A VNAR-based brain shuttle for payload conjugation and gene therapy

Abstract

The present disclosure relates to brain shuttles based on single domain VNAR antibodies that target receptors at the blood-brain barrier, which transport essential metabolites including the transferrin receptor TfR1 and the amino acid transporter by expanding on the payload diversity modifications in an Ig chain or Fc domain to allow site-specific conjugation via an accessible sulfhydryl group present on a surface exposed cysteine. Potential payloads that could be delivered via receptor-mediated transport using VNAR antibodies to TfR1 include oligonucleotides, polypeptides, cytotoxins, enzymes, proteins, peptides, small molecules, radioisotopes, fluorophores, polymers, lipids, nanoparticles, liposomes, capsids, and viruses.

Description

A VNAR-based brain shuttle for payload conjugation and gene therapyCROSS REFERENCE TO RELATED APPLICATION

[0001] This PCT application claims priority to provisional application U.S. Serial No. 63 / 277,590, filed November 6, 2024, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains sequences and includes a Sequence Listing which is hereby incorporated by reference in its entirety.FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to brain shuttles based on single domain VNAR antibodies that target receptors at the blood-brain barrier, which transport essential metabolites including the transferrin receptor TfRl and the amino acid transporter, by expanding on the payload diversity modifications in an Ig chain or Fc domain to allow sitespecific conjugation via an accessible sulfhydryl group present on a surface exposed cysteine. Potential payloads that could be delivered via receptor-mediated transport using VNAR antibodies to TfRl include oligonucleotides, polypeptides, cytotoxins, enzymes, proteins, peptides, small molecules, radioisotopes, fluorophores, polymers, lipids, nanoparticles, liposomes, capsids, and viruses.BACKGROUND OF THE DISCLOSURE

[0004] Using a variety of in vitro and in vivo selection approaches, single domain VNARs to TfRl, for example, have been identified that can shuttle therapeutic payloads across the brain capillary endothelium forming the impermeable blood brain barrier (“BBB”) (see, e.g., WO20 15 / 200883; WO2016 / 077840; WO2018 / 031424; WO2019 / 089395; W02020 / 056327). VNARs to TfRl that function in vivo as effective BBB shuttles have remarkably different pharmacokinetic, potency and side-effect profiles than those found for monoclonal antibodies to the same receptor. When these VNARs are fused to Fc domains, to immunoglobulins (Igs) or other Ig domains, herein collectively referred to as VNAR antibodies, they are also effective brain shuttles to deliver biomolecules, e.g. therapeutic and diagnostic agents, into the brain.

[0005] Brain shuttles based on VNAR antibodies that target receptors at the blood-brain barrier (BBB), such as receptors which transport essential metabolites including the transferrin receptor TfRl and the amino acid transporter CD98 are also being developed (Hasler, Rutkowski et al. 2016, Wicher, Szary et al. 2019, Rutkowski, Walsh et al. 2021, Stocki, Szary et al. 2021, Clarke, Stocki et al. 2022, Stocki, Szary et al. 2022) and have been shown to efficiently delivery different payloads to the central nervous system (CNS).

[0006] To expand on the payload diversity modifications, the Fc domains in VNAR antibodies have been modified to allow site-specific conjugation via an accessible sulfhydryl group present on a surface exposed cysteine. Potential payloads that can be delivered via receptor-mediated transport using the VNAR antibodies include oligonucleotides, polypeptides, cytotoxins, enzymes, proteins, peptides, small molecules, radioisotopes, fluorophores, polymers, lipids, nanoparticles, liposomes, capsids, and viruses.

[0007] These brain shuttle constructs are needed to carry a variety of different payloads for efficient brain delivery and activity at the site of action in the CNS. Additionally, such brain shuttle constructs when directed to TfRl can be used to deliver payloads across the retinal, gut mucosal or tumor barriers that use the TFR-mediated transport system (Harel, Rubinstein et al. 2011, Steeg 2021, Ramsay, Lajunen et al. 2023).

[0008] The present disclosure thus addresses the need for a variety of VNAR antibodies which efficiently cross the BBB (or other membranes) and release a therapeutic cargo in vivo.SUMMARY

[0009] The present disclosure relates to brain shuttles based on single domain VNAR antibodies against cell membrane receptors, especially those found at the blood-brain barrier but also in the GI tract and other cellular membranes. In accordance herewith, one aspect is directed to a single chain VNAR antibody which comprises a BBB-shuttling VNAR domain fused to the N-terminus of a mammalian immunoglobulin (Ig) chain, wherein said the Ig chain of the VNAR antibody is effector-attenuated and capable of site-specific conjugation at a surface accessible cysteine. In some embodiments, the Ig chain is from or is an IgM, IgA, IgG, IgE chain, a single chain Fv, an Fab fragment, or an Fc domain. In any of these the embodiments, the Ig chain is an Fc domain chain from an IgG light chain or a heavy chain.

[0010] In another aspect, the embodiments of the disclosure provide a single chain VNAR antibody which comprises a VNAR domain fused to the N-terminus of a human IgGl FcLALA-PG-SC domain comprising L13A, L14A, P108G and S221C substitutions (using the numbering system in Table 1) or the substitutions which are positionally and structurally equivalent thereto; and wherein the VNAR antibody is effector-attenuated and capable of site-specific conjugation at the S221C cysteine.

[0011] In any of the embodiments herein, the VNAR antibody comprises a BBB-shuttling VNAR domain, wherein the domain is(a) a TfR-binding VNAR domain capable of specifically binding to a human TfR-1 without substantially interfering with transferrin binding to and / or transport by said human TfR-1,(b) a TfR-binding VNAR domain capable of specifically binding to a human TfR-1 without substantially interfering with transferrin binding to and / or transport by said human TfR-1 and capable of cross reacting with mouse TfR 1, or(c) a TfR-binding VNAR domain capable of binding human TfR-1 with an EC50 ranging from about 1 nM to about 800 nM.

[0012] In any of the embodiments herein, the VNAR antibody comprises a BBB-shuttling VNAR domain, wherein the domain is(a) the TfR-binding VNAR domain designated as Clone C or one of its variants,(b) the TfR-binding VNAR domain designated as Clone H or one of its variants,(c) the TfR-binding VNAR domain designated as Clone 8 or one of its variants(d) TXB4,(e) TXP1, or(f) a CD98-binding VNAR domain.

[0013] In some embodiments of any of the VNAR antibodies herein, the VNAR domain is a Type II VNAR domain represented by the formula, from N to C terminus, FW1-CDR1- FW2-HV2-FW2’-HV4-FW3-CDR3-FW4, wherein CDR1 has an amino acid sequence of DSNCALSS (SEQ ID NO: 1) and CDR3 has an amino acid sequence of VVGTWCMSWRDV (SEQ ID NO: 10), and wherein said VNAR domain is capable of specifically binding to human TfRl without substantially interfering with transferrin binding to and / or transport by human TfRl . In some of these embodiments, the VNAR domain comprises an amino acid sequence of any one of sequences in Table 3. These VNAR antibodies include those with the cognate CDR1 / CDR3 pair for TXP1, TXP1 and TXP1 where the VNAR scaffold has been deimmunized.

[0014] In some embodiments of any of the VNAR antibodies herein, the VNAR domain is a Type II VNAR domain represented by the formula, from N to C terminus, FW1-CDR1- FW2-HV2-FW2’-HV4-FW3-CDR3-FW4, wherein CDR1 has an amino acid sequence of DSNCALSS (SEQ ID NO: 1) and CDR3 has an amino acid sequence of VQYPQYPNYFWCDV (SEQ ID NO: 11), and wherein the VNAR domain is capable of specifically binding to human and mouse TfRl without substantially interfering with transferrin binding to and / or transport. In some of the embodiments, the VNAR domain comprises an amino acid sequence of ARVDQTPQTITKETGESLTINCVLRDSNCALSSTYWYRKKSGSTNEENISKGGRYVET VNSGSKSFSLRINDLTVEDSGTYRCNVVQYPQYPNYFWCDVYGDGTAVTVNA (SEQ ID NO: 2). This sequence is that of the VNAR domain TXB4. These VNAR antibodies include those with the cognate CDR1 / CDR3 pair for TXB4 and TXB4.

[0015] In a further aspect, embodiments of the disclosure relate to bispecific VNAR antibodies which comprise a dimer of any of the single chain VNAR antibodies of the disclosure.

[0016] In still further aspects, embodiments of the disclosure include nucleic acids encoding any of the VNAR antibodies herein, vectors comprising those nucleic acids and host cells comprising such vectors.

[0017] In an aspect, the disclosure provides conjugates of any of the VNAR antibodies with at least one heterologous agent, including diagnostic or therapeutic agents, wherein the conjugates are joined to the antibody via the sulfhydryl of the site-specific cysteine residue.

[0018] In some embodiments, the agent is selected from the group consisting of oligonucleotides, polypeptides, cytotoxins, enzymes, proteins, peptides, small molecules, radioisotopes, fluorophores, polymers, lipids, nanoparticles, liposomes, capsids, and viruses.

[0019] In some embodiments, the agent is an oligonucleotide. In any of the embodiments hereof, the oligonucleotide targets an ASO or siRNA of Table 4 or Table 5. In any of the embodiments, the oligonucleotide targets any of the diseases listed in Table 4 or Table 5.

[0020] In some embodiments hereof, the oligonucleotide is anyone of ASO(MAPT), ASObio(MAPT) or siRNA(HPRT) of Table 9, or an oligonucleotide of Table 4 or Table 5. In any of the embodiments hereof, the oligonucleotide has one or more of the modifications in Tables Y and Z which are useful to enhance binding affinity, reduce immunogenicity, andimprove nuclease resistance. In any of the embodiments hereof, the oligonucleotide is anyone of those in Table 11 or Table 12.

[0021] In some embodiments hereof, the agent is PEGbio (described in the examples).

[0022] In another aspect, embodiments include pharmaceutical compositions comprising any VNAR antibody conjugate of the present disclosure.

[0023] Another aspect hereof includes methods of medical treatment which comprises administering a therapeutically-effective amount of the pharmaceutical composition of the disclosure to deliver a diagnostic or therapeutic agent to the brain of a mammalian subject in need thereof. The disclosure includes embodiments using a VNAR antibody conjugate of the disclosure for the preparation of a medicament to deliver a diagnostic or therapeutic agent to the brain of a mammalian subject in need thereof.

[0024] In another aspect, embodiments of the disclosure relate to methods of targeting delivery of a payload to brain parenchymal tissue in a mammal which comprises administering a VNAR antibody conjugate of the disclosure to said mammal. The disclosure includes embodiments using a VNAR antibody conjugate of the disclosure for targeting delivery of a payload to brain parenchymal tissue in a mammal.

[0025] A still further method of the disclosure is directed to a method of delivering a therapeutic or diagnostic agent across the blood brain barrier by administering a VNAR antibody conjugate of the disclosure to a subject for a time and in an amount effective to treat or diagnose a CNS disease or condition.

[0026] A still further method of the disclosure is directed to method of delivering a therapeutic or diagnostic agent to the gastrointestinal (GI) tract by administering a VNAR antibody conjugate of the disclosure to a subject for a time and in an amount effective to treat or diagnose a GI disease or condition.

[0027] In yet another aspect, the disclosure provides a method of producing a VNAR antibody conjugate which comprises (a) treating a VNAR antibody of the disclosure in solution with a reducing agent under conditions sufficient to reduce solvent accessible (surface-exposed) cysteine residues while retaining interchain, structural disulphate bridges in the VNAR and Ig or Fc domains, (b) quenching the reducing agent with an oxidizing agent, (c) adding a maleimide-conjugating agent to said solution, (d) incubating for a time sufficient and at a temperature to form stable thioether bonds and produce said VNAR antibodyconjugate, and (e) separating the VNAR antibody conjugate from the unconjugated VNAR antibody. In some embodiments, the maleimide-conjugating agent is maleimide-PEGl 1 -Biotin or a maleimide-oligonucleotide.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1. Cartoon depicting the dimeric brain shuttle for site-specific conjugation. The molecule is composed of a VNAR domain against TfRl (black circles) genetically fused to an Fc domain of IgG, which contains LALA-PG mutations to remove effector function (dotted regions) and the SC mutation that substitutes a surface exposed serine to cysteine for site-specific conjugation (crossed regions).

[0029] Figure 2. Human TfRl (hTfRl) binding of TXP1 brain shuttle fused to wild type Fc (TXP1 WT) or TXP1 fused to Fc domain with effector attenuated function LALA-PG as well as SC substitution (TXP1 LALA-PG-SC). The reactivity was assessed by ELISA with absorbance measured at 450 nm and 4-parametric non-linear regression was used to calculate EC50 values (Table 6). Data presented as OD at 450 nm (mean ±SD, n = 3).

