid in detection of host cells transformed with the desired polynucleotide sequences.

[0067] A host cell includes cells stably or transiently transfected, transformed, transduced or infected with one or more expression vectors expressing all or a portion of the TfR binding VHH binding polypeptides described herein. According to some embodiments, a host cell may be stably transfected, transformed, transduced or infected with an expression vector expressing VHH polypeptide of the TfR binding polypeptides as described herein. In some embodiments, a host cell may be transiently transfected, transformed, transduced or infected with an expression vector expressing VHH polypeptide of the TfR binding polypeptides described herein. The TfR binding VHH polypeptides may be produced in mammalian cells such as CHO, NSO, HEK293 or COS cells according to techniques well known in the art.

[0068] Medium, into which the TfR binding or ALB binding VHH polypeptides has been secreted, may be purified by conventional techniques, such as mixed-mode methods of ion-exchange and hydrophobic interaction chromatography. For example, the medium may be applied to and eluted from a Protein A or G column using conventional methods; mixed-mode methods of ion-exchange and hydrophobic interaction chromatography may also be used. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. Various methods of protein purification may be employed, and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182: 83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0069] The TfR (transferrin receptor protein 1) binding VHH polypeptides can be synthesized and purified by methods known in the art. Different recombinant expression organisms can be utilized, including but not limited to bacterial, yeast and mammalian cell lines. A host cell, such asChinese hamster ovarian cells (CHO), can be transiently or stably transfected with an expression system for secreting proteins. Clarified media containing the secreted protein, can be purified using commonly used techniques. In some instances, VHH polypeptides is generated in a mammalian cell expression system using CH0K1 cell derivatives (Lonza Biologies Inc). A cDNA sequence encoding the respective VHH amino acid sequence is sub-cloned into GS-containing expression plasmid backbone (pEE12.4-based plasmids, Lonza Biologies Inc.). The cDNA sequence is fused in frame with the coding sequence of a signal peptide sequence, METDTLLLWVLLLWVPGSTG (SEQ ID NO: 68) to enhance secretion of the VHH into the tissue culture medium. In certain instances, a C-terminal GGGGQGGGGQGGGGQGGGGQGGGGQYPYDVPDYA (SEQ ID NO: 69)) amino acid sequence motif (HA-tag) or a single Cysteine residue are added to the VHH sequences and encoded in the respective cDNA sequence. Expression is driven by the viral CMV promoter.

[0070] The VHH polypeptides secreted into the media from the CHO cells can be purified by Protein A affinity chromatography followed by size-exclusion chromatography. The VHH polypeptide from harvested media is captured onto MabSelect PrismA resin (Cytiva). The resin then is briefly washed with a running buffer, such as a phosphate-buffered saline (PBS; pH 7.2) or a running buffer containing Tris, to remove non-specifically bound material. In some cases, salt may be needed in the wash buffer to retain the VHH polypeptide on the column before elution. The VHH fusion protein is eluted from the resin with a low pH solution, such as 10 mM citric acid pH 3. Fractions containing the VHH polypeptide are pooled. The pH may be neutralized by adding a base such as IM Tris pH 8.0. The VHH polypeptide protein may be further purified by size exclusion chromatography using resins such as Superdex75 (Cytiva) with isocratic elution in PBS, pH 7.2.Conjugates comprising TfR binding VHH polypeptides

[0071] In another aspect, provided herein are conjugates comprising TfR binding VHH polypeptides described herein and at least one agent, which can be a therapeutic agent, and which are conjugated in any order. In some embodiments, the conjugates comprise a TfR binding VHH polypeptide as described herein and one agent, which can be a therapeutic agent, and which are conjugated in any order. In some embodiments, the conjugates comprise a TfR binding VHH polypeptide as described herein and two agents, wherein at least one of the agents is a therapeuticagent, and all of which are conjugated in any order. In some embodiments, the conjugates comprise a TfR binding VHH polypeptide as described herein and three agents, wherein at least two of the agents are therapeutic agents, and all of which are conjugated in any order. In some embodiments, the conjugates comprise a TfR binding VHH polypeptide as described herein and three agents, wherein at least one of the agents is a polypeptide which is a half-life extender, and all of which are conjugated in any order. In some embodiments, the conjugates comprise a TfR binding VHH polypeptide as described herein and three agents, wherein one of the agents is a polypeptide which is a half-life extender, and two of the agents are therapeutic agent, and all of which are conjugated in any order.

[0072] In some embodiments, the agent is: a polypeptide, including without limitation an antigen binding VHH domain or polypeptide; a peptide; a nucleic acid molecule, including without limitation double stranded (ds)RNA such as siRNA, saRNA, antisense oligonucleotide (ASO), and a guide RNA; a dye; a small molecule; nanoparticles (NPs); lipid NPs; a liposome; an exosome; an antibody, an antibody Fc portion or antigen binding fragments thereof; a viral particle, or any combination thereof.

[0073] In certain embodiments, the conjugates of the invention comprise at least one TfR binding VHH polypeptide, a first agent, which in some embodiments extends the half-life of the TfR binding VHH polypeptide, and a second agent, which can be a therapeutic agent, and which are conjugated in any order A non-limiting embodiment is a conjugate comprising: [TfR binding VHH polypeptide], [Fc portion of an antibody] and a [second agent, e.g. a therapeutic agent] conjugated in any order, with optional linker between the TfR and Fc, and the Fc and the second agent. Anon-liraiting embodiment is a conjugate comprising: [TfR binding VHH polypeptide], [albumin binding polypeptide], [second agent, e.g. a therapeutic agent] conjugated in any order, with optional linker between the TfR and the albumin binding polypeptide, and the albumin binding polypeptide and the second agent. In some embodiments, the second agent is: a polypeptide, including without limitation an antigen binding VHH domain or polypeptide; a peptide, a nucleic acid molecule, including without limitation double stranded (ds)RNA such as siRNA, saRNA, antisense oligonucleotide (ASO), and a guide RNA; a dye; a small molecule; nanoparticles (NPs); lipid NPs; a liposome, an exosome, an antibody, an antibody Fc portion or antigen binding fragments thereof; a viral particle, or any combination thereof. In someembodiments, the second agent is a double stranded RNA (dsRNA), e.g. an siRNA. In some embodiments, the dsRNA comprises a sense strand and an antisense stand, wherein the antisense strand is complementary to a target mRNA of interest.

[0074] In some embodiments of TfR binding VHH conjugates, a polypeptide is linked to the 3’ end of the sense strand of the dsRNA. In some embodiments, the polypeptide is linked to the 5’ end of the sense strand of the dsRNA. In some embodiments, the protein polypeptide is linked to an internal position of the sense strand of the dsRNA. In some embodiments, the polypeptide is linked to the 3’ end of the antisense strand of the dsRNA. In some embodiments, the polypeptide is linked to an internal position of the antisense strand of the dsRNA.

[0075] TfR binding VHH conjugates can be formed by any suitable means, including without limitation chemical conjugation, recombinant / genetic fusion and / or combination thereof TfR binding VHH conjugates can comprise any agent, including therapeutic, imaging and / or functional agents, and / or combinations thereof. In some embodiments the agent is: a polypeptide, including without limitation an antigen binding VHH domain or polypeptide; a peptide; a nucleic acid molecule, including without limitation double stranded (ds)RNA such as siRNA, saRNA, antisense oligonucleotide (ASO), and a guide RNA; a dye; a small molecule; nanoparticles (NPs); lipid NPs; a liposome; an exosome; an antibody, an antibody Fc portion or antigen binding fragments thereof; a viral particle, or any combination thereof.

[0076] In some embodiments, the conjugates comprise at least one polypeptide comprising at least one cysteine residue which can be used for conjugation. The approach of including cysteines, including engineered cysteines, as a means for conjugation has been described in WO 2018 / 232088. In some embodiments, the conjugates comprise a TfR binding VHH polypeptide comprising one or more cysteines at an internal VHH residue(s). In some embodiments, the TfR binding VHH polypeptides described herein comprise one or more native cysteine residues, which can be used for conjugation. For example, in some embodiments, the TfR binding VHH polypeptides described herein comprises a native cysteine in the VHH chain, which can be used for conjugation. In some embodiments, the TfR binding VHH polypeptides described herein comprise one or more engineered cysteine residues for conjugation. In some embodiments, the TfR binding VHH polypeptides described herein comprise a cysteine residue at the C-terminal end of the VHH polypeptide. In some embodiments, the TfR binding VHH polypeptides described herein comprisea cysteine residue at the N-terminal end of the VHH polypeptide. In some embodiments, the conjugates comprise an agent which is a polypeptide, e.g. without limitation an ALB binding VHH, comprising one or more cysteines at an internal VHH residue(s). In some embodiments, the agent polypeptides comprise one or more native cysteine residues, which can be used for conjugation. For example, in some embodiments, the ALB binding VHH polypeptides described herein comprises a native cysteine in the VHH chain, which can be used for conjugation. In some embodiments, the agent polypeptides described herein comprise one or more engineered cysteine residues for conjugation. In some embodiments, the ALB binding VHH polypeptides comprise a cysteine residue at the C-terminal end of the VHH polypeptide. In some embodiments, the ALB binding VHH polypeptides comprise a cysteine residue at the N-terminal end of the VHH polypeptide.

[0077] In some embodiments, the agent is linked to the TfR binding VHH polypeptides through a linker. In some embodiments, the linker is a chemical linker. In some embodiments, the linker is a Mal-Tet-TCO linker, SMCC linker, or GDM linker (structures of embodiments of these linkers shown in Table 3).

[0078] The conjugates described herein can be made by a variety of procedures known to one of ordinary skill in the art, some of which are illustrated in the preparations and examples described herein, e.g., in Example 6A and 6B. One of ordinary skill in the art recognizes that the specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare conjugates. The product of each step can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. The reagents and starting materials are readily available to one of ordinary skill in the art.

[0079] In some embodiments of preparing the TfR binding conjugates, a polypeptide(s) with native or engineered cysteines can be first treated with a reducing agent, e.g., DTT, and then re-oxidized with an oxidizing agent, e.g., DHAA. For example, a TfR binding VHH polypeptide can be first treated with a reducing agent, e.g., DTT, and then re-oxidized with an oxidizing agent, e.g., DHAA. The resulting oxidized polypeptides are then incubated with a linker functionalized agent, e.g., linker-dsRNA, to produce the conjugates.

[0080] In some embodiments, conjugation is by a genetic fusion. In these embodiments, the linker is a genetic / recombinant linker Such strategy can be used when the agent is a peptide or polypeptide. In such a case, a nucleic acid molecule encoding the TfR binding VHH polypeptide is recombinantly engineered as a fusion nucleic acid comprising a sequence encoding the TfR binding VHH polypeptide and the agent. In some embodiments, the agent is half-life extender protein, e.g. without limitation Fc or albumin binding protein. In some embodiments, the fusion nucleic acid further comprises a nucleic acid sequence which encodes a peptide linker connecting the TfR binding VHH polypeptide and the agent. The fusion nucleic acid is introduced and expressed in any suitable expression system, to produce the conjugate. This conjugate can be further modified to comprise additional agent(s), e.g. without limitation nucleic acid molecules such as siRNA and / orASO.

[0081] In some embodiments, conjugation of the TfR binding polypeptide and the agent is at a lysine residue, which can be engineered and / or naturally present in the amino acid sequence. In some embodiments, conjugation of the TfR binding polypeptide and the agent is at a glutamine residue, which can be engineered and / or naturally present in the amino acid sequence.

[0082] In other embodiments, conjugation is by enzymatic reaction. In some embodiments, sitespecific conjugation onto the VHH polypeptide can be performed using the transglutaminase enzyme (TGase). TGase catalyzes the formation of a stable isopeptidic bond between (i) the side chain of a glutamine residue inserted in a tag sequence specifically recognized by the TGase and (ii) an amino-functionalized donor substrate.

[0083] In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is glycine rich linker. In some embodiments, the glycine rich linker is located at the ('- terminal end of the VHH polypeptide. Non-limited embodiments of glycine rich linker include: (G(x)S)n, (G(X)Q)n, wherein x=l, 2, 3, 4, or 5, and n=l, 2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein n and x are independently selected. In some embodiments, the Gly linker may comprise a glycine repeat of 2-9, for example 2, 3, 4, 5, 6 or 7 glycine residues. Non-limiting examples of specific glycine linkers include Gly3, Gly4, Gly 5 or SerGlySerGly(n) wherein n=l, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0084] In some embodiments, provided herein are TfR binding VHH conjugates of Formula I: [[first agent] -linker 1](n)-[TfR VHH]-[linker 2-[ second agent]] <m), wherein the [TfR VHH]comprises any one of the TfR binding VHH embodiments described herein; the linker 1 and the linker 2 which linkers conjugate each agent to the [TfR VHH] are optional; and a [first agent] and a [second agent], wherein m and n are independent, n=0, 1, 2, 3, 4 or 5, m=0, 1, 2, 3,4 or 5. In embodiments where n=l, 2, 3, 4 or 5, each [first agent] comprises a linker 1. In embodiments where m=l, 2, 3, 4, or 5, each agent comprises a linker 2. In some embodiments, the [first agent], the [second agent] and the [TfR VHH] are conjugated in any order.

[0085] In certain aspects provided are TfR binding VHH conjugates of the following Formula I:(I) [[first agent]-linker l](n)-[TfR VHH]-[linker 2-[second agent]] (m), wherein the [TfR VHH] comprises any one of the TfR binding VHH embodiments described herein; the linker 1 and linker 2 are optional; and a [first agent] and a [second agent], wherein m and n are independent, n=0, 1, 2 or 3, m=0, 1, 2 or 3.

[0086] In some embodiments of Formula (I), at least one of the first or the second agent is a therapeutic agent. In some embodiments of Formula (I), the first and / or the second agent is a polypeptide; a peptide; a nucleic acid molecule; a dye; a small molecule; nanoparticles (NPs); lipid NPs; a liposome; an exosome; an antibody, an antibody Fc portion or antigen binding fragments thereof; viral particle; or any combination thereof. In some embodiments of Formula (I), the first agent is conjugated via the linker 1 and / or the second agent is conjugated via the linker 2 to the TfR binding VHH. In some embodiments, wherein n=0 and m=l, wherein when m=l the second agent is a nucleic acid molecule. In some embodiments, wherein n=0 and m=2, wherein one [second agent] is a half-life extender and the other [second agent] is a nucleic acid molecule. In some embodiments of Formula (I), wherein when n=l, the [first agent] is a nucleic acid molecule, and m=2, wherein one [second agent] is a half-life extender and the other [second agent] is a nucleic acid molecule. In some embodiments of Formula (I), wherein when n=l, the [first agent] is a nucleic acid molecule, and m=2, wherein one [second agent] is a half-life extender and the other [second agent] is a nucleic acid molecule, the other [second agent] is conjugated to the C terminal or an internal cysteine of the half-life extender: [nucleic acid molecule, e g. siRNA]-linker l-[TfR VHH]-linker 2-a [half-life extender]-linker 3-[nucleic acid molecule, e.g. siRNA], wherein the linker 3 is selected from Table 3.

[0087] In some embodiments, provided are TfR binding VHH conjugates of Formula 11(a) or 11(b):11(a) [second agent] -linker l-[TfR VHH] -linker 2- [ALB VHH] or11(b) [TfR VHH]-linker 1-[ALB VHH]-linker 2-[second agent] wherein the [TfR VHH] comprises any one of the TfR binding VHH described herein and [ALB VHH] comprises albumin binding VHH; the linker 1 or the linker 2 is optional; and a second agent, which is a therapeutic agent.

[0088] TfR binding VHH conjugates comprising an ALB binding VHH can be formed by any suitable means, including without limitation chemical conjugation, genetic / recombinant fusion and / or combination thereof Polypeptides in the TfR binding VHH conjugates can be conjugated to any agent, including therapeutic, imaging and / or functional agents, and / or combinations thereof. In some embodiments the agent is a polypeptide, including without limitation an antigen binding VHH domain or polypeptide; a peptide; a nucleic acid molecule, including without limitation double stranded (ds)RNA such as siRNA, saRNA, antisense oligonucleotide (ASO), and a guide RNA; a dye; a small molecule; nanoparticles (NPs); lipid NPs; a liposome; an exosome; an antibody, an antibody Fc portion or antigen binding fragments thereof; a viral particle, or any combination thereof.

[0089] In some embodiments, provided herein are conjugates of Formula 11(a) or 11(b) comprising a [second agent], e.g. an siRNA comprising a sense stand and an antisense strand; and wherein a linker 1 and a linker 2, are optionally absent, wherein the TfR binding VHH polypeptide comprises complementarity determining regions CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, CDR3 comprise sequences selected from one of the following sequences: SEQ ID NO: 1; SEQ ID NO: 8; SEQ ID NO: 13; SEQ ID NO: 20; SEQ ID NO: 23; SEQ ID NO: 28; SEQ ID NO: 32; SEQ ID NO: 35; SEQ ID NO: 38; SEQ ID NO: 41; SEQ ID NO: 44; SEQ ID NO: 47; SEQ ID NO: 49; SEQ ID NO: 52; SEQ ID NO: 64; or SEQ ID NO: 66.

[0090] In some embodiment provided is a conjugate of Formula 11(a), comprising [a nucleic acid molecule, e.g. siRNA]-linker 1 selected from Table 3-[TfR VHH]-linker 2 is a genetic linker-[ALB VHH], wherein the TfR binding VHH polypeptide comprises complementarity determining regionsCDR1, CDR2, and CDR3, wherein the CDR1, CDR2, CDR3 comprise sequences selected from one of the following sequences: SEQ ID NO: 1; SEQ ID NO: 8; SEQ ID NO: 13; SEQ ID NO: 20; SEQ ID NO: 23; SEQ ID NO: 28; SEQ ID NO: 32; SEQ ID NO: 35; SEQ ID NO: 38; SEQ ID NO: 41; SEQ ID NO: 44; SEQ ID NO: 47; SEQ ID NO: 49; SEQ ID NO: 52; SEQ ID NO: 64; or SEQ ID NO: 66.

