Protein conjugates

Abstract

This invention relates to novel protein conjugates that can be used to label red blood cells (RBCs) with one or more enzymes. The conjugates and / or the labelled RBCs can be used to deliver the one or more enzymes in therapeutic contexts. The invention also relates to RBC labelled with the protein conjugates and therapeutic methods using the protein conjugates and / or labelled RBCs.

Description

[0001] NOVEL PROTEIN CONJUGATES

[0002] TECHNICAL FIELD

[0003] This invention relates to novel protein conjugates that can be used to surface label red blood cells (RBCs) with one or more enzymes. The conjugates and / or the surface labelled RBCs can be used to deliver the one or more enzymes in therapeutic contexts. The invention also relates to RBCs surface labelled with the protein conjugates and therapeutic methods using the protein conjugates and / or surface labelled RBCs.

[0004] BACKGROUND

[0005] A variety of diseases and disorders, especially in humans, involve a reduced amount and / or activity of a specific enzyme. Enzyme replacement therapies face a variety of challenges, such as a short in vivo half-life of the enzyme, lack of targeted action and the patient generating an immune response against the enzyme. Conventional enzyme therapies often face rapid clearance from the body within hours or a day, toxicity at high doses and patient non-compliance due to the requirement for frequent dosing due to rapid drug clearance.

[0006] Several approaches for delivering enzymes have been tested, including encapsulation in red blood cells (RBCs) or other lipid-based delivery systems. Encapsulation of enzymes in RBCs has some disadvantages, including the need for a release mechanism and damage to the RBCs. Some researchers have proposed genetically modifying RBC precursors to generate genetically modified RBCs, but that can be expensive and require a higher regulatory burden.

[0007] The surfaces of RBCs have also been labelled with a variety of molecules, such as erythropoietin (EPO; WO 2020 / 132234), Von Willebrand factor (vWF; US 2022 / 0348637), Factor 8 (US 2022 / 0089685) and thrombomodulin (WO 2019 / 113224), and the surface labelled RBCs have been used to deliver those molecules in vivo. The use of RBCs to deliver these molecules can improve their pharmacodynamics and pharmacokinetics because RBCs circulate throughout the body and last for approximately 120 days.

[0008] The surfaces of RBCs have been labelled previously using traditional non-specific chemical modifications, but this can impair protein functions on the RBC, leading to a reduced RBC lifespan. Other approaches have utilised antibody- or peptide-mediated methods, showing promising results in treating conditions like stroke and heart disease in mouse models but to our knowledge no enzymes have been successfully attached to RBC using an antibodyenzyme fusion protein. For example, the anti-thrombolytic thrombomodulin has been attached to human RBCs using single-chain variable fragments (scFvs, e.g., WO 2019 / 113224). However, this is not an enzyme and the method may not fully leverage the long circulation time of RBCs as the work did not illustrate the stability of the interaction and relied on attachment to a less abundant protein. When attempts were made to use scFvs to attach to the higher abundant band 3 or GPA proteins on the RBC surface this caused a disturbance in deformability illustrating this method has limitations. It is also unknown if an enzyme can be fused directly to an antibody and retain enzyme functionality and whether this enzymes activity would remain once attached to a RBC by such an antibody. This is particularly the case for enzymes that are multimeric.

[0009] Strategies like biotin-streptavidin linking, such as the attachment of another anti- thrombolytic, tissue-type plasminogen activator (tPA), to RBCs from animal blood (Armstead WM et al., J Cereb Blood Flow Metab. 2009 Aug;29(8): 1463-74. doi: 10.1038 / jcbfm.2009.61. Epub 2009 May 13), is another method previously used method for non-specific binding to RBC, but as well as lack of specificity this is likely to be immunogenic and so unsuitable for repeat dosing.

[0010] There is therefore a need for improved delivery of enzymes in a variety of therapeutic contexts and for improved methods for targeted, stable surface labelling RBCs with minimal immunogenicity for delivery in vivo.

[0011] SUMMARY OF THE INVENTION

[0012] The inventors have surprisingly shown that the surfaces of RBCs can be labelled with one or more enzymes using protein conjugates comprising the one or more enzymes and antibodies specific for a target antigen on the surface RBCs. In particular, the inventors have surprisingly demonstrated the enzymes in the conjugates of the invention remain functional and can even form functional multimers despite the presence of the antibody. They have also surprisingly shown the RBCs remain viable and functional when surface labelled with the conjugates of the invention.

[0013] In the non-limiting Examples, the inventors combine a RBC specific nanobody, IH4, that targets glycophorin A (GPA), a protein exclusively found on the surface of RBCs, fused to therapeutic enzymes. The IH4 nanobody is preferable because of its high affinity and specificity to GPA, and the inventors have shown that the binding to RBCs persists and does not affect RBC functionality. Therefore, the exemplar nanobody is IH4 but it is possible to extend this to other RBC-specific antibodies which also could be used to attach therapeutics. Importantly, nanobodies are known to be well tolerated by the immune system and have been used in therapeutics previously, such as the nanobody targeting the spike protein of SARS-CoV-2. Furthermore, it has also been shown that attachment to RBCs can also induce tolerance to immunogenic proteins which is another attractive feature of using this methodology to couple proteins to the outside of the cell.

[0014] The invention contemplates two ways in which the surfaces of RBCs can be labelled with the protein conjugates of the invention : (1) a non-covalent approach using antibodies or variants thereof alone for potentially reversible and temporary labelling and (2) a covalent approach for stable, long-term labelling, ideal for applications requiring persistent modification. The choice of method will depend on the specific application, offering flexibility to meet various therapeutic or diagnostic needs. In some scenarios non-covalent conjugation will be advantageous whilst in others covalent will be. The inventors also provide a beta-swapped antibody variant for facilitating covalent attachment in (2).