[0030] Figure 3. Gel shift assay of the conjugation process. TXP1 LALA-PG-SC was used for conjugation with maleimide-PEGi i-biotin (PEGbio) and subsequently analysed by nonreducing SDS-PAGE. Unconjugated (-) versus conjugated (+) samples were compared with streptavidin incubated (+) samples. Open arrow indicates the size of conjugated protein, dotted arrow indicates unbound streptavidin and black filled arrow indicates a mass shift resulting from streptavidin binding to successfully conjugated TXP1 LALA-PG-SC with PEGbio.

[0031] Figure 4. Human TfRl (hTfRl) binding of unconjugated and PEGbio conjugated TXP1 LALA-PG-SC using Fc detection method. The reactivity was assessed by ELISA with absorbance measured at 450 nm and 4-parametric non-linear regression was used to calculate EC50 values (Table 7). Data presented as OD at 450 nm (mean ±SD, n = 3).

[0032] Figure 5. Human TfRl (hTfRl) binding of unconjugated and PEGbio conjugated TXP1 LALA-PG-SC using streptavidin detection method. The reactivity was assessed by ELISA with absorbance measured at 450 nm and 4-parametric non-linear regression was used to calculate EC50 values (Table 7). Data presented as OD at 450 nm (mean ±SD, n = 3).

[0033] Figure 6. Gel shift assay of the conjugation process. TXB4 LALA-PG-SC was used for conjugation with ASO modified at 3’ with biotin (ASObio) and subsequently analysed bynon-reducing SDS-PAGE. Unconjugated (-) versus conjugated (+) samples were compared with streptavidin incubated (+) samples. Open arrow indicates the size of conjugated protein, dotted arrow indicates unbound streptavidin and black filled arrow indicates a mass shift resulting from streptavidin binding to successfully conjugated TXB4 LALA-PG-SC with ASObio.

[0034] Figure 7. Mouse TfRl (mTfRl) binding of unconjugated and ASObio conjugated TXB4 LALA-PG-SC using Fc detection method. The reactivity was assessed by ELISA with absorbance measured at 450 nm and 4-parametric non-linear regression was used to calculate EC50 values (Table 8). Data presented as OD at 450 nm (mean ±SD, n = 4).

[0035] Figure 8. Mouse TfRl (mTfRl) binding of unconjugated and ASObio conjugated TXB4 LALA-PG-SC using streptavidin detection method. The reactivity was assessed by ELISA with absorbance measured at 450 nm and 4-parametric non-linear regression was used to calculate EC50 values (Table 8). Data presented as OD at 450 nm (mean ±SD, n = 4).

[0036] Figure 9. Internalisation by model mouse brain endothelial cells bEnd.3. The cells were incubated for 1 hour with either the isotype control antibody or TXB4-ASO conjugate at 0.1 pM concentration at 37°C. The cells were washed and stained followed by a fluorescence readout. The data presented as the average spot intensity (mean ±SD, n = 3).

[0037] Figure 10. Knock down assessment using neuronal mouse Neuro 2a (N2a) cells. Tau (MAPT) KD relative to PPIA expression was assessed in Neuro 2a cells transfected using RNAiMAX reagent with 100 nM TXB4-ASO after 3 -day incubation. TaqMan method was used for gene expression quantification and data were normalised to PPIA expression (mean ±SD, n = 3).

[0038] Figure 11. Stability of the conjugated TXB4-ASObio in PBS. The stability was assessed by incubating the compound at 100 nM concentration at 37°C for 7 days followed by mouse TfRl binding. The ELISA based binding was performed using either human Fc detection antibody or streptavidin for either protein or oligonucleotide detection, respectively. Data presented as normalised OD at 450 nm (mean ±SD, n = 2).

[0039] Figure 12. Stability of the conjugated TXB4-ASObio in mouse serum. The stability was assessed by incubating the compound at 100 nM concentration in mouse serum at 37°C for 7 days followed by mouse TfRl binding. The ELISA based binding was performed using either human Fc detection antibody or streptavidin for either protein or oligonucleotide detection, respectively. Data presented as normalised OD at 450 nm (mean ±SD, n = 2).

[0040] Figure 13. Brain levels of unconjugated and ASObio conjugated TXB4 LALA-PG- SC in mice. The animals were dosed by IV with 25 nmol / kg of either the isotype control antibody, unconjugated TXB4 LALA-PG-SC or TXB4-ASObio. The animals were sacrificed at 18-hour timepoint following cardiac perfusion. The brain homogenates were prepared and used for quantitative ELISA (mean ±SD, n = 3). Significance was determined via two-tailed, unpaired t-test with **** p < 0.0001.

[0041] Figure 14. Plasma levels of unconjugated and ASObio conjugated TXB4 LALA- PG-SC in mice. The animals were dosed by IV with 25 nmol / kg of either the isotype control antibody, unconjugated TXB4 LALA-PG-SC or TXB4-ASObio. The animals were sacrificed at 18-hour timepoint. The plasmas were prepared and used for quantitative ELISA (mean ±SD, n = 3).

[0042] Figure 15. Tissue levels of unconjugated and ASObio conjugated TXB4 LALA- PG-SC in mice. The animals were dosed by IV with 25 nmol / kg of either the isotype control antibody, unconjugated TXB4 LALA-PG-SC or TXB4-ASObio. The animals were sacrificed at 18-hour timepoint following cardiac perfusion. The tissue homogenates were prepared and used for quantitative ELISA (mean ±SD, n = 3).

[0043] Figure 16. Analytical SEC quality control of TXPl-siRNA conjugate. SEC analysis was applied to the conjugation product that underwent two-step purification process. The observed mass shift between unconjugated and siRNA conjugated TXP1 LALA-PG-SC was observed and attributed to the applied modification. SEC allowed purity assessment to be >95%. In this figure, TXP1 ( — ) represents unconjugated TXP1 LALA-PG-SC.

[0044] Figure 17. SDS-PAGE analysis of siRNA conjugated TXP1 LALA-PG-SC. The purified conjugated product was analysed under non-reducing (N) and reducing (R) conditions where a double band indicated successful DARI conjugation of TXP1 to siRNA.

[0045] Figure 18. Human TfRl (hTfRl) binding of unconjugated and siRNA conjugated TXP1 LALA-PG-SC. The reactivity was assessed by ELISA with absorbance measured at 450 nm and 4-parametric non-linear regression was used to calculate EC50 values (Table 7). Data presented as normalised OD at 450 nm (mean ±SD, n = 3). In this figure, TXP1 (-•-) represents unconjugated TXP1 LALA-PG-SC.

[0046] Figure 19. Cyno TfRl (cTfRl) binding of unconjugated and siRNA conjugated TXP1 LALA-PG-SC. The reactivity was assessed by ELISA with absorbance measured at 450 nm and 4-parametric non-linear regression was used to calculate EC50 values (Table 7).Data presented as normalised OD at 450nm (mean ±SD, n = 3). In this figure, TXP1 (-•-) represents unconjugated TXP1 LALA-PG-SC.

[0047] Figure 20. HPRT KD levels in Tg hTfRl expressing mice following TXP1 -siRNA treatment. The animals were dosed by IV with 1 mg / kg (siRNA molar equivalents) on days 0 and 7. The animals were sacrificed on day 21, and brain tissues were assessed for gene expression using quantitative PCR. The expression was normalised to GAPDH and compared to the control group (mean ±SD, n = 4). Significance was determined via two-tailed, unpaired t-test with **** p < 0.0001.

[0048] Figure 21. Knock down assessment using neuronal human SH-SY5Y cells. Tau (MAPT) KD relative to PPIA expression was assessed in the cells transfected using RNAiMAX reagent with TXP1 -siRNA MAPT2 at the indicated concentration range after 3- day incubation. TaqMan method was used for gene expression quantification and data were normalised to PPIA expression (mean ±SD, n = 2).

[0049] Figure 22. Knock down assessment using neuronal mouse Neuro 2a (N2a) cells. Tau (MAPT) KD relative to PPIA expression was assessed in Neuro 2a cells transfected using RNAiMAX reagent with TXP1 -siRNA MAPT2 at the indicated concentration range after 3-day incubation. TaqMan method was used for gene expression quantification and data were normalised to PPIA expression (mean ±SD, n = 2).

[0050] Figure 23. Knock down assessment using neuronal human SH-SY5Y cells. Alpha- synuclein (SNCA) KD relative to PPIA expression was assessed in the cells transfected using RNAiMAX reagent with TXP1 -siRNA SNCA at the indicated concentration range after 3- day incubation. TaqMan method was used for gene expression quantification and data were normalised to PPIA expression (mean ±SD, n = 2).DETAILED DESCRIPTION OF THE DISCLOSURE

[0051] In order that the present disclosure may be more readily understood, certain terms are defined below. Additional definitions may be found within the detailed description of the disclosure.

[0052] Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components).

[0053] The singular forms “a,” “an,” and “the” include the plurals unless the context clearly dictates otherwise.

[0054] The term “including” is used to mean “including but not limited to.” “Including” and “including but not limited to” are used interchangeably.

[0055] The terms “patient,” “subject,” and “individual” may be used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as humans, primates, livestock animals (e.g., cows, pigs), companion animals (e.g., dogs, cats) and rodents (e.g., mice and rats).

[0056] The term “non-human mammal” means a mammal which is not a human and includes, but is not limited to, a mouse, rat, rabbit, pig, cow, sheep, goat, dog, primate, or other non-human mammals typically used in research. As used herein, “mammals” includes the foregoing non-human mammals and humans.

[0057] As used herein, “treating” or “treatment” and grammatical variants thereof refer to an approach for obtaining beneficial or desired clinical results. The term may refer to slowing the onset or rate of development of a condition, disorder or disease, reducing or alleviating symptoms associated with it, generating a complete or partial regression of the condition, or some combination of any of the above. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, reduction or alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable."Treatment" can also mean prolonging survival relative to expected survival time if not receiving treatment. A subject (e.g., a human) in need of treatment may thus be a subject already afflicted with the disease or disorder in question. The term “treatment” includes inhibition or reduction of an increase in severity of a pathological state or symptoms relative to the absence of treatment, and is not necessarily meant to imply complete cessation of the relevant disease, disorder or condition.

[0058] As used herein, the terms "preventing" and grammatical variants thereof refer to an approach for preventing the development of, or altering the pathology of, a condition, disease or disorder. Accordingly, "prevention" may refer to prophylactic or preventive measures. For the purposes of this disclsoure, beneficial or desired clinical results include, but are not limited to, prevention or slowing of symptoms, progression or development of a disease,whether detectable or undetectable. A subject (e.g., a human) in need of prevention may thus be a subject not yet afflicted with the disease or disorder in question. The term “prevention” includes slowing the onset of disease relative to the absence of treatment, and is not necessarily meant to imply permanent prevention of the relevant disease, disorder or condition. Thus “preventing” or “prevention” of a condition may in certain contexts refer to reducing the risk of developing the condition, or preventing or delaying the development of symptoms associated with the condition.

[0059] As used herein, an "effective amount," "therapeutically-effective amount" or "effective dose" is an amount of a composition (e.g., a therapeutic composition or agent) that produces at least one desired therapeutic effect in a subject, such as preventing or treating a target condition or beneficially alleviating a symptom associated with the condition.

[0060] A physiologically-acceptable solution for use in an amount and for a time sufficient to effectively reduce a circulating concentration of the plurality of polypeptides is also referred to herein as a perfusate. The amount of perfusate and time of perfusion depends on the non-human mammal and can be readily determined by those of skill in the art. For example, with a mouse, using a volume of perfusate approximately lOx the blood volume of the mouse is effective at reducing the circulating concentration of polypeptides. Likewise, any volume of perfusate that reduces the circulating concentration of the plurality of polypeptides by about 10%, 25%, 50% or more (relative to the theoretical concentration of the plurality of polypeptides) being delivered is considered effective at reducing the circulating concentration of that plurality.

[0061] As used herein, a “VNAR domain” or “VNAR” has the general structure, from N to C terminus, given by the formula FW1-CDR1-FW2-HV2-FW2’-HV4-FW3-CDR3-FW4, wherein the FWs are framework regions, CDRs are complementarity determining regions and HVs are hypervariable regions, which as taken together form the variable domain of a shark IgNAR (“VNAR”). The CDR3 in naturally-occurring VNARs is of heterogeneous size, ranging from about 7 to about 32 amino acid residues in length. The VNAR domains of the disclosure can optionally have a His-Tag (or other convenient tag for purification purposes). In some cases, such tags are removable. VNAR domains are categorized into four principal isotypes (Type I, II, III, and IV) based on the number and position of non-canonical cysteine residues, in addition to the two canonical cysteines. These extra cysteines form additional disulfide bonds which determine the structure and binding properties of the VNARdomain. Typical VNAR domains have amino acid residues (aa) 1-25 as FW1; aa 26-32 as CDR1; aa 33-43 as FW2; aa 44-52 as HV2; aa 53-85 as FW3; and aa 61-65 as HV4, followed by a CDR3 of variable length and an FW4 of 11 residues starting as XGXG).

