Improved transferrin receptor binding proteins

Abstract

Transferrin receptor binding polypeptides, and their use, are provided that have an amino acid sequence at least 50%, identical to the amino acid sequence of SEQ ID NO: 1, wherein (a) the polypeptide is identical to SEQ ID NO: 1 at residues corresponding to residues 20, 23. 24, 27, 28, 31, 32, 34, 35. 36, 37, 74, 77, 78, 81, 82. 84, 85, and 88 in SEQ ID NO: 1; and (b) the polypeptide is substituted relative to SEQ ID NO: I as shown in Table 1 at least at 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34. 35, or more residues relative to SEQ ID NO: 1.

Description

[0001]Improved Transferrin Receptor Binding Proteins ^ Sequence Listing Statement A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on May 6th, 2024 having the file name “23-^ 0492-WO_SequenceListing” and is 32.0 kb in size. Background Human Transferrin Receptor (hTfR) transports transferrin, the major carrier of iron in the body, across the blood brain barrier (BBB) via receptor-mediated transcytosis. This^ process can be exploited to deliver therapeutic payloads into the brain parenchyma that would otherwise be blocked by the BBB. Thus, hTfR is an attractive target candidate for the development of BBB traversing vehicles. Summary ^ In one aspect, the disclosure provides transferrin receptor binding polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, or 98%, identical to the amino acid sequence of SEQ ID NO:1, wherein (a) the polypeptide is identical to SEQ ID NO:1 at residues corresponding to residues 20, 23, 24, 27, 28, 31, 32, 34, 35, 36, 37, 74, 77, 78, 81, 82, 84, 85, and 88 in SEQ ID^ NO:1; and (b) the polypeptide is substituted relative to SEQ ID NO:1 as shown in Table 1 at least at 5, 6, 7, 8, 9, 10, 11, 12, 13, 1415, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or more residues relative to SEQ ID NO:1. In one embodiment, the polypeptide is identical to SEQ ID NO:1 at residues corresponding to residues 7, 9, 11, 13, 16, 18, 20-21, 23-24, 27-28, 30-32, 34-37, 41, 47, 49-51, 56, 58, 60, 65-66, 68, 70-71, 74-75, 77-^ 78, 80-82, 84-85, and 87-88; and optionally identical to SEQ ID NO:1 at residues corresponding to residues 3-5 in SEQ ID NO:1. In another embodiment, the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all 14 substitutions relative to SEQ ID NO:1 selected from the group consisting of Q6K, A8F, E10K, R14E, E19K, R29K, A33S, R42E, S48A, R52K, R53K, A64E, L76E, and R79K. In a further embodiment, the ^ polypeptide comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, ^ 94%, 96%, 98%, or 100% identical to the amino acid sequence selected from SEQ ID NO:2- 7, wherein residues in parentheses are optional and may be present or absent. In one embodiment, the polypeptide comprises one or more additional functional domains. In non-limiting embodiments, the one or more functional domains may be selected^ from the group consisting of detection domains, stabilization domains, therapeutic moieties, diagnostic moieties and drug delivery vehicles. In another non-limiting embodiment, the one or more functional domains may comprise a stabilization domain, including but not limited to polyethylene glycol (PEG), albumin, hydroxyethyl starch (HES), conformationally disordered polypeptide sequence composed of the amino acids Pro, Ala, and / or Ser ('PASylation'), and a^ mucin diffusivity polypeptide composed of amino acids Lys and Ala, with or without Glu. In one embodiment, the disclosure provides nucleic acids encoding the transferrin receptor binding polypeptide of any embodiment or combination of embodiments herein. In another embodiment, the disclosure provides expression vectors comprising the nucleic acid of any embodiment or combination of embodiments herein, operatively linked to a promoter.^ In a further embodiment, the disclosure provides host cell comprising the transferrin receptor binding polypeptide, the nucleic acid, and / or the expression vector of any embodiment or combination of embodiments herein. In one embodiment, the disclosure provides pharmaceutical compositions, comprising the polypeptide, the nucleic acid, the expression vector, or the host cell of any embodiment or combination of embodiments herein, and a^ pharmaceutically acceptable carrier. The disclosure also provides methods for using the polypeptide, the nucleic acid, the expression vector, the host cell, and / or the pharmaceutical composition of any embodiment or combination of embodiments herein, including but not limited to treating or limiting arenavirus infection; delivery of therapeutics for treating tumors; and fusion to therapeutics^ such as biologicals (including but not limited to protein, nucleic acid, and antibody therapeutics) to increase serum half-life of the therapeutic. Description of the Figures Figure 1. Design of improved binders. Binding of 2DS25.5 (PDB 6wrw) on the left^ and Alphafold2 models of the three optimized variants. Interface residues are indicated in sticks and were not redesigned. Figure 2. Biolayer interferometry assays. A graph showing that the three improved designs still bind to the ectodomain of the human Transferrin receptor, using a biolayer interferometry assay. ^ Detailed Description / Claims All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several^ well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol.185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), “Guide to Protein Purification” in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al.