[0015] The attachment of the enzyme to an antibody, such as a nanobody, means that the enzyme will be non-covalently attached to the RBCs and the inventors have shown that IH4 nanobody association with RBCs lasts a prolonged time. The attachment to RBCs will extend the circulation time of enzymes in the bloodstream, offering an improvement over traditional enzyme replacement therapies which are usually just injected as recombinant proteins and therefore often rapidly removed and degraded, necessitating frequent injections. The antibody attachment of enzymes to RBC as treatment is likely to be vastly more cost effective, is less complicated (the recombinant protein is produced in E.coli using established methodology) and this approach has less regulatory barriers than genetic modification of red blood cells during development. Importantly, the inventors have shown using thymidine phosphorylase as an example, that the coupling to nanobodies works for dimeric enzymes. This opens up the potential for combinations of enzymes working together to produce therapeutic effects or combined enzyme reactions.

[0016] In certain scenarios, this non-covalent approach may be more advantageous for optimally extending enzyme activity without unnecessary prolongation, especially in instances where prolonged enzyme treatment is unnecessary or undesired. There are some situations, including some diseases or disorders, where excessive long-lasting treatment is not required or where it is advantageous for the therapeutic enzyme(s) to unbind from the RBCs and / or be cleared from the circulation. The duration of labelling of the surfaces of the RBCs can be altered by increasing the affinity of the antibody or variant thereof.

[0017] The covalent approach of the invention, where protein conjugate is modified to facilitate covalent binding to RBCs, is advantageous in situations where long-lasting treatment is needed and / or maintenance of the one or more enzymes in the circulation is preferred.

[0018] The inventors envisage there are two options for how to utilise this technology. One is where the conjugate of the invention is injected into the bloodstream and then the nanobody becomes rapidly associated with the RBCs. The other is a more standard approach for attachment to RBCs, where RBCs are removed from the body first, the conjugate of the invention is bound to the RBCs, and then the RBCs are reinjected. A key advantage of the invention is the RBCs can be surface labelled in vitro or ex vivo, when the RBCs are surface labelled before the RBCs are administered to the patient, or in vivo, when the protein conjugate itself is administered to the patient such that it surface labels the patient's endogenous RBCs. The surface labelling of RBCs using the protein conjugates of the invention also overcomes the disadvantages of standard enzyme replacement therapy (ERP), encapsulation of enzymes in RBCs and the genetic modification of RBCs.

[0019] The concept of attaching enzymes to RBCs aims to significantly improve their pharmacokinetic profile of injected recombinant therapeutics. By linking enzymes to RBCs, their half-life may even extend to the RBCs lifespan of up to 4-months. This feature is particularly advantageous for chronic metabolic diseases like thymidine phosphorylase- deficiency (MNGIE) and adenosine deaminase-deficiency (SCID), where sustained therapeutic action is needed, potentially reducing the frequency of doses. In addition, linking enzymes to RBCs could confine their distribution to the bloodstream, minimising non-specific tissue penetration and toxicity while lowering the maximum concentration required for effectiveness.

[0020] Nanobodies are particularly advantageous as they are not typically immunogenic due to their small size and high sequence homology to human proteins, which reduces their likelihood of triggering an immune response. This makes them an ideal choice for linking enzymes directly to unmodified RBCs under mild physiological conditions, preserving both enzyme activity and RBC integrity, and eliminating the need for a release mechanism. Moreover, the exemplified nanobody technique targets GPA, a protein specifically found on RBCs, thereby streamlining the administration process by not only enhancing scalability and reducing production costs but also simplifying the therapeutic application. Unlike traditional methods that involve extracting, modifying and reintroducing RBCs (these could be autologous RBCs; allogenic RBCs sourced from a commercial blood bank; or artificially manufactured universal donor RBCs), the therapeutic fused to the nanobody could be administered directly with the attachment of enzymes occurring in vivo within the patient's bloodstream, using the patient's own RBCs. This direct and non-invasive approach eliminates multiple steps involved in other approaches and significantly reduces the complexity and cost of treatment. As a result, this method offers significant advantages in terms of scalability and efficiency, cost effective because the proteins can be made in bacteria, potentially transforming therapeutic RBC-based delivery to improve drug efficacy, medical outcomes, and patient experiences.

[0021] The invention provides a protein conjugate comprising (a) one or more antibodies or variants thereof which specifically bind to one or more antigens on the surface of red blood cells (RBCs) and (b) one or more enzymes and / or variants thereof.

[0022] The invention also provides a protein conjugate comprising (a) an antibody or a variant thereof which specifically binds to an antigen on the surface of red blood cells (RBCs) and (b) one or more enzymes and / or variants thereof.

[0023] The invention also provides: an oligomeric construct comprising two or more protein conjugates of the invention; a population of red blood cells (RBCs) surface labelled with a protein conjugate of the invention and / or an oligomeric construct of the invention; a pharmaceutical composition comprising one or more protein conjugates of the invention, an oligomeric construct of the invention or a population of RBCs of the invention and a pharmaceutically or physiologically acceptable diluent and / or carrier; a method of treating or preventing disease or disorder in a subject, comprising administering to the subject one or more protein conjugates of the invention, an oligomeric construct of the invention, a population of RBCs of the invention or a pharmaceutical composition of the invention; and one or more protein conjugates of the invention, an oligomeric construct of the invention, a population of RBCs of the invention or a pharmaceutical composition of the invention for use in a method of treating or preventing a disease or disorder in a subject.