[0062] As used herein, binding to the target of interest is called specific binding, while binding to other sites is called nonspecific binding. As used herein, a binding moiety, specific binding moiety, antibody or VNAR domain that “specifically binds” to its target does so selectively or preferentially. Such moieties, antibodies and VNARS can exhibit specific binding to multiple targets such as occurs when one of these entities exhibits species cross reactivity.

[0063] As used herein, the terms “VNAR antibody,” “VNAR-Fc fusion,” and “VNAR-Fc fusion protein” are used interchangeably, and include, but are not limited to antibodies that have a VNAR domain as their variable region and a non-IgNAR constant regions derived from the Fc fragments of IgG, IgM, IgA and IgE. In other words, the non-IgNAR constant region of a VNAR antibody include the Fc portion of conventional antibodies, whether joined by chemical linkers or joined as fusion proteins with or without amino acid linking regions. Further, VNAR antibodies can be monovalent or bivalent. In some contexts, the term “VNAR antibody” is used to refer to a VNAR domain fused to any immunoglobulin (Ig) chain, e.g., a light chain or heavy chain, or fragments of Ig chains such as those associated with Fab fragments and the like.

[0064] As used herein, the term “TfR,” “TfRl” or “TfR-1” refers to a mammalian transferrin receptor- 1 (in context as a protein or a nucleic acid), unless the context indicates that it refers specifically to human TfR-1 (see, e.g., UniProt P02786 TFRI Human) or mouse TfR-1.

[0065]

[0066] The term "CD98 heavy chain" or "CD98hc" as used herein, refers to any native CD98hc from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. CD98hc is also known by the names, inter alia, SLC3 A2, 4F2, 4F2hc, Mdul, LylO, Mdvl, Frpl, Mgp2, Mgp2hc, NACAE, 4T2, 4T2hc, and TROP4. The amino acid sequence of human CD98hc is provided in GenBank® Accession Nos. NP_001012680.1 (isoform b), NP_002385.3 (isoform c), NP_001012682.1 (e), and NP_001013269.1 (isoform f), respectively, and for murine CD98hc ("mCD98hc"), in GenBank® Accession No.: NP_001154885.1 (isoform a) and NP_032603.3 (isoform b)

[0067] Abbreviations used herein for conventional antibodies include: VL, variable region, light chain; VH, variable region, heavy chain; CL, light chain of; HC, heavy chain.VNAR Antibodies and Conjugates ThereofA. VNAR Antibodies

[0068] The present disclosure provides single chain VNAR antibodies comprising a BBB- shuttling VNAR domain fused to the N-terminus of a mammalian immunoglobulin (Ig) chain, wherein the Ig chain of the VNAR antibody is effector-attenuated and capable of sitespecific conjugation at a surface accessible cysteine. The VNAR antibodies can be single chain molecules, that is having a single VNAR domain, sometimes referred to as monospecific, or can be dimers of single chain antibodies and provide two VNAR domains as in a conventional antibody (albeit with two chains instead of the four chains of a conventional antibody), and are referred to herein as bispecific. As used herein, in a bispecific VNAR antibody, the VNAR domains can be the same or different. In some embodiments the VNAR domains of a bispecific VNAR antibodies have the same specificity, that is they are against the same target.

[0069] In some embodiments, the VNAR antibodies of the disclosure have an Ig chain which is from or is an IgM, IgA, IgG, IgE chain, a single chain Fv, an Fab fragment, or an Fc domain. In some embodiments, the Ig chain is the Fc domain of an immunoglobulin heavy chain. In some embodiments the Ig chain is one or more constant regions of an immunoglobulin heavy chain or light chain. In some embodiments, the Ig chain is an Fc domain chain from an IgG heavy chain, and preferably from IgGl .

[0070] In any of the embodiments hereof, the Ig chain is a mammalian Ig chain. In some embodiments the Ig chain is a human Ig chain, a cynomolgus macaque Ig chain or a murine Ig chain.

[0071] In some embodiments, the Fc domain used for conjugation is LALA-PG-SC and includes mutations which (a) attenuate Fc effector function - LALA-PG (L13A, L14A, P108G) (Lo, Kim et al. 2017) and (b) introduce a surface exposed cysteine as a suitable conjugation site (S221C) (Stimmel, Merrill et al. 2000). Table 1 provides the amino acid sequences for wild type (WT) and the LALA-PG-SC variant of human IgGl Fc domains. The mutations for attenuation of the effector function are depicted in bold and underlined (L13A, L14A, P108G) as is the substitution of the surface exposed serine to cysteine (S221C). TheLALA-PG-SC Fc domain can be used with any VNAR domain, including any one of those disclosed herein.Table 1. Human IgGl Fc domain sequences

[0072] In some embodiments, the disclosure provides single-chain VNAR antibodies which comprises a BBB-shuttling VNAR domain fused to the N-terminus of a human IgGl Fc LALA-PG-SC domain comprising L13A, L14A, P108G and S221C substitutions (using the numbering system in Table 1) or is an Fc domain from another immunoglobulin isotype having structurally and positionally equivalent substitutions, wherein the VNAR antibody is effector-attenuated and capable of site-specific conjugation at the S221C cysteine or its equivalent.

[0073] In a preferred embodiment, the single chain VNAR antibody comprises a BBB- shuttling VNAR domain fused to the N-terminus of a human IgGl Fc LALA-PG-SC domain comprising L13A, L14A, P108G and S221C substitutions. In these embodiments, the VNAR antibody is effector-attenuated and capable of site-specific conjugation at the S221C cysteine.

[0074] As used herein, a “BBB-shuttling VNAR domain” is a VNAR domain with binding specificity for a membrane transporter found on the endothelial or other cells of the BBB and which is capable of being endocytosed or transcytosed across the BBB in a manner that does not generally interfere with or impair the transport of the ligand binding and / or ligand transport properties of the transporter. Additionally, these VNAR domains can act as shuttles to transport other molecules (sometimes referred to as “cargo” or “payloads”) across the BBB and into the brain. Many such VNAR domains have been isolated and characterized and arefurther capable of delivering therapeutic levels of such cargo or payloads into the brain, including VNAR domains with specificity for TfRl and CD98, and many of those are more fully described below.

[0075] Thus, in some embodiments, the VNAR domains of the disclosure are BBB- shuttling VNAR domains that are independently one or more of (a) TfR-binding VNAR domains capable of specifically binding to a human TfRl without substantially interfering with transferrin binding to and / or transport by said human TfRl, (b) TfR-binding VNAR domains capable of specifically binding to a human TfRl without substantially interfering with transferrin binding to and / or transport by said human TfRl and capable of cross reacting with mouse TfRl, or (c) TfR-binding VNAR domains capable of binding human TfRl with an EC50 ranging from about 1 nM to about 800 nM.

[0076] In some embodiments, the domains of the disclosure are BBB-shuttling VNAR domains that are independently one or more of (a) the TfR-binding VNAR domains designated as Clone C or one of its variants described in WO2018 / 031424 and WO2019 / 089395, respectively; (b) the TfR-binding VNAR domains designated as Clone H or one of its variants described in WO2018 / 031424 and WO2019 / 089395, respectively; (c) the TfR-binding VNAR domains designated as Clone 8 or one of its variants described in W02020 / 056327; (d) TXB4 (also known as Clone 18 and described in W02020 / 056327; (e) the CD98-binding VNAR domains described in WO2020 / 246288; (f) the TfR-binding VNAR domains described in W02016 / 077840 as capable of BBB shuttling; (g) the TfR-binding VNAR domain variants of Clone C specifically designated as variants 7, 13, 14, 16, 18, 25, 30, 31, and 34 in WO2019 / 089395; and (h) the VNAR-txpl (or txpl) domain described in WO2022 / 103769 and the related VNAR domains with deimmunized scaffolds described therein, Any of the other forgoing VNAR domains can also have deimmunized scaffold as described in W02023 / 023166. The amino acid sequences of these VNAR domains have been published in the foregoing applications and are expressly incorporated herein by reference. These and other specific VNAR domains suitable, independently alone or in combination, for use in the present VNAR antibodies and conjugates are also provided in Table 2 and Table 3.

[0077] The TfR-binding VNAR domain designated here as TXB4 is a single domain shark antibody that binds to TfRl on these cells and can carry therapeutic antibodies across BBB to the brain parenchyma. The VNAR domain amino acid sequence for Clone TXB4 is:ARVDQTPQTITKETGESLTINCVLRDSNCALSSTYWYRKKSGSTNEENISKGGRYVET VNSGSKSFSLRINDLTVEDSGTYRCNVVQYPQYPNYFWCDVYGDGTAVTVNA (SEQ ID NO. 2).

[0078] Clone C is a human and mouse TfR-binding VNAR obtained by in vivo selection of brain penetrating phages as described in Examples 1 and 2 of Inti. Appln. No. PCT / US2017 / 045592, filed August 4, 2017 (now WO2018 / 031424). The VNAR domain amino acid sequence for Clone C is:ARVDQTPQTITKETGESLTINCVLRDSNCALSSTYWYRKKSGSTNEENISKGGRYVET VNSGSKSFSLRINDLTVEDSGTYRCNVVQYPSYNNYFWCDVYGDGTAVTVN (SEQ ID NO. 3). Suitable variants of Clone C are described in WO2019 / 098395 and in Table 2.

[0079] Clone H is a human and mouse TfR-binding VNAR obtained by in vivo selection of brain penetrating phages as described in Examples 1 and 2 of Inti. Appln. No.PCT / US2017 / 045592, filed August 4, 2017 (now WO2018 / 031424). The VNAR domain amino acid sequence for Clone H is:ARVDQTPQTITKETGESLTINCVLRDSNCELSSTYWYRKKSGSTNEESISKGGRYVET VNSGSKSFSLRINDLVVEDSGTYRCNVQQFPSSSNGRYWCDVYGGGTAVTVNA (SEQ ID NO. 4). Suitable variants of Clone H are described in WO2019 / 098395 and in Table 2

[0080] Clone 8 described in W02020 / 056327 is a human TfR-1 binding VNAR. The VNAR domain amino acid sequence for Clone 8 is: ARVDQTPQTITKETGESLTINCVLRDSNCALPSTYWYRKKSGSTNEESISKGGRYVET VNSGSKSFSLRINDLTVEDSGTYRCKVIAQLSSILRGCNYRKHDVYGDGTAVTVNA (SEQ ID NO. 5). Suitable variants of Clone 8 are described in W02020 / 056327 and in Table 2

[0081] The VNAR domain TXP1 binds to human and macaque TfRl and is described in U.S. Serial No. 63 / 112,314, filed November 11, 2020 (now WO2022 / 103769; it is also referred to therein as VNAR-txpl). The amino acid sequence of TXP1 is ARVDQTPQTITKETGESLTINCVLRDSNCALSSTYWYRKKSGSTNEENISKGGRYV ETVNSGSKSFSLKINDLTVEDSGTYRCNVVGTWCMSWRDVYGGGTAVTVNA (SEQ ID NO. 6).

[0082] Clone F12 is a Type IV VNAR domain capable of specifically binding to human and murine CD98hc as described in WO2019 / 246288. The VNAR domain amino acid sequence for Clone F12 is:ARVDQTPQTITKEEGESLTINCVLRVHGRALASTSWYRKKSGSTREETISKGGRYVET VNSGSKSFSLRINDLTVEDSGTYRCNVYGLSFGDIEGVKKIDVYGDGTAVTVNA (SEQ ID NO. 7). Suitable variants of Clone F12 and other CD98hc-binding VNARs are described in WO2019 / 246288 and in Table 2.TABLE 2. Exemplary BBB-shuttling VNARs

[0083] The foregoing VNAR domains can be fully identified based on their VNAR isotype and their cognate CDR1 / CDR3 pair. For example, TXP1 is a Type II VNAR domain with a CDR1 having the amino acid sequence DSNCALSS (SEQ ID NO: 1), and a CDR3 having the amino acid sequence WGTWCMSWRDV (SEQ ID NO: 10). TXB4 is a Type II VNAR domain with a CDR1 having the amino acid sequence DSNCALSS (SEQ ID NO: 1) , and a CDR3 having the amino acid sequence VQYPQYPNYFWCDV (SEQ ID NO: 11).In some embodiments, the VNAR domain comprises an amino acid sequence shown in Table 3. The second VNAR domain or in Table 3 is referred to herein as TXP1 (and may also be referred to as VNAR-txpl). The third and fourth two lines of Table 3 are variants of TXP1 with deimmunized scaffolds. These VNAR domains were obtained and characterized as described in W02023 / 023166.Table 3. TfR-binding VNAR Domains

[0084] The VNAR domains of the disclosure can optionally have a His-Tag (or other convenient tag for purification purposes). In some cases, such tags are removable.

[0085] In a preferred embodiment, the VNAR antibody comprises a TX1, or a deimmunized variant of TXP1, fused at the N-terminal end of an hFc IgGl LALA-PG-SC to form the VNAR-Fc fusion designated herein as TXP1 LALA-PG-SC. The hFc domain of TXP1 has attenuated effector function (AEF) and carries a series of mutations L13A, L14A, P108G (Lo, Kim et al. 2017) and (b) a surface exposed cysteine as a suitable conjugation site (S221C) (Stimmel, Merrill et al. 2000). The numbering system used herein is based on the Fc domain sequence in Table 1.