^ 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2ndEd. (R.I. Freshney.1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp.109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), Dang, B. et al. SNAC-tag for sequence-specific chemical protein cleavage. Nat. Methods 16, 319–322 (2019), and the Ambion 1998 Catalog (Ambion, Austin, TX). ^ As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I),^ leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Any N-terminal methionine residue in any polypeptide of the disclosure may be present or may be deleted. In all embodiments of the polypeptides disclosed herein 1, 2, 3, 4,^ or 5 residues may be deleted from the N-terminus and / or the C-terminus of the polypeptide while retaining activity. All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise. Unless the context clearly requires otherwise, throughout the description and the^ claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below” and ^ words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application. In a first aspect, the disclosure provides transferrin receptor binding polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,^ 90%, 92%, 94%, 96%, or 98%, identical to the amino acid sequence of SEQ ID NO:1, wherein (a) the polypeptide is identical to SEQ ID NO:1 at residues corresponding to residues 20, 23, 24, 27, 28, 31, 32, 34, 35, 36, 37, 74, 77, 78, 81, 82, 84, 85, and 88 in SEQ ID NO:1; and ^ (b) the polypeptide is substituted relative to SEQ ID NO:1 as shown in Table 1 at least at 5, 6, 7, 8, 9, 10, 11, 12, 13, 1415, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or more residues relative to SEQ ID NO:1. >2DS25.5 ^ DEEEIQKAIEELLRKGVSEEEAAIIIVQRFNVAVVVVVQDERQGKHISEYIRRYIPEADVILFANLVV IKVETHELSTRVWEAAQKAY (SEQ ID NO:1; 88 amino acids) The polypeptide of SEQ ID NO:1 (2DS25.5) is a previously described transferrin receptor binding protein (see PCT application publication WO / 2022 / 026555, incorporated by^ reference herein in its entirety). The polypeptides of the present disclosure have substantially improved expression levels and solubility relative to those disclosed in WO / 2022 / 026555, and can thus be more efficiently coupled to therapeutic cargos for blood brain barrier (BBB) traversal. Residues 20, 23, 24, 27, 28, 31, 32, 34, 35, 36, 37, 74, 77, 78, 81, 82, 84, 85, and 88 in^ SEQ ID NO:1 are in the polypeptide-transferrin receptor protein binding interface, and were retained in polypeptides of the present disclosure. Table 1 shows the location of substitutions relative to 2DS25.5 residues in exemplary polypeptides of the disclosure, and does not show the residues that are maintained relative to SEQ ID NO:1 as recited in (a) above. There are 3 columns in Table 1 of substitutions in an^ exemplary polypeptide of the disclosure relative to SEQ ID NO:1 (combo35_M0; combo35_M3; and combo35_M5). The amino acid position number relative to SEQ ID NO:1 is in the first column, and permissible substitutions at a given residue can be identified by looking across a given row in columns combo35_M0; combo35_M3; and combo35_M5. ^ For example, amino acid position 1 in SEQ ID NO:1 is D, and it can be substituted with N or S. Those of skill in the art will understand other permissible substitutions based on Table 1. ^ Table 1: Redesigned residues (“-“ indicates that residue is the same as in original) ^ In one embodiment, the polypeptide is identical to SEQ ID NO:1 at residues^ corresponding to residues 7, 9, 11, 13, 16, 18, 20-21, 23-24, 27-28, 30-32, 34-37, 41, 47, 49- ^ 51, 56, 58, 60, 65-66, 68, 70-71, 74-75, 77-78, 80-82, 84-85, and 87-88; and optionally identical to SEQ ID NO:1 at residues corresponding to residues 3-5 in SEQ ID NO:1. In another embodiment, the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all 14 substitutions relative to SEQ ID NO:1: Q6K, A8F, E10K, R14E, E19K, R29K, A33S, R42E,^ S48A, R52K, R53K, A64E, L76E, and / or R79K. In specific embodiments, the polypeptide comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% identical to the amino acid sequence selected from SEQ ID NO:2-7, wherein residues in parentheses are optional and may be present or absent. The sequences of SEQ ID NO:2-7 are shown in Table 2. ^ Table 2 The transferrin receptor binding polypeptides of the disclosure may comprise additional residues. In some embodiments, the polypeptides may comprise additional residues at the N-terminus and / or C-terminus of the polypeptides. Any additional residues^ may be added as deemed appropriate for an intended purpose. In various non-limiting embodiments, the polypeptide may further comprise a functional domain. The polypeptides may comprise any additional functional domain(s), including but not limited