[0024] DESCRIPTION OF THE FIGURES

[0025] Figure 1: This figure shows the predicted protein structure of dimeric IH4-TP (Figure 1A) and TP-IH4 (Figure IB) generated by AlphaFold3, with overall confidence scores of 0.71 and 0.72, respectively. In both structures, TP is shown in black and IH4 in grey.

[0026] Figure 2: This figure illustrates the expression and purification process of the IH4-TP fusion protein. IH4-TP was expressed in a soluble form in SHuffle T7 cells and purified using the His-tag via a Ni-NTA resin (Figure 2A), followed by size exclusion chromatography (Figure 2B). Figure 2C shows the collection fractions from the three largest peaks analysed by SDS- PAGE, followed by Coomassie blue staining. Fractions corresponding to the third peak were retained and combined. The calculated molecular weight of IH4-TP is 61.5 kDa.

[0027] Figure 3: Size exclusion chromatograms of all IH4-containing constructs. Peaks corresponding to correctly folded protein are highlighted for clarity.

[0028] Figure 4: SDS-PAGE and Western blot analysis of all IH4-containing constructs. SDS-PAGE was performed and proteins in the gel were either stained with Coomassie blue (Figure 4A) or transferred to a membrane and detected using an anti-His antibody (Figure 4B). The calculated molecular weight of IH4 is 14.7 kDa, IH4-TP is 61.5 kDa, TP-IH4 is 61.8 kDa, IH4-GFP is 40.8 kDa and IH4-SC003 is 26.8 kDa.

[0029] Figure 5: Size exclusion chromatograms of all SpyTagT003-containing constructs. Peaks corresponding to correctly folded protein are highlighted for clarity. Figure 6: SDS-PAGE and Western blot analysis of all SpyTag003-containing constructs. SDS-PAGE was performed and proteins in the gel were either stained with Coomassie blue (Figure 6A) or transferred to a membrane and detected using an anti-His antibody (Figure 6B). The calculated molecular weight of ST003-TID is 38.9 kDa and SC003-GFP is 30.4 kDa.

[0030] Figure 7. Flow cytometry analysis of IH4 and IH4-TP fusion proteins interaction with RBCs. RBCs were incubated with purified IH4, IH-TP or TP-IH4, and binding activity was detected using an APC-conjugated anti-His antibody, quantified as mean fluorescence intensity (MFI). Data analysis was performed using FlowJo vl0.7 software. Representative flow cytometry histograms are shown, with control RBCs incubated with the anti-His antibody alone displayed in grey. Binding affinity was estimated by fitting the calculated MFI values versus protein concentration to a one-site specific binding model. The estimated Kd values for IH4, IH4-TP and TP-IH4 were 139 nM, 203 nM and 284 nM, respectively, with corresponding Bmax values of 73, 390 and 317 MFI, respectively. Error bars correspond to standard deviation.

[0031] Figure 8. Flow cytometry analysis of IH4 and IH4-TP fusion proteins interaction with BEL-A cells. BEL-A cells were incubated with purified IH4, IH4-TP, or TP-IH4, and binding activity was detected using an APC-conjugated anti-His antibody. Data analysis was performed using FlowJo vl0.7 software. Representative flow cytometry histograms show binding to unedited BEL-As (solid lines) and GPA KO BEL-As (dashed lines), compared to control BEL- As incubated with the anti-His antibody alone (filled in grey).

[0032] Figure 9. Preliminary assessment of stability and attachment lifetime of TP-IH4 on RBCs. TP-IH4 was incubated with 2 x 106RBCs at a concentration of 1 pM, and the presence of bound TP-IH4 was periodically measured. The cells were collected by centrifugation and detecting using an APC-conjugated anti-His antibody. The experiments were conducted in PBSAG at 4°C, room temperature, and 37°C over a period of up to 8 days. Data analysis was performed using FlowJo vl0.7 software. Representative flow cytometry histograms are shown with control RBCs incubated with the anti-His antibody alone filled in grey.

[0033] Figure 10. Representative deformability index profiles are shown for untreated RBCs (dotted lines), RBCs treated with IH4 or IH4-TP (grey lines) at the indicated concentrations, and RBC treated with the monoclonal anti-GPA antibody, Brie 256 (black lines), which is known to increase erythrocyte rigidity (black lines). Data were obtained using an Automated Rheoscope Cell Analyser (ARCA).

[0034] Figure 11. Flow cytometry analysis was performed to confirm the binding of IH4 and IH4-TP to RBCs under the same conditions used for deformability measurements. RBCs were prepared at a concentration of 2 x 106cells and proteins were added at the indicated concentrations. The analysis was conducted on 0.2 x 106cells labelled with APC-conjugated anti-His antibody. Flow cytometry data were analysed using FlowJo vl0.7 software. Representative flow cytometry histograms are shown, with control RBCs incubated with the anti-His antibody alone displayed in grey.

[0035] Figure 12. Thymidine phosphorylase activity assessed for TP alone and two IH4-TP fusion proteins across a range of concentrations (0-10 pM) following the addition of thymidine and incubation for 1 hour at 37°C, as described in the methods section. Representative absorbance spectra are shown, with the blank sample (reaction buffer only) shown as a black dashed line, the reaction with thymidine but without protein shown as a black solid line, and reactions with varying protein concentration shown by grey solid and dotted lines.

[0036] Figure 13. Percentage activity of the two IH4-TP fusion proteins compared to TP Alone. The activity of IH4-TP and TP-IH4 fusion proteins was compared to TP alone at various protein concentrations. Activities were measured by A300 nm, with thymine formation calculated using a molar extinction coefficient difference of 3.4 x 103IJmol / cm. Enzyme activity, expressed as nanomoles of thymine formed / hr / mg, is shown as a percentage of TP alone. Data points show mean values with error bars indicating standard deviation.