[0086] One advantage of TXP1 is that it allows for efficient brain penetration at low therapeutic doses.

[0087] In another aspect of the disclosure the single chain VNAR antibodies can be expressed recombinantly and purified. In any of the embodiments hereof, purification can be done under conditions which lead to isolation of single chains or dimers (see, e.g., Fig. 1), which dimers, for convenience, may be are referred to herein as bispecific VNAR antibodies (e.g., a moiety having two VNARs on a single Fc domain). Generally, single chain VNAR antibodies are obtained under certain reducing conditions and bispecific VNAR antibodies are obtained under non-reducing conditions. Single chain VNAR antibodies of different specificities can be mixed to produce true bispecific molecules, meaning a single Fc domain having two VNARs with each VNAR having a different binding target.B. VNAR antibody Conjugates and Production thereof1. Conjugates with Heterologous Agents

[0088] VNAR antibodies of the disclosure are conjugated via the site-specific surface accessible cysteine residue to one or more heterologous agents, preferably a therapeutic and / or diagnostic agent, to produce VNAR antibody conjugates.

[0089] This conjugation occurs via the sulfhydryl group using maleimide chemistry as described in Example 2 and is done in a manner and under conditions that preserve the structural disulfide bonds in the VNAR and Fc / Ig domains while allowing reaction of the surface thiol with the maleimide. Other maleimide bioconjugation methods are reviewed in Ravasco, Faustino 2018. Any compatible conjugation chemistry known in the art may also be used.

[0090] The heterologous agents that can be conjugated oligonucleotides, polypeptides, cytotoxins, enzymes, proteins, peptides, small molecules, radioisotopes, fluorophores,polymers, lipids, nanoparticles, liposomes, capsids, and viruses compatible with maleimide conjugation techniques.

[0091] In any of the embodiments hereof, the agents are chemotherapeutics such as cytostatic drugs, cytotoxins, radioisotopes, chelators, enzymes, nucleases, nucleic acids such as DNA, RNA or mixed nucleic acid oligonucleotides, including siRNAs, shRNAs, microRNAs, aptamers and the like; immunomodulators such as therapeutic antibodies, antibody and antibody-like fragments, inflammatory and anti-inflammatory cytokines, antiinflammatory agents, radiotherapeutics, photoactive agents, diagnostic markers and the like.2. Oligonucleotides as Heterologous Agents

[0092] Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) can act as heterologous agents for treating central nervous system (CNS) disorders by targeting diseasecausing genes to modulate mRNA to either decrease toxic protein levels or increase beneficial protein production. Use of these technologies is being investigated for a wide range of neurodegenerative and neurodevelopmental diseases, including amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Parkinson’s disease (PD) and Alzheimer's disease (AD).

[0093] In accordance with the disclosure, in any of the embodiments herein (including, but not limited to, conjugates, methods, pharmaceutical compositions, etc.) a VNAR antibody of the disclosure is conjugated to heterologous agent, wherein the agent is an oligonucleotide. Oligonucleotides are nucleic acid molecules and may be single, double or more stranded RNA and / or DNA molecules, and analogs and derivatives thereof. Oligonucleotides of the disclosure include, but are not limited to, small regulatory RNA such as shRNA, miRNA, siRNA and the like. The size of oligonucleotides can vary but range from about 10 to about 100 bases, and more typically range from about 10 to about 30 nucleotides.

[0094] Table 4 and Table 5 provide sequences of mRNA targets for ASO and siRNA therapy, respectively, for the indicated CNS diseases except for the last line of Table 5, which provides an siRNA for targeting HRPT.Table 4. mRNA targets for ASO therapy in the CNSTable 5. mRNA targets for siRNA therapy in the CNS

[0095] Sense and antisense sequences are shown in 5’ to 3’ orientation. All or a potion of these sequences sufficient to modulate mRNA expression can be synthesised, modified as needed, e.g., to increase stability and / or cell penetrability, and conjugated to a VNAR antibody of the disclosure. Methods of conjugation are known in the art and any method suitable for conjugation via the exposed surface cysteine if a VNAR antibody of the disclosure can be used. For example, a NH2C6 linker can be added to 5’ end of sense strand and the oligonucleotide joined via mal eimide chemistry.

[0096] In any of the embodiments hereof, the heterologous agent is a modified antisense oligonucleotide (ASO) such as ASO (MAPT), ASO (MAPT) or ASObio (MAPT) of Table 9. (see Examples 5-7).a. ASO modifications

[0097] Gapmer antisense oligonucleotides (ASOs) are typically composed of a central DNA “gap” flanked by chemically modified nucleotides to optimize stability, affinity, and safety while enabling RNase H-mediated cleavage. The central gap (usually 8-10 DNA nucleotides) remains unmodified to allow RNase H activity. The flanking regions (commonly 3-5 nucleotides on each side) are heavily modified with 2'-O-substitutions such as 2'-O- methoxyethyl (2'-M0E), 2'-O-methyl (2'-OMe), or locked nucleic acids (LNA) to enhance binding affinity, reduce immunogenicity, and improve nuclease resistance. The entire backbone typically uses phosphorothioate (PS) linkages for stability and protein interactions. Additional conjugates like GalNAc or fatty acids may be attached at the termini for targeted delivery and improved pharmacokinetics. Table Y provides modifications found in and useful for Gapmer ASOs and ASOs. Anyone, all or combination of modifications are contemplated for use in the oligonucleotides of the disclosure.Table Y: Gapmer ASO Structure and Modificationsb. siRNA Modifications

[0098] siRNA designs typically use extensive chemical modifications to optimize stability, reduce immune activation, and maintain RNAi potency. siRNAs are 21-23 nucleotide duplexes with a guide (antisense) and a passenger (sense) strand, often featuring two- nucleotide 3' overhangs (UU or TT) for efficient RNA-induced silencing complex (RISC) loading. The antisense strand, which directs target cleavage, can be modified with 2'-0Me throughout most positions, combined with strategic 2'-F substitutions at selected pyrimidines — commonly in the seed region (e.g., position 2) and sometimes mid / distal positions (e.g., 14 and 16) — to enhance stability without impairing Ago2 compatibility. The sense strand is usually more heavily modified, often alternating 2'-0Me and 2'-F across its length to reduce immunogenicity and improve duplex integrity. Additional features include vinyl phosphonate (VP) at the antisense 5' end for exonuclease protection, phosphorothioate (PS) linkages at the first and last two positions of each strand for nuclease resistance, and 2'- OMe-modified overhangs for extra stability. For targeted delivery, conjugates such as GalNAc or cholesterol are typically attached to the sense strand. Table Z provides modifications found in and useful with siRNA oligonucleotides. Anyone, all or combination of modifications are contemplated for use in the oligonucleotides of the disclosure.Table Z: siRNA Structure and Modifications3. Other Heterologous Agents

[0099] Other heterologous agents which may be used in conjunction with any one of the above embodiments may comprise, e.g., one or more biologically active molecules and / or imaging agents. Exemplary biologically active molecules which may be transported into a TfR-positive cell (when using a BBB-shuttling VNAR domain against TfRl) include, e.g., toxins for targeted TfR-positive cell death (useful e.g., in certain hyperproliferative diseases or disorders such as cancers or aberrant proliferative conditions). Other exemplary biologically active molecules which may be transported in association with a VNAR antibody conjugate of the disclosure include, e.g., polypeptides, such as an antibody or antibody fragment; a therapeutic peptide such as a hormone, cytokine, growth factor, enzyme, antigen or antigenic peptide, transcription factor, or any functional domain thereof. Other exemplary biologically active molecules which may be transported into a TfR-positive cell in association with a VNAR antibody conjugate of the disclosure include, e.g., nucleic acid molecules, such as an oligonucleotide (e.g., single, double or more stranded RNA and / or DNA molecules, and analogs and derivatives thereof); small regulatory RNA such as shRNA, miRNA, siRNA and the like; and a plasmid or fragment thereof.

[0100] Exemplary polypeptides which may be therapeutically beneficial when administered as a heterologous agent for TfR-mediated transport across the BBB or other TfR-containing cell membrane include but are not limited to: a brain derived neurotrophic factor (BDNF), a bone morphogenic protein (e.g., BMP-1 through BMP-7, BMP8a, BMP8b, BMP10 and BMP 15), a ciliary neurotrophic factor (CNF), an epidermal growth factor (EGF), erythropoietin, a fibroblast growth factor (FGF), a glial derived neurotrophic factor (GDNF), a heptocyte growth factor, an interleukin (e.g., IL-1, IL-4, IL-6, IL-10, IL-12, IL-13, IL-15, IL-17), a nerve growth factor (NGF), a neurotrophin (e.g., NT-3 and NT-4 / 5), a neurturin, a neuregulin, a platelet derived growth factor (PDGF), a transforming growth factor (e.g., TGF- alpha and TGF-beta), apolipoprotein E (ApoE), a vasoactive intestinal peptide, artemin,persephin, netrin, neurotensin, GM-GSF, cardiotrophin-1, stem cell factor, midkine, pleiotrophin, a saposin, a semaporin, leukemia inhibitory factor, and the like.

[0101] Exemplary therapeutic antibodies or fragments that may be transported across the BBB or other TfR-containing cell membrane as a heterologous biologically active agent of the disclosure include but are not limited to: antibodies for neurodegeneration including anti- Abeta, anti-Tau, anti-alpha-synuclein anti-Trem2, anti-C9orf7 dipeptides, anti-TDP-43, antiprion protein C, anti-huntingtin, anti-nogo A, anti-TRAIL (tumor necrosis factor-related apoptosis-inducing ligand); antibodies for neuro-oncology including anti-HER2, anti-EGF, anti-PDGF, anti-PDl / PDLl, anti-CTLA-4, anti-IDO, anti-LAG-3, anti-CD20, anti-CD19, anti-CD40, anti-OX40, anti-TIM3, anti -toll-like receptors; antibodies for neuroinflammation including anti-TNF, anti-CD138, anti-IL-21, anti-IL-22; antibodies to viral diseases of the brain including anti-West Nile virus, anti-Zika, anti-HIV, anti-CMVanti-HSV and the like.

[0102] Exemplary enzymes that may be transported across the BBB or other TfR- containing cell membrane as a heterologous biologically active agent of the disclosure include but are not limited to: alpha-L-iduronidase, iduronate-2-sulfatase, N-acetyl- galactosamine-6-sulfatase, arylsulfatase B, acid alpha-glucosidase, tripeptidyl-peptidase 1, acid sphingomyelinase glucocerebrosidase and heparan sulfamidase.

[0103] Also included as exemplary biologically active agents are small molecules comprising chemical moieties (such as a therapeutic small molecule drugs); carbohydrates; polysaccharides; lipids; glycolipids and the like. Exemplary embodiments of such small molecule therapeutic agents include certain cancer drugs, such as daunorubicin, doxorubicin, and other cytotoxic chemical agents including microtubule inhibitors, topoisomerase inhibitors, platins, alkylating agents, and anti-metabolites all of which may beneficially be administered across the BBB at lower overall systemic doses than by IV administration. Other small molecule therapeutic agents may include corticosteroids, NSAIDs, COX-2 inhibitors, small molecule immunomodulators, non-steroidal immunosuppressants, 5-amino salicylic acid, DMARDs, hydroxychloroquine sulfate, and penicillamine. LD- ribofuranosyl- l,2,4-triazole-3 carboxamide, 9-2-hydroxy-ethoxy methylguanine, adamantanamine, 5-iodo-2'- deoxyuridine, trifluorothymidine, interferon, adenine arabinoside, protease inhibitors, thymidine kinase inhibitors, sugar or glycoprotein synthesis inhibitors, structural protein synthesis inhibitors, attachment and adsorption inhibitors, and nucleoside analogues such as acyclovir, penciclovir, valacyclovir, and ganciclovir, among others. Small moleculetherapeutic agents which may be used according to the disclosure also include bevacizumab, cisplatin, irinotecan, methotrexate, temozolomide, taxol and zoledronate. Certain antiinflammatory agents may be useful biologically active molecules. Fluoxetine, for example, reportedly inhibits MMP-2, MMP-9 and MMP-12 expression associated with blood-brain barrier disruption and inflammatory reactions after spinal cord injury, which may be used according to the disclosure to protect blood-brain barrier and to inhibit deleterious inflammatory responses in spinal cord injury and central nervous system disease. Other nonlimiting examples of therapeutic antibodies which may be beneficially transported across the BBB include anti-CD133, anti-CD137, anti-CD27, anti-VEGF, anti-EGRFvIII, anti-IL-15 and anti-IL13R.

[0104] Exemplary embodiments of an imaging agent as an associated heterologous agent include agents that comprise at least one of a metal such as a paramagnetic metal, a radionuclide such as a radioisotope, a fluorochrome or fluorophor, an energy emitting particle, a detectable dye, and an enzyme substrate.