[0091] In some embodiment provided is a conjugate of Formula 11(a), comprising [a nucleic acid molecule, e.g. siRNA]-linker 1 selected from Table 3-[TfR VHH]-linker 2 is a genetic linker-[ALB VHH], wherein the TfR binding VHH polypeptide comprises: a. CDR1 comprises SEQ ID NO: 2, CDR 2 comprises SEQ ID NO: 3 and CDR3 comprises SEQ ID NO: 4; b. CDR1 comprises SEQ ID NO: 9, CDR 2 comprises SEQ ID NO: 10 and CDR3 comprises SEQ ID NO: 11; c. CDR1 comprises SEQ ID NO: 14, CDR 2 comprises SEQ ID NO: 15 and CDR3 comprises SEQ ID NO: 16; d. CDR1 comprises SEQ ID NO: 14, CDR 2 comprises SEQ ID NO: 21 and CDR3 comprises SEQ ID NO: 16; e. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 26; f. CDR1 comprises SEQ ID NO: 29, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 30; g. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33; h. CDR1 comprises SEQ ID NO: 36, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33; i. CDR1 comprises SEQ ID NO: 39, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33; j. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 42 and CDR3 comprises SEQ ID NO: 33; k. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 45 and CDR3 comprises SEQ ID NO: 33;l. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 21 and CDR3 comprises SEQ ID NO: 33; m. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 50 and CDR3 comprises SEQ ID NO: 33; n. CDR1 comprises SEQ ID NO: 53, CDR 2 comprises SEQ ID NO: 54 and CDR3 comprises SEQ ID NO: 33, wherein CDR1 and CDR2 comprise any combination of amino acids XI and X2 in CDR1, wherein Xl= Q or T and X2= A or S, and X3, X4, and X5 in CDR2, wherein X3= N or S, X4= A, G or T, and X5= R or Y; o. CDR1 comprises SEQ ID NO: 53, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33, wherein CDR1 comprises any combination of amino acids XI and X2, wherein XI = Q or T and X2= A or S; or p. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 54 and CDR3 comprises SEQ ID NO: 33, wherein CDR2 comprises any combination of amino acids X3, X4, and X5, wherein X3= N or S, X4= A, G or T, and X5= R or Y.

[0092] In some embodiment provided is a conjugate of Formula 11(b), [TfR VHH]-linker 1 is a genetic linker-[ALB VHH]-linker 2 is selected from Table 3 -[ a nucleic acid molecule, e.g. siRNA], wherein the TfR binding VHH polypeptide comprises complementarity determining regions CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, CDR3 comprise sequences selected from one of the following sequences: SEQ ID NO: 1; SEQ ID NO: 8; SEQ ID NO: 13; SEQ ID NO: 20; SEQ ID NO: 23; SEQ ID NO: 28; SEQ ID NO: 32; SEQ ID NO: 35; SEQ ID NO: 38;SEQ ID NO: 41; SEQ ID NO: 44; SEQ ID NO: 47; SEQ ID NO: 49; SEQ ID NO: 52; SEQ ID NO: 64; or SEQ ID NO: 66.

[0093] In some embodiment provided is a conjugate of Formula 11(b), [TfR VHH]-linker 1 is a genetic linker-[ALB VHH]-linker 2 is selected from Table 3 -[ a nucleic acid molecule, e g. siRNA], wherein the TfR binding VHH polypeptide comprises: a. CDR1 comprises SEQ ID NO: 2, CDR 2 comprises SEQ ID NO: 3 and CDR3 comprises SEQ ID NO: 4; b. CDR1 comprises SEQ ID NO: 9, CDR 2 comprises SEQ ID NO: 10 and CDR3 comprises SEQ ID NO: 11;c. CDR1 comprises SEQ ID NO: 14, CDR 2 comprises SEQ ID NO: 15 and CDR3 comprises SEQ ID NO: 16; d. CDR1 comprises SEQ ID NO: 14, CDR 2 comprises SEQ ID NO: 21 and CDR3 comprises SEQ ID NO: 16; e. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 26; f. CDR1 comprises SEQ ID NO: 29, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 30; g. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33; h. CDR1 comprises SEQ ID NO: 36, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33; i. CDR1 comprises SEQ ID NO: 39, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33; j. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 42 and CDR3 comprises SEQ ID NO: 33; k. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 45 and CDR3 comprises SEQ ID NO: 33; l. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 21 and CDR3 comprises SEQ ID NO: 33; m. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 50 and CDR3 comprises SEQ ID NO: 33; n. CDR1 comprises SEQ ID NO: 53, CDR 2 comprises SEQ ID NO: 54 and CDR3 comprises SEQ ID NO: 33, wherein CDR1 and CDR2 comprise any combination of amino acids XI and X2 in CDR1, wherein Xl= Q or T and X2= A or S, and X3, X4, and X5 in CDR2, wherein X3= N or S, X4= A, G or T, and X5= R or Y; o. CDR1 comprises SEQ ID NO: 53, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33, wherein CDR1 comprises any combination of amino acids XI and X2, wherein XI = Q or T and X2= A or S; orp. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 54 and CDR3 comprises SEQ ID NO: 33, wherein CDR2 comprises any combination of amino acids X3, X4, and X5, wherein X3= N or S, X4= A, G or T, and X5= R or Y.

[0094] In some embodiments, provided are conjugates which comprise a TIR binding VHH polypeptide and two agents, wherein one agent is a half-life extending polypeptide, for example without limitation an ALB binging VHH polypeptide. In some embodiments, where one agent is an ALB binding VHH polypeptide, the second agent is a therapeutic agent, e.g. a nucleic acid therapeutic agent such siRNA. In some embodiments, the siRNA is conjugated to either TfR binding VHH polypeptide, or ALB binding VHH polypeptide, or the both VHH polypeptides.

[0095] In some embodiments, the linker 1 or the linker 2 is present and selected from: a Mal-Tet- TCO linker, SMCC linker, or GDM linker (e.g. Table 3). In some embodiments, the linker 1 or the linker 2 is absent.

[0096] In some embodiments of TfR binding VHH conjugates, a polypeptide is linked to the 3’ end of the sense strand of the siRNA. In some embodiments, the polypeptide is linked to the 5’ end of the sense strand of the siRNA. In some embodiments, the protein polypeptide is linked to an internal position of the sense strand of the siRNA. In some embodiments, the polypeptide is linked to the 3’ end of the antisense strand of the siRNA. In some embodiments, the polypeptide is linked to an internal position of the antisense strand of the siRNA. The polypeptide is TfR binding VHH and / or an ALB binding VHH.Nucleic acid molecules

[0097] In some embodiments, the dsRNA comprises an antisense strand complementary to a target mRNA of choice. In some embodiments, the ds RNA is an siRNA. In some embodiments, the sense strand and the antisense strand of the dsRNA are each 15-30 nucleotides in length, e.g., 20-25 nucleotides in length. In some embodiments, the dsRNA has a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides. In some embodiments, the sense strand and antisense strand of the dsRNA may have overhangs at either the 5’ end or the 3’ end (i.e., 5’ overhang or 3’ overhang). For example, the sense strand and the antisense strand may have 5’ or 3’ overhangs of 1to 5 nucleotides or 1 to 3 nucleotides. In some embodiments, the antisense strand comprises a 3’ overhang of two nucleotides.

[0098] The dsRNA can include modifications. The modifications can be made to one or more nucleotides of the sense and / or antisense strand or to the internucleotide linkages, which are the bonds between two nucleotides in the sense or antisense strand. For example, some 2’- modifications of ribose or deoxyribose can increase RNA or DNA stability and half-life. Such 2’- modifications can be 2’-fluoro, 2’-O-methyl (i.e., 2’-methoxy), or 2'-O-alkyl.

[0099] In some embodiments, one or more nucleotides of the sense strand and / or the antisense strand are independently modified nucleotides, which means the sense strand and the antisense strand can have different modified nucleotides. In some embodiments, each nucleotide of the sense strand is a modified nucleotide. In some embodiments, each nucleotide of the antisense strand is a modified nucleotide. In some embodiments, the modified nucleotide is a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide, or 2'-O-alkyl modified nucleotide. In some embodiments, each nucleotide of the sense strand and the antisense strand is independently a modified nucleotide, e.g., a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide, or 2'- O-alkyl modified nucleotide.

[0100] In some embodiments, the sense strand has four 2'-fluoro modified nucleotides, e.g., at positions 7, 9, 10, 11 from the 5’ end of the sense strand. In some embodiments, the other nucleotides of the sense strand are 2'-O-methyl modified nucleotides. In some embodiments, the antisense strand has four 2'-fluoro modified nucleotides, e.g., at positions 2, 6, 14, 16 from the 5’ end of the antisense strand. In some embodiments, the other nucleotides of the antisense strand are 2'-O-methyl modified nucleotides.

[0101] In some embodiments, the sense strand has three 2'-fluoro modified nucleotides, e.g., at positions 9, 10, 11 from the 5’ end of the sense strand. In some embodiments, the other nucleotides of the sense strand are 2'-O-methyl modified nucleotides. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 5, 7, 14, 16 from the 5’ end of the antisense strand. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e g., at positions 2, 5, 8, 14, 16 from the 5’ end of the antisense strand. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 3, 7,14, 16 from the 5’ end of the antisense strand. In some embodiments, the other nucleotides of the antisense strand are 2'-O-methyl modified nucleotides.

[0102] In some embodiments, the 5’ end of the antisense strand has a phosphate analog, e.g., 5’- vinylphosphonate (5 ’-VP).

[0103] In some embodiments, the sense strand or the antisense strand comprises an abasic moiety or inverted abasic moiety. In some embodiments, the sense strand comprises an abasic moiety at any position, e.g. without limitation position 10.StructureStructure 1(abasic)Structure 2(iAb)“5”’ and “3”’ indicate the 5’ to 3’ direction of the nucleotide sequences which incorporates the moiety.

[0104] In some embodiments, the sense strand and the antisense strand have one or more modified intemucleotide linkages. In some embodiments, the modified intemucleotide linkage is phosphorothioate linkage. In some embodiments, the sense strand has four or five phosphorothioate linkages. In some embodiments, the antisense strand has four or five phosphorothioate linkages. In some embodiments, the sense strand and the antisense strand each has four or five phosphorothioate linkages. In some embodiments, the sense strand has four phosphorothioate linkages and the antisense strand has five phosphorothioate linkages.

[0105] The sense strand and antisense strand of dsRNA can be synthesized using any nucleic acid polymerization methods known in the art, for example, solid-phase synthesis by employing phosphoramidite chemistry methodology (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA), H-phosphonate, phosphortriester chemistry, or enzymatic synthesis. Automated commercial synthesizers can be used, for example, MerMade™ 12 from LGC Biosearch Technologies, or other synthesizers from BioAutomation or Applied Biosystems. Phosphorothioate linkages can be introduced using a sulfurizing reagent such as phenylacetyl disulfide or DDTT (((dimethylaminomethylidene) amino)- 3H-l,2,4-dithiazaoline-3-thione). It is well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products to synthesize modified oligonucleotides or conjugated oligonucleotides.

[0106] Purification methods can be used to exclude the unwanted impurities from the final oligonucleotide product. Commonly used purification techniques for single stranded oligonucleotides include reverse-phase ion pair high performance liquid chromatography (RP-IP- HPLC), capillary gel electrophoresis (CGE), anion exchange HPLC (AX-HPLC), and size exclusion chromatography (SEC). After purification, oligonucleotides can be analyzed by mass spectrometry and quantified by spectrophotometry at a wavelength of 260 nm. The sense strand and antisense strand can then be annealed to form a dsRNA.Table 2. Exemplary amino acid sequences of TfR binding VHH polypeptides linked to an albumin (ALB) binding VHH polypeptide and listing of CDRs (North numbering).

[0107] In some embodiments, any one of the amino acid sequences described in Table 2 may comprise a C-terminal cysteine residue. In embodiments, where there is no C-terminal cysteine residue, a cysteine residue is engineered at an internal position. In some embodiments, the cysteine residue is used to conjugate the VHH polypeptide to an agent. Conjugation is optionally through a linker, with non-limiting embodiments of linkers as described herein, e g. Table 3.Table 3. Non-limiting embodiments of exemplary linker structures. In this table, A is an agent as described herein; P is a target binding VHH polypeptide, e.g. a TfR binding VHH polypeptide and / or TfR VHH binding polypeptide further conjugated to an albumin binding protein, which in some embodiments is an albumin binding VHH polypeptide.* One skilled in the art would recognize that the sulfur connection can be part of a cysteine of P.Pharmaceutical Compositions

[0108] In another aspect, provided herein are pharmaceutical compositions comprising any of the TfR binding VHH polypeptides or conjugates described herein and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can also comprise one or more pharmaceutically acceptable excipient, diluent, or carrier. Pharmaceutical compositions can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 23rd edition (2020), A. Loyd et al., Academic Press).Method of Treatment and Therapeutic Use

[0109] In another aspect, provided herein are methods of delivering an agent, for example a therapeutic agent, across a cell membrane, in a patient in need thereof. In some embodiments, provided herein are methods of treating a disease or disorder, in a patient in need thereof. Such methods comprise administering to the patient a therapeutically effective amount of the TfR binding VHH polypeptide comprising an agent (e.g. a therapeutic agent), a conjugate as described herein or a pharmaceutical composition comprising any of these. In embodiments where the agent is a nucleic acid, e.g. siRNA or ASO targeting an mRNA of interest, the methods treat a disease or disorder which benefits from modulating the level the target mRNA of interest.

[0110] Compositions comprising TfR binding VHH polypeptide comprising an agent, a conjugate as described herein or a pharmaceutical composition comprising any of these are administered by any suitable route of administration. Non-limiting examples include by parenteral route such as subcutaneous, intravenous, or intramuscular using pharmaceutical preparations suitable for such administration.Definitions

[0111] As used herein, the terms “a,” “an,” “the,” and similar terms used in the context of the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.

[0112] The term “antigen binding domain”, as used herein, refers to a portion of an antibody or antibody fragment that binds an antigen or an epitope of the antigen.

[0113] “Antibody,” as used herein, refers to a molecule that binds an antigen. Embodiments of an antibody include a monoclonal antibody, polyclonal antibody, human antibody, humanized antibody, chimeric antibody, heterodimeric antibody, bispecific or multispecific antibody, or conjugated antibody. The antibodies can be of any class (e.g., IgG, IgE, IgM, IgD, IgA), and any subclass (e.g., IgGl, IgG2, IgG3, IgG4).Embodiments of the present disclosure also include antibody fragments or antigen-binding fragments that, as used herein, comprise at least a portion of an antibody retaining the ability to specifically interact with an antigen or an epitope of the antigen, such as Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, scFab, disulfide-linked Fvs (sdFv), a Fd fragment.

[0114] The term “antigen binding domain”, as used herein, refers to a portion of an antibody or antibody fragment that binds an antigen or an epitope of the antigen. For example. “TfR binding domain” refers to a portion of an antibody or antibody fragment that binds TfR or an epitope of TfR.

[0115] As used herein, “antisense strand” means a single- stranded oligonucleotide that is complementary to a region of a target sequence. Likewise, and as used herein, “sense strand” means a single-stranded oligonucleotide that is complementary to a region of an antisense strand.

[0116] The terms “bind” and “binds” as used herein are intended to mean, unless indicated otherwise, the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, which results in proximity of the two proteins or molecules as determined by common methods known in the art.

[0117] An “effective amount” refers to an amount necessary (for periods of time and for the means of administration) to achieve the desired therapeutic result. An effective amount of a protein or conjugate may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the protein or conjugate to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the protein or conjugate are outweighed by the therapeutically beneficial effects.

[0118] As referred to herein, the term “epitope” refers to the amino acid residues, of an antigen, that are bound by an antibody or an antigen binding VHH polypeptide or VHH antibody. An epitope can be a linear epitope, a conformational epitope, or a hybrid epitope. The term “epitope”may be used in reference to a structural epitope. A structural epitope, according to some embodiments, may be used to describe the region of an antigen which is covered by an antibody or antigen binding protein. In some embodiments, a structural epitope may describe the amino acid residues of the antigen that are within a specified proximity (e.g., within a specified number of Angstroms) of an amino acid residue of the antibody or antigen binding protein. The term “epitope” may also be used in reference to a functional epitope. A functional epitope, according to some embodiments, may be used to describe amino acid residues of the antigen that interact with amino acid residues of the antibody or antigen binding protein in a manner contributing to the binding energy between the antigen and the antibody or antigen binding protein.

[0119] An epitope can be determined according to different experimental techniques, also called “epitope mapping techniques.” It is understood that the determination of an epitope may vary based on the different epitope mapping techniques used and may also vary with the different experimental conditions used, e.g., due to the conformational changes or cleavages of the antigen induced by specific experimental conditions. Epitope mapping techniques are known in the art (e.g., Rockberg and Nilvebrant, Epitope Mapping Protocols: Methods in Molecular Biology Humana Press, 3rded. 2018), including but not limited to, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, species swap mutagenesis, alanine- scanning mutagenesis, hydrogen-deuterium exchange (HDX) and cross-blocking assays.

[0120] As used herein, “modified internucleotide linkage” means an internucleotide linkage having one or more chemical modifications when compared with a reference internucleotide linkage having a phosphodiester bond. A modified internucleotide linkage can be a non-naturally occurring linkage. In some embodiments, the modified internucleotide linkage is phosphorothioate linkage.

[0121] As used herein, “modified nucleotide” refers to a nucleotide having one or more chemical modifications when compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. A modified nucleotide can have, for example, one or more chemical modification in its sugar, nucleobase, and / or phosphate group. Additionally, or alternatively, a modified nucleotide can have one or more chemical moi eties conjugated to a corresponding reference nucleotide. In some embodiments, the modified nucleotide is a 2'-fluoro modifiednucleotide, 2'-O-methyl modified nucleotide, or 2'-O-alkyl modified nucleotide. In some embodiments, the modified nucleotide has a phosphate analog, e g., 5’-vinylphosphonate. In some embodiments, the modified nucleotide has an abasic moiety or inverted abasic moiety.

[0122] The term “% sequence identity” or “percentage sequence identity” with respect to a reference nucleic acid sequence is defined as the percentage of nucleotides, nucleosides, or nucleobases in a candidate sequence that are identical with the nucleotides, nucleosides, or nucleobases in the reference nucleic acid sequence, after optimally aligning the sequences and introducing gaps or overhangs, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software programs, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987, Supp. 30, section 7.7.18, Table 7.7.1), and including BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), Clustal W2.0 or Clustal X2.0 software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Percentage of “sequence identity” can be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the nucleic acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical nucleotide, nucleoside, or nucleobase occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. The output is the percent identity of the subject sequence with respect to the query sequence.

[0123] The term “polypeptide” or “protein”, as used herein, refers to a polymer of amino acid residues. The term applies to polymers comprising naturally occurring amino acids and polymers comprising one or more non-naturally occurring amino acids. A polypeptide which extends the half-life of the TIR -binding VHII polypeptides is “half-life extender.”

[0124] In the context of the invention, the term TfR is used to designate the TfRl homologue.

[0125] The following examples are offered to illustrate, but not to limit, the claimed inventions.