[0037] Figure 14. Thymidine phosphorylase activity assessed for IH4-TP bound at the RBC surface. Following binding of a ~22-fold molar excess of IH4-TP to an increasing number of RBCs, cells were pelleted, resuspended and used in endpoint TP activity assays. TP alone, which was not expected to bind to RBCs, served as a control to determine the residual activity of non-specifically bound protein. Representative absorbance spectra are shown, with untreated RBCs (no protein) shown by a black dashed line and with reactions with increasing number of cells, as indicated, shown by grey solid lines.

[0038] Figure 15. Flow cytometry analysis confirming the binding of IH4-TP to RBCs in cell-surface bound TP activity assays. Flow cytometry was performed to confirm the binding of IH4-TP to RBCs and to verify that no binding was observed with TP alone under the same conditions used for cell-surface bound TP activity assays. Following the binding of IH4-TP to RBCs, 0.2 x 106cells were labelled with APC-conjugated anti-His antibody and analysed by flow cytometry the using FlowJo vl0.7 software. Representative flow cytometry histograms are shown, with control RBCs (without protein) incubated with the anti-His antibody alone displayed in grey.

[0039] Figure 16. Flow cytometry analysis confirming the binding and specificity of IH4-SC to GPA. Purified IH4 or IH4-SC were incubated with RBCs (left panel) or BEL-A cells (solid line) and GPA KO BEL-A cells (dotted line) (right panel) at the indicated concentrations. Binding activity was detected using an APC-conjugated anti-His antibody. Representative flow cytometry histograms are shown, with control RBCs (no protein) incubated with the anti-His antibody alone displayed in grey. Flow cytometry data were analysed using FlowJo vl0.7 software.

[0040] Figure 17. Retention of isopeptide bond formation between IH4-SC and ST-GFP. IH4-SC and ST-GFP were mixed at a molar ratio of 5: 10 pM, respectively, in PBS for a total of 90 minutes at room temperature. Samples were analysed periodically after quenching the reaction by the addition of SDS-PAGE sample buffer and boiling, by SDS-PAGE with Coomassie staining, alongside unreactive controls (IH4-SC and ST-GFP alone). As ST-GFP is added in excess, the band corresponding to unreactive ST-GFP is visible in the reaction samples, whereas all SC-IH4 bound to ST-GFP is used up and therefore not visible.

[0041] Figure 18. Confirmation of assembly of IH4-SC-ST-GFP / ST-TID fusion proteins prior to binding RBCs. IH4-SC and ST-GFP or ST-TID were mixed at a molar ratio of 1:2, respectively, at the indicated concentrations for 1 hour at room temperature in PBSAG. The formation of the full-length fusion proteins, comprising IH4 and either GFP or TID, was confirmed by SDS-PAGE analysis.

[0042] Figure 19. Flow cytometry analysis confirming the binding of full-length fusion proteins, comprising IH4 and either GFP or TID to RBCs. Following the assembly of fusion proteins via the SpyCatcher-SpyTag system, the reaction mixtures were incubated with RBCs. Binding was detected using an APC-conjugated anti-His antibody or, in the case of GFP-containing reactions, by intrinsic GFP fluorescence. Representative flow cytometry histograms are shown, with control RBCs (no protein) incubated with the anti-His antibody alone displayed in grey. Control reactions included IH4-SC and either ST-GFP or ST-TID alone. Flow cytometry data were analysed using FlowJo vl0.7 software.

[0043] Figure 20. Schematic representation of the structural organisation of two IH4-beta-swapped variants, called here IH4BS1 and IH4BS2, resulting in the flipped orientation of the N and C- termini of IH4. In IH4BS1, the first beta-strand (shown in grey) is relocated to the opposite end of the nanobody in reverse orientation. After optimising amino acid residues in the newly positioned beta-strand, the final beta-swapped strand is depicted in striped grey. In IH4BS2, the beta-strand adjacent to the N-terminal strand (shown in black) is duplicated. Amino acid residues were selected for enhanced charge and hydrogen-bond complementarity, favoring those with a higher propensity for beta-sheet formation. The newly positioned final strand is shown in black stripes. Molecular dynamics (MD) simulations were performed for 100 ns using the final IH4-beta-swapped sequences, with the final frame's model presented, highlighting the newly positioned beta-strand in each variant.

[0044] Figure 21. Flow cytometry analysis of IH4BS1 interaction with RBCs. RBCs were incubated with purified IH4BS1, and binding activity was detected using an APC-conjugated anti-His antibody, quantified as mean fluorescence intensity (MFI). Data analysis was performed using FlowJo vl0.7 software. Representative flow cytometry histograms are shown, with control RBCs incubated with the anti-His antibody alone displayed in grey. Binding affinity was estimated by fitting the calculated MFI values versus protein concentration to a one-site specific binding model. The estimated Kd values for IH4BS1 is 7.7 pM with corresponding Bmax values of 9.5. Error bars correspond to standard deviation.

[0045] Figure 22. Flow cytometry analysis confirming the specificity of IH4BS1 to GPA. Purified IH4BS1 was incubated with differentiated BEL-A cells (solid line) and GPA KO BEL-A cells (dotted line) at the indicated concentrations. BEL-A cells were first differentiated for 7 days to increase GPA expression levels to comparable to those found in RBCs, which naturally express higher levels of GPA. Binding activity was detected using an APC-conjugated anti- His antibody. Representative flow cytometry histograms are shown, with control BEL-As (no protein) incubated with the anti-His antibody alone displayed in grey. Data analysis was performed using FlowJo vl0.7 software.