[0105] Further examples of biologically active agents include small molecules, including therapeutic agents, in particular those with low blood-brain barrier permeability. Some examples of these therapeutic agents include cancer drugs, such as daunorubicin, doxorubicin, and toxic chemicals which, because of the lower dosage that can be administered by this method, can now be more safely administered. For example, a therapeutic agent can include bevacizumab, irinotecan, zoledronate, temozolomide, taxol, methotrexate, and cisplatin.

[0106] In another embodiment, the therapeutic agent can include a broad-spectrum antibiotic (e.g., cefotaxime, ceftriaxone, ampicillin and vancomycin); an antiviral agent (e.g., acyclovir); acetazolamide; carbamazepine; clonazepam; clorazepate dipotassium; diazepam; divalproex sodium; ethosuximide; felbamate; fosphenytoin sodium; gabapentin; lamotrigine; levetiracetam; lorazepam; oxcarbazepine; phenobarbital; phenytoin; phenytoin sodium; pregabalin; primidone; tiagabine hydrochloride; topiramate; trimethadione; valproic acid; zonisamide; copaxone; tysabri; novantrone; donezepil HCL; rivastigmine; galantamine; memantine; levodopa; carbidopa; parlodel, permax, requip, mirapex; Symmetrel; artane; cogentin; eldepryl; and deprenyl. Antiviral compounds are also beneficial therapeutic agents that can be delivered using a VNAR antibody conjugate of the disclosure, especially for cases in which the virus uses TfR transport as its route of entry into infected cells.

[0107] Numerous other examples of biologically active agents may be used in association with a VNAR antibody conjugate of the disclosure, appropriate selection of which will be apparent to the skilled artisan depending on the condition, disease or disorder to be treated.

[0108] Yet other examples of a biologically active agent which may be used according to the present disclosure is an antigenic peptide. Antigenic peptides may provide immunological protection when imported by cells involved in an immune response. Other examples include immunosuppressive peptides (e.g., peptides that block autoreactive T cells, such peptides being known in the art).

[0109] An imaging agent, as used herein, may be any chemical substance which may be used to provide a signal or contrast in imaging. A signal enhancing domain may be an organic molecule, metal ion, salt or chelate, a particle (e.g., iron particle), or a labeled peptide, protein, glycoprotein, polymer or liposome. For example, an imaging agent may include one or more of a radionuclide, a paramagnetic metal, a fluorochrome, a dye, and an enzyme substrate.

[0110] For x-ray imaging, the imaging agent may comprise iodinated organic molecules or chelates of heavy metal ions of atomic numbers 57 to 83. In certain embodiments, the imaging agent is I125labeled IgG (see, e.g., M. Sovak, ed., "Radiocontrast Agents," Springer- Verlag, pp. 23-125 (1984).

[0111] For ultrasound imaging, an imaging agent may comprise gas-filled bubbles or particles or metal chelates where the metal ions have atomic numbers 21-29, 42, 44 or 57-83. See e.g., Tyler et al., Ultrasonic Imaging, 3, pp. 323-29 (1981) and D. P. Swanson, "Enhancement Agents for Ultrasound: Fundamentals," Pharmaceuticals in Medical Imaging, pp. 682-87. (1990) for other suitable compounds.

[0112] For nuclear radiopharmaceutical imaging or radiotherapy, an imaging agent may comprise a radioactive molecule. In certain embodiments, chelates of Tc, Re, Co, Cu, Au, Ag, Pb, Bi, In and Ga may be used. In certain embodiments, chelates of Tc-99m may be used. See e.g., Rayudu GVS, Radiotracers for Medical Applications, I, pp. 201 and D. P. Swanson et al., ed., Pharmaceuticals in Medical Imaging, pp. 279-644 (1990) for other suitable compounds.

[0113] For ultraviolet / visible / infrared light imaging, an imaging agent may comprise any organic or inorganic dye or any metal chelate.

[0114] For MRI, an imaging agent may comprise a metal-ligand complex of a paramagnetic form of a metal ion with atomic numbers 21-29, 42, 44, or 57-83. In certain embodiments, the paramagnetic metal is selected from: Cr(III), Cu(II), Dy(III), Er(III) and Eu(III), Fe(III), Gd(III), Ho(III), Mn(II and III), Tb(III). A variety of chelating ligands useful as MRI agents are well known in the art.Methods Of Producing VNAR Antibodies

[0115] The VNAR antibodies of the disclosure may be manufactured by standard synthetic methods, by use of recombinant expression systems, or by any other suitable method. Thus, these antibodies may be synthesized in a number of ways, including, e.g., methods comprising: (1) synthesizing the VNAR domain using standard solid-phase or liquid-phase methodology, either stepwise or by fragment assembly, and isolating and purifying the domain and covalently or non-covalently linking it to already purified Ig chain or Fc domain; (2) expressing a nucleic acid construct that encodes a fusion of the VNAR domain and Ig chain / Fc domain in a host cell and recovering the expression product from the host cell or host cell culture; or (3) cell-free in vitro expression of such a nucleic acid construct and recovering the expression product; or by any combination of the methods of (1), (2) or (3)

[0116] Accordingly, the present disclosure also provides methods for producing a VNAR antibody of the disclosure according to above recited methods; a nucleic acid molecule encoding part or all of a VNAR antibody of the disclosure, a vector comprising at least one nucleic acid of the disclosure, expression vectors comprising at least one nucleic acid of the disclosure capable of producing a VNAR antibody of the disclosure when introduced into a host cell, and a host cell comprising a nucleic acid molecule, vector or expression vector of the disclosure. .

[0117] VNAR antibody of the disclosure may be prepared using recombinant techniques well known in the art. In general, methods for producing polypeptides by culturing host cells transformed or transfected with a vector comprising the encoding nucleic acid and recovering the polypeptide from cell culture are described in, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989); Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995).

[0118] A nucleic acid encoding a desired polypeptide may be inserted into a replication vector for further cloning (amplification) of the DNA or for expression of the nucleic acid into RNA and protein. A multitude of cloning and expression vectors are publicly available.

[0119] Expression vectors capable of directing transient or stable expression of genes to which they are operably linked are well known in the art. The vector components generally include, but are not limited to, one or more of the following: a heterologous signal sequence or peptide, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, each of which is well known in the art. Optional regulatory control sequences, integration sequences, and useful markers that can be employed are known in the art.

[0120] Any suitable host cell may be used to produce conjugates of the disclosure. Host cells may be cells stably or transiently transfected, transformed, transduced or infected with one or more expression vectors which drive expression of a polypeptide of the disclosure. Suitable host cells for cloning or expressing nucleic acids of the disclosure include prokaryote, yeast, or higher eukaryote cells. Eukaryotic microbes such as filamentous fungi yeast, Arabidopsis, and other plant and animal eukaryotic host cells that may be grown in liquid culture are suitable cloning or expression hosts for vectors. Suitable host cells for the expression of glycosylated polypeptides may also be derived from multicellular organisms.

[0121] Creation and isolation of host cell lines producing a VNAR antibody of the disclosure can be accomplished using standard techniques known in the art. Mammalian cells are preferred host cells for expression of peptides. Particularly useful mammalian cells include, inter alia, HEK 293, NSO, DG-44, and CHO cells, but any other suitable host cell may be used according to the disclosure. Preferably, the VNAR antibodies are secreted into the medium in which the host cells are cultured, from which the VNAR antibodies may be recovered or purified.

[0122] When a polypeptide is produced in a recombinant cell other than one of human origin, it is typically free of polypeptides of human origin. In certain embodiments, it is advantageous to separate a polypeptide away from other recombinant cell components such as host cell polypeptides to obtain preparations that are of high purity or substantially homogeneous. As a first step, culture medium or cell lysates may be centrifuged to remove particulate cell debris and suitable protein purification procedures may be performed. Such procedures include, inter alia, fractionation (e.g., size separation by gel filtration or chargeseparation by ion-exchange column); ethanol precipitation; Protein A Sepharose columns to remove contaminants such as IgG; hydrophobic interaction chromatography; reverse phase HPLC; chromatography on silica or on cation-exchange resins such as DEAE and the like; chromatofocusing; electrophoretic separations; ammonium sulfate precipitation; gel filtration using, for example, Sephadex beads such as G-75. Any number of biochemical purification techniques may be used to increase the purity of a conjugate of the disclosure.Monitoring Receptor Binding and Cell Internalization

[0123] Receptor-binding activity of the VNAR antibody or antibody conjugate is determined by the binding specificity of the VNAR domain. The following illustrates determining such activity for VNAR domains against Tfirl, but can be generalized for other receptor binding assessments such as for VNAR domains against CD98hc.

[0124] Hence, TfR-binding activity (also referred to herein as “TfR bioactivity”) may be determined by one or more assays described in the Examples herein, or by any other suitable method in the art, including well-known immunoassays, such as for example the ELISAs or variations thereon described in the Examples. Any other binding assay which directly or indirectly measures the binding of a VNAR antibody of the disclosure to a cell surface TfR, or alternatively, which measures the ability of the VNAR antibody or VNAR antibody conjugate comprising such a moiety of the disclosure to compete for binding to TfR in the presence of a different TfR binding compound (such as an anti-TfR antibody) such as by a competitive inhibition assay, may be used. Preferably, a selected assay measures the effect of a TfR-specific VNAR comprising such a moiety on its ability to transport a heterologous agent or biomolecule across the membrane of a TfR-positive cell. In certain embodiments, the TfR-positive cell is one which transports a heterologous agent across the blood brain barrier (BBB). In certain embodiments, the TfR-positive cell is one which transports a heterologous agent across cells of the gastrointestinal tract. In certain embodiments, binding of the TfR binding moiety to TfR is measured by monitoring internalization of a VNAR antibody or VNAR antibody conjugate of the disclosure into TfR-positive cells or cell type. In vivo assays of TfR bioactivity include, but are not limited to those described in the Examples herein.

[0125] Other test systems to assess TfR binding and functional activity include, for example: Surface plasmon resonance to determine affinity and off-rates; using radiolabeledor fluorescent tagged molecule or GFP fusion proteins in in vitro or in vivo animal studies including binding and internalization in tumor cell lines, immortalized endothelial cell lines or primary cells expressing TfR; in vitro transcytosis in capillary endothelial cells and cells lines; and permeability assay using Caco-2 and MDCK epithelial cell lines; in situ perfusion models and immunohistochemical or immunofluorescent staining of tissue sections; optical or PET animal imaging; standard PK and tissue distribution assays; and measuring one or more biological effects of a heterologous agent (drug cargo or payload) in normal animals or disease animal models.Methods of Treatment Using VNAR Antibody Conjugates

[0126] The present disclosure provides VNAR antibodies or VNAR antibody conjugates of the disclosure for use, alone or in combination with one or more additional therapeutic agents, in a pharmaceutical composition, for treatment or prophylaxis of neurodegenerative diseases, cancers, GI diseases or conditions. The methods comprises administering a therapeutically-effective amount of VNAR antibody, a VNAR antibody conjugate of the disclosure or pharmaceutical composition comprising a VNAR antibody or VNAR antibody conjugate of the disclosure to a mammalian subject in need thereof for a time and in an amount effective to treat the disease or condition.

[0127] In some embodiments, these molecules are administered intravenously, intramuscularly, subcutaneously, intraarterially, intracranially or intrathecally. In an embodiment, the VNAR antibody conjugate is preferably administered intravenously. The routes of administration can be determined by those of skill in the art and lead to accumulation of the conjugate in the brain of the mammalian subject to thereby treat the disease or condition.

[0128] The methods of the disclosure can be used to treat neurodegenerative diseases or conditions that include but are not limited to, Parkinson’s disease, an acute or chronic neurological injury or wound, amyotrophic lateral sclerosis (ALS), or Alzheimer’s disease (AD).

[0129] In some embodiments, the therapeutic is a VNAR antibody conjugate with an antisense oligonucleotide (ASOs) for treating neurological diseases. Such ASOs are designed to bind to the RNAs encoded by a target gene associated with the specific disease and thereby suppress expression by catalyzing degradation of those RNAs or to elevate expression bycorrecting faulty RNA splicing. ASOs can be used for treating amyotrophic lateral sclerosis, Huntington’s disease, Alzheimer’s disease, Parkinson’s, disease, Angelman syndrome as well as for the treatment of spinocerebellar ataxias, sporadic forms of amyotrophic lateral sclerosis, infantile seizure disorders, and neurodevelopmental disorders.

[0130] In accordance with the foregoing, the VNAR antibody conjugates can be used the preparation of a medicament to treat a neurodegenerative disease or condition in a mammalian subject in need thereof.