[0126] Scaffold to demonstrated shuttling of TV7-Fc:

[0127] SEQ ID NO: 76 (TfR VHH-04-linker-hIgG4PAA)

[0128] EVKLVESGGGLVQAGGSLRLSCAASGPAFSAYAMAWFRQAPGKEREFVGAVSGNT NRTRYADSVKGRFTVSRDNTKNTMYLQMNSLKPDDTATYYCARHYYYSTSGFFVDYSRV DEFDYWGQGTQVTVSSGGGGSENLYFQGGGGGSESKYGPPCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC S VMHEALHNHYTQK SLSL SLGK

[0129] SEQ ID NO: 77 (hIgG4PAALK stump)

[0130] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMfSRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TfSK AKGQPREPQ VYTLPP SQEEMTKNQ VSLTCLVKGF YP SDIAVEWE SNGQPENNYKTTP PVLDSDGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0131] SEQ ID NO: 78 (Linker)

[0132] GGGGSENLYFQGGGGG

[0133] SEQ ID NO: 79 (hIgG4PAA Fc)

[0134] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMfSRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0135] SEQ ID NO: 97 (e GF P-4.05 kb -AAV Transgene)cattcgccattcaggctgcaaataagcgttgatattcagtcaattacaaacattaataacgaagagatgacagaaaaattttcatt ctgtgacagagaaaaagtagccgaagatgacggtttgtcacatggagttggcaggatgtttgattaaaaacataacaggaagaa aaatgccccgctgtgggcggacaaaatagttgggaactgggaggggtggaaatggagtttttaaggattatttagggaagagtgac aaaatagatgggaactgggtgtagcgtcgtaagctaatacgaaaattaaaaatgacaaaatagtttggaactagatttcacttatc tggttcggatctcctagagcttacattattgaagcatttatcagggttattgtctcagacctgcaggcagctgcgcgctcgctcgc tcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgca gagagggagtggccaactccatcactaggggttcctgcggccgcacgcgttcgtcgactactagtgggtaccagagctccct aggttctagaaccggtgacgtctcccatggtgaagcttggatctgaattcggtacctagttattaatagtaatcaattacggggtcat tagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcc cattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaa ctgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggc attatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgag ccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgat gggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtg cggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcga agcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccgg ctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacg gcttgtttcttttctgtggctgcgtgaaagccttgaggggctccgggagctagagcctctgctaaccatgttcatgccttcttctttttc ctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattcctcgaagatccgaagggaaagtcttcc acgactgtgggatccgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctgga cggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttc atctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgct accccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaa ggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcat cgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgac aagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactac cagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaa agaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgt acaagtaacaattgttaattaagtttaaaccctcgaggccgcaagcttatcgataatcaacctctggattacaaaatttgtgaaag attgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatgg ctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgca ctgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctcccta ttgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgtt gtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcg gccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacga gtcggatctccctttgggccgcctccccgcatcgataccgtcgactagagctcgctgatcagcctcgactgtgccttctagttgcc agccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaa attgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaa tagcaggcatgctggggagagatccacgataacaaacagcttttttggggtgaacatattgactgaattcccgtgcggaccgctc tagactgttttaactctccaggttacttggctaattttatggtcctccctcctactttcactgctttcagttccaccttagccctgagct gttctttctctttagtactggctgtggcataagacccttgcaaacatgaaatcagttggaagaaattctgaatttcacctgcctccaa ttaaatctcatggactttctttccctccaatttgtataattgtttgatcttgaccaatcccttctttctcttttataatttccaaaggtttttg cgttgtgtttttggttggtttgttgttttaatgtttggggttagtattgatatcatttttcttgaagctctatacttcattctcccagtttttaag aattgtctgtcatttcttccagtcagctgtgacactgtactgatcttgaaagattaagacagatatcattgtttctcagtgtagaaatggtgaccaatagaaaacagaccatgtaaatgaaaataccactgattatacattatccagcctcttctactttttttttttttttacttaa aaggtaccttattgttttattttccagaaaggaaagataaaaaaataactattttacaatttggtatgagtcttggtgtcaacaacttt ggaattcagtaaaacaatttgaggttaaagtaaaaagcatgattactaaagagcaccttgaatctgttgctgaacaccctcatatt ggtgactaagtttcatcttttccctgaccatgaacatgaatttaatgaatgaaacacgatagaaaaattaaataaatgatagttatt agctattatgaaaaatgtattccaaatggcaagctcaaggatgtgatatcacttaacacaattcagcctcatcattttgactcatag acattaaaagagtaaacgaaagactagataaactcacagatgttaaacactaaagactttagtgagccattaaaatgattggag ccattttctcaaagcttccggaccgagcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgct cgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcg cgcagctgcctgcaggcatgcaagctgtagccaaccactagaactatagctagagtcctgggcgaacaaacgatgctcgcct tccagaaaaccgaggatgcgaaccacttcatccggggtcagcaccaccggcaagcgccgcgacggccgaggtcttccgatct cctgaagccagggcagatccgtgcacagcaccttgccgtagaagaacagcaaggccgccaatgcctgacgatgcgtggaga ccgaaaccttgcgctcgttcgccagccaggacagaaatgcctcgacttcgctgctgcccaaggttgccgggtgacgcacaccg tggaaacggatgaaggcacgaacccagttgacataagcctgttcggttcgtaaactgtaatgcaagtagcgtatgcgctcacgc aactggtccagaaccttgaccgaacgcagcggtggtaacggcgcagtggcggttttcatggcttgttatgactgtttttttgtacagt ctatgcctcgggcatccaagcagcaagcgcgttacgccgtgggtcgatgtttgatgttatggagcagcaacgatgttacgcagca gcaacgatgttacgcagcagggcagtcgccctaaaacaaagttaggtggctcaagtatgggcatcattcgcacatgtaggctcg gccctgaccaagtcaaatccatgcgggctgctcttgatcttttcggtcgtgagttcggagacgtagccacctactcccaacatca gccggactccgattacctcgggaacttgctccgtagtaagacattcatcgcgcttgctgccttcgaccaagaagcggttgttggcg ctctcgcggcttacgttctgcccaggtttgagcagccgcgtagtgagatctatatctatgatctcgcagtctccggcgagcaccgg aggcagggcattgccaccgcgctcatcaatctcctcaagcatgaggccaacgcgcttggtgcttatgtgatctacgtgcaagca gattacggtgacgatcccgcagtggctctctatacaaagttgggcatacgggaagaagtgatgcactttgatatcgacccaagta ccgccacctaacaattcgttcaagccgagatcggcttcccggccgcggagttgttcggtaaattgtcacaacgccgcgaatata gtctttaccatgcccttggccacgcccctctttaatacgacgggcaatttgcacttcagaaaatgaagagtttgctttagccataa caaaagtccagtatgctttttcacagcataactggactgatttcagtttacaactattctgtctagtttaagactttattgtcatagttt agatctattttgttcagtttaagactttattgtccgcccacacccgcttacgcagggcatccatttattactcaaccgtaaccgatttt gccaggttacgcggctggtctgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttc ctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccaca gaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctg gcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggact ataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcc tttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggct gtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgact tatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcc taactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctctt gatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaag aagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaa ggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttacca atgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacg atacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaat aaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccggga agctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggt atggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcgg tcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgcca tccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactc tcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagc gtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatact cttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaat aggggttccgcgcacatttccccgaaaagtgccacctgaaattgtaaacgttaatattttgttaaaattcgcgttaaatttttgttaa atcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgt tccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcc cactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccc cgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcg ctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtc

[0136] In the nucleic acid sequence of SEQ ID NO: 97, the ITR is bolded; CAG promoter is underlined; the signal peptide is bolded and underlined; eGFT is italicized.

[0137] SEQ ID NO: 98 (eGFP amino acid sequence)LDGDVNGHKF S VSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCF SRYPDH MKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILG HKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDN HYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKEXAMPLESEXAMPLE 1: Discovery of TfRl (transferrin receptor protein 1) binding VHH polypeptides

[0138] To discover VHH polypeptides that can bind to TfRl orthologs of different species, a llama was immunized with both human and mouse TfRl extracellular domain (ECD) protein every 2 weeks over a 12-week period. Immunogens used were human TfRl ECD (ECD region AA 89-760, UNIPROT ID: P02786) and mouse TfRl ECD (ECD region AA 89-763, UNIPROT ID: Q62351) with a N-terminal 6X His tag. PBMC’s were harvested from llama blood and enriched for binding to human TfR ECD protein by MACS sorting. Total RNA was isolated and converted to cDNA.VHH sequences were amplified by PCR and cloned into a M13 phage display vector. Phage clones were captured to nitrocellulose filters coated with an anti-M13 antibody and displayed VHH’s screened for binding to biotinylated human TfR ECD protein.

[0139] Phage clones that bound to human TfR ECD on the filters were eluted into IX TBS buffer, VHH cDNA sequences amplified by PCR and sequenced. Sequence confirmed clones were tested for binding to human, cynomolgus monkey and mouse TfR ECD protein in phage ELISAs. A total of 13 unique VHH sequences were identified by ELISA to bind human, cynomolgus and mouse TfR ECD proteins. All 13 VHH cDNA sequences were cloned as fusions to the N-terminus of a human IgG4 Fc into a GS-containing expression plasmid backbone (pEE12.4-bas. The cDNA sequence was fused in frame with the coding sequence of a signal peptide sequence, METDTLLLWVLLLWVPGSTG (SEQ ID NO:68) to enhance secretion of the VHH into the tissue culture medium. VHH fusion proteins were transiently expressed in a CHOKl-derived cell line and purified by Protein A affinity chromatography followed by size-exclusion chromatography. Purified VHH fusion proteins were tested for binding and internalization on human and mouse TfR expressing hCMEC and bEND.3 cells by FACS using DL650 or pHAb labelled secondary antibodies, respectively. Six of the 13 VHH fusion proteins were confirmed to bind and internalize on both cell lines.

[0140] Binding of the VHH fusion proteins to human TfR expressing hCMEC cells was also assessed in the presence and absence of human transferrin. Three of the six VHH fusion proteins did not compete with human transferrin binding to TfR indicating their binding to TfR does not interfere with TfR function (transferrin binding and iron transport). HDX-MS (hydrogen deuterium exchange mass spectrometry) was used to determine the epitopes of the VHH fusion proteins as well as human transferrin on human TfR ECD protein. This experiment demonstrated that the three VHH fusion proteins bind to a different epitope than transferrin. This further confirmed the findings from the cell binding competition experiment showing that the three VHH fusion proteins of interest do not compete with transferrin binding to human TfR.EXAMPLE 2: Engineered humanized TfR binding VHH polypeptides

[0141] The TfR binding VHH sequences (From Example 1) were humanized by grafting llama CDR1, CDR2 and CDR3 (CDR numbering according to North) into a human VH3-23 framework while maintaining select llama amino acid sequence positions 37, 44, 45, 47, 49 in framework 2 and position 84 in framework 3 (amino acid numbering according Kabat). The VHH CDRs were engineered to equalize binding affinities to human, cynomolgus monkey, and in some cases mouseTfR. Amino acid changes were also made to generate VHH polypeptides with favorable developability characteristics for recombinant production, CMC and / or therapeutic use.

[0142] VHH cDNA sequences were cloned in a GS-containing expression plasmid backbone (pEE 12.4-based plasmids, Lonza Biologies Inc.). The cDNA sequence was fused in frame with the coding sequence of a signal peptide sequence, METDTLLLWVLLLWVPGSTG (SEQ ID NO: 68). To facilitate functional characterizations, in certain instances, a C-terminal 6x Histidine, a (G4Q)5YPYDVPDYA amino acid sequence motif (HA-tag) (SEQ ID NO: 69), or a single Cysteine residue were added to VHH sequences and encoded in the respective cDNA sequence.

[0143] For generating the VHH polypeptides via transient transfection, CHOK1 -derived cells were cultured in a bioreactor up to densities between 35-50 x 106vc / mL. The cells were transfected with the recombinant expression plasmids using a PELbased method. Briefly, the appropriate volume of CHOKl-derived suspension cells at a density of 20 x 106cells / mL was transferred in shake flasks, and both PEI and recombinant plasmid DNA are added to the cells. Cells were incubated in a suspension culture at 32°C for 7-days. At the end of the production period, cells were removed by centrifugation and the VHH polypeptide was purified from the clarified medium.

[0144] The VHH polypeptide secreted into the media from the CHO cells was purified by Protein A affinity chromatography followed by size-exclusion chromatography. Specifically, the VHH polypeptide from harvested media was captured onto MabSelect PrismA resin (Cytiva). The resin then was briefly washed with a running buffer, such as a phosphate-buffered saline (PBS; pH 7.2) or a running buffer containing Tris, to remove non-specifically bound material. In some cases, salt was added in the wash buffer to retain the VHH polypeptide on the column before elution. The VHH polypeptide was eluted from the resin with a low pH solution, such as 10 mM citric acid pH 3. Fractions containing the VHH polypeptide were pooled. The pH was neutralized by adding a base such as IM Tris pH 8.0. The VHH polypeptide was further purified by size exclusion chromatography using resins such as Superdex75 (Cytiva) with isocratic elution in PBS, pH 7.2.EXAMPLE 3: Cell-based binding affinity of TfR VHH polypeptides (no payload) to human, cyno and mouse cell lines expressing TfR

[0145] The binding affinity of TfR VHH molecules (without payload) to human, cyno and mouse cell lines expressing TfR was assayed using the following procedure.

[0146] Human TfRl expressing EFO-21 cells (eClipse, Germany), cynomolgus TfRl expressing MDCK cells (MDCK-cyTfR-pCMVzeo, BN20-2390), and mouse TfRl expressing BV-2 (ATCC) cells were detached from culture flasks with accutase (Coming). 100,000 cells per well were transferred to 96-well round bottom polypropylene plate and washed twice in cold BD Pharmingen Stain Buffer (BD; referred to herein as FACS buffer). To assess cell viability, cells were treated with LIVE / DEAD™ Fixable Blue (Invitrogen) and incubated for 20 minutes on ice, in the dark. To block Fc receptors, cells were treated with TruStain FcX™ PLUS (Biolegend, for BV-2 cells) or Human TruStain FcX™ (Biolegend, for EFO-21 and Cyno TfR MDCK cells) and incubated for 20 minutes on ice, in the dark. Cells were washed twice in cold FACS buffer. TfR VHH molecules diluted in FACS buffer were added to cells and incubated for 30 minutes on ice, in the dark. To generate concentration / dose response curves, final concentrations of 1000, 333.3, 111.1, 37.0, 12.3, 4.11, 1.37, 0.46nM of TfR VHH polypeptides that contained a C-terminal HA-tag were used. Cells were washed twice in cold FACS buffer then incubated for 30 minutes with DyLight 650 Anti-HA Tag (Abeam). Cells were washed and resuspended in FACS for flow cytometry collection. Cell events were acquired on a ZE5 flow cytometer (Biorad) and analyzed in FlowJo (BD). EC50 values were calculated using the GraphPad Prism 4-parameter nonlinear fit model for curves that reached full sigmoidal profiles. EC50 values of weak binders and / or instable curve fits due to outliers were extrapolated based on experimental conditions used and are indicated with a * in the table 4 below.Table 4. Binding of TfR binding VHHs (no payload) to cell lines expressing human, cynomolgus or mouse TfRl.EXAMPLE 4: TfR binding VHH polypeptides and Transferrin binding to TfR

[0147] Discovery TfR binding VHH polypeptides were tested for binding to human TfRl expressing cell lines in the presence and absence of human Transferrin. No significant difference in binding was observed demonstrating that VHH binding to TfR does not interfere with Transferrin binding to TfR and iron transport.

[0148] HDX-MS (hydrogen deuterium exchange mass spectrometry) was used as an orthogonal experimental method to verify that the TfR binding discovery VHH polypeptides bind to a different epitope than transferrin.

[0149] Additional TfR binding VHH polypeptides as described herein can be tested either in a competition assay to show that there is no difference in binding of the TfR binding VHH polypeptides to TfR in presence and absence of transferrin.

[0150] Alternatively, or in addition, the epitope(s) of the different TfR binding VHH polypeptides can be characterized by HDX-MS (hydrogen deuterium exchange mass spectrometry) to show that the TfR binding VHH polypeptides bind a different epitope than where transferrin binds to TfR.EXAMPLE 5: Expression of TfR binding VHHs with free Cysteines in different positions for chemical conjugations of payloads

[0151] To make TfR binding VHH polypeptides amenable for chemical conjugation to different payloads, a single Cysteine amino acid was added at the C-terminus of either the TfR binding VHH or at the C-terminus of a fusion protein containing the TfR binding VHH fused to an albuminbinding (ALB) VHH. A cDNA sequence encoding the respective TfR VHH polypeptide or TfR VHH fusion polypeptide amino acid sequence was synthesized and cloned into an expression plasmid backbone essentially as described above in Example 2.

[0152] The VHH fusion protein secreted into the media from the CHO cells was purified by Protein A affinity chromatography followed by size-exclusion chromatography. Specifically, the VHH fusion protein from harvested media was captured onto MabSelect PrismA resin (Cytiva). The resin then was briefly washed with a running buffer, such as a phosphate-buffered saline (PBS; pH 7.4) or a running buffer containing Tris, to remove non-specifically bound material. In some cases, NaCl was added in the wash buffer to retain the VHH molecule on the column before elution. The VHH fusion protein was eluted from the resin with a low pH solution, such as 10 mM citric acid pH 3. Fractions containing the VHH fusion protein were pooled, and the pH was neutralized by adding Tris base to pH 5. The VHH fusion protein was further purified by size exclusion chromatography using resins such as Superdex75 (Cytiva) with isocratic elution in Acetate buffer, pH 5 with NaCl and EDTA.EXAMPLE 6A: Preparation of HPRT siRNA molecules and conjugates comprising TfR binding VHH polypeptides.

[0153] Oligonucleotides were synthesized on a Bioautomation Mermade-12 instrument using standard phosphoramidite chemistry and were purified by ion exchange chromatography. A 3’ C6- NH2 substituent was installed on the single sense strands using Phthalimido Amino C6 LCAA CPG 500 A (ChemGenes). Conjugation of the C6-NH2-oligonucleotide to SMCC was accomplished by addition of excess SMCC in acetonitrile to a solution of oligonucleotide and 30 equivalents of NaHCCf in water. Upon completion of the reaction, the mixture was acidified with dilute aqueous HC1 and either centrifuged in a 3,000 molecular weight-cutoff spin filter or processed via tangential flow filtration. Complementary single strands were annealed in water at room temperature to create dsRNA comprising SMCC linker 1 at the 3’ end of the sense strand.

[0154] In some instances, an Alexa Fluor 647-NHS Ester Dye was conjugated to the 3’ of an antisense strand C6-NH2 oligonucleotide by addition of excess AF647-NHS dye in DMSO to a solution of oligonucleotide and 20x borate buffer in water. Upon completion of the reaction, the mixture was centrifuged in a 3,000 molecular weight-cutoff spin filter and then purified by AEX chromatography. Complementary single strands were annealed in water at room temperature.