to detection domains, stabilization domains, therapeutic moieties, diagnostic moieties and drug delivery ^ vehicle. The functional domains may be added as a translational fusion with the polypeptide, or may be chemically coupled to the polypeptide. Any suitable chemical coupling may be used, including but not limited to covalent linkage to a cysteine residue. For example, any surface amino acid residue in the polypeptide not present at or near the binding interface can^ be mutated to cysteine (see above for positions of interface residues). In one embodiment, the one or more additional functional domains are present at the N and / or C terminus of the polypeptide as a translational fusion. In one embodiment, the one or more functional domains comprises a stabilization domain , including but not limited to polyethylene glycol (PEG), albumin, hydroxyethyl starch (HES), conformationally disordered polypeptide sequence^ composed of the amino acids Pro, Ala, and / or Ser ('PASylation'), and / or a mucin diffusivity polypeptide composed of amino acids Lys and Ala, with or without Glu. In another embodiment, the functional domain may comprise a helical repeat protein. This embodiment results in a polypeptide with a longer residency time in the blood. In non- limiting embodiments, the helical repeat proteins comprises an amino acid sequence at least^ 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 8-13. The amino acid sequences of SEQ ID NO:8-13 are provided in Table 3. Table 3 7^ ^ In another aspect, the disclosure provides nucleic acids encoding the polypeptide of any embodiment or combination of embodiments of the disclosure. The nucleic acid sequence may comprise single stranded or double stranded RNA or DNA in genomic or cDNA form, or^ DNA-RNA hybrids, each of which may include chemically or biochemically modified, non- natural, or derivatized nucleotide bases. Such nucleic acid sequences may comprise additional sequences useful for promoting expression and / or purification of the encoded peptide or chimeric molecular construct, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and^ secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptide or fusion protein of the disclosure. In a further aspect, the disclosure provides expression vectors comprising the nucleic acid of any aspect of the disclosure operatively linked to a suitable control sequence, such as a^ promoter. “Expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. “Control sequences” operably linked to the nucleic acid sequences of the disclosure are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they^ function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence are considered “operably linked” to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of^ any type, including but not limited plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible ^ promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA. In various embodiments, the expression vector may comprise a plasmid, viral-based vector, or any other suitable expression vector. ^ In another aspect, the disclosure provides host cells that comprise the polypeptide, nucleic acid or expression vector (i.e.: episomal or chromosomally integrated) disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably engineered to incorporate the expression vector of the disclosure, using techniques including but not limited to bacterial transformations, calcium phosphate co-^ precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. In another aspect, the disclosure provides pharmaceutical compositions, comprising the polypeptide, the recombinant nucleic acid, the expression vector, or the recombinant host cell of any of any embodiment or combination of embodiments, and a pharmaceutically^ acceptable carrier. The pharmaceutical compositions of the disclosure can be used, for example, in the methods of the disclosure described herein. The pharmaceutical composition may further comprise (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and / or (g) a buffer. In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a^ histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. The pharmaceutical composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose. In certain embodiments, the pharmaceutical composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate,^ thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a bulking agent, like glycine. In yet other embodiments, the pharmaceutical composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate- 60, polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan^ trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof. The pharmaceutical composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood. Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the ^ pharmaceutical composition additionally includes a stabilizer, e.g., a molecule which, when combined with a protein of interest substantially prevents or reduces chemical and / or physical instability of the protein of interest in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and^ arginine hydrochloride. The polypeptide, nucleic acid, expression vector, or cell of any embodiment or combination of embodiments herein may be the sole active agent in the pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for an intended use. ^ In a further aspect, the disclosure provides methods for using, or a use of the polypeptide, the recombinant nucleic acid, the expression vector, the recombinant host cell, and / or the pharmaceutical composition of any embodiment or combination of embodiments of the disclosure, for any suitable purpose including but not limited to those disclosed herein. In various embodiments, the purpose includes, but is not limited to, treating or limiting^ arenavirus infection; delivery of therapeutics for treating tumors; and fusion to therapeutics such as biologicals (including but not limited to protein, nucleic acid, and antibody therapeutics) to increase serum half-life of the therapeutic. The TfR apical domain (where the polypeptides of the disclosure bind) also serves as the site for the entry of new world arenaviruses into cells (Abraham et al.2010, Nat. Struct.^ Mol. Biol.17, 438–444 (2010); Clark et al.2018; Nat. Commun.9, 1884 (2018).). A number of these viruses such as Machupo, Junín, Guanarito and Sabiá viruses cause hemorrhagic fevers with high fatality rates. Hence, the polypeptides of the disclosure may block viral entry the same way antibodies that bind to the apical domain can block viral entry. TfR is overexpressed in a number of tumors (Daniels-Wells, T. R. and Penichet, M. L.^ Transferrin receptor 1: a target for antibody-mediated cancer therapy. Immunotherapy 8, 991- 994 (2016)) raising the possibility of targeted therapy using the disclosed polypeptides as targeting module. Similarly since, TfR is expressed throughout the body, the disclosed polypeptides may be exploited as a general delivery platform. Finally, TfR binding proteins have been suggested to be useful as recycling factors to^ increase the lifetime of biologics (including but not limited to protein, nucleic acid, and antibody therapeutics) in serum. Like the Fc receptor, TfR continuously cycling between the cell surface and endocytotic vesicles as part of its natural function to deliver serum Tf into cells. Fusion of biologics to Tf has increased their serum lifetime. Fusions of biologics to the ^ disclosed polypeptides could likewise lead to increased lifetime of the biologic. Thus, in another embodiment, the disclosure provides fusion proteins, comprising: (a) the transferrin receptor binding polypeptide of any embodiment or combination of embodiments herein; and ^ (b) a protein therapeutic. In one embodiment, the protein therapeutic comprises a therapeutic antibody or antigen-binding fragment or portion thereof. The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While the specific embodiments of, and^ examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. Examples ^ The human transferrin receptor is expressed on the blood-brain-barrier (BBB) and can shuttle interacting proteins across the BBB to deliver molecules such as therapeutics into the brain parenchyma. We de novo designed a small protein, 2DS25.5, that interacts with TfR and is transported across the BBB in brain-on-chip models. Here we present variants of 2DS25.5 that are redesigned and have substantially improved expression levels and solubility. These^ designs can be used to efficiently couple to therapeutic cargos and have improved BBB traversal properties. Results In order to create improved transferrin receptor binding proteins, we started with^ 2DS25.5, which is a previously designed protein that has been evolved in vitro to bind the human transferrin receptor with an equilibrium dissociation constant of 50 nM in biolayer interferometry assays. We used a deep learning algorithm to redesign all residues of 2DS25.5 with exception of the residues that directly interact with the transferrin receptor (Figure 1). The structures of the sequences that were obtained were next predicted using^ AlphaFold2TM(Jumper et al, 2021) (Figure 1). Designs with an average plDDT of >=90 and rmsd of < 1 Å to the crystal structure of the design 2DS25.5 (Sahtoe et al, 2021) were ordered as synthetic genes and expressed in E.coli with an C-terminal polyhistidine tag followed by purification using immobilized metal affinity chromatography and size exclusion chromatography (SEC). Design combo35_M0 (SEQ ID NO:2 and 5), combo35_M3 (SEQ ID ^ NO:3 and 6) and combo35_M5 (SEQ ID NO:4 and 7) expressed better and had monodispersed peaks in SEC compared to 2DS25.5 (Sahtoe et al, 2021). We tested if these designs could still bind the human transferrin receptor ectodomain in biolayer interferometry assays and confirmed that all three designs could still bind tightly (Figure 2). ^ In summary, we designed optimized variants of 2DS25 that express better and have improved solubility compared to the original design. ^ References Dauparas et al 2022, science.org / doi / 10.1126 / science.add2187. ^ Sahtoe et al 2021, pnas.org / doi / 10.1073 / pnas.2021569118. Jumper et al 2021, nature.com / articles / s41586-021-03819-2. ^