[0046] Figure 23: Size exclusion chromatograms of IH4-beta swapped constructs fused with SPM (the self-processing module (SPM) from the FrpA protein of Neisseria meningitis'). Peaks corresponding to correctly folded protein are highlighted for clarity.

[0047] Figure 24. SDS-PAGE analysis of purified IH4BS-SPM constructs. IH4BS1-SPM1 and IH4BS2-SPM were expressed in SHuffle T7 Express cells and purified using the same protocol as for IH4 and IH4-protein fusion proteins, but in HBS buffer. Following purification, successful cleavage of the SPM domain was induced by adding 2 mM CaC in HBS buffer and incubating the mixture for 1 hour at 37°C. Additionally, for IH4BS2-SPM, a Western blot analysis was performed using an HRP-conjugated anti-His antibody to confirm successful cleavage of the SPM domain.

[0048] Figure 25. Flow cytometry analysis of the binding of IH4BS-SPM variants to RBCs. Purified IH4BS1-SPM1 and IH4BS2-SPM (black dotted line) were incubated with RBCs alongside their corresponding constructs without the SPM domain (black line) and WT IH4 (grey line) at a concentration of 10 pM in HBS buffer for 1 hour at 37°C. The cells were then washed. In samples where the NeissLock component was activated, Ca2+was added, and the mixture was further incubated for 1 hour at 37°C, followed by an additional wash. Binding was detected using an AF647-conjugated anti-VHH antibody. Representative flow cytometry histograms are shown, with control RBCs (no protein) incubated with the anti-VHH antibody alone displayed in grey. Data analysis was performed using FlowJo vl0.7 software. The table shows the measured MFI values from the histograms for each condition, providing a clearer representation of the binding levels.

[0049] Figure 26: This figure illustrates the possible structure, expression and purification of the IH4-ADA fusion protein. The predicted protein structure of IH4-ADA as generated by AlphaFold3, with ADA shown in dark grey, and IH4 is shown in light grey. The model shows side and front views. Beneath is a simplified cartoon illustration showing a possible structure side and front views with ADA shown in dark grey, and IH4 is shown in light grey. (Figure 26A). IH4-ADA was expressed in a soluble form in BL21 (DE3) cells, purified using the His- tag via a Ni-NTA resin, and further purified by size exclusion chromatography (SEC). The process was followed and analysed using SDS-PAGE on a 4-20% gel, visualised with Coomassie blue staining (Figure 26B). Peak 2 protein sample was transferred to a membrane and detected using an anti-His antibody (Figure 26C). Figure 26D shows the SEC chromatogram for IH4-ADA with the two major protein peaks highlighted, and with Peak 2 corresponding to correctly folded protein. The calculated molecular weight of IH4-ADA is 55.3 kDa.

[0050] Figure 27: Flow cytometry analysis of the IH4-ADA fusion protein interaction with RBCs. RBCs were incubated with purified IH4-ADA, and binding activity was detected using an AF647-conjugated anti-Alpaca IgG VHH antibody, quantified as mean fluorescence intensity (MFI). Data analysis was performed using FlowJo vl0.7 software. Representative flow cytometry histograms are shown with control RBCs incubated with the anti-VHH antibody alone displayed in grey. Binding affinity was estimated by fitting the calculated MFI values versus protein concentration to a one-site specific binding model. The estimated Kd values for IH4-ADA are 220 nM with a corresponding Bmax value of 19 MFI.

[0051] Figure 28: Flow cytometry analysis of the IH4-ADA fusion protein interaction with BEL-A cells. BEL-A cells were incubated with purified IH4-ADA, and binding activity was detected using an AF647-conjugated anti-Alpaca IgG VHH antibody. Data analysis was performed using FlowJo vl0.7 software. Representative flow cytometry histograms show binding to unedited BEL-As (solid lines) and GPA KO BEL-As (dashed lines), compared to control BEL- As incubated with the anti-VHH antibody alone (filled in grey).

[0052] Figure 29: Enzymatic activity of ADA and IH4-ADA measured in solution. Figure 29A) ADA activity was assessed by spectroscopically monitoring the continuous decrease in absorbance at 265 nm as adenosine was converted to inosine. Reaction kinetics for free ADA (black dashed line) and IH4-ADA (black solid line) in solution were initiated by adding 100 pM adenosine to 1ml of PBS pH 7.4 containing 10 nM protein and measured over a 5- min period. Control reactions (grey dashed line for ADA and grey dotted line for IH4-ADA) were performed by substituting PBS for adenosine. Figure 29B) Inhibition studies of IH4- ADA in solution were conducted under the same conditions with the addition of either no inhibitor (solid line), 250 nM EHNA (dashed line) or 10 mM 1,10-phenanthroline (dotted line). Figure 29C) Michaelis-Menten kinetics were evaluated for ADA (circles) and IH4-ADA produced with (squares) and without (triangles) ethanol supplementation. Reaction rates were measured at a fixed protein concentration (10 nM) and varying adenosine concentrations (0-100 pM). The data were analysed using GraphPad Prism and fitted to the Michaelis-Menten model (dashed, solid, and dotted lines) to estimate kinetic parameters: Km values of 26.7 pM (ADA), 20.5 pM (IH4-ADA with ethanol), and 13.7 pM (IH4-ADA without ethanol); Vmax values of 53.7 pM / min, 45.4 pM / min, and 28.8 pM / min, respectively.