[0155] Sense strands having SMCC linker 1 were annealed to antisense strands having AF647- NHS dye at their 3’ end to create dsRNA comprising SMCC linker 1 at the 3’ end of the sense strand and AF647-NHS dye at the 3’ end of the antisense strand.Scheme A:Table 5. HPRT siRNA and Conjugate.Abbreviations - “m” indicates 2’-0Me; “f’ indicates 2’-fluoro; indicates phosphorothioate linkage; “VP” indicates 5’-vinylphosphate; “S” means the sense strand; “AS” means the antisense strand.Preparation of TfR binding VHHs for conjugation of dsRNA

[0156] TfR binding VHH polypeptides that has a single Cysteine amino acid incorporated (as described in Example 5) were prepared for chemical conjugations in the following manner: For the VHH polypeptides the pH was increased using 1 / 10 volume addition of IM Tris pH8. After pH adjustment, the VHH polypeptides were reduced with 10 molar equivalents reducing agent tris(2- carboxyethyljphosphine (Thermo Scientific Bond-Breaker™ TCEP Solution, Neutral pH) at room temperature for 10-20 minutes, followed by desalting to remove reducing agent quickly.

[0157] Conjugation of HPRT dsRNA onto VHHs was done immediately after reducing agent removal in order to prevent VHH polypeptide dimer reformation using the following methods.Conjugation Scheme

[0158] The conjugation method utilized the SMCC-functionalized HPRT dsRNA for conjugating onto the introduced cysteine amino acid of the TfR binding VHH polypeptide. For this method, VHHs were prepared similarly as above to make the cysteine available for conjugation by undergoing a reduction and desalting process. This is immediately followed by incubating the SMCC-HPRT dsRNA with the VHHs at 1.2 molar equivalents for 2hr conjugation at room temperature. In the scheme below, the oval represents a VHH polypeptide, e.g. VHH control polypeptide, TfR binding VHH polypeptide and / or TfR VHH binding polypeptide fusion with an albumin binding VHH, comprising the cysteine used for conjugation. In some instances (not depicted in the scheme), the dsRNA has a AF647-NHS dye at the 3’ end of the antisense strand.

[0159] Scheme B for HPRT dsRNA VHH conjugation via SMCC linker:

[0160] Conjugation was monitored using anion exchange chromatography. A 1 mL PO OS™ GoPure™ XQ Pre-packed Column, 0.5 x 5 cm was utilized with the following method. Flow rate of 0.5 mL / min, Buffer A: 20mM Acetate pH5.0, Buffer B: 20 mM Acetate pH 5.0 + 1.0 M NaCl, at room temperature.Table 6. HPLC gradient used to asses HPRT dsRNA conjugation to VHH.

[0161] Drug / dsRNAto VHH / protein ratio (DAR) was calculated based on peak area % from the anion exchange (AEX) chromatogram. Post conjugation of HPRT dsRNA to the VHH, excess dsRNA and unconjugated VHH was removed by further purification.

[0162] Preparative anion exchange chromatography and preparative size exclusion chromatography (SEC) was utilized for purification of the final conjugate. Anion exchange, e.g., ThermoFisher POROS™ XQ, was used with starting buffer of 20mM TRIS pH 7.0 and eluting with 20 column volume gradient with a buffer containing 20mM TRIS pH 7.0 and IM NaCl.Preparative SEC was performed using Cytiva Superdex® 200 in IX PBS pH 7.2 under an isocratic condition. In some cases, for instance when TfR VHH is not expressed with the albumin binding VHH, Mab Select PrismA resin (Cytiva) in place of POROS™ XQ is used as the primary purification step. The conjugate is captured on the column under high salt conditions (20mM TRIS pH 7.0 and IM NaCl), washed and eluted by removing the salt using 20mM TRIS pH 7.0 buffer followed by SEC polishing.

[0163] These resulted in purified TfR binding VHH-HPRT dsRNA conjugate devoid of excess dsRNA and minimal unconjugated protein. The resulting conjugate profile was analyzed by analytical anion exchange for final DAR quantitation.EXAMPLE 6B: Additional linkers and schemes for preparing conjugates comprising TfR binding VHH polypeptides

[0164] Certain abbreviations are defined as follows: “ACN” refers to acetonitrile; “aAEX” refers to analytical anion exchange; “AS” refers to antisense strand; “DAR” refers to drug / siRNAto antibody / protein ratio; “DCM” refers to dichloromethane; “DHAA” refers to dehydroascorbic acid; “dsRNA” refers to double stranded ribonucleic acid; “DTT” refers to dithiothreitol; “h” refers to hours; “HPLC” refers to high-performance liquid chromatography; “LC / MS” refers to liquid chromatography mass spectrometry; “LTQ / MS” refers to linear ion trap mass spectrometer; “min” refers to minutes; “MSPT” refers to 4-(5-methylsulfonyl-lH-tetrazole-lyl)phenol; “MW” refers to molecular weight; “MWCO” refers to molecular weight cut-off; “NHS” refers to N- hydroxysuccinimide; “OD” refers to 4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2-yl)phenol; “PBS” phosphate-buffered saline; “PEG” refers to polyethylene glycol; “RNAi” refers to RNA interference; “rpm” refers to revolutions per minute; “SEC” refers to size exclusion chromatography; “siRNA” refers to small interfering RNA; “SMCC” refers to succinimidyl-4-(N- mal eimidom ethyl )cy cl ohexane-1 -carboxylate; “SS” refers to sense strand; “TCO” refers to trans- cyclo-octene; “TfR” refers to transferrin receptor; “THF” refers to tetrahydrofuran; “TRIS” refers to tris(hydroxymethyl)aminomethane; “UPLC” refers to ultra performance liquid chromatography; and “UV” refers to ultraviolet.Scheme 1Q is 1,3,4-oxadiazole or 1 f-tetrazole.

[0165] Scheme 1 depicts the synthetic route to the intermediates that will be used to form the final MSPT and OD linkers shown in Table 3.

[0166] Scheme 1, step A depicts the methylation of the thiol on compound (1) using iodomethane and a suitable base such as DIEA in a solvent such as THF to give compound (2). Step B shows an alkylation of compound (2) with Z< / 7-butyl 2-(2-(2-bromoethoxy)ethoxy)acetate using a base such as potassium carbonate in a solvent such as acetone to give compound (3). Step C shows the oxidation of compound (3) with hydrogen peroxide and ammonium molybdate (VI) tetrahydrate in a solvent such as EtOH followed by an acidic deprotection using an acid such as TFA in a solvent such as DCM to give compound (4). Note that in the case of the I / 7-tetrazole, the deprotection took place during the oxidation step. Step D depicts a coupling of compound (4) and 1- hydroxypyrrolidine-2, 5-dione using EDCI in a solvent system such as DCM and THF to give compound (5).Scheme 2

[0167] Scheme 2, step A shows the coupling of compound (6) and isoindoline-1, 3-dione using DIAD and tributyl phosphine in a solvent such as THF to give compound (7). Step B depicts the phosphorylation of compound (7) with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite using a base such as DIEA in a solvent such as DCM to give compound (8).Preparation 14-(5-(Methylthio)-l,3,4-oxadiazol-2-yl)phenol

[0168] A solution of 4-(5-mercapto-l,3,4-oxadiazol-2-yl)phenol (3.00 g, 15.4 mmol) in THF (50 mb) is cooled to 0 °C. DIEA (3.46 mL, 20.1 mmol) is added then stirred for 5 minutes before adding iodomethane (2.85 g, 20.1 mmol) dropwise over a period of 1 minute. The mixture is stirred at 0 °C for 5 minutes, and then stirred at ambient temperature for 2 hours. After this time, the mixture is diluted with DCM (100 mL) and washed with saturated aqueous NH4CI (pH was adjusted to ~5 by adding citric acid solution, 2 x 50 mL). The organic layer is separated, dried over sodium sulfate, and concentrated in vacuo to give the title compound as a pale-yellow solid (520 mg, 97%). ES / MS m / z: 209 (M+H).

[0169] The compound in Table 7 is prepared in a manner essentially analogous to that found in Preparation 1.Table 7. Structure of preparation 2.Preparation 3 / cvz-Butyl 2-(2-(2-(4-(5-(methylthio)-l,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate

[0170] In a pressure vessel, / c77-butyl 2-(2-(2-bromoethoxy)ethoxy)acetate (5.5 g, 20 mmol) and potassium carbonate (4.2 g, 30 mmol) are added to 4-(5-(methylthio)-l,3,4-oxadiazol-2-yl)phenol (3.3 g, 15 mmol) in acetone (60 mL). The pressure vessel is sealed and heated at 70 °C for 5 hours with vigorous stirring. After this time, the mixture is cooled to ambient temperature. The mixture is filtered while washing through with EtOAc / DCM. The filtrate is concentrated in vacuo and purified via silica gel column chromatography eluting with 0-100% EtOAc / DCM to give the title compound as a white solid (4.8 g, 74%). ES / MS m / 'z: 411 (M+H).

[0171] The compound in Table 8 is prepared in a manner essentially analogous to that found in Preparation 3.Table 8. Structure of preparation 4.Preparation 5 tert-Butyl 2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate

[0172] Ze / 7-Butyl 2-(2-(2-(4-(5-(methylthio)-l,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate (5.20 g,12.7 mmol) is dissolved in EtOH (100 mL) and cooled to 5-10 °C. Then, 30% hydrogen peroxide (10 mL, 97 mmol) is added, followed by ammonium molybdate (VI) tetrahydrate (501 mg, 0.405 mmol). After two hours of vigorous stirring, additional 30% hydrogen peroxide (15 mL, 145.5 mmol) and ammonium molybdate (VI) tetrahydrate (1 g, 0.910 mmol) are added. The mixture is stirred for 6 hours, then diluted with DCM (150 mL) and washed with saturated aqueous sodium chloride solution. The organic phase is separated, dried over sodium sulfate, and concentrated in vacuo. The resulting residue is triturated with MeOH to provide the first lot of the title compound. The solvent from the mother liquor is concentrated in vacuo and purified via silica gel column chromatography eluting with 0-100% EtOAc / DCM to give additional product as white solid. The recovered materials are combined to give the title compound as a white solid (5.3 g, 90%). ES / MS m / z-. 387 (M+H-tBu).

[0173] The compound in Table 9 is prepared in a manner essentially analogous to that found in Preparation 5.Table 9. Structure of preparation 6.

[0174] Note that the conditions are analogous, but the t-butyl group is removed in the process.Preparation 72-(2-(2-(4-(5-(Methylsulfonyl)-l,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetic acid

[0175] TFA (20 mL, 12.0 mmol) is added to a solution of tert-butyl 2-(2-(2-(4-(5 -(methyl sulfonyl)- l,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate (5.60 g, 12.0 mmol) in DCM (60 mL). The mixture is stirred at ambient temperature for 2 hours, concentrated in vacuo, and purified via silica gel column chromatography eluting with 0-100% EtOAc / DCM to give the title compound (4.12g, 82%). ES / MS m / . 387 (M+H).Preparation 82,5-Dioxopyrrolidin-l-yl-2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2- yl)phenoxy)ethoxy)ethoxy)acetate

[0176] EDCI (1.60 g, 10.3 mmol) is added to a solution of 2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4- oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetic acid (3.00 g, 7.38 mmol) and 1-hydroxypyrrolidine- 2, 5-dione (1.19 g, 10.3 mmol) in DCM (50 mL) and THF (70 mL). Another 20 mL of DCM is added to bring the mixture into a solution followed by stirring at ambient temperature for 12 hours. After this time, concentrated in vacuo and purified via silica gel column chromatography eluting with 0-100% EtOAc / DCM to give the title compound (2.61g, 65%). ES / MS m / z 484 (M+H).

[0177] The compound in Table 10 is prepared in a manner essentially analogous to that found in Preparation 8.Table 10. Structure of preparation 9.Preparation 102-(((2R, 3R,4R,5R)-5-(2, 4-Dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3 -hydroxy -4- methoxytetrahydrofuran-2-yl)methyl)isoindoline- 1,3-dione

[0178] A solution of l-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran- 2-yl)pyrimidine-2,4(lH,3H)-dione (30 g, 120 mmol), isoindoline-1, 3-dione (21 g, 140 mmol), DIAD (27 mL, 140 mmol), tributyl phosphine (36 mL, 150 mmol), and THF (300 mL) is stirred at ambient temperature for 12 h. The crude reaction is filtered, concentrated in vacuo, and purified via silica gel flash chromatography eluting with 0-100% EtOAc / hexanes to give the title compound as a white solid (6.0 g, 13%).Preparation 11

[0179] 2-Cyanoethyl ((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-2-((l,3- dioxoisoindolin-2-yl)methyl)-4-methoxytetrahydrofuran-3-yl) diisopropylphosphoramidite

[0180] A solution of 2-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3-hydroxy- 4-methoxytetrahydrofuran-2-yl)methyl)isoindoline-l, 3-dione (3.00 g, 7.74 mmol), 2-cyanoethyl- N,N-diisopropylchlorophosphoramidite (2.47 mL, 11.6 mmol), DIEA (4.05 mL, 23.2 mmol), and DCM (40 mL) is stirred at ambient temperature. After 1 hour, additional 2-cyanoethyl-N,N- diisopropylchlorophosphoramidite (0.82 mL, 3.8 mmol) is added. After 1 hour, the crude reaction is poured into a slurry of silica gel (15 g) in 30 mL of 1% TEA / DCM, concentrated in vacuo to a dry powder, and purified via silica gel flash chromatography eluting with 40-100% EtOAc / hexanes (0.5% TEA) to give the title compound as a white foam (3.70 g, 81%).JH NMR (d6-DMSO) d11.4 (br s, 1 H), 7.96-7.78 (m, 5H), 5.83 (dd, 1H), 5.71 (dd, 1H), 4.46-3.47 (m, 9H), 3.39 (s, 1.5H), 3.35 (s, 1.5H), 2.82-2.73 (m, 2H), 1.16-0.97 (m, 12H).31P NMR (d6-DMSO) d 149.7, 149.4.Preparation 123’ Oxadiazole linker-functionalized sense strand

[0181] A 40 mL Falcon tube is charged with single sense strand RNA (7.66 mg, 3.00 mL, 1.07 pmol) and 3 mL of PBS 7.4 (lOx). 2,5-Dioxopyrrolidin-l-yl-2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4- oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate (10.4 mg, 1.07 mL, 21.4 pmol) and ACN (1 mL) is added. The mixture is vortexed for 3 minutes, then shook at 900rpm at ambient temperature for another hour. After this time, the mixture is concentrated in vacuo then de-salted using a 3K spin filter (Fisher biologies, 4500rpm, 3x 1 hour ). The optical density measurement of the product solution (average of 3 measurements, 20x dilution) was 6.42 OD / mL, concentration = 628 pmol / L, 4.72 mg / mL, total 1.65 mL, 7.80 mg.

[0182] The compound in Table 11 below is prepared in a manner essentially analogous to that found in Preparation 12.Table 11. Structure of preparation 13.Preparation 145’ Oxadiazole linker-functionalized sense strand

[0183] A sense strand (0.0013 mmol in 0.470 mL water) synthesized using conditions found in the protocols below is added to 20X borate buffer (0.071 mL), then is treated with a solution of 2,5- dioxopyrrolidin-l-yl-2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2- yl)phenoxy)ethoxy)ethoxy)acetate (0.0064 g, 0.0131 mmol) in MeCN (0.470 mL). The solution is shaken for 30 mins at 40 °C. The solution is then diluted to 20 mL using RNAse free water to bring concentration of organic solvent to < 10%. Excess NHS ester is removed using 20 mL 3K MWCO centrifugal spin tubes at 3500x for ~30 minutes. The oligonucleotides are rinsed with RNAse free water three times. After removing NHS ester, 1 mL of RNAse free water is added then aspirated lOx and the retentate is transferred to a 5 mL falcon tube. This is repeated until complete transfer of oligo by measuring concentration of compound on filter via nanodrop. The final oligonucleotide isanalyzed for concentration (nano drop at A260), characterized by IP-RP, LCMS for mass purity, and UPLC for UV-purity. ES / MS (m / z): 7324.03 (M+H).

[0184] The compound in Table 12 below is prepared in a manner essentially analogous to that found in Preparation 14.Table 12. Structure of preparation 15.Linker-functionalization of dsRNA

[0185] A freshly prepared solution of (2,5-dioxopyrrolidin-l-yl) 4-[(2,5-dioxopyrrol-l- yl)methyl]cyclohexanecarboxylate (185 mg, 0.542 mmol) in THF (50 mL) is added to single sense strand (44 mL, 0.0528 mmol; OD / mL of 250.4, or -1200 pM (-8.8 mg / mL)) in 0.2M phosphate buffer (44 mL). Vbrtexed vigorously for 2 minutes, and then shook at ambient temperature at 900 rpm for 2 h total. Analysis by LTQ shows about 94-95% conversion. Will be followed by acidification to pH~4 with 20-30 drops of 5N HC1, and then removal of organics in a Genevac concentrator. Is desalted by centrifugal filtration on a 3K spin filter (4 x 4000 rpm, 30 min), and pool the retentates. The optical density measurement of the solution (average of 3 measurements, lOx dilution) is 266 equating to 1.3mM and a total of 316 mg. Extinction coefficient is 204.12. LTQ / MS m / z 7358.Linker-RNA Duplex

[0186] The nanodrop concentrations of aqueous solutions of each strand (average of 3x) are measured as SS = 1322pM and AS = 1108pM. The sense strand and antisense strand are annealed to form a dsRNA. 32 mL of SS and 36.2 mL of AS are mixed and shook for 30 min at 30 °C. Theamount of residual SS strand is measured until completion and required adding an additional 360 pL of AS. Removed endotoxins by filtering through a 0.45 pM filter. The resulting 75 mL of solution measured (Nanodrop™ Lite, 5x average, lOx dilution) 217 OD / mL equating to 575pM and a total of 653 mg. LTQ / MS m / z 7358,7825; UV purity 99+%.Conjugation of dsRNA to TfR binding or ALB binding VHH polypeptides

[0187] Site-specific native or engineered cysteine amino acid residues in the TfR binding or ALB binding VHH polypeptides are used to conjugate dsRNA. Cysteines can be engineered into the primary amino acid sequence of the TfR binding or ALB binding VHH polypeptides. The approach of introducing cysteines as a means for conjugation has been described in WO 2018 / 232088, which is both incorporated by reference in its entirety and incorporated specifically in relation to conjugation via cysteine residues.

[0188] For engineered cysteine conjugation, the TfR binding or ALB binding VHH polypeptides are first reduced with 40 molar equivalents reducing agent dithiothreitol (DTT) at 37 °C for two hours, followed by desalting to remove reducing agent via dialysis or desalting columns. This is followed by re-oxidation of the TfR binding or ALB binding VHH polypeptide to reform the structural disulfides with 10 molar equivalent dehydroascorbic acid (DHAA) incubation at ambient temperature for two hours. A follow up desalting is performed to remove oxidizing agent.