[0131] In yet other aspects, VNAR antibody conjugates of the disclosure can be used in treating a brain or CNS disease, condition, injury or disorder, such as, for example, neurodegenerative diseases, neuronal injury, stroke, genetic disorders, psychiatric disorders, developmental disorders, inflammation, infection or damage, and brain cancers, spinal cord injury (SCI) and traumatic brain injury (TBI). In certain embodiments, a brain disorder is selected from epilepsy, meningitis, encephalitis including HIV Encephalitis, progressive multifocal leukoencephalopathy, neuromyelitis optica, multiple sclerosis, late-stage neurological trypanosomiasis, amyotrophic lateral sclerosis (ALS), progressive bulbar palsy (PBP), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), Alzheimer's disease, Parkinson's disease, Huntington’s disease, De Vivo disease, and any type of tumor, cancer or hyperproliferative disease in the brain or CNS.Pharmaceutical Compositions

[0132] The present disclosure further provides pharmaceutical compositions comprising a VNAR antibody or VNAR antibody conjugate of the disclosure, or a pharmaceutically acceptable salt or solvate thereof, according to the disclosure, together with a pharmaceutically acceptable carrier, excipient or vehicle.

[0133] Accordingly, the present disclosure further provides a pharmaceutical composition comprising a VNAR antibody or VNAR antibody conjugate of the disclosure comprising a TfR-specific VNAR domain, such as TXP1 or TXB4, as well as variant and derivative compounds thereof. Certain embodiments of the pharmaceutical compositions of the disclosure are described in further detail below.

[0134] The present disclosure also provides pharmaceutical compositions comprising a VNAR antibody or VNAR antibody conjugate of the disclosure for use in treating, ameliorating or preventing one or more diseases, conditions, disorders or symptoms relatingto B cells and immunoglobulin production, as described in further detail below. Each such disease, condition, disorder or symptom is envisioned to be a separate embodiment with respect to uses of a pharmaceutical composition according to the disclosure.Formulations, Administration and Dosing

[0135] A VNAR antibody or VNAR antibody conjugate of the disclosure, or salts thereof, may be formulated as pharmaceutical compositions prepared for storage or administration, which typically comprise a therapeutically effective amount of a compound of the disclosure, or a salt thereof, in a pharmaceutically acceptable carrier.

[0136] The therapeutically effective amount of these molecules will depend on the route of administration, the type of mammal being treated, and the physical characteristics of the specific mammal under consideration. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical arts. This amount and the method of administration can be tailored to achieve optimal efficacy, and may depend on such factors as weight, diet, concurrent medication and other factors, well known to those skilled in the medical arts. The dosage sizes and dosing regimen most appropriate for human use may be guided by the results obtained by the present disclosure, and may be confirmed in properly designed clinical trials.

[0137] An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Such considerations are known to the skilled person. The term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). For example, sterile saline and phosphate- buffered saline at slightly acidic or physiological pH may be used. pH buffering agents may be phosphate, citrate, acetate, tri s / hydroxymethyl)aminom ethane (TRIS), N- Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, which is a preferred buffer, arginine, lysine, or acetate or mixturesthereof. The term further encompasses any agents listed in the US Pharmacopeia for use in animals, including humans.

[0138] The term “pharmaceutically-acceptable salt” refers to the salt of the compounds. As used herein a pharmaceutically-acceptable salt retains qualitatively a desired biological activity of the parent compound without imparting any undesired effects relative to the compound. Salts include pharmaceutically acceptable salts such as acid addition salts and basic salts. Acid addition salts include salts derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphorous, phosphoric, sulfuric, hydrobromic, hydroiodic and the like, or from nontoxic organic acids such as aliphatic mono- and di-carboxylic acids, phenylsubstituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Examples of basic salts include salts where the cation is selected from alkali metals, such as sodium and potassium, alkaline earth metals such as calcium and magnesium, and ammonium ions+N(R3)s(R4), where R3and R4independently designate optionally substituted Ci-6-alkyl, optionally substituted C2-6-alkenyl, optionally substituted aryl, or optionally substituted heteroaryl, and more specifically, the organic amines, such as N, N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like. Other examples of pharmaceutically acceptable salts are described in "Remington's Pharmaceutical Sciences", 17th edition. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, U.S.A., 1985 and more recent editions, and in the Encyclopaedia of Pharmaceutical Technology.

[0139] " Treatment" is an approach for obtaining beneficial or desired clinical results. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treatment" is an intervention performed with the intention of preventing the development or altering the pathology of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures in certain embodiments. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. By treatment is meant inhibiting orreducing an increase in pathology or symptoms when compared to the absence of treatment, and is not necessarily meant to imply complete cessation of the relevant condition.

[0140] The pharmaceutical compositions can be in unit dosage form. In such form, the composition is divided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparations, for example, packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms. It may be provided in single dose injectable form, for example in the form of a pen. Compositions may be formulated for any suitable route and means of administration.

[0141] Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and transdermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Subcutaneous or transdermal modes of administration may be particularly suitable for the compounds described herein.

[0142] An acceptable route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal (e.g., topical administration of a cream, gel or ointment, or by means of a transdermal patch). "Parenteral administration” is typically associated with injection at or in communication with the intended site of action, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrastemal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal administration.

[0143] In another aspect, the present disclosure provides a composition, e.g., a pharmaceutical composition, comprising one or a combination of different VNAR antibodies or VNAR antibody conjugates of the disclosure, or an ester, salt or amide of any of the foregoing, and at least one pharmaceutically acceptable carrier.

[0144] Pharmaceutical compositions of the disclosure may be administered alone or in combination with one or more other therapeutic or diagnostic agents. A combination therapy may include a VNAR antibody or VNAR antibody conjugate of the disclosure combined with at least one other therapeutic agent selected based on the particular patient, disease orcondition to be treated. Examples of other such agents include, inter alia, a cytotoxic, anticancer or chemotherapeutic agent, an anti-inflammatory or anti-proliferative agent, an antimicrobial or antiviral agent, growth factors, cytokines, an analgesic, a therapeutically active small molecule or polypeptide, a single chain antibody, a classical antibody or fragment thereof, or a nucleic acid molecule which modulates one or more signaling pathways, and similar modulating therapeutics which may complement or otherwise be beneficial in a therapeutic or prophylactic treatment regimen.

[0145] As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically acceptable, i.e., compatible, solvents, dispersion media, coatings, antimicrobial agents, isotonic and absorption delaying agents, and the like. In certain embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on selected route of administration, the VNAR polypeptide-comprising compound or component may be coated in a material or materials intended to protect the compound from the action of acids and other natural inactivating conditions to which the active TfR binding VNAR moiety may encounter when administered to a subject by a particular route of administration.

[0146] A pharmaceutical composition of the disclosure also optionally includes a pharmaceutically acceptable antioxidant. Exemplary pharmaceutically acceptable antioxidants are water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0147] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyloleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0148] TfR selective binding moieties and compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. Isotonic agents, such as sugars, sodium chloride, and the like into the compositions, may also be desirable. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as, aluminum monostearate and gelatin.

[0149] Exemplary pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Such media and reagents for pharmaceutically active substances are known in the art. The pharmaceutical compositions of the disclosure may include any conventional media or agent unless any is incompatible with the active TfR specific binding compound. Supplementary active compounds may further be incorporated into the compositions.

[0150] Therapeutic compositions are typically sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, alcohol such as ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or any suitable mixtures. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by use of surfactants according to formulation chemistry well known in the art. In certain embodiments, isotonic agents, e.g., sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride may be desirable in the composition. Prolonged absorption of injectable compositions may be brought about by including in the composition an agent that delays absorption for example, monostearate salts and gelatin.

[0151] Solutions or suspensions used for intradermal or subcutaneous application typically include one or more of: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such asacetates, citrates or phosphates; and tonicity adjusting agents such as, e.g., sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide, or buffers with citrate, phosphate, acetate and the like. Such preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0152] Sterile injectable solutions may be prepared by incorporating a VNAR antibody or VNAR antibody conjugate of the disclosure in the required amount in an appropriate solvent with one or a combination of ingredients described above, as required, followed by sterilization microfiltration. Dispersions may be prepared by incorporating the active compound into a sterile vehicle that contains dispersion medium and other ingredients, such as those described above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient in addition to any additional desired ingredient from a sterile-filtered solution thereof.

[0153] When a therapeutically effective amount of a VNAR antibody or VNAR antibody conjugate of the disclosure is administered by, e.g., intravenous, cutaneous or subcutaneous injection, the binding agent will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. Methods for preparing parenterally acceptable protein solutions, taking into consideration appropriate pH, isotonicity, stability, and the like, are within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection will contain, in addition to binding agents, an isotonic vehicle such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicle as known in the art. A pharmaceutical composition of the present disclosure may also contain stabilizers, preservatives, buffers, antioxidants, or other additives well known to those of skill in the art.

[0154] The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending on a variety of factors, including the subject being treated, and the particular mode of administration. In general, it will be an amount of the composition that produces an appropriate therapeutic effect under the particular circumstances. Generally, out of one hundred percent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, from about 0.1 per cent to about 70 per cent, or from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.

[0155] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the particular circumstances of the therapeutic situation, on a case by case basis. It is especially advantageous to formulate parenteral compositions in dosage unit forms for ease of administration and uniformity of dosage when administered to the subject or patient. As used herein, a dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms depends on the specific characteristics of the active compound and the particular therapeutic effect(s) to be achieved, taking into consideration and the treatment and sensitivity of any individual patient.

[0156] For administration of a VNAR antibody or VNAR antibody conjugate of the disclosure therewith, the dosage range will generally be from about 0.0001 to 100 mg / kg, and more usually 0.01 to 5 mg / kg, of the host body weight. Exemplary dosages may be 0.25 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight or 10 mg / kg body weight or within the range of 1-10 mg / kg. An exemplary treatment regime is a once or twice daily administration, or a once or twice weekly administration, once every two weeks, once every three weeks, once every four weeks, once a month, once every two or three months or once every three to 6 months. Dosages may be selected and readjusted by the skilled health care professional as required to maximize therapeutic benefit for a particular subject. Intervals between single dosages can be, for example, 2-5 days, weekly, monthly, every two or three months, every six months, or yearly. Intervals between administrations can also be irregular. In some methods, dosage is adjusted to achieve a plasma antagonist concentration of about 1-1000 pg / ml and in some methods about 25-300 pg / ml. Dosage regimens for a VNAR antibody or VNAR antibody conjugate of the disclosure include intravenous administration of 1 mg / kg body weight or 3 mg / kg body weight with the compound administered every two to four weeks for six dosages, then every three months at 3 mg / kg body weight or 1 mg / kg body weight.

[0157] In certain embodiments, two or more VNAR antibodies or VNAR antibody conjugates with different binding properties may be administered simultaneously orsequentially, in which case the dosage of each administered compound may be adjusted to fall within the ranges described herein.

[0158] In certain embodiments, a VNAR antibody or VNAR antibody conjugate of the disclosure may be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the VNAR antibody or VNAR antibody conjugate in the subject or patient. The dosage and frequency of administration may vary depending on whether the treatment is therapeutic or prophylactic (e.g., preventative), and may be adjusted during the course of treatment. In certain prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a relatively long period of time. Some subjects may continue to receive treatment over their lifetime. In certain therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient may be switched to a suitable prophylactic dosing regimen.

[0159] Actual dosage levels of the VNAR antibody or VNAR antibody conjugate alone or in combination with one or more other active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without causing deleterious side effects to the subject or patient. A selected dosage level will depend upon a variety of factors, such as pharmacokinetic factors, including the activity of the particular TfR specific binding compound or composition employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject or patient being treated, and similar factors well known in the medical arts.

[0160] Administration of a "therapeutically effective dosage" of a VNAR antibody or VNAR antibody conjugate of the disclosure may result in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.

[0161] A VNAR antibody, VNAR antibody conjugate or composition of the present disclosure may be administered via one or more routes of administration, using one or more of a variety of methods known in the art. As will be appreciated by the skilled worker, the route and / or mode of administration will vary depending upon the desired results. Routes of administration include, e.g., intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.

[0162] In other embodiments, A VNAR antibody, VNAR antibody conjugate or composition of the present disclosure may be administered by a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0163] As described elsewhere herein, an active compound may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0164] Therapeutic compounds or compositions of the disclosure may be administered with one or more of a variety of medical devices known in the art. For example, in one embodiment, a therapeutic composition of the disclosure may be administered with a needleless hypodermic injection device. Examples of well-known implants and modules useful in the present disclosure are in the art, including e.g., implantable micro-infusion pumps for controlled rate delivery; devices for administering through the skin; infusion pumps for delivery at a precise infusion rate; variable flow implantable infusion devices forcontinuous drug delivery; and osmotic drug delivery systems. These and other such implants, delivery systems, and modules are known to those skilled in the art.

[0165] While some embodiments of the disclosure have been described by way of illustration, it will be apparent that the disclosure can be put into practice with many modifications, variations and adaptations, and with the use of numerous equivalents or alternative solutions that are within the scope of persons skilled in the art, without departing from the spirit of the disclosure or exceeding the scope of the claims.