[0053] Figure 30: Enzymatic activity of IH4-ADA bound to the RBC surface. Figure 30A) Flow cytometry analysis was performed to confirm the binding of IH4-ADA. Proteins were added to 10 x 106RBCs at the indicated concentrations. Binding was assessed on 0.2 x 106cells using an AF647-conjugated anti-Alpaca IgG VHH antibody. Flow cytometry data were analysed using FlowJo vl0.7 software. Representative flow cytometry histograms are shown, with control RBC, incubated with the anti-VHH antibody alone, displayed in grey. Figure 30B) Enzymatic activity of bound IH4-ADA measured using the remaining RBCs. Reaction rates were determined as the initial velocity (Vo) and converted to pM / min. Figure 30C) The reaction rates of IH4-ADA in solution at known concentrations were used to calculate the effective bound concentration of IH4-ADA on RBCs. The data were analysed using GraphPad Prism, and a linear regression equation was fitted: Rate = (2.26 x concentration) + 0.1187.

[0054] Figure 31 : The enzymatic activity of IH4-ADA bound to RBCs under saturable conditions, by incubating 2.7 uM IH4-ADA with 10 x 106RBCs, was further analysed across a range of adenosine concentrations (0-100 pM).

[0055] Figure 32: Osmotic resistance of untreated RBCs and RBCs with bound IH4-ADA under saturating conditions. Osmotic resistance was analysed by measuring viable cell counts using flow cytometry after incubating RBCs with decreasing concentrations of NaCI. Comparisons were made between untreated RBCs (circles, dotted line) and RBCs with bound IH4-ADA under saturating binding conditions (squares, solid line).

[0056] Figure 33: This figure shows the predicted protein structure of ADAMTS13-IH4 generated by AlphaFold3, with an overall confidence score (pTM) of 0.6. ADAMTS13 MDTCS is shown in dark grey, and IH4 is shown in light grey.

[0057] Figure 34: This figure illustrates the expression and purification process of the ADAMTS13 fusion protein. ADAMTS13 was expressed in HEK293 cells, purified using the His-tag using Ni-NTA resin (Figure 34A). The peak corresponding to protein elution are highlighted for clarity. Fractions of the eluted peak were dialysed into PBS and spin concentrated. SDS- PAGE gels were performed and proteins either stained with Coomassie blue (Figure 34B) or transferred to a PVDF membrane and detected with an anti-His (Figure 34C), anti-VHH (Figure 34D) or anti-ADAMTS13 (Figure 34E) antibody. The calculated molecular weight of ADAMTS13-IH4 is ~84.5kDa, with an apparent molecular weight of ~100kDa on SDS-PAGE gels.

[0058] Figure 35: Flow cytometry analysis of ADAMTS13-IH4 fusion protein interaction with RBCs. RBCs were incubated with purified ADAMTS13-IH4, and binding activity was detected using a Vio515-conjugated anti-ADAMTS13 antibody, quantified as median fluorescence intensity (MFI). Data analysis was performed using FlowJo vlO.10 software. Representative flow cytometry histograms are shown, with control RBCs incubated with no ADAMTS13-IH4 and anti-ADAMTS13 antibody alone, displayed in grey (Figure 35A). Binding affinity was estimated by fitting the calculated MFI values versus protein concentration to a one-site specific binding model. The estimated Kd value for ADAMTS13-IH4 was 692.8nM (Figure 35B).

[0059] Figure 36: Flow cytometry analysis of ADAMTS13-IH4 fusion proteins interacting with normal and GPA Knock out (KO) BEL-A cells. Differentiated BEL-A cells were incubated with purified ADAMTS13-IH4 and binding activity detected with an APC-conjugated anti-His antibody. Data analysis was performed using FlowJo vlO.10 software. Representative flow cytometry histograms show binding to control BEL-As (solid lines) and GPA KO BEL-As (dashed lines), compared to control BEL-As incubated with the anti-ADAMTS13 antibody alone (filled in grey).

[0060] Figure 37: FRETS-vWF73 activity assay of ADAMTS13-IH4 fusion protein in solution. The ADAMTS13-IH4 was diluted in PBSAG+2mM CaCh and tested for activity against a modified vWF fragment, vWF73, that emits fluorescence as the M1605-Y1606 scissile bond is cleaved. Fluorescence emission was collected every 2 minutes over 2 hours at 37°C (Figure 37A). lOmM EDTA was added as a negative control. BAX930 is a commercially available recombinant ADAMTS13 protein used here as a positive control in comparison to molar concentration ADAMTS13-IH4 (Figure 37B). Initial rates of reaction were plotted against ADAMTS13-IH4 concentration and Michaelis-Menten curve fit in GraphPad Prism (Figure 37C). The reciprocal of each of these were plotted in Lineweaver Burke graph and a linear regression line (Y=0.3255X+0.01824) fit to find Km = 17.86nM and Vmax=54.8au / second (Figure 37D).

[0061] Figure 38: FRETS-vWF73 activity assay of ADAMTS13-IH4 fusion protein bound to the RBC surface. 7.2e5 RBC were bound with 150nM ADAMTS13-IH4 in PBSAG for 1 hour at room temperature. After washing, cells were resuspended in PBSAG+2mM CaC with FRETS- vWF73 and kinetic data on fluorescence emission collected as before (Figure 38A). After 2 hours, RBCs were washed in PBSAG and binding of ADAMTS13-IH4 detected via flow cytometry with an Vio515 conjugated anti-ADAMTS13 antibody (Figure 38B). Figure 39: This figure shows the predicted protein structure of the ADAMTS13-SpyTag construct, generated using AlphaFold3, with an overall confidence score (pTM) of 0.67. The ADAMTS13 MDTCS is shaded in grey, and the SpyTag sequence shaded black.

[0062] Figure 40: This figure illustrates the expression and purification process of the ADAMTS13- SpyTag fusion protein. ADAMTS13 was expressed in HEK293 cells, purified using the His-tag via a Ni-NTA resin. The peak corresponding to protein elution are highlighted for clarity.