[0189] Conjugation of dsRNA onto T1R binding or A LB binding VHH polypeptides is done using the following methods. In the conjugation schemes below, the oval represents a TfR binding VHH polypeptide or a TfR binding VHH polypeptide conjugated to a half-life extender molecule, e.g. ALB binding VHH.Conjugation Scheme 1

[0190] The first conjugation method utilized the 3’SS oxadiazole (OD) -functionalized dsRNA for conjugating onto the engineered cysteine of the Tf binding or ALB binding VHH polypeptides. For this method, TfR binding or ALB binding VHH polypeptide is prepared similarly as above to make the engineered thiol available for conjugation by undergoing a reduction and oxidation process of the TfR binding or ALB binding VHH polypeptides. This is followed by incubating theOD-dsRNA with the TfR binding or ALB binding VHH polypeptides at 1.2 to 2 molar equivalents for overnight conjugation at ambient temperature.

[0191] TfR binding or ALB binding VHH polypeptide conjugation with 3’ OD linkerConjugation Scheme 2

[0192] The second conjugation method utilized the 5’SS oxadiazole (OD) -functionalized dsRNA for conjugating onto the engineered cysteine of the TfR binding or ALB binding VHH polypeptides. For this method, TfR binding or ALB binding VHH polypeptide is prepared similarly as above to make the engineered thiol available for conjugation by undergoing a reduction and oxidation process of the TfR binding or / VLB binding VHH polypeptides. This is followed by incubating the OD-dsRNA with the TfR binding or ALB binding VHH polypeptides at 1.2 to 2 molar equivalents for overnight conjugation at ambient temperature.

[0193] TfR binding or ALB binding VHH polypeptide conjugation with 5’ OD linkerConjugation Scheme 3

[0194] The third conjugation method utilized the 3’SS tetrazole (MSPT) -functionalized dsRNA for conjugating onto the engineered cysteine of the TfR binding or ALB binding VHH polypeptides. For this method, TfR binding or ALB binding VHH polypeptide is prepared similarly as above to make the engineered thiol available for conjugation by undergoing a reduction and oxidation process of the TfR binding or ALB binding VHH polypeptides. This is followed by incubating the MSPT-dsRNA with the TfR binding or ALB binding VHH polypeptides at 1.2 to 2 molar equivalents for overnight conjugation at ambient temperature.

[0195] TfR binding or ALB binding VHH polypeptide conjugation with 3’MSPT linkerConjugation Scheme 4

[0196] The fourth conjugation method utilized the 5’SS tetrazole (MSPT) -functionalized dsRNA for conjugating onto the engineered cysteine of the TfR binding or ALB binding VHH polypeptides. For this method, TfR binding or ALB binding VHH polypeptide is prepared similarly as above to make the engineered thiol available for conjugation by undergoing a reduction and oxidation process of the TfR binding or A LB binding VHH polypeptides. This is followed by incubating the MSPT-dsRNA with the TfR binding or .ALB binding VHH polypeptides at 1.2 to 2 molar equivalents for overnight conjugation at ambient temperature.SMCC-functionalization of sense strand RNA

[0198] To a 50 mL conical tube containing amino-functionalized sense strand oligonucleotide appended to a C6-amino chain via a 3 ’-terminal phosphorothiolate ester, as a solution in water (8.42 mL, 0.023 mmol, 19.857 mg / mL), is added sodium bicarbonate powder (59 mg, 0.702 mmol). The mixture is briefly vortexed and sonicated to dissolve the bicarbonate. A freshly prepared solution of (2,5-dioxopyrrolidin-l-yl) 4-[(2,5-dioxopyrrol-l- yl)methyl]cyclohexanecarboxylate (96 mg, 0.281 mmol) in acetonitrile (6.32 mL) is then added to the bicarbonate-oligo solution, for example, dsRNA-48-PS-C6-amino (8.42 mL, 0.023 mmol, 19.857 mg / mL in water) and vortexed for 30 seconds. Then, the reaction is allowed to proceed for 4 hours with shaking at ambient temperature at 300 rpm, at which point temperature control on a ThermoMixer® C took the reaction mixture down to 10 °C for 15 hours. At this point, LTQ-MS analysis is performed to determine full conversion. At full conversion, the reaction is quenched to pH 5 using IN HC1 (621 pL, 0.621 mmol). The quenched reaction mixture is then concentrated to approximately 1 / 2 volume using a GeneVac™ centrifugal evaporator and the resultant precipitatecontaining suspension is filtered using a 0.22 micron Steri-Flip® apparatus to remove precipitate, rinsing once with 5 mL of nuclease-free water. The resulting clear solution containing oligo is then diluted to approximately 55 mL with 20% acetonitrile in nuclease-free water and concentrated using a CentriCon® ultrafiltration apparatus (3000 MWCO regenerated cellulose membrane). Following passage of all the volume through the Centricon®, two more 55 mL portions of 20% acetonitrile in nuclease-free water are passed through the CentriCon® to rinse the material, and finally one passage of 55 mL pure Milli-Q® water to remove residual acetonitrile. The retentate is then recovered by inverting the Centricon® apparatus on the included recovery cup. The Centricon® apparatus is then washed and aspirated twice with 800 pL nuclease-free water in each of the two filtration pores (1.6 mL total per wash), and the combined rinsate and retentate are passed through a 50k MWCO filter, which was rinsed one more time with 5 mL nuclease-free water. Finally, the desired compound is measured for concentration using a NanoDrop™ apparatus (OD260 - calculated extinction coefficient: 216.09 mmol-lcm-1) to give the desired compound (sense strand with appended C6-amino-SMCC (SS-RNA-SMCC)) as a solution of 9.77 mg / mL in 13.219 mL (129 mg, 68.1%). LTQ-MS: observed deconvoluted m / z = 7361.7, calculated mass 7361.17, mass purity 91.37%.SMCC-dsRNA Duplex

[0199] To a conical tube containing SS-RNA-SMCC, with appended C6-Amino-SMCC (12.05 mL, 0.016 mmol, 1.328 mmol / L), was added its corresponding antisense RNA strand, with 5’-E- vinyl phosphonate, (0.0165 mmol, 2.619 mmol / L). The solutions is shaken at 25 °C for 30 minutes to give the desired SMCC-functionalized dsRNA (SMCC-dsRNA), then refrigerated to 10 °C for storage. The annealed solutions are sampled for LTQ purity and UPLC non-denaturing chromatography. Analysis via non-denaturing UPLC (run at 10 °C) shows a major single peak of 92% purity. LTQ-MS: (Antisense strand observed deconvoluted m / z = is measured and compared to calculated mass; Sense strand observed deconvoluted m / z = is measured and compared to calculated mass).Conjugation Scheme for SMCC-dsRNA

[0200] The typical conjugation method utilizes the SMCC-functionalized dsRNA for conjugating onto the engineered cysteine of the TfR binding or ALB binding VHH polypeptides. For this method, TfR binding or ALB binding VHH polypeptide is prepared similarly as above to make the engineered thiol available for conjugation by undergoing a reduction and oxidation process of the TfR binding or ALB binding VHH polypeptides. This is followed by incubating the SMCC-dsRNA at 1.2 molar equivalents with the TfR binding or ALB binding VHH polypeptides for overnight conjugation at room temperature.

[0201] Optionally, following conjugation a maleimide hydrolysis step can be done to secure the linker-payload in terminal stage and avoid deconjugation during human body circulation via retro- Michael addition. This succinimide ring hydrolysis process is done by elevating the conjugate pH to 9.0 using 50mM Arginine (stock solution of 0.7M arginine, pH 9.0 was used) and incubating the solution at 37 °C for 20 hours. The hydrolysis state of the maleimide is confirmed by LCMS characterization of +18Da that is incurred by the water addition to the succinimide ring.

[0202] Step la: TfR binding or ALB binding VHH polypeptide conjugation with SMCC linker

[0203] Step lb: TfR binding or ALB binding VHH polypeptide conjugation with SMCC linker ring opening

[0204] Synthesis of Mal-Tet-TCO and GDM linkers and conjugation of Mal-Tet-TCO- or GDM linker-functionalized dsRNA to the engineered cysteine of the TfR binding or ALB binding VHH polypeptides have been described in WO 2024 / 036096.

[0205] Conjugation is monitored using analytical anion exchange chromatography. AProPac™ SAX- 10 HPLC Column, 10pm particle, 4mm diameter, 250mmlength is utilized with the following method. Flow rate of 1 mL / min, Buffer A: 20mM TRIS pH 7.0, Buffer B: 20 mM TRIS pH 7.0 + 1.5M NaCl, at 30 °C.

[0206] Drug(agent) / siRNA to antibody / protein ratio (DAR) is calculated based on peak area % from the analytical anion exchange (aAEX) chromatogram.

[0207] Post conjugation of dsRNAto the TfR binding or ALB binding VHH polypeptide, excess dsRNA and unconjugated protein is removed by further purification. Either preparative size exclusion chromatography (SEC) or preparative anion exchange chromatography is utilized for purification of the final conjugate. Preparative SEC is performed using Cytiva Superdex® 200 in IX PBS pH 7.2 under an isocratic condition. Alternatively, anion exchange, e.g., ThermoFisher POROS™ XQ, was used with starting buffer of 20mM TRIS pH 7.0 and eluting with 20 column volume gradient with a buffer containing 20mM TRIS pH 7.0 and IM NaCl. These resulted in purified TfR binding or ALB binding VHH polypeptide-dsRNA conjugate devoid of excess dsRNA and minimal unconjugated protein. The resulting conjugate profile is analyzed by analytical anion exchange for final DAR quantitation.EXAMPLE 7: Binding of TfR binding VHH conjugates to HPRT siRNA to human TfR expressing cells.

[0208] The binding affinity of TfR binding VHH polypeptide-siRNA-AF647 conjugates for cells expressing human TfRl was assayed using the following procedure.

[0209] EFO-21 (DSMZ, Germany) cells were detached from culture flasks with Accutase (Coming). 50,000 cells per well were transferred to 96-well round bottom polypropylene plate and washed twice in cold FACS buffer (PBS- / - with 2% heat inactivated FBS, 2mM EDTA, and 0.1% NaN3). TfR binding VHH polypeptide-siRNA-AF647 conjugate molecules and non-binding controls (VHbbl polypeptide-siRNA-AF647) were then diluted in FACS buffer and added to cells and incubated for 330 minutes on ice in the dark. To generate concentration / dose response curves, conjugates were prepared at a top concentration of lOuM and diluted threefold serially ten times. Events were acquired on a ZE5 flow cytometer (Biorad) and analyzed in Flowlo (BD). EC50 values were calculated by plotting geometric mean fluorescent intensity values (gMFIs) for single cell non-debris events in GraphPad Prism 9.5.0 and analyzing curves using the GraphPad Prism 4- parameter nonlinear fit model for test article concentration range of 370nM to 0.5 InM.Table 13. Cell-based binding of TfR VHH siHPRT AF647 conjugates to a human cell line expressing TfR.EXAMPLE 8: In vitro knockdown of human HPRT mRNA levels in EFO-21 cells with TfR VHHs conjugated to HPRT siRNA

[0210] Knockdown of human HPRT mRNA expression levels by the TfR binding VHH molecule conjugated to HPRT siRNA was assayed using the following procedure.

[0211] On day 1, EFO-21 cells (eClipse, Germany) were added to 96-well plates at 8000 cells per well in cell culture growth media. On day 2, the culture media was replaced with ACCELL media (Dharmacon) and the TfR binding VHH-HPRT siRNA conjugates were added directly to the well. To generate concentration / dose response curves, final concentrations of 1000, 200, 40, 8, 1.6, 0.32, 0.06, O.OlnM of TfR binding VHH-HPRT siRNA conjugates were used. On day 5, treated cells were lysed directly in the 96-well plate using Cells-to-CT™ Bulk Lysis Reagents (Tnvitrogen). cDNA was created from lysates using Cells-to-CT™ Bulk RT Reagents (Invitrogen) using the following steps in a thermocycler: 37 °C for 1 hour, 95 °C for 5 minutes, and 4 °C hold. cDNAs were used immediately or stored frozen. Quantitative Polymerase Chain Reaction (qPCR) performed using TaqMan Fast Advanced Master Mix (Applied Biosystems) with HPRT (Hs02800695_ml) primer. The following cycle temperatures and times were used: 50 °C for 2minutes, 95 °C for 20 seconds, 40 cycles of 95C for 1 second and 60 °C for 20 seconds. The human HPRT levels were normalized to human GAPDH (Applied Biosystems) and represent relative knockdown of human HPRT mRNA expression as compared to vehicle-treated control cells. IC50 values were calculated using the GraphPad Prism 4-paramater nonlinear fit model.Table 14. In vitro knockdown of human HPRT mRNA levels in EFO-21 cells with TfR VHHs conjugated to HPRT siRNA.EXAMPLE 9: In vivo activity of TfR binding VHH polypeptide conjugates to HPRT siRNA

[0212] The activity of TfR-binding protein conjugated HPRT siRNA was tested in transgenic mice expressing chimeric TfR with a human extracellular domain. Six- to eight-week-old hTfR transgenic mice were divided into groups of 4 mice (2 males and 2 females) each. Each mouse received a single dose of TfR-binding protein conjugated HPRT siRNA at 66nmole / kg by intravenous administration. Two groups of 4 mice received Phosphate-buffered saline (PBS) as negative control and those animals were sacrificed on days 1 and 84. The group of PBS-treatedmice sacrificed on day 1 constituted the comparator PBS group on day 7, while the group of PBS- treated mice sacrificed on day 84 constituted the comparator PBS group on day 84. The test article-treated mice were sacrificed on days 7, and 84 post-treatment, and gastrocnemius muscle and heart tissues were collected for real-time quantitative Polymerase Chain Reaction (qPCR).

[0213] Total RNA from the muscle tissue was isolated using Trizol™ (Invitrogen) reagent followed by purification of RNA using Purelink™ RNA mini kit (Invitrogen). cDNA was synthesized from muscle and heart 89 tissue RNA using ABI High-Capacity cDNA Archive Kit (ThermoFisher Scientific) using the following steps in a thermocycler: 25C for 10 minutes, 37C for 2 hours, and 4C hold. cDNA samples were used immediately or stored frozen, and qPCR was performed using TaqMan Fast Advanced Master Mix to measure the amount of HPRT mRNA using the mouse primer probe sets (Mm03024075_ml, Applied Biosystems). The following cycle temperatures and times were used: 50C for 2 minutes, 95C for 20 seconds, 40 cycles of 95C for 1 second and 60C for 20 seconds. The mouse HPRT levels were normalized to mouse GAPDH (Mm99999915_gl, Applied Biosystems) and the results represented as percent knockdown of mouse HPRT mRNA compared to PBS-administered control.Table 15. In vivo knockdown of HPRT mRNA levels with TfR-binding VHH conjugated HPRT siRNAs in hTfR transgenic mice.

[0214] ND-Knockdown not detected.EXAMPLE 10: Preparation of Acvr2a siRNA molecules and conjugates comprising VHH polypeptides.

[0215] The sequence of antisense oligonucleotides were designed using 15 to 50 nucleotides of the following ACVR2A transcript (SEQ ID NO: 74), where T nucleotides are replaced by U nucleotides, and where one or more nucleotides and one or more internucleotide linkages are optionally further modified as described herein.

[0216] Single strands (sense and antisense) of the dsRNA duplexes were synthesized on solid support via a MerMade™ 12 (LGC Biosearch Technologies). The sense strands were synthesized using phthalamido amino C6 Icaa CPG 500 A (Chemgenes) or standard support (LGC Biosearch Technologies) whereas the antisense strands used standard support (LGC Biosearch Technologies). The oligonucleotides were synthesized via phosphoramidite chemistry at either 5, 10, or 50 pmol scales.

[0217] Standard reagents were used in the oligo synthesis, where 0.1M xanthane hydride in pyridine was used as the sulfurization reagent and 20% DEA in ACN was used as an auxiliary wash post synthesis. All monomers were made at 0. IM in ACN and contained a molecular sieves trap bag.

[0218] The oligonucleotides were cleaved and deprotected (C / D) at 45 °C for 20 hours. The sense strands were C / D from the CPG using cold 50% (methylamine / ammonia hydroxide 28-30%) at RT for 3 hrs, whereas 3% DEA in ammonia hydroxide (28-30%, cold) was used for the antisense strands. C / D was determined complete by IP-RP LCMS when the resulting mass data confirmedthe identity of sequence. Dependent on scale, the CPG was fdtered via 0.45 um PVDF syringeless filter, 0.22 um PVDF Steriflip® vacuum filtration or 0.22 um PVDF Stericup® Quick release. The CPG was back washed / rinsed with either 30% EtOH / RNAse free water then filtered through the same filtering device and combined with the first filtrate. This was repeated twice. The material was then divided evenly into 50 mL falcon tubes to remove organics via Genevac™. After concentration, the crude oligonucleotides were diluted back to synthesized scale with RNAse free water and filtered either by 0.45 pm PVDF syringeless filter, 0.22 pm PVDF Steriflip® vacuum filtration or 0.22 pm PVDF Stericup® Quick release.

[0219] The crude oligonucleotides were purified via AKTA™ Pure purification system using anion-exchange (AEX). For AEX, an ES Industry Source™ 15Q column with MPA: 20mM NaH2PO4, 15% ACN, pH 7.4 and MPB: 20 mM NaH2PO4, IMNaBr, 15% ACN, pH 7.4. Fractions which contained a mass purity greater than 85% without impurities >5% where combined.

[0220] The purified oligonucleotides were desalted using 15 mL 3K MWCO centrifugal spin tubes at 3500xg for ~30 min. The oligonucleotides were rinsed with RNAse free water until the eluent conductivity reached < 100 usemi / cm. After desalting was complete, 2-3 mL of RNAse free water was added then aspirated lOx, the retainment was transferred to a 50 mL falcon tube, this was repeated until complete transfer of oligo by measuring concentration of compound on filter via nanodrop. The final oligonucleotide was then nano filtered 2x via 15 mL 100K MWCO centrifugal spin tubes at 3500xg for 2 min. The final desalted oligonucleotides were analyzed for concentration (nanodrop at A260), characterized by IP-RP LC / MS for mass purity and UPLC for UV-purity.Table 16. Oligonucleotide Synthesis Reagents.Table 17. Phosphorami dites.