Claims

We claim:

1. A transferrin receptor binding polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, or 98%, identical to the amino acid sequence of SEQ ID NO:1, wherein ^ (a) the polypeptide is identical to SEQ ID NO:1 at residues corresponding to residues 20, 23, 24, 27, 28, 31, 32, 34, 35, 36, 37, 74, 77, 78, 81, 82, 84, 85, and 88 in SEQ ID NO:1; and (b) the polypeptide is substituted relative to SEQ ID NO:1 as shown in Table 1 at least at 5, 6, 7, 8, 9, 10, 11, 12, 13, 1415, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,^ 30, 31, 32, 33, 34, 35, or more residues relative to SEQ ID NO:

1.

2. The transferrin receptor binding polypeptide of claim 1, wherein the polypeptide is identical to SEQ ID NO:1 at residues corresponding to residues 7, 9, 11, 13, 16, 18, 20-21, 23- 24, 27-28, 30-32, 34-37, 41, 47, 49-51, 56, 58, 60, 65-66, 68, 70-71, 74-75, 77-78, 80-82, 84-^ 85, and 87-88; and optionally identical to SEQ ID NO:1 at residues corresponding to residues 3-5 in SEQ ID NO:

1.

3. The transferrin receptor binding polypeptide of claim 1 or 2, wherein the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all 14 substitutions relative to SEQ ID^ NO:1 selected from the group consisting of Q6K, A8F, E10K, R14E, E19K, R29K, A33S, R42E, S48A, R52K, R53K, A64E, L76E, and R79K.

4. The transferrin receptor binding polypeptide of any one of claims 1-3, comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%^ identical to the amino acid sequence selected from SEQ ID NO:2-7.

5. The transferrin receptor binding polypeptide of any one of claims 1-3, comprising an amino acid sequence at least 90% identical to the amino acid sequence selected from SEQ ID NO:2-7, wherein residues in parentheses are optional and may be present or absent. ^ 6. The transferrin receptor binding polypeptide of any one of claims 1-3, comprising an amino acid sequence at least 95% identical to the amino acid sequence selected from SEQ ID NO:2-7. ^The transferrin receptor binding polypeptide of any one of claims 1-6, comprising one or more additional functional domains.

8. The transferrin receptor binding polypeptide of claim 7, wherein the one or more^ additional functional domains are covalently bound to the polypeptide via linkage to a cysteine residue.

9. The transferrin receptor binding polypeptide of claim 7 or 8 wherein the one or more additional functional domains are present at the N and / or C terminus of the polypeptide.^ 10. The transferrin receptor binding polypeptide of any one of claims 7-9, wherein the one or more functional domains are selected from the group consisting of detection domains, stabilization domains, therapeutic moieties, diagnostic moieties and drug delivery vehicles. ^ 11. The transferrin receptor binding polypeptide of any one of claims 7-10, wherein the one or more functional domains comprises a stabilization domain selected from the group consisting of polyethylene glycol (PEG), albumin, hydroxyethyl starch (HES), conformationally disordered polypeptide sequence composed of the amino acids Pro, Ala, and / or Ser ('PASylation'), and a mucin diffusivity polypeptide composed of amino acids Lys^ and Ala, with or without Glu. 12 A nucleic acid encoding the transferrin receptor binding polypeptide of any one of claims 1-11. ^ 13. An expression vector comprising the nucleic acid of claim 12 operatively linked to a promoter.

14. A host cell comprising the transferrin receptor binding polypeptide, the nucleic acid, and / or the expression vector of any one of claims 1-13. ^ 15. A pharmaceutical composition, comprising the polypeptide, the nucleic acid, the expression vector, or the host cell of any one of claims 1-14, and a pharmaceutically acceptable carrier. ^16. A method for using, or a use of the polypeptide, the nucleic acid, the expression vector, the host cell, and / or the pharmaceutical composition of any one of claims 1-15, for any suitable purpose including but not limited to those disclosed herein. ^ 17. The method or use of claim 16, wherein the purpose is selected from the group consisting of treating or limiting arenavirus infection; delivery of therapeutics for treating tumors; and fusion to therapeutics such as biologicals (including but not limited to protein, nucleic acid, and antibody therapeutics) to increase serum half-life of the therapeutic. ^ 18. A method for treating or limiting arenavirus infection, comprising administering to a person having or at risk of an arenavirus infection an amount of the polypeptide, the nucleic acid, the expression vector, the host cell, and / or the pharmaceutical composition of any one of claims 1-15 to treat or limit the arenavirus infection. ^ 19. A fusion protein, comprising: (a) the transferrin receptor binding polypeptide of any one of claims 1-11; and (b) a protein therapeutic. ^ ^