[0166] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.EXAMPLES

[0167] The examples presented herein represent certain embodiments of the present disclosure. However, it is to be understood that these examples are for illustration purposes only and do not intend, nor should any be construed, to be wholly definitive as to conditions and scope of this disclosure. The examples were carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail.EXAMPLE 1. Production and Purification of VN AR Antibodies for Payload ConjugationA. VNAR Antibody Constructs

[0168] A VNAR domain against human or mouse TfRl (TXP1 and TXB4, respectively;Table 3) was fused to the N-terminus of a human IgGl Fc domain as shown schematically in Fig- 1 to form a VNAR antibody. The Fc domain used for conjugation is referred to as LALA-PG-SC (Table 1) and includes mutations which (a) attenuate Fc effector function - LALA-PG (L13A, L14A, P108G) (Lo, Kim et al. 2017) and (b) introduce a surface exposed cysteine as a suitable conjugation site (S221C) (Stimmel, Merrill et al. 2000). Table 1 provides the amino acid sequences for wild type (WT) and the LALA-PG-SV variant of human IgGl Fc domains. The mutations for attenuation of the effector function are depictedin bold and underlined (L13A, L14A, P108G) as is the substitution of the surface exposed serine to cysteine (S221C).

[0169] To test the brain shuttle compatibility of a VNAR with a LALA-PG-SC Fc domain, the VNAR antibodies were cloned in the Exp293F expression system (ThermoFisher) and used for protein production following the manufacturer's instructions. Transiently transfected cells were grown for 5 days, collected and centrifuged at 2000 rpm for 10 minutes. Supernatants were filtered using 0.22 pm membrane filters and loaded onto a Protein A- affinity column (HiTrap MabSelect SuRe; Cytiva) pre-equilibrated with PBS, pH 7.4. The bound VNAR antibodies were eluted with 0.1 M glycine, pH 2.7 and subsequently purified by size exclusion chromatography (SEC) using HiLoad 26 / 600 Superdex 200 pg column (Cytiva) and PBS pH 7.4 as mobile phase. Collected peak fractions were pooled and concentrated on Protein Concentrators PES, 10K MWCO (Pierce), filtered using a 0.22 pm syringe filter and stored at -80°C.

[0170] Characterization of the purified VNAR antibodies showed no difference in expression and purification yields between TXP1 fused to either WT or LALA-PG-SC Fc domains. Further, TXP1 WT as well as TXP1 LALA-PG-SC were tested for binding to human TfRl by ELISA (Fig. 2). The calculated EC50 affinities were comparable at 4.2 nM and 4.1 nM for TXP1 WT and TXP1 LALA-PG-SC, respectively (Table 6).

[0171] For the ELISA, high-binding plates (Greiner) were coated with 100 pl of 5 pg / ml purified recombinant human TfRl diluted in PBS and incubated overnight at 4°C. Plates were washed 3x with 300 pl of PBST (PBS with 0.1% Tween20) and blocked with 2% BSA in PBST for 1 hr at room temperature. Next, the plates were washed and incubated with serially diluted TXP1 WT or TXP1 LALA-PG-SC. After 1 hr incubation, the plates were washed and incubated for 1 hr with 1 :5000 diluted detection antibody, anti-hFc-HRP (Sigma) or with 1 : 1000 diluted streptavidin-HRP (Merck Millipore), before a final wash. The signal was developed using HRP substrate in colorimetric reaction with 1-Step™ Ultra TMB- ELISA Substrate Solution (ThermoFisher) and stopped with 1% HC1 solution. The plates were analysed using a microplate reader (ThermoFisher) with reading at 450 nm. Four- parametric non-linear regression analysis was used to define EC50 values (Prism).Table 6. ELISA binding affinity of hTfRl to VNAR TXP1 WT and TXP1 LALA-PG-SCExample 2. PEG-biotin ConjugationA. Conjugation

[0172] The conjugation process followed the method described by (Bai et al. 2020) with the below described modifications. To remove glutathione (GSH) and / or free cysteine caps from the surface exposed cysteine, the purified VNAR antibodies were reduced with tris(2- carboxy ethyl) phosphine (TCEP) at 1 : 50 molar ratio using 0.5 M stock pH 7.2 for 1 hr at 37°C. To quench TCEP and reestablish any reduced structural disulfide bridges, dehydroascorbic acid was subsequently added to the samples at 1 : 100 molar ratio using 20 mg / ml stock in DMSO and incubated further for 90 min at 25°C. The VNAR antibodies were buffer-exchanged to PBS supplemented with 2 mM EDTA, pH 6.8 using a HiPrep 26 / 10 desalting column and concentrated to approximately 2 mg / ml using Protein Concentrator PES, 30K MWCO (Pierce). The VNAR antibodies were conjugated with maleimide-PEGll- Biotin compound (PEGbio; ThermoFisher) which allowed formation of stable thioether bonds during overnight incubation at room temperature. To remove excess unconjugated compound, the samples were buffer exchanged to PBS, pH 7.4 using Protein Concentrators PES, 10K MWCO (Pierce).B. Results

[0173] TXP1 LALA-PG-SC was used for the conjugation process with a test compound containing maleimide arm for conjugation to the cysteine sulfhydryl group, PEG to improve solubility and biotin to allow detection with streptavidin (maleimide-PEGl 1 -Biotin, ThermoFisher). The conjugation method included gentle reduction of the surface exposed cysteine to remove caps such as glutathione (GSH) that form during protein expression in tissue culture and that could inhibit site-specific conjugation. The reduction conditions with TCEP were optimised to remove the caps but to retain structural disulfide bridges in bothVNAR and Fc domains. Subsequently, the reducing agent was quenched with an oxidating agent that also allowed recreation of any reduced structural disulfides before buffer exchange and PEGbio conjugation. After an overnight reaction, excess the compound was removed by buffer exchange and the products were analysed on a non-reducing SDS-PAGE gel. A mass shift was observed on the gel following incubation with streptavidin that interacted with biotin, confirming successful conjugation of PEGbio with TXP1 LALA-PG-SC (Fig. 3); no conjugation was observed with TXP1 WT. The lack of gel shift with the TXP1 WT control for the process indicated that the conjugation with TXP1 LALA-PG-SC is predominantly via the surface exposed cysteine and thus site-specific.

[0174] To confirm retention of binding specificity after site-specific conjugation, TfRl binding was assessed by ELISA using conjugated and unconjugated TXP1 LALA-PG-SC. First, binding to human TfRl was detected with anti-Fc antibodies (Fig. 4) with calculated EC50 affinities of at 1.7 nM and 2.1 nM for unconjugated and conjugated TXP1 LALA-PG- SC, respectively (Table 7). Second, the same ELISA plate was used with streptavidin for biotin detection of the conjugate (Fig. 5) and showed an EC50 affinity of 0.9 nM for PEGbio conjugated TXP1 LALA-PG-SC but no detection of unconjugated TXP1 LALA-PG-SC (Table 7). The results showed successful site-specific conjugation of these brain shuttle constructs using maleimide chemistry with no effect on antibody binding to the target.Table 7. Binding affinity of hTfRl to unconjugated and conjugated TXP1 LALA-PG-SCExample 3. Oligonucleotide ConjugationA. Conjugation

[0175] For oligonucleotide conjugation, VNAR antibodies were prepared as described for the PEGbio conjugation above. To produce 5’-maleimido-modified oligonucleotides suitable for conjugation with the protein constructs, 5’-amino-modified oligonucleotides (ASO or siRNA) were prepared by mixing with sulfo-SMCC at 1 : 100 molar ratio in PBS pH 6.8 overnight at room temperature with gentle agitation followed by buffer exchange to PBS supplemented with 2 mM EDTA, pH 6.8 using a HiPrep 26 / 10 desalting column.

[0176] Next, the VNAR antibodies were incubated with freshly prepared reactive 5’- maleimido-modified oligonucleotide at 1 : 1.4 molar ratio overnight at room temperature. The reaction was diluted 10-fold with PBS pH 7.4 before being loaded onto an anion exchange column (HiTrap Q HP) and gradient eluted using PBS pH 7.4 supplemented with 2 M NaCl. The selected peaks were further purified using size exclusion chromatography (SEC) Superdex 200 26 / 600 with PBS pH 7.4 as mobile phase. The eluted constructs were concentrated using Protein Concentrator PES, 30K MWCO (Pierce), filtered using a 0.22 pm syringe filter and stored at -80°C.B. Results

[0177] The mouse brain shuttle, TXB4 LALA-PG-SC, was conjugated to an anti-sense oligonucleotide (ASO) that was additionally biotinylated at its 3’ end to allow easy detection (ASObio). The conjugation was followed by a two-step purification process using an ion exchange step to remove unconjugated protein, and SEC to remove any residual unreacted oligonucleotide. This conjugate is designated herein as TXB4-ASObio. The conjugate TXB4-ASObio was subsequently detected in a gel mass shift assay by loading 10-30 pg total protein in loading buffer and resolving by SDS-PAGE under non-reducing conditions (ThermoFisher). The TXB4-ASObio reacted with added streptavidin during incubation preceding non-reducing, native SDS-PAGE analysis, showed a significant mass shift, confirming successful conjugation (Fig. 6).

[0178] To confirm binding to mouse Tfrl by TXB4 LALA-PG-SC before and after conjugation with ASObio, and the constructs were tested using ELISA methods. First, binding to mouse TfRl was detected with anti-Fc antibodies (Fig. 7), resulting in calculated EC50 affinities of 0.16 nM and 1.4 nM for unconjugated and ASObio conjugated TXB4LALA-PG-SC, respectively (Table 8). Second, the same ELISA plate was used with streptavidin for biotin detection of the conjugate (Fig. 8) and showed an EC50 of 0.28 nM for ASObio conjugated TXB4 LALA-PG-SC but no detection of unconjugated TXB4 LALA- PG-SC (Table 8). Consequently, the results showed successful ASObio conjugation of the TXB4 LALA-PG-SC brain shuttle construct using maleimide chemistry with no effect on antibody binding to the target.Table 8. Binding affinity of mTfRl to unconjugated and conjugated TXB4 LALA-PG-SCExample 4. In Vitro Internalization

[0179] The in vitro activity of TXB4-ASObio was assessed using a cell internalisation assay. For this assay, mouse brain endothelial cells, bEnd.3, were seeded at 5,000 cells / well in 100 pL DMEM complete culture medium in 96-well pClear microplate and cultured for 1 day. The cells were incubated with the test VNAR antibodies at the final 0.1 pM concentration for 1 hour at 37°C incubator (5% CO2). The cells were washed with PBS, fixed with 4% PFA for 15 minutes, washed again and permeabilized with 0.1% saponin in PBS supplemented with 5% goat serum. Cells were incubated with anti -human IgG Alexa488-conjugated secondary antibody (Thermo Fisher) for 1 hour in the dark followed by a nuclear stain. Internalisation was measured by fluorescent microscopy and compared to VNAR isotype control antibody (G12, which has a non-cell binding, non-specific VNAR domain fused to an Fc LALA-PG-SC domain). Fluorescence was measured using Celllnsight NXT (Thermo Scientific) and analysed using HCS studio software. The assay showed TXB4-ASObio conjugate to be internalised by the cells (Fig. 9).Example 5. MAPT Knockdown Activity

[0180] Knock-down (KD) activity of TXB4-ASObio conjugate was tested in mouse Neuro 2A (N2a) cells with an ASO designed to specifically target microtubule associated protein tau (MAPT) gene expression (Table 9).

[0181] Table 9 provides the sequences of the ASO (DeVos, Goncharoff et al. 2013) and siRNA (Malecova, Burke et al. 2023) used for conjugation with brain shuttles. In the sequences, the symbol * is a phosphorothioate backbone linkage, + is a locked nucleic acid (LNA) to a subsequent nucleotide base. For the ASOs, all cytosines are methylated. For the siRNA, Xo is 2’O-methyl on X base, Xf is 2’fluoro on X base, dX is a deoxynucleotide, iB is inverted abasic, vp is vinylphosphonate, m is 2 ’methoxy-ethyl and s is phosphorothioate. The 3’C6SSC6dT moiety is six carbons disulfide linked to six carbon followed by a terminal dT nucleotide.

[0182] For the KD activity assay, Neuro2A cells were seeded at 250,000 / well in a 12 well plate (1.0 mL volume) and cultured in EMEM with 2mM glutamine, 1% NEAA and 10% FBS for 4 days to reach 80% confluency. The KD was performed with 100 nM concentration of the test VNAR antibodies using RNAiMAX transfection reagent (Thermo Scientific). After 3 additional days of incubation, the cells were collected, RNA isolated and cDNA prepared. The samples were used for qPCR assessment using TaqMan method and quantified for MAPT gene expression using RT-PCT CFX instrument (BioRad). The expression was normalised to the PPIA housekeeping gene expression and showed approximately 50% reduction in comparison to the untreated cells (Fig. 10).