[0063] Figure 41 : SDS-PAGE and Western blot analysis of the ADAMTS13-SpyTag construct. SDS- PAGE gels were performed and proteins either stained with Coomassie blue (Figure 43A) or transferred to a PVDF membrane and detected with an anti-His antibody (Figure 43B). The calculated molecular weight of ADAMTS13-SpyTag is ~72.9kDa, with an apparent molecular weight of ~100kDa on SDS-PAGE gels.

[0064] Figure 42: ADAMTS13-SpyTag was incubated 2: 1 with SpyCatcher-IH4. Isopeptide bond formation was confirmed by running on SDS-PAGE gel, with proteins transferred to a PVDF membrane and detected ADAMTS13-SpyTag, SpyCatcher-IH4 and the resulting complexes with anti-His antibody (Figure 42A). Remaining sample was bound to 2e5 RBC in 30|iL PBSAG for 1 hour at RT and after washing, presence of ADAMTS13 was detected on the RBC surface thorough flow cytometry with an anti-ADAMTS13 antibody conjugated to Vio515 (Figure 42B), or anti-VHH antibody conjugated to Alexa-647 (Figure 42C). A negative control of ADAMTS13-SpyTag detected with anti-ADAMTS13 antibody (Figure 42D) and a positive control of IH4-SC detected with anti-VHH (Figure 42E) were included.

[0065] Figure 43: The specificity of the ADAMTS13-ST-IH4-SC fusion was assessed on BEL-A control cells and BEL-A cells previously CRISPR edited to produce a GPA-null phenotype (Figure 43A). ADAMTS13-ST was also tested alone (Figure 43B). ADAMTS13 presence was detected with an anti-ADAMTS13 primary antibody, with an anti-rabbit PE-conjugated secondary. Representative flow cytometry histograms show binding to control BEL-As (solid lines) and GPA KO BEL-As (dashed lines), compared to control BEL-As incubated with the antibodies alone (filled in grey).

[0066] Figure 44: The activity of ADAMTS13-SpyTag alone in solution was assessed with FRETS- vWF73 assay, using a modified vWF fragment that fluoresces at 450nm when the scissile bond is cleaved. Samples (0nM-50nM) were run in IOOJJL PBSAG+2mM CaCh in black plates for 2 hours at 37°C. lOmM EDTA was used as a negative control by preventing ADAMTS13 function. 20nM BAX930 recombinant ADAMSTS13 was used as a positive control.

[0067] Figure 45: FRETS-vWF73 activity assay of ADAMTS13-SpyTag-IH4-SpyCatcher RBC surface binding. 7.2e5 RBC were bound with 150nM ADAMTS13-SpyTag-IH4-SpyCatcher in PBSAG for 1 hour at room temperature. After washing in PBSAG, cells were resuspended in PBSAG with FRETS-vWF73 in a black plate and kinetic data on fluorescence emission collected for 2 hours at 37°C, as before (Figure 45A). After 2 hours, RBCs were washed in PBSAG and binding of ADAMTS13 detected via flow cytometry with an Vio515 conjugated anti- ADAMTS13 antibody (Figure 45B).

[0068] Figure 46: This figure shows A) the ADAMTS13-CA52 protein construct design and B) the AlphaFold predicted protein structure of ADAMTS13-CA52 fusion protein, with an overall confidence score (pTM) of 0.59. The ADAMTS13 is shown in black, and the CA52 nanobody in grey, with the 3 CDR loops shown in a darker shade of grey.

[0069] Figure 47: This figure shows the expression and protein purification of ADAMTS13-CA52 from HEK293T cells. Figure 47A shows the AKTA elution of the ADAMTS13-CA52 construct from a Nickel-affinity column. The peak corresponding to protein elution is highlighted for clarity. The protein was subjected to SDS-PAGE and either stained with Coomassie blue (Figure 47B), or transferred to PVDF membrane and protein detected with anti-His (Figure 47C), anti-VHH (Figure 47D), or anti-ADAMTS13 (Figure 47E) antibodies. The calculated molecular weight of ADAMTS13-CA52 is ~84kDa, with an apparent molecular weight of ~105kDa on SDS-PAGE gels due to glycosylation.

[0070] Figure 48: Flow cytometry analysis of ADAMTS13-CA52 fusion protein interaction with RBCs. RBCs were incubated with the purified ADAMT13-CA52 and binding activity was detected with a Vio515-conjugated anti-ADAMTS13 antibody (Figure 48A-B), or an Alexa-647 conjugated anti-VHH antibody (Figure 48C-D). Flow cytometry readout was quantified as media fluorescence intensity (MFI). Representative flow cytometry histograms are shown, with control RBCs incubated with only anti-ADAMTS13 antibody displayed in grey (Figure 48A and C). Binding affinity was estimated by fitting the calculated MFI values (with background removed) versus protein concentration to a one-site specific binding model (Figure 48B and D). The estimated Kd value for ADAMTS13-CA52 was 27.22nM with anti- ADAMTS13, and 15.01nM with anti-VHH.

[0071] Figure 49:The specificity of ADAMTS13-CA52 fusion protein was assessed using BEL-A control cells and BEL-A cells CRISPR edited to produce a duffy-null phenotype. The BEL-A cells were differentiated for 7-days and then were incubated with purified ADAMTS13-CA52 and binding activity detected with an Alexa-647-conjugated anti-VHH antibody. Representative flow cytometry histograms show binding to unedited BEL-As (solid lines) and Duffy knock-out BEL-As (dashed lines), compared to unedited BEL-As incubated with the anti-VHH antibody alone (filled in grey).