[0221] ACVR2A Transcript, SEQ ID NO:74 gtctgggctt ccgaatatgt tttatgacgg ttgattttac accaggaggt ttgtctccgaggaagaccca gggaactgga tatctagcga gaacttcctc cggattcccc ggcgcctcgggaaaatggga gctgctgcaa agttggcgtt tgccgtcttt cttatctcct gttcttcaggtgctatactt ggtagatcag aaactcagga gtgtcttttc tttaatgcta attgggaaaaagacagaacc aatcaaactg gtgttgaacc gtgttatggt gacaaagata aacggcggcattgttttgct acctggaaga atatttctgg ttccattgaa atagtgaaacaaggttgttggctggatgat atcaactgct atgacaggac tgattgtgta gaaaaaaaag acagccctgaagtatatttt tgttgctgtg agggcaatat gtgtaatgaa aagttttctt attttccggagatggaagtc acacagccca cttcaaatcc agttacacct aagccaccct attacaacatcctgctctat tccttggtgc cacttatgtt aattgcgggg attgtcattt gtgcattttg ggtgtacagg catcacaaga tggcctaccc tcctgtactt gttccaactc aagacccaggaccaccccca ccttctccat tactaggttt gaaaccactg cagttattag aagtgaaagcaaggggaaga tttggttgtg tctggaaagc ccagttgctt aacgaatatg tggctgtcaa aatatttcca atacaggaca aacagtcatg gcaaaatgaa tacgaagtct acagtttgcctggaatgaag catgagaaca tattacagtt cattggtgca gaaaaacgag gcaccagtgttgatgtggat ctttggctga tcacagcatt tcatgaaaag ggttcactat cagactttcttaaggctaat gtggtctctt ggaatgaact gtgtcatatt gcagaaacca tggctagaggattggcatat ttacatgagg atatacctgg cctaaaagat ggccacaaac ctgccatatctcacagggac atcaaaagta aaaatgtgct gttgaaaaac aacctgacag cttgcattgctgactttggg ttggccttaa aatttgaggc tggcaagtct gcaggcgata cccatggacaggttggtacc cggaggtaca tggctccaga ggtattagag ggtgctataa acttccaaagggatgcattt ttgaggatag atatgtatgc catgggatta gtcctatggg aactggcttctcgctgtact gctgcagatg gacctgtaga tgaatacatg ttgccatttg aggaggaaattggccagcat ccatctcttg aagacatgca ggaagttgtt gtgcataaaa aaaagaggcctgtttaaga gattatggc agaaacatgc tggaatggca atgctctgtg aaaccattgaagaatgttgg gatcacgacg cagaagccag gttatcagct ggatgtgtag gtgaaagaattacccagatg cagagactaa caaatattat taccacagag gacattgtaa cagtggtcacaatggtgaca aatgttgact ttcctcccaa agaatctagt ctatgatggt tgcgccatctgtgcacacta agaaatggga ctctgaactg gagctgctaa gctaaagaaa ctgcttacagtttattttct gtgtaaaatg agtaggatgt ctcttggaaa tgttaagaaa gaagaccctttgttgaaaaa tgttgctctg ggagacttac tgcattgccg acagcacaga tgtgaaggacatgagactaa gagaaacctt gcaaactcta taaagaaact tttgaaaaag tgtacatgaagaatgtagcc ctctccaaat caaggatctt ttggacctgg ctaatggagt gtttgaaaactgacatcaga tttcttaatg tctgtcagaa gacactaatt ccttaaatga actactgctattttttttaa atcaaaaact tttcatttca gattttaaaa agggtaactt gtttttattgcatttgctgt tgtttctata aatgactatt gtaatgccaa tatgacacag cttgtgaatgtttagtgtgc tgctgttctg tgtacataaa gtcatcaaag tggggtacag taaagaggcttccaagcatt actttaacct ccctcaacaa ggtatacctc agttccacgg ttgctaaattataaaattga aaacactaac aaaatttgaa taataaatcg atccatgttt tgtaacaaattcactgtgtt atttaaggaa aaaaaggtaa gctatgctta gtgccaacaa taagtggccattcgtaaagc agtgttttag catttcttgt gctggcttgt aatgtaggga aaaaaagtgctgttttttga aaagatggtg tcatttcccc cttcttccca tgttttaaag ccccatcttatatccagttc ccaaaatttg catacttacc taagtatttt ttttaggtgt gctgtgtttggggaatattt gaaaatttaa agcatgattt aaaatttttt aaagtgagct gtgacactggaaagctcttc attttatctt ttaaaataga gttttttcta tttatatatg taaaattgtagtgtatttct tttcaccaaa cagtgtgtgg gacattcttt atcactgttt taggatcacctcaggaagtg tcgttaccca gaattcccca ctgtctgcta tgagacttgt aactttatcactatacttct gcttggtgcc atcttgtcag agtaatattt gatgtctgtg atatgtaaagaattatccta ggataaagat attaaacttt aagcagattt cagatgttac tgctttaaaacaaatcaggg ataacaaatt aaacgtataa cttaaaatat gcaatgacat ttagaggtaaccaatgttga tataggtagc atagcctagc ctcctcccca aaattgcttt tacaactaacactgatacta atttaggata gttcatgcct tatccttgct aagaaaatgg aattgatggtaggcaggtgc taaagtgct ttcaaaacaa tattacgtta gaatacaatt ggattcttcctcaaatttat acaggccaaa aagtaaaaca ttaattttct gaatttccag attaccaatcaattaatcaa caaatagcca gtattatgct gtgtatttct gtcaggtcat tttaaaatccatgttaattt tataaaagaa ttttttacat gtcactgtca ggagctcact gtgaatgtgt tgtcttcaaa tggttattta accacacagt acactacatt ttacatatat gtacgtaatctctgggaata gtaaattaat tatgttattt ataaacaata cataggtcaa cagactttaagcagggagga aaagaagagt aatagcgtct gtgtgctgca gaccattcag aactgtcacgtgtgtcccca tggtctcatt cattgtattc ctagcaattc ccttttcaat gttgagttcacctctttatt tcacaaagta cttggtctct caatttcttg atctggtttt gcttccatttaaaaactaat caagaaggga aaatattgag aatgtgcata caagaaaatc attaatttcctgaagatgaa tttctacctg ttgtgaacat ttaactttct ttttaaaagt taaacaaaaataaacaaggg atattatgat gaatgtttgg cttatgtgag tactagagat aaaatttttaaacccagtta ttcacaatat aaaatgtttt caagttagaa aaaattttta gaaatcctgggtattgtatt taactgtagc taaccaattt taaaacttgt attcttttga gaactattattaatagaaaa actttttata agcagtaaaa taagaatgtt ccagtgacta cctgtccttatacctagtct tgttaaaact ttcttttgca gggtatttag tgtttggttt acagtcagtgcagagtgggc aagttaacag aaagtttgag ctagagatac tggaaaaaaa aaagatcaaagaatgagaaa aatggtgatc cattttgggg caaactgaga ccccccaaat aactctttcctcatgtgtat ggtgctcctc atgactcgtc ttgtattttg cctttctgat acccatcagaactgctgctg ctctaactta tactctttac cttgcccaga tctccgcgta aggaatgctttatgatcaac ttgccatagg actgatggat taaccagtgt tcggctttat ttgaagtctatgccctgcac agctcttgta tgtattttag atgctagaag tttttttagc atgtgatgtgtgattcttgt ttgaattcta ggtaccttgt gaattccaga aaaagagact gtgcttcacgattgttagtc ccatgaactt gcactatcta tctttcatgg tgatgttttgaaaatacaatcaggaaaaaa cccaacacct ttggaattta aaatagaatc atatcatgaa atttaaaaagaatctcttct gttgcatttc ctcaccccta agtaacagct acatttaagt aaaatgcaggtggtagggga aaaaaaacca tggcgagatg gtggtttagt ggaataaact gattactggtttttttgttt tttttttttt ttttaaagaa agaagcttca tcacagatac tttccagtttctcttttata cttttttgaa agattacttt ttaggaacat ttggtatgat atgcataaaattatttatcc atttatgggc aaaatgatac aagtagcatc ttgattgaac atcatttacctcagatattc aaccagcagt acgtttttta tgcagtctca acccatatcc catttgttacctctcagaat attggtaagc agttattttc gctttactct gtatttcttg tgttttgggcacaggttatt gtactactgt caaatcgtac ttgctatttt ttctgcaagt atttaacagaaagcttaaaa tccccataaa accccacctt ggataagtga ttgttaaata ttgtacaaataaaatgtatg ctatccccat tccatcccca agttaaataa aaaaatgaat acgg

[0222] TfR binding VHH polypeptides and conjugates to Acvr2a siRNA oligonucleotides were generated and prepared essentially as described above in Examples 5 and 6 except for the conjugation reaction which is carried out for 48hr, due to slower reaction kinetics with the MSPT linker instead of SMCC.EXAMPLE 11: In vitro knockdown of human Acvr2a mRNA levels in EFO-21 cells with TfR VHH conjugated to Acvr2a siRNA

[0223] Knockdown of human Acvr2a mRNA expression levels by TfR binding VHH conjugate conjugates to Acvr2a siRNA was assayed using the following procedure.

[0224] On day 1, EFO-21 cells (eClipse, Germany) were added to 96-well plates at 8000 cells per well in cell culture growth media. On day 2, the culture media was replaced with ACCELL Media (Dharmacon) and the TfR binding VHH-Acvr2a siRNA linker 13 conjugates were added directly to the well. To generate concentration / dose response curves, final concentrations of 1000, 200, 40, 8, 1.6, 0.32, 0.06, O.OlnM of TfR binding VHH-Acvra2a siRNA conjugates were used. On day 5, treated cells were lysed directly in the 96 well plate using Cells-to-CT™ Bulk Lysis Reagents (Invitrogen). cDNA was synthesized from lysates using Cells-to-CT™ Bulk RT Reagents (Invitrogen) using the following steps in a thermocycler: 37 °C for 1 hour, 95 °C for 5 minutes, and 4 °C hold. cDNA samples were used immediately or stored frozen. Quantitative Polymerase Chain Reaction (qPCR) performed using TaqMan Fast Advanced Master Mix (Applied Biosystems) with Acvr2a (Hs00155658 ml) primers. The following cycle temperatures and times were used: 50 °C for 2 minutes, 95 °C for 20 seconds, 40 cycles of 95 °C for 1 second and 60 °C for 20 seconds. The human Acvr2a mRNA levels were normalized to human GAPDH (Applied Biosystems) mRNA to represent the relative knockdown of human Acvr2a mRNA as compared to vehicle-treated control cells. IC50 values were calculated using the GraphPad Prism 4-parameter nonlinear fit model.Table 18. In vitro knockdown of human Acyr2a mRNA in EFO-21 cells with TfR VHH conjugates to Acyr2a siRNA.Table 19. Acvr2a siRNA Sequence.Abbreviations - “m” indicates 2’-0Me; “f” indicates 2’-fluoro; indicates phosphorothioate linkage; “VP” indicates 5’-vinylphosphate; “S” means the sense strand; “AS” means the antisense strand.EXAMPLE 12: In vivo knockdown of human Acvr2a mRNA levels in human TfR transgenic mice with TfR VHH conjugates to Acvr2a siRNA

[0225] The activity of TfR-binding VHH conjugates to Acvr2a siRNA with linker 13 were tested in transgenic mice expressing chimeric TfR with a human extracellular domain. Six- to eight- week-old female hTfR transgenic mice were divided into groups of 6 mice each. Every mouse received a single dose of TfR-binding VHH conjugated Acvr2a siRNA at 33 and 132nmole / kg by subcutaneous administration. One group of 6 mice received Phosphate-buffered saline (PBS) asnegative control. Mice were sacrificed on day 14 post-treatment, and tibialis anterior muscle was collected for real-time quantitative Polymerase Chain Reaction (qPCR).Total RNA from the muscle tissue was isolated using Trizol™ (Invitrogen) reagent followed by purification of RNA using Purelink™ RNA mini kit (Invitrogen). cDNA was synthesized from tibialis anterior muscle RNA using ABI High-Capacity cDNA Archive Kit (ThermoFisher Scientific) using the following steps in a thermocycler: 25 °C for 10 minutes, 37 °C for 2 hours, and 4 °C hold. cDNA samples were used immediately or stored frozen, and qPCR was performed using TaqMan Fast Advanced Master Mix to measure the amount of Acvr2a mRNA using the mouse primer probe sets (Acvr2a Mm00725448_sl, Applied Biosystems). The following cycle temperatures and times were used: 50 °C for 2 minutes, 95 °C for 20 seconds, 40 cycles of 95 °C for 1 second and 60 °C for 20 seconds. Results are presented as a percentage knockdown of Acvr2a relative to PBS -administered control normalized to mouse RplpO (primer probe set Mm00725448_sl).Table 20. In vivo knockdown of Acvr2a mRNA levels with TfR -binding VHH conjugated Acvr2a siRNAs in in tibialis anterior muscle of hTfR transgenic mice.ND-Knockdown not detectedEXAMPLE 13: Generation of TfR binding VHH protein fusions to deliver protein cargos (TfR VHH-04-Fc 1-arm)

[0226] cDNA sequence encoding a polypeptide amino acid sequence including TfR VHH 04 polypeptide of (SEQ ID NO: 13) which was fused to the N-terminus of hIgG4PAAFc (SEQ ID NO: 79) via a GGGGSENLYFQGGGGG linker (SEQ ID NO: 78), herein referred to as TfR VHH- Fc fusion protein (SEQ ID NO: 76) was synthesized and cloned into an expression plasmid backbone and transfected into CHO cells essentially as described above in Example 2. The TfR VHH-Fc fusion protein homodimer secreted into the media from the CHO cells was purified by Protein A affinity chromatography followed by size-exclusion chromatography. Specifically, the VHH-Fc fusion protein homodimer from harvested media was captured onto MabSelect PrismA resin (Cytiva). The resin then was briefly washed with a running buffer, such as a phosphate- buffered saline (PBS; pH 7.4) or a running buffer containing Tris, to remove non-specifically bound material. In some cases, NaCl was added in the wash buffer to retain the VHH-Fc molecule on the column before elution. The VHH-Fc fusion protein homodimer was eluted from the resin with a low pH solution, such as 10 mM citric acid pH 3. Fractions containing the VHH-Fc fusion protein homodimer were pooled, and the pH was neutralized by adding Tris base to pH 5. The VHH-Fc fusion protein homodimer was further purified by size exclusion chromatography using resins such as Superdex75 (Cytiva) with isocratic elution in Acetate buffer, pH 5 with NaCl and EDTA.

[0227] To generate monovalent presentation, the VHH-Fc fusion protein homodimer (comprising SEQ ID NO:76) was reduced at room temperature in PBS supplemented with 50mM of 2-MEA in the presences of equal molarity of hIgG4PAALK stump protein homodimer (SEQ ID NO: 77) for 4 hours. The reaction mixture was subsequently subjected to extensive dialysis against PBS at 4 °C to yield the desired heterodimer of monovalent VHH-Fc fusion protein (SEQ ID NO: 76) and monovalent hIgG4PAALK stump protein (SEQ ID NO: 77).EXAMPLE 14: In vivo delivery of protein cargos by TfR binding VHH fusions to the brain

[0228] CNS exposure of the heterodimer of monovalent VHH-Fc fusion protein (comprising SEQ ID NO: 76) and monovalent hIgG4PAALK stump protein (SEQ ID NO: 77) was evaluated in NHPs. In brief, NHPs were administered a single 2.3 mg / kg intravenous injection of themonovalent TfR VHH-Fc fusion protein or 5 mg / kg of an isotype control human IgG antibody. The exposure of the test articles in multiple CNS compartments was measured at 24 hours (N=2 per treatment and timepoint). Brains were serially extracted with PBS buffer followed by RIPA buffer. The fraction of test article in interstitial fluid (ISF) was derived from the concentration of compound in PBS extract and the brain tissue associated compound fraction was derived from the concentration of compound in the RIPA buffer extract. Brain extract concentrations in PBS buffer and RIPA buffer were determined by enzyme-linked immunoassays (ELISAs). CNS concentrations of the monovalent TfR VHH-Fc fusion protein were markedly higher than that of the isotype control human IgG antibody dosed at a molar equivalence. In particular the RIPA extracts demonstrated dramatic increase in TfR VHH-Fc fusion protein levels as compared to the non-TfR VHH shuttled isotype control antibody.Table 21. Assessment of monovalent TfR binding VHH fusion protein concentrations in NHP brain.EXAMPLE 15: Preparation of MALAT-1 ASO molecules and conjugates comprising VHH polypeptides.

[0229] MALAT1 antisense oligonucleotide (ASO) with a SMCC linker was synthesized as described above in Example 6. Conjugation of the C6-NH2-oligonucleotide to SMCC was accomplished by addition of 20 molar excess of SMCC in acetonitrile to a solution of oligonucleotide and 25 equivalents of NaHCOs in water. Upon completion of the reaction, themixture was acidified with dilute aqueous HC1 and either centrifuged in a 3,000 molecular weightcutoff spin filter or processed via tangential flow filtration.

[0230] Scheme A:Table 22. MALAT1-ASO SequenceAbbreviations - “m” indicates 2’-OMe; indicates phosphorothioate linkagePreparation ofTfR binding VHHs for conjugation of MALAT1-ASO

[0231] TfR binding VHH polypeptides (for exampleTfR VHH-03 — linker-ALB VHH-Cys, SEQ ID NO: 56) that have a single Cysteine amino acid incorporated (as described in Example 5) were prepared for chemical conjugations in the following manner: For the VHH polypeptides the pH was increased using 1 / 10 volume addition of IM Tris pH8. After pH adjustment, the VHH polypeptides were reduced with 10 molar equivalents reducing agent tris(2- carboxyethyljphosphine (Thermo Scientific Bond-Breaker™ TCEP Solution, neutral pH) at room temperature for 10-20 minutes, followed by desalting to remove reducing agent quickly.

[0232] Conjugation of MALAT1-ASO onto VHHs was done immediately after reducing agent removal in order to prevent VHH polypeptide dimer reformation using the following methods.Conjugation Scheme

[0233] The conjugation method utilized the SMCC-functionalized ASO for conjugating onto the introduced cysteine amino acid of the TfR binding VHH polypeptide. For this method, VHH popypepetides were prepared similarly as above to make the cysteine available for conjugation by undergoing a reduction and desalting process. This is immediately followed by incubating the SMCC-MALAT1 ASO with the VHHs at 1.2 molar equivalents for 2hr conjugation at room temperature. In the scheme below, the oval represents a VHH polypeptide, e.g. VHH control polypeptide, TfR binding VHH polypeptide and / or TfR VHH binding polypeptide fusion with an albumin binding VHH, comprising the cysteine used for conjugation.

[0234] Scheme B for single strand oligonucleotide conjugation to TfR VHHs via SMCC linker:

[0235] Conjugation was monitored using analytical anion exchange chromatography. A ProPac™ SAX- 10 HPLC Column, 10pm particle, 4mm diameter, 250mm length was utilized with the following method. Flow rate of 1 mL / min, Buffer A: 20mM TRIS pH 7.0, Buffer B: 20 mM TRIS pH 7.0 + 2M NaCl, at ambient temperature.Table 23. HPLC gradient used to asses single strand oligonucleotide (ASO) conjugation to TfR binding VHHs.