[0183] The stability of the conjugated oligonucleotide on TXB4-ASObio was determined after incubation for 7 days at 37°C at 100 nM concentration in either PBS (Fig. 11) or mouse serum (Fig. 12). In both cases, the conjugated TXB4-ASObio was assessed by ELISA for binding to mouse TfRl followed by detection with either human Fc antibodies or streptavidin. The analysis showed that the oligonucleotide conjugation remained stable for the period of 7 days.Table 9. ASO and siRNA SequencesExample 6. In Vivo Activity of Mouse TXB4 targeting MAPT

[0184] The biological activity of TXB4-ASObio was assessed in vivo in mice. Animals were IV dosed at 25 nmol / kg and the brains were extracted following cardiac perfusion to remove residual blood from the brain as generally described in WO2021 / 102276. The homogenates were prepared and quantified by ELISA.

[0185] For the tissue ELISA, sample preparation and the ELISA protocol followed the methods described before (Stocki, 2021). In brief, milk-blocked supernatants recovered from homogenized tissues or plasma were analyzed by ELISA using human specific anti-hFc capture (Thermo Fisher) and detection (Sigma) antibodies. The absolute concentrations were determined from standard curves prepared individually for each fusion protein using 4- parametric regression analysis.

[0186] Unconjugated TXB4 LALA-PG-SC as well as the TXB4-ASObio conjugate was shown to penetrate the brain at 8.5 nM and 10.5 nM concentrations, respectively (Fig. 13) while isotype control antibody showed 0.47 nM concentration in the brain. Plasma levels showed 153 nM for isotype control and 59 nM and 74 nM concentrations for TXB4 LALA- PG-SC and TXB4-ASObio conjugate, respectively (Fig. 14). Brain selectivity wasconfirmed by biodistribution analysis that included liver, kidney, spleen, lung, heart and femur muscle. No increase over isotype control was observed in any of the tested organs except for the brain for unconjugated TXB4 LALA-PG-SC. The brain selectivity was reduced for TXB4-ASObio conjugate with up to a 4.5-fold level increase observable in lung and heart (Fig. 15).Example 7. In Vivo Activity of Human TXP1-LALA-PG-SC targeting HPRT

[0187] TXP1 LALA-PG-SC was conjugated to an siRNA targeting HPRT (Table 9; (Malecova, Burke et al. 2023) followed by two-step purification. Quality control using analytical SEC showed desired a mass shift in comparison to unconjugated TXP1 LALA-PG- SC that resulted from the siRNA conjugation with overall >95% purity (Fig. 16). Because the conjugation process used a 1 : 1.4 of TXP1 LALA-PG-SC to siRNA ratio, the final product contained single siRNA per protein dimer, referred to as DARI (drug to antibody ratio of 1) as verified by SDS-PAGE analysis (Fig. 17). The follow up affinity testing based on ELISA showed that the siRNA conjugation had no negative impact on either human TfRl (hTfRl) or cynomolgus macaque TfRl (cTfRl) binding by TXP1, producing EC50 affinity values in the range 1.7-2.8 nM (Table 10, Fig. 18 and Fig. 19).

[0188] The conjugated TXP1 -siRNA was tested for gene KD in vivo using Tg mice expressing human TfRl. The animals were dosed at 1 mg / kg (siRNA molar equivalent) by IV on days 0 and 7. The tissues were collected on day 21 and assessed for gene expression by quantitative PCR. The HPRT expression was normalised to GAPDH expression before comparison to the untreated control group and showed 66% reduction confirming successful brain delivery and feasibility of the therapeutic approach used (Fig. 20).Table 10. Binding affinity of hTfRl and cTfRl to unconjugated and siRNA-conjugated TXP1 LALA-PG-SCExample 8. Further Knockdown Activity Experiments

[0189] For the KD activity assay, human neuronal SH-SY5y cells or mouse Neuro2A (N2a) cells were seeded at 250,000 / well in a 12 well plate (1.0 mL volume) and cultured in EMEM with 2mM glutamine, 1% NEAA and 10% FBS for 4 days to reach 80% confluency. The KD was performed with 100 nM concentration of the test VNAR antibodies using RNAiMAX transfection reagent (Thermo Scientific). After 3 additional days of incubation, the cells were collected, RNA isolated and cDNA prepared. The samples were used for qPCR assessment using TaqMan method and quantified for gene expression using RT-PCT CFX instrument (BioRad). The expression was normalised to the PPIA housekeeping gene expression.

[0190] Fig. 21 shows the KD activity of TXP1 LALA-PG-SC conjugated to siRNA targeting MAPT (TXP1 -siRNA MAPT2) in SH-SY5Y cells. The sequence of the siRNA is shown in Table 5 (SEQ ID NOS: ) and was used with the modifications shown in Table 11. Conjugation to the VNAR antibody was via a NH2C6 linker added to 5’ end of the sense strand.

[0191] Fig. 22 shows the KD activity of TXP1 -siRNA MAPT2 in N2a cells.

[0192] Fig. 23 shows the KD activity of TXP1 LALA-PG-SC conjugated to siRNA targeting alpha-synuclein (SNCA) (TXP1 -siRNA SNCA) in SH-SY5Y cells. The sequences of the siRNA for SNCA is shown in Table 5 (SEQ ID NOS: ) and was used with the modification shown in Table 12. Conjugation to the VNAR antibody was via a NH2C6 linker added to 5’ end of the sense strand.

[0193] In Table 11 and Table 12, show the sense and antisense sequences in 5’ to 3’ orientation (from top to bottom). Abbreviations used: m - 2'-O-methyl, f- 2'-fluoro, s - phosphorothioate, VP - vinyl phosphonate, s - phosphorothioate.Table 11. The sequences of siRNA for MAPT knockdown with modifications.Table 12. The sequences of siRNA for SNCA knockdown with modifications.

[0194] The EC50 was calculated for siRNA MAPT potency in SH-SY5Y (see, Fig. 21), N2a (see, Fig. 22) and SNCA potency in SH-SY5Y (see, Fig. 23). Four parametric non-linear regression was used for EC50 value calculation. The results are shown in Table 13.Table 13. The knock down potency of siRNA as assessed in vitro cell based assays.REFERENCES

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Claims

We claim:

1. A single chain VNAR antibody which comprises a BBB-shuttling VNAR domain fused to the N-terminus of a mammalian immunoglobulin (Ig) chain, wherein said the Ig chain of said VNAR antibody is effector-attenuated and capable of site-specific conjugation at a surface accessible cysteine.

2. The VNAR antibody of claim 1, wherein said Ig chain is an IgM, IgA, IgG, IgE chain, a single chain Fv, an Fab fragment, or an Fc domain, or a portion thereof.

3. The VNAR antibody of claim 1 or 2, wherein said Ig chain is an Fc domain chain from an IgG light chain or a an IgG heavy chain.

4. A single chain VNAR antibody which comprises a VNAR domain fused to the N- terminus of a human IgGl Fc LALA-PG-SC domain comprising L13A, L14A, P108G and S221C substitutions (using the numbering system in Table 1) or having substitutions which are positionally equivalent thereto; and wherein said VNAR antibody is effector-attenuated and capable of site-specific conjugation at the S221C cysteine or positionally equivalent thereto.

5. The VNAR antibody of any one of claims 1-4, wherein said BBB-shuttling VNAR domain is(a) a TfR-binding VNAR domain capable of specifically binding to a human TfR-1 without substantially interfering with transferrin binding to and / or transport by said human TfR-1,(b) a TfR-binding VNAR domain capable of specifically binding to a human TfR-1 without substantially interfering with transferrin binding to and / or transport by said human TfR-1 and capable of cross reacting with mouse TfR 1, or(c) a TfR-binding VNAR domain capable of binding human TfR-1 with an EC50 ranging from about 1 nM to about 800 nM.

6. The VNAR antibody of any one of claims 1-5, wherein said BBB-shuttling VNAR domain is(a) the TfR-binding VNAR domain designated as Clone C or one of its variants,(b) the TfR-binding VNAR domain designated as Clone H or one of its variants,(c) the TfR-binding VNAR domain designated as Clone 8 or one of its variants(d) TXB4,(e) TXP1, or(f) a CD98-binding VNAR domain.

7. The VNAR antibody of any one of claims 1-5, wherein said VNAR domain is a Type II VNAR domain represented by the formula, from N to C terminus, FW1-CDR1-FW2-HV2- FW2’-HV4-FW3-CDR3-FW4, wherein CDR1 has an amino acid sequence of DSNCALSS (SEQ ID NO: 1) and CDR3 has an amino acid sequence of VVGTWCMSWRD V (SEQ ID NO: 10), and wherein said VNAR domain is capable of specifically binding to human TfRl without substantially interfering with transferrin binding to and / or transport by human TfRl.

8. The VNAR antibody of Claim 7, wherein said VNAR domain comprises an amino acid sequence of ARVDQTPQTITKETGESLTINCVLRDSNCALSSTYWYRKKSGSTNEENISKGGRYV ETVNSGSKSFSLKINDLTVEDSGTYRCNVVGTWCMSWRDVYGGGTAVTVNA (SEQ ID NO. 6).

9. The VNAR antibody of Claim 7, wherein said VNAR domain comprises an amino acid sequence of any one of the sequences in Table 2 or Table 3, and preferably one of the sequences in Table 3.

10. The VNAR antibody of any one of claims 1-5, wherein said VNAR domain is a Type II VNAR domain represented by the formula, from N to C terminus, FW1-CDR1-FW2- HV2-FW2’-HV4-FW3-CDR3-FW4, wherein CDR1 has an amino acid sequence of DSNCALSS (SEQ ID NO: 1) and CDR3 has an amino acid sequence of VQYPQYPNYFWCDV (SEQ ID NO: 11), and wherein said VNAR domain is capable of specifically binding to human TfRl without substantially interfering with transferrin binding to and / or transport by human TfRl .

11. The VNAR antibody of Claim 10, wherein said VNAR domain comprises an amino acid sequence of ARVDQTPQTITKETGESLTINCVLRDSNCALSSTYWYRKKSGSTNEENISKGGRYVET VNSGSKSFSLRINDLTVEDSGTYRCNVVQYPQYPNYFWCDVYGDGTAVTVNA (SEQ ID NO: 2).

12. A bispecific VNAR antibody which comprises a dimer of the single chain VNAR antibody of any one of claims 1-11.

13. A nucleic acid encoding the VNAR antibody of any one of claims 1-11.

14. A vector comprising a nucleic acid of Claim 13.

15. A host cell comprising the vector of Claim 14.

16. The VNAR antibody of any one of claims 1-12 conjugated to at least one diagnostic or therapeutic agent via the sulfhydryl of the surface accessible cysteine, the site-specific cysteine residue at S221C or its equivalent.

17. The VNAR antibody of Claim 16, wherein said agent is selected from the group consisting of oligonucleotides, polypeptides, cytotoxins, enzymes, proteins, peptides, small molecules, radioisotopes, fluorophores, polymers, lipids, nanoparticles, liposomes, capsids, and viruses.

18. The VNAR antibody of Claim 17, wherein said agent is an oligonucleotide.

19. The VNAR antibody of Claim 18, wherein said oligonucleotide targets an ASO or siRNA of Table 4 or Table 5.

20. The VNAR antibody of Claim 18, wherein said agent is ASO (MAPT), ASObio (MAPT) or ASObio (MAPT) of Table 9 or an oligonucleotide of Table 11 or Table 12.

21. A pharmaceutical composition comprising a VNAR antibody conjugate of any one of claims 16-20.

22. A method of medical treatment which comprises administering a therapeutically- effective amount of the pharmaceutical composition of Claim 21 to deliver a diagnostic or therapeutic agent to the brain of a mammalian subject in need thereof.

23. Use of a VNAR antibody conjugate of any one of claims 16-20 for the preparation of a medicament to deliver a diagnostic or therapeutic agent to the brain of a mammalian subject in need thereof.

24. A method of targeting delivery of a payload to brain parenchymal tissue in a mammal which comprises administering a VNAR antibody conjugate of any one of claims 16-20.

25. Use of a VNAR antibody conjugate of any one of claims 16-20 for targeting delivery of a payload to brain parenchymal tissue in a mammal.

26. A method of delivering a therapeutic or diagnostic agent across the blood brain barrier which comprises administering a VNAR antibody conjugate of any one of claims 16-20 to a subject for a time and in an amount effective to treat or diagnose a CNS disease or condition.

23. A method of delivering a therapeutic or diagnostic agent to the gastrointestinal (GI) tract which comprises administering a VNAR antibody conjugate of any one of claims 16-20 to a subject for a time and in an amount effective to treat or diagnose a GI disease or condition.

27. A method of producing an VNAR antibody conjugate which comprises treating the VNAR antibody of any one of claims 1-12 in solution with a reducing agent under conditions sufficient to reduce solvent accessible (surface-exposed) cysteine residues while retaining interchain, structural disulphate bridges in the VNAR and Ig or Fc domains, quenching aid reducing agent with an oxidizing agent, adding a maleimide-conjugating agent to said solution, incubating for a time sufficient and at a temperature to form stable thioether bondsand produce said VNAR antibody conjugate, and separating said VNAR antibody conjugate from unconjugated VNAR antibody.

28. The method of claim 27, wherein said maleimide-conjugating agent is maleimide- PEG11 -Biotin or a maleimide-nucleotide.

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