[0236] Drug / ASO to VHH polypeptide ratio (DAR) was calculated based on peak area % from the analytical anion exchange (aAEX) chromatogram.

[0237] Post conjugation of ASO to the TfR binding VHHs, excess ASO and unconjugated protein was removed by further purification. Either preparative size exclusion chromatography (SEC) or preparative anion exchange chromatography was utilized for purification of the final conjugate. Preparative SEC was performed using Cytiva Superdex® 75 in IX PBS pH 7.2 under an isocratic condition. Alternatively, anion exchange, e.g., ThermoFisher POROS™ XQ, was used with starting buffer of 20mM TRIS pH 7.0 and eluting with 20 column volume gradient with a buffer containing 20mM TRIS pH 7.0 and 2M NaCl. These resulted in purified TfR TIH-MALATl ASO conjugates devoid of excess oligonucleotide and minimal unconjugated protein. The resulting conjugate profile was analyzed by analytical anion exchange for final DAR quantitation Drug / ASO to VHH polypeptide ratio (DAR) was calculated based on peak area % from the anion exchange (AEX) chromatogram.EXAMPLE 16: In vitro knockdown of MALAT-1 mRNA levels in mouse cortical neuronal cells with TfR VHH conjugates to MALAT-1 ASO

[0238] Mouse primary cortical neurons were isolated from wild type C57BL6 mouse embryos at embryonic day 18 (El 8). Cells were plated in poly-D-lysine coated 96-well plates at a density of 40,000 cells / well and cultured in NbActivl (BrainBits, LLC) containing 1%Antibiotic / Antimycotic (Corning) for 7 days at 37 °C in a tissue culture incubator in a humidified chamber with 5% CO2. On day 7, half of the medium was removed from each well and 2x concentration of TfR binding VHH-MALAT1 ASO conjugates in culture media with 2% FBS was added for treatment and incubated with cells for additional 7 days. At the end of treatment, RT- qPCR was performed to quantify targeted mRNA levels using TaqMan Fast Advanced Cell-to-CT kit. Specifically, cells were lysed, cDNA was generated on Mastercycler X50a (Eppendorf), and qPCR was carried out on QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Gene expression levels of the MALAT1 were normalized by ACTB using respective probes (ThermoFisher).

[0239] Results provided in Table 24 demonstrate that the exemplified TfR binding VHH-MALAT1 ASO conjugate successfully knocks down mouse MALAT1 mRNA levels.Table 24. In vitro potency TfR binding VHH conjugates to MALAT1 ASO for reducing mouse MALAT1 mRNA levels in mouse cortical neuroEXAMPLE 17: In vivo activity of TfR binding VHH polypeptide conjugates to MALAT-1 ASO

[0240] To determine the efficacy of the TfR binding VHH-MALAT1 ASO conjugates, a study is performed in mice with a single intravenous (IV) dose of 10 mg / kg ASO dose and compared against the PBS control group (n=4 per group). 28 days following initial dosing, mice are perfused under anesthesia and sacrificed, then hemibrain and gastrocnemius muscle is collected and processed for assessment of MALAT1 gene expression changes by RT-qPCREXAMPLE 18: Preparation of small molecule (e.g. fluorescent dye) conjugates to TfR VHH polypeptides.

[0241] TfR binding VHH polypeptides that have a single Cysteine amino acid incorporated (as described in Example 5) were prepared for chemical conjugations essentially as described above in Example 6A.Conjugation Scheme

[0242] The conjugation method utilized the small molecule maleimide functionalized AZDye647 (Vector Laboratories, SKU: FP-1122) for conjugating onto the introduced cysteine amino acid of the TfR binding VHH polypeptide. For this method, VHHs were prepared similarly as above to make the cysteine available for conjugation by undergoing a reduction and desalting process. This is immediately followed by incubating the Maleimide AZDye647 small molecule with the VHH’s at 2 molar equivalents for Ihr conjugation at room temperature. In the scheme below, the oval represents a VHH polypeptide, e g. VHH control polypeptide, TfR binding VHH polypeptide and / or TfR VHH binding polypeptide fusion with an albumin binding VHH, comprising the cysteine used for conjugation.Scheme for small molecule AZDye647 conjugation via maleimide linker:

[0243] Conjugation was monitored using Time of Flight (TOF) mass spectrometry.Time of Flight Mass Spectrometry Method:

[0244] Eight (8) pg of the sample was injected onto a Poroshell 300sb-C3 2.1x2.5 mm, 5 pM column (Agilent Part# 821075-924). Buffer A was made up of 0.1% trifluoroacetic acid (TFA) inwater while buffer B comprised of 0.1% trifluoroacetic acid (TFA) in acetonitrile (ACN). The column was equilibrated in 10% B buffer prior to sample injection followed by a wash with 10% B and elution using 80% B. The average DAR was determined by calculating the contribution from each individual DAR species from the fractional percentage multiplied by the DAR number for each contributing species.

[0245] Post conjugation of Mai eimide AZDye647 to the TfR binding VHHs, excess small molecule was removed by further purification. Preparative SEC was performed using Cytiva Superdex® 75 in IX PBS pH 7.2 under an isocratic condition. This resulted in purified TfR binding VHH-AZDye647 conjugate devoid of excess small molecule and minimal unconjugated protein. The resulting conjugate profile was analyzed by TOF for final DAR quantification.Example 19: TfR binding VHH polypeptide small molecule conjugate binding to human TfR expressing cells.

[0246] The binding affinity of the TfR binding VHH polypeptide small molecule conjugate for cells expressing human TfRl was assayed using the following procedure.

[0247] EFO-21 (eClipse, Germany) cells were detached from culture flasks with accutase (Coming). 100,000 cells per well were transferred to 96-well round bottom polypropylene plate and washed twice in cold BD Pharmingen Stain Buffer (BD; referred to herein as FACS buffer). TfR binding VHH polypeptide small molecule diluted in FACS buffer was added to cells and incubated for 1 hour on ice, in the dark. To generate concentration / dose response curves, final concentrations of 1000, 333.33, 111.11, 37.04, 12.35, 4.11, 1.37, 0.46nM of TfR binding VHH polypeptide small molecule conjugate was used. Cells were washed twice in cold FACS buffer. To assess cell viability, cells were treated with DAPI solution (Thermo, 62248) and incubated for 5 minutes on ice, in the dark. Cells were washed once and resuspended in FACS for flow cytometry collection. Cell events were acquired on a ZE5 flow cytometer (Biorad) and analyzed in FlowJo (BD). EC50 values were calculated using the GraphPad Prism 4-parameter nonlinear fit model.Table 25. Cell-based binding of TfR VHH polypeptide small molecule conjugate to cells expressing human TfRl.EXAMPLE 20: In vivo delivery of TfR binding VHH polypeptide conjugated small molecule to various tissues

[0248] The delivery of the small molecule (AZDye647) conjugate by the TfRbinding VHH to various tissues was tested in transgenic mice expressing chimeric TfR with a human extracellular domain. hTfR transgenic female mice (N=3) received a single dose of TfR VHH conjugated small molecule AZDye647 at 10 mg / kg by subcutaneous administration. The animals were euthanized 24 hr post dose and brain, dorsal root ganglion (DRG), liver, spleen, heart, gonadal white adipose tissue (gWAT), intestine, pancreatic islet, gastrocnemius, or tibialis were isolated. Subsequently, isolated tissues were immediately imaged using an inverted LSM 980 Confocal Microscope (Carl Zeiss).Table 26. Mean fluorescent intensity (MFI) values measured in a tissue area of interest.EXAMPLE 21: Preparation of TfR binding VHH decorated AAV particles carrying GFP as payloadCloning & vector design strategy

[0249] A vector containing the AAV9 VP1 capsid sequence was engineered to contain a stuffer sequence flanked by two PaqCI restriction sites at amino acid position 454 of the capsid protein. The restriction site was oriented so that the enzyme’s recognition site lays within the stuffer sequence, while the cut site is located adjacent to the stuffer sequence allowing for scarless cloning. Such a design does not only facilitate VHH insertion cloning, but also facilitates the cloning of VHH libraries from immune and / or naive sources.

[0250] The TfR VHHs selected for AAV display (Table 27), were ordered as genes from IDT containing compatible adapter sequences and flanked by linker sequences. The N-terminus of the VHH contained a (G4S)5 or a (G4S)3 linker and the C-terminus a G4A linker. The longer linker at the start of the VHH allows for an upright presentation perpendicular from the AAV particle’s surface.Table 27. TfR VHHs selected for AAV display.

[0251] Both VHH gene fragments and vector were digested using PaqCI and ligated using T4 DNA ligase. DH5alpha cells were transformed and selected transformant were screened using Sanger sequencing.TfR binding VHH decorated AAV9 particles formation (with GFP as pay load).

[0252] TfR binding VHH decorated AAV9 particles were generated using VPC 2.0 cells (ThermoFisher, #A49784), expanded to 200 mL with a viability of 96% at a density of 4.86 x 106cells / mL, and diluted to 3 x 106cells / mL in Viral Production Medium (ThermoFisher, #A4817901) supplemented with 4 mM GlutaMAX. AAV MAX Enhancer (2 mL; ThermoFisher, #A5O515) was added, and cells were incubated at 37°C with 8% CO? in an orbital shaker. For transfection, a total of 600 pg plasmid DNA (3 pg / mL) was prepared in 20 mL Viral Plex Complexation Buffer at a plasmid ratio of 0.5: 1 :2 (pHelper: pEGFP-4.05kb: pNLRap2-Cap9). To assemble AAV9 via a split vector system, pSMAl (encoding VP2 and VP3) was combined with either pSMBC pNLRep2- Cap9-v2 PaqCI 454 DO_gbTV6F. Ic9_aav or pSMBC pNLRep2-Cap9-v2 PaqCI 454 DO_gbTV7G. Ic6_aav_short at a 1 : 1 ratio. The DNA mixture was combined with Viral Plex Complexation Buffer, followed by the addition of 600 pL AAV-MAX Transfection Booster and 1200 pL AAV-MAX Transfection Reagent. The transfection complex was incubated at room temperature for 20-30 min before being added to cells, which were then incubated at 37°C with 8% CO2 for 72 hours. For viral particle harvest, 22 mL of lysis buffer was added, followed by the addition of Pierce™ Universal Nuclease (Pierce, #88700; final concentration 90 U / mL) and 1 M MgCL (final concentration 2 mM), and incubated at 37°C for 4 hours. The lysate was centrifuged at 4000g for 30 min, and the supernatant was filtered for downstream purification. The lysate wasadjusted to 350 mM NaCl and pH 7.8 using 1 M Tris-HCl (pH 8.0) to a final volume of 100 mL, yielding 8-60 x 1013vector genomes (vg). The sample (400 mL) was loaded onto a 5 mL AAV9 affinity column at 2.0 mL / min using an AKTAPURE system, eluted with 14 mM citric acid, 0.5% sucrose, and 0.1% poloxamer 188 (pH 2.5), and immediately neutralized with Tris buffer. The final formulation buffer consisted of 20 mM phosphate, 350 mM NaCl, 0.5% sucrose, and 0.1% poloxamer 188 (pH 7.8). Capsid titer was determined using the Progen AAV9 Xpress ELISA (Progen, #PRAAV9XP), while viral genome titer was quantified by digital PCR (dPCR) using the Qiagen CGT Viral Vector Lysis and CGT dPCR Assay (Qiagen, #250230, #250256) targeting the EGFP sequence.EXAMPLE 22: Binding of TfR binding VHH decorated AAV particles carrying GFP as payload to soluble human TfR extracellular domain in ELISAs

[0253] The binding of TfR binding VHH decorated AAV9 particles was assessed in an ELISA. The assay was conducted using Streptavidin (SA) coated High Binding Capacity (HBC) 96-well plates (PI15500), a 0.05% Polyoxyethylene sorbitan (PBST) solution, biotinylated human Transferrin Receptor extracellular domain protein (hTfR), CaptureSelect™ Horseradish Peroxidase (HRP) conjugated Anti-AAV9 antibody (Progen, #7303332100), TMB Ultra One-Step Enzyme-Linked Immunosorbent Assay (ELISA) substrate Solution (Pierce #34029), and ELISA Stop Solution (Thermo Scientific, #N600). Initially, the plates were subjected to three washing cycles with PBST 0.05%. Subsequently, the experimental plate was coated with 50 pl of 50 nM hTfR ECD protein, while the SA control plate received PBST, followed by incubation for 30 minutes with orbital shaking. After an additional three washing steps with PBST 0.05%, 50 pl of AAV9 capsids at an initial concentration of 5E11 capsids / ml were added to each well, subsequently subjected to serial 1 :3 dilutions across eight data points. The plates were then incubated for one hour with shaking. Post-incubation, the plates underwent three further washing cycles with PBST 0.05%, after which 50 pl of anti-AAV9-HRP, diluted 1: 10,000 in PBST, was added and incubated for another hour with shaking. Following an additional washing step, 50 pl of TMB substrate was introduced and allowed to react for 2-5 minutes. The reaction was terminated by adding ELISA stop solution, and optical density readings were taken at 450 nm.Table 28. Binding of TfR binding VHH decorated AAV particles carrying GFP as payload to human TfR ECD proteinEXAMPLE 23: Preparation of TfR binding VHH decorated lipid nanoparticles (LNPs) carrying Alexafluor647 conjugated to HPRT siRNA as cargo

[0254] LNP formation and encapsulation of nucleic acids according to the present disclosure may be accomplished substantially as set forth below. Briefly, by way of exemplification, siRNA that inhibits translation of HPRT, is encapsulated with exemplified LNPs of the present disclosure. Lipids were combined in 200-proof ethanol in the specific ratios of 2.25mol% DMG-PEG (2kDa), 0.25mol% DSPE-PEG-Maleimide (2kDa), 45 mol% LP-01 (9Z,12Z-octadecadienoic acid, 3-[4,4- bis(octyloxy)-l-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester), 43.5mol% cholesterol, and 9mol% DSPC (distearoylphosphatidylcholine). If the lipids did not fully dissolve, the process was repeated. The RNA solution was prepared in water and mixed with sodium citrate buffer. The solution was then mixed with aqueous and lipid solutions using microfluidic channels. The parameters used were flow rate volume ratio to 3 : 1 (RNAdipid), total flow rate 12 mL / min. Buffer exchange was performed by preparing PD-10 columns to exchange ethanol and citrate buffer to 0.1 M PBS.

[0255] Exemplified LNPs of the present disclosure may have a targeting moiety conjugated. VHH polypeptides (in this example, TfR VHH-06 (SEQ ID NO: 20), functionalized with a reduced thiol, were presented to the LNPs at a molar ratio of 4: 1 (maleimide-PEG-DSPE:VHH polypeptide) for 4 hours. Isotype lipid nano particle controls may be made in the same manner but with nontargeting analogs of the same VHH or other structures. Bare lipid nanoparticle controls were also made in the same manner, but with functionalized lipid-PEG molecules replaced by non-functionalized lipid-PEG molecules (e.g., no reactive group associated with the PEG molecule or other hydrophilic polymer).EXAMPLE 24: Binding of TfR binding VHH decorated lipid nanoparticles (LNPs) carrying Alexafluor647 conjugated to HPRT siRNA as cargo to human TfR by ELISA

[0256] The binding of TfR-binding protein conjugated LNPs were tested in an ELISA with immobilized human TfR ECD protein. A stock solution was prepared of human TfR. ECD protein in PBS. With the £moiar and Mw of target protein, the concentration of protein stock solution was determined. After calibrating the protein stock concentration, working solution was prepared by diluting the stock solution to 10 ug / mL protein with IX PBS. Human TfR ECD protein solution was added to ELISA plates, plates covered and incubated at 4 °C for overnight. Human TfR ECD protein solution was removed, and casein solution was added as a blocking agent to each well and the plates were incubated at room temperature. LNPs were prepared in serial dilutions with 3 replicates with a concentration series from 10,000 ng / mL to 30 ng / mL of RNA. LNPs samples were added to the blocked ELISA plates and incubated at room temperature with mild shaking on the orbital shaker for 1 hr. Plates were washed 3 times with PBS and treated with Triton X buffer to disrupt LNPs and disperse the Al exafl our647-HPRT siRNA in the well. Fluorescence of the cargo from bound LNPs was measured in a plate reader.Table 29. Binding to human TfR ECD protein of TfR-binding VHH decorated and non-decorated LNPs carrying Alexafluor 647 conjugated HPRT siRNAs as cargo (Relative fluorescence units = RFU).

Claims

WHAT IS CLAIMED IS:

1. A Transferrin Receptor 1 (TfR) binding variable domain of a heavy chain only antibody (VHH) (TfR binding VHH) comprising three complementarity determining regions CDR1, CDR2 and CDR3 in any one of the following sequences: SEQ ID NO: 1; SEQ ID NO: 8; SEQ ID NO: 13; SEQ ID NO: 20; SEQ ID NO: 23; SEQ ID NO: 28; SEQ ID NO: 32; SEQ ID NO: 35; SEQ ID NO: 38; SEQ ID NO: 41; SEQ ID NO: 44; SEQ ID NO: 47; SEQ ID NO: 49; SEQ ID NO: 52; SEQ ID NO: 64; or SEQ ID NO: 66.

2. The TfR binding VHH of claim 1, wherein: a. CDR1 comprises SEQ ID NO: 2, CDR 2 comprises SEQ ID NO: 3 and CDR3 comprises SEQ ID NO: 4; b. CDR1 comprises SEQ ID NO: 9, CDR 2 comprises SEQ ID NO: 10 and CDR3 comprises SEQ ID NO: 11; c. CDR1 comprises SEQ ID NO: 14, CDR 2 comprises SEQ ID NO: 15 and CDR3 comprises SEQ ID NO: 16; d. CDR1 comprises SEQ ID NO: 14, CDR 2 comprises SEQ ID NO: 21 and CDR3 comprises SEQ ID NO: 16; e. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 26; f. CDR1 comprises SEQ ID NO: 29, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 30; g. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33; h. CDR1 comprises SEQ ID NO: 36, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33; i. CDR1 comprises SEQ ID NO: 39, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33; j. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 42 and CDR3 comprises SEQ ID NO: 33;k. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 45 and CDR3 comprises SEQ ID NO: 33; l. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 21 and CDR3 comprises SEQ ID NO: 33; m. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 50 and CDR3 comprises SEQ ID NO: 33; n. CDR1 comprises SEQ ID NO: 53, CDR 2 comprises SEQ ID NO: 54 and CDR3 comprises SEQ ID NO: 33; o. CDR1 comprises SEQ ID NO: 53, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33; or p. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 54 and CDR3 comprises SEQ ID NO: 33.

3. The TfR binding VHH of claim 1 or 2, wherein: a. CDR1 consists essentially of SEQ ID NO: 2, CDR 2 consists essentially of SEQ ID NO: 3 and CDR3 consists essentially of SEQ ID NO: 4; b. CDR1 consists essentially of SEQ ID NO: 9, CDR 2 consists essentially of SEQ ID NO: 10 and CDR3 consists essentially of SEQ ID NO: 11; c. CDR1 consists essentially of SEQ ID NO: 14, CDR 2 consists essentially of SEQ ID NO: 15 and CDR3 consists essentially of SEQ ID NO: 16; d. CDR1 consists essentially of SEQ ID NO: 14, CDR 2 consists essentially of SEQ ID NO: 21 and CDR3 consists essentially of SEQ ID NO: 16; e. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 25 and CDR3 consists essentially of SEQ ID NO: 26; f. CDR1 consists essentially of SEQ ID NO: 29, CDR 2 consists essentially of SEQ ID NO: 25 and CDR3 consists essentially of SEQ ID NO: 30; g. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 25 and CDR3 consists essentially of SEQ ID NO: 33; h. CDR1 consists essentially of SEQ ID NO: 36, CDR 2 consists essentially of SEQ ID NO: 25 and CDR3 consists essentially of SEQ ID NO: 33;i. CDR1 consists essentially of SEQ ID NO: 39, CDR 2 consists essentially of SEQ ID NO: 25 and CDR3 consists essentially of SEQ ID NO: 33; j. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 42 and CDR3 consists essentially of SEQ ID NO: 33; k. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 45 and CDR3 consists essentially of SEQ ID NO: 33; l. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 21 and CDR3 consists essentially of SEQ ID NO: 33; m. CDR1 consists essentially of SEQ ID NO: 24, CDR 2 consists essentially of SEQ ID NO: 50 and CDR3 consists essentially of SEQ ID NO: 33; or n. CDR1 consists essentially of SEQ ID NO: 53, CDR 2 consists essentially of SEQ ID NO: 54 and CDR3 consists essentially of SEQ ID NO: 33; o CDR1 comprises SEQ ID NO: 53, CDR 2 comprises SEQ ID NO: 25 and CDR3 comprises SEQ ID NO: 33; or р. CDR1 comprises SEQ ID NO: 24, CDR 2 comprises SEQ ID NO: 54 and CDR3 comprises SEQ ID NO: 33.

4. The TfR binding VHH of claim 1, 2 or 3 wherein: a. CDR1 consists of SEQ ID NO: 2, CDR 2 consists of SEQ ID NO: 3 and CDR3 consists of SEQ ID NO: 4; b. CDR1 consists of SEQ ID NO: 9, CDR 2 consists of SEQ ID NO: 10 and CDR3 consists of SEQ ID NO: 11; с. CDR1 consists of SEQ ID NO: 14, CDR 2 consists of SEQ ID NO: 15 and CDR3 consists of SEQ ID NO: 16; d. CDR1 consists of SEQ ID NO: 14, CDR 2 consists of SEQ ID NO: 21 and CDR3 consists of SEQ ID NO: 16; e. CDR1 consists of SEQ ID NO: 24, CDR 2 consists of SEQ ID NO: 25 and CDR3 consists of SEQ ID NO: 26; f. CDR1 consists of SEQ ID NO: 29, CDR 2 consists of SEQ ID NO: 25 and CDR3 consists of SEQ ID NO: 30;g. CDR1 consists of SEQ ID NO: 24, CDR 2 consists of SEQ ID NO: 25 and CDR3 consists of SEQ ID NO: 33; h. CDR1 consists of SEQ ID NO: 36, CDR 2 consists of SEQ ID NO: 25 and CDR3 consists of SEQ ID NO: 33; i. CDR1 consists of SEQ ID NO: 39, CDR 2 consists of SEQ ID NO: 25 and CDR3 consists of SEQ ID NO: 33; j. CDR1 consists of SEQ ID NO: 24, CDR 2 consists of SEQ ID NO: 42 and CDR3 consists of SEQ ID NO: 33; k. CDR1 consists of SEQ ID NO: 24, CDR 2 consists of SEQ ID NO: 45 and CDR3 consists of SEQ ID NO: 33; l. CDR1 consists of SEQ ID NO: 24, CDR 2 consists of SEQ ID NO: 21 and CDR3 consists of SEQ ID NO: 33; m. CDR1 consists of SEQ ID NO: 24, CDR 2 consists of SEQ ID NO: 50 and CDR3 consists of SEQ ID NO: 33; or n. CDR1 consists of SEQ ID NO: 53, CDR 2 consists of SEQ ID NO: 54 and CDR3 consists of SEQ ID NO: 33; o. CDR1 consists of SEQ ID NO: 53, CDR 2 consists of SEQ ID NO: 25 and CDR3 consists of SEQ ID NO: 33; or p. CDR1 consists of SEQ ID NO: 24, CDR 2 consists of SEQ ID NO: 54 and CDR3 consists of SEQ ID NO: 33.

5. The TfR binding VHH of any one of claims 1-4, wherein the TfR binding VHH comprises an amino acid sequence which is at least 90% identical to SEQ ID NO: 1, 6, 8, 13, 18, 20, 23, 28, 32, 35, 38, 41, 44, 47, 49, 52, 64, 66, 75, 80, or 81.

6. The TfR binding VHH of any one of claims 1-5, wherein the TfR binding VHH comprises or consists essentially of an amino acid sequence of SEQ ID NO: 1, 6, 8, 13, 18, 20, 23, 28, 32, 35, 38, 41, 44, 47, 49, 52, 64, 66, 75, 80, or 81.

7. The TfR binding VHH of any one of claims 1-6, wherein the TfR binding VHH binds to human and non-human primate (NHP) TfR, and optionally to mouse TfR.

8. The TfR binding VHH of any one of claims 1-7, wherein the TfR binding VHH is conjugated to at least one agent.

9. The TfR binding VHH of any one of claims 1-8, wherein the TfR binding VHH is conjugated to one agent.

10. The TfR binding VHH of any one of claims 1-9, wherein the TfR binding VHH is conjugated to two agents.

11. The TfR binding VHH of any one of claims 8-10, wherein the agent is a polypeptide; a peptide; a nucleic acid molecule; a dye; a small molecule; nanoparticles (NPs); lipid NPs; a liposome; an exosome; an antibody, an antibody Fc portion or antigen binding fragments thereof; a viral particle; or any combination thereof.

12. The TfR binding VHH of any one of claims 8- 11, wherein the agent is conjugated to the TfR binding VHH directly.

13. The TfR binding VHH of any one of claims 8-11, wherein the agent is conjugated to the TfR binding VHH via a linker.

14. The TfR binding VHH of any one of claims 8-13, wherein the agent is a nucleic acid molecule.

15. The TfR binding VHH of claim 14, wherein the agent is an siRNA.

16. The TfR binding VHH of any one of claims 13-15, wherein the linker is a SMCC linker.

17. The TfR binding VHH of claim 16, wherein the linker is a SMCC linker conjugated to the antisense strand of the siRNA.

18. The TfR binding VHH of claim 16, wherein the linker is a SMCC linker conjugated to the sense strand of the siRNA.

19. The TfR binding VHH of any one of claims 8-13, wherein the agent is a half-life extender.

20. The TfR binding VHH of claim 19, wherein the half-life extender is an albumin (ALB) binding polypeptide.

21. The TfR binding VHH of claim 20, wherein the albumin binding polypeptide is an albumin (ALB) binding VHH which binds to human serum albumin.

22. The TfR binding VHH of claim 21, wherein the ALB binding VHH comprises three complementarity determining regions, CDR1, CDR2, and CDR3, wherein CDR1 comprises SEQ ID NO: 61; CDR2 comprises SEQ ID NO:62; and CDR3 comprises SEQ ID NO:63 or SEQ ID NO: 71.

23. The TfR binding VHH of any one of claims 21-22, wherein the ALB binding VHH comprises SEQ ID NO: 60 or SEQ ID NO: 70, and optionally comprises a cysteine at the C- terminal end.

24. The TfR binding VHH of any one of claims 21-22, wherein the ALB binding VHH comprises SEQ ID NO: 60 or SEQ ID NO: 70, without the two C-terminal proline amino acids [PP], and optionally comprises a cysteine at the C-terminal end.

25. The TfR binding VHH of any one of claims 19-24, wherein the C-terminal residue of the half-life extender is conjugated either directly or via a linker to the N-terminal residue of the TfR binding VHH.

26. The TfR binding VHH of any one of claim 19-24, wherein the C-terminal residue of the TfR binding VHH is conjugated either directly or via a linker to the N-terminal residue of the half-life extender.

27. The TfR binding VHH of claims 25 or 26, wherein the linker is a glycine rich linker.

28. The TfR binding VHH of claim 27, wherein the glycine rich linker comprises the amino acid sequence of (G4Q)n(SEQ ID NO:59), wherein n=l, 2, 3, 4 or 5.

29. The TfR binding VHH of claim 10, wherein the first agent is a half-life extender which is an ALB binding VHH and wherein the second agent is an siRNA, wherein the siRNA comprises a sense strand and an antisense strand.

30. The TfR binding VHH of claim 29, wherein the ALB binding VHH is conjugated via a glycine rich linker to the TfR binding VHH, and wherein the siRNA is conjugated via achemical linker to (1) a C terminal cysteine residue in the ALB binding VHH or (2) a C terminal cysteine residue in the TfR binding VHH.

31. A conjugate of the following formula:[[first agent]-[linker 1 ]]<n)-[TfR VHH]-[[linker 2]- [second agent]](m) wherein the [TfR VHH] comprises the TfR binding VHH of any one of claims 1-7; the linker 1 and linker 2 are optional; wherein m and n are independent, n=0, 1, 2 or 3, and m=0, 1, 2 or 3.

32. The conjugate of claim 31, wherein the first and / or the second agent is a polypeptide; a peptide; a nucleic acid molecule; a dye; a small molecule; nanoparticles (NPs); lipid NPs; a liposome; an exosome; an antibody, an antibody Fc portion or antigen binding fragments thereof; a viral particle; or any combination thereof.

33. The conjugate of claim 32, wherein the first agent is conjugated via the linker 1 and / or the second agent is conjugated via the linker 2 to the TfR VHH.

34. The conjugate of any one of claims 31-33, wherein n=0 and m=l, wherein when m=l the second agent is a nucleic acid molecule.

35. The conjugate of any one of claims 31-33, wherein n=0 and m=2, wherein one [second agent] is a half-life extender and the other [second agent] is a nucleic acid molecule.

36. The conjugate of any one of claims 31-33, wherein when n=l, the [first agent] is a nucleic acid molecule, and m=2, wherein one [second agent] is a half-life extender and the other [second agent] is a nucleic acid molecule.

37. The conjugate of any one of claims 34-36, wherein the nucleic acid molecule of [the first agent] is an siRNA and the nucleic acid molecule of the [second agent] is an siRNA conjugated to the TfR binding VHH, wherein each siRNA comprises a sense strand and an antisense strand.

38. The conjugate of any one of claims 34-37, wherein the nucleic acid molecule is an siRNA conjugated to a cysteine residue in the TfR binding VHH, wherein the siRNA comprises a sense strand and an antisense strand.

39. The conjugate of claim 35, wherein the C-terminal residue of the TfR binding VHH is conjugated either directly or via the linker 2 to the N-terminal residue of the half-life extender.

40. The conjugate of any one of claims 31-33, wherein when n=l, the [first agent] is a nucleic acid molecule, and m=l, wherein the [second agent] is a half-life extender further comprising an siRNA, wherein the siRNA is conjugated to a cysteine residue of the half-life extender.

41. The conjugate of any one of claims 35-40, wherein the half-life extender is an albumin (ALB) binding polypeptide.

42. The conjugate of any one of claims 35-41, wherein the ALB binding polypeptide is an ALB binding VHH comprising three complementarity determining regions, CDR1, CDR2, and CDR3, wherein CDR1 comprises SEQ ID NO: 61; CDR2 comprises SEQ ID NO:62; and CDR3 comprising SEQ ID NO:63 or SEQ ID NO: 71.

43. The TfR binding VHH of any one of claims 35-42, wherein the ALB binding VHH comprises SEQ ID NO: 60 or SEQ ID NO: 70, and optionally comprises a cysteine at the C- terminal end.

44. The TfR binding VHH of any one of claims 35-42, wherein the ALB binding VHH comprises SEQ ID NO: 60 or SEQ ID NO: 70 without the two C-terminal [PP] amino acids, and optionally comprises a cysteine at the C-terminal end.

45. The conjugate of any one of claims 31, 33-43, wherein the linker 1 or the linker 2 is a linker comprising an amino acid sequence of (G4Q)n (SEQ ID NO:59), wherein n= 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

46. The TfR binding VHH of claim 45, wherein the linker 1 or the linker 2 comprises the amino acid sequence of (G4Q)n(SEQ ID NO:59), wherein n=l, 2, 3, 4 or 5.

47. The conjugate of any one of claims 31 or 33-43, wherein the linker 1 or the linker 2 is a SMCC linker.

48. The conjugate of any one of claims 37-47, wherein the linker 1 or the linker 2 is a SMCC linker conjugated to the antisense strand of the siRNA agent.

49. The conjugate of any one of claims 37-47, wherein the linker 1 or the linker 2 is a SMCC linker conjugated to the sense strand of the siRNA agent.

50. A conjugate of the following formulae:(a) [second agent]-linker l-[TfR VHH]-linker 2-[ALB VHH] or(b) [TfR VHH]-linker 1-[ALB VHH]-linker 2- [second agent] wherein the [TfR VHH] comprises the TfR binding VHH of any one of claims 1-7 and the [ALB VHH] comprises an albumin binding VHH; and the linker 1 or the liner 2 is optional.

51. The conjugate of claim 50, wherein the second agent is a polypeptide; a peptide; a nucleic acid molecule; a dye; a small molecule; nanoparticles (NPs); lipid NPs; a liposome; an exosome; an antibody, an antibody Fc portion or antigen binding fragments thereof; a viral particle; or any combination thereof52. The conjugate of claim 50 or 51, wherein the second agent is conjugated via the linker 1 to the TfR binding VHH or via the linker 2 to the ALB VHH.

53. The conjugate of any one of claims 50-52, wherein the second agent is a nucleic acid molecule.

54. The conjugate of claim 53, wherein the nucleic acid molecule is an siRNA conjugated via the linker 1 to the TfR binding VHH, wherein the siRNA comprises a sense strand and an antisense strand.

55. The conjugate of any one of claims 53-54, wherein the nucleic acid molecule is conjugated via the linker 1 to a cysteine residue in the TfR binding VHH56. The conjugate of claim 53, wherein the nucleic acid molecule is an siRNA conjugated via the linker 2 to the ALB binding VHH, wherein the siRNA comprises a sense strand and an antisense strand.

57. The conjugate of any one of claims 53 or 56, wherein the nucleic acid molecule is an siRNA conjugated via the linker 2 to a cysteine residue in the ALB binding VHH, wherein the siRNA agent comprises a sense strand and an antisense strand.

58. The conjugate of any one of claims 50-57, wherein the ALB binding VHH comprises three complementarity determining regions, CDR1, CDR2, and CDR3, wherein CDR1 comprises SEQ ID NO: 61; CDR2 comprises SEQ ID NO:62; and CDR3 comprising SEQ ID NO:63 or SEQ ID NO: 70.

59. The conjugate of any one of claims 50-58, wherein the ALB binding VHH comprises SEQ ID NO: 60 or SEQ ID NO: 70, and optionally comprises a cysteine at the C-terminal end.

60. The conjugate of any one of claims 50-58, wherein the ALB binding VHH comprises SEQ ID NO: 60 or SEQ ID NO: 70 without the two C-terminal [PP] amino acids, and optionally comprises a cysteine at the C-terminal end.

61. The conjugate of any one of claims 50-60, wherein the second agent is conjugated to a polypeptide comprising SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 or SEQ ID NO: 58.

62. The conjugate of any one of claim 54-61, wherein the siRNA is conjugated to a polypeptide comprising SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID N: 57 or SEQ ID NO: 58.

63. The conjugate (a) of any one of claims 50- 62, wherein the linker l is a SMCC linker.

64. The conjugate (a) of any one of claims 54-62, wherein the linker l is a SMCC linker conjugated to an antisense strand of an siRNA agent and the linker 2 is a glycine rich linker.

65. The conjugate (a) of any one of claims 50- 62, wherein the linker l is a SMCC linker conjugated to a sense strand of an siRNA agent and the linker 2 is a glycine rich linker.

66. The conjugate (b) of any one of claims 50- 62, wherein the linker 1 is glycine rich linker and the linker 2 is a SMCC linker conjugated to an antisense strand of an siRNA agent.

67. The conjugate (b) of any one of claims 50-62, wherein the linker 1 is glycine rich linker and the linker 2 is a SMCC linker conjugated to a sense strand of an siRNA agent.

68. The conjugate (a) or (b) of any one of claims 64-67, wherein the glycine rich linker comprises an amino acid sequence of (G- Qjn (SEQ ID NO:59), wherein n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

69. The conjugate (a) or (b) of any one of claims 64-68, wherein the linker 1 or the linker 2 comprises the amino acid sequence of (G4Q)n(SEQ ID NO:59), wherein n=l, 2, 3, 4 or 5.

70. A pharmaceutical composition comprising the TfR binding VHH of any one of claims 1-30 or the conjugate of any one of claims 31-69 and a pharmaceutically acceptable carrier or excipient.

71. A method of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 70.

72. A method for delivering an agent to a subject comprising administering to a subject in need thereof a therapeutically effective amount of pharmaceutical composition of claim 70.

73. The method of claim 70 or 71, wherein the pharmaceutical composition is administered systemically; subcutaneously or intravenously.

74. The TfR binding VHH of any one of claims 1-30, the conjugate of claims 31-69 or the pharmaceutical composition of claim 70 for use in therapy.

75. Use of the TfR binding VHH of any one of claims 1-30 or the conjugate of claims 31-69 in the manufacture of a medicament for treatment of a disease or disorder.

76. The TfR binding VHH of any one of claims 1-30 or the conjugate of claims 31-69 for use in the treatment of a disease or disorder in a subject, or for delivering a therapeutic agent to a subject.

77. A nucleic acid molecule comprising a first nucleic acid sequence encoding the TfR binding VHH of any one of claims 1-30.

78. The nucleic acid molecule of claim 77 further comprising a second nucleic acid sequence encoding any one of a protein agent, an antibody or antigen binding fragment thereof, an antigen binding VHH, or a combination thereof, wherein the second nucleic acid sequence is operably linked to the fist nucleic acid sequence.

79. The nucleic acid molecule of claim 78, wherein the second nucleic acid sequence encodes an ALB binding VHH.

80. An expression vector comprising the nucleic acid molecule of claim 77, 78 or 79.

81. A host cell comprising the nucleic acid molecule of claim 77, 78 or 79.

82. A host cell comprising the expression vector of claim 80.

83. The host cell of claim 80 or 81, wherein the host cell is a mammalian cell or a yeast cell.