[0072] Figure 50: This figure shows a FRETS-vWF73 activity assay of ADAMTS13-CA52 fusion protein in solution. The ADAMTS13-CA52 was diluted in PBSAG+2mM CaCh and tested for activity against a modified vWF fragment, vWF73. lOmM EDTA was added as a negative control

[0073] Figure 51 : This figure shows the FRETS-vWF73 activity of ADAMTS13-CA52 fusion protein bound to RBC surface. FRETS-vWF73 and kinetic data on fluorescence emission collected as before (Figure 51A). After 2 hours, RBCs were washed in PBSAG and binding of ADAMTS13- IH4 detected via flow cytometry with an Vio515 conjugated anti-ADAMTS13 antibody (Figure 51B), and Alexa-647 conjugated anti-VHH antibody (Figure 51C).

[0074] Figure 52: This figure shows the deformability measurements of RBCs bound with ADAMTS13-CA52 measured using ARCA. Figure 52A confirms maximal ADAMTS13-CA52 binding does not cause decreased deformability of RBC as measured by the ARCA. BRIC256 (anti-GPA antibody known to induce deformation) was used as a positive control. After binding with the protein, cells remain a similar overall size (Figure 52B). The presence of ADAMTS13-IH4 binding to the RBC was confirmed by flow cytometry with an anti- ADAMTS13 antibody conjugated to Vio515 fluorophore (Figure 52C), or anti-VHH antibody conjugated to Alexa647.

[0075] Figure 53: This figure shows A) the protein construct design, and B) the predicted protein structure of a bivalent IH4-IH4-GFP fusion protein generated by AlphaFold, with an overall confidence score (pTM) of 0.5. This includes an SGGGSG linker between the 2 IH4 nanobodies, and a GSS linker between the C-terminal IH4 nanobody and the N-terminus of the GFP protein. The GFP protein used is msGFP2. The GFP is shown in black, and the IH4 nanobodies in grey, with the SGGGSG linker in a darker grey colour.

[0076] Figure 54:This figure shows the expression and protein purification of a bivalent IH4-IH4- GFP. Figure 54A shows the size exclusion elution of the IH4-GFP-GFP. The highlighted peak corresponding to protein elution is highlighted for clarity. SDS-PAGE gels were conducted on the protein and stained with Coomassie blue (Figure B), or transferred to PVDF membrane and protein detected with anti-VHH (Figure C). The calculated molecular weight of IH4-IH4- GFP is ~54.3kDa, with an apparent molecular weight of ~60kDa on SDS-PAGE gels.

[0077] Figure 55: Figure shows flow cytometry analysis of the bivalent IH4-IH4-GFP fusion protein interaction with RBCs. 2e5 RBCs were incubated with purified IH4-IH4-GFP and binding activity was detected by flow cytometry for GFP directly (Figure 55A-B), or an Alexa-647 conjugated anti-VHH antibody (Figure C-D). Flow cytometry readout was quantified as media fluorescence intensity (MFI). Representative flow cytometry histograms are shown, with control RBCs incubated with no protein displayed in grey (Figure 55A and C). Binding affinity was estimated by fitting the calculated MFI values (with background removed) versus protein concentration to a one-site specific binding model (Figure B and D). The estimated Kd value for IH4-IH4-GFP was 465.3pM by GFP, and 225. lpM with anti-VHH. Figure 56: Flow cytometry analysis of IH4-IH4-GFP bivalent fusion protein interacting with BEL-A cells. 2e5 day 7 differentiated BEL-A cells were incubated with purified IH4-IH4-GFP in PBSAG for 1 hour at RT, and binding activity detected on flow cytometry via GFP fluorescence. Data analysis was performed using FlowJo vlO.10 software. Representative flow cytometry histograms show binding to unedited BEL-As (solid lines) and GPA KO BEL- As (dashed lines), compared to control BEL-As incubated in PBSAG alone (filled in grey).

[0078] Figure 57: Figure shows the resilience of IH4-GFP compared to IH4-IH4-GFP binding after conducting multiple washes. 5e5 RBC were bound to 5 nM of protein at room temperature for 1 hour, washed and resuspended in PBSAG. Cells were incubated at room temperature, 4°C or 37°C for 1 hour before samples were taken for flow cytometry analysis of GFP expression. The remaining samples were pelleted and resuspended in fresh PBSAG (a wash). This was repeated hourly for 7 total washes. Representative flow cytometry histograms show GFP expression on the RBC across washes (Figure 57A). The median fluorescence intensity values were taken and plotted in GraphPad Prism as the percentage of the initial measured binding (i.e. 1 wash), with mean and standard deviation of MFI shown (Figure 57B).

[0079] Figure 58: Figure shows the effect of binding IH4-IH4-GFP to RBC on deformability as measured by ARCA. 2e6 cells were bound with IH4-IH4-GFP in PBSAG for 1 hour. RBC were washed and, immediately prior to measurement, resuspended in PVP for ARCA analysis. Figure 58A confirms up to 5nM IH4-IH4-GFP can be used to bind RBC in this experiment before alterations in deformability are observed. After binding with the protein, cells retain a similar overall area (Figure 58B). The presence of IH4-IH4-GFP binding to the RBC was confirmed by flow cytometry. Representative histograms show GFP levels of IH4-IH4-GFP bound RBCs (Figure C).

[0080] Figure 59: This figure shows A) the bivalent IH4-IH4-ADA protein construct design, and (B) the predicted protein structure of the IH4-IH4-ADA fusion protein generated by AlphaFold, with an overall confidence score (pTM) of 0.6. This includes an SGGGSG linker separating the two IH4 nanobodies, and a GSS linker between the C-terminal IH4 nanobody and the N- terminus of the ADA protein. The ADA is shown in black, and the IH4 nanobodies in grey, with the SGGGSG linker in a darker grey colour.

[0081] Figure 60: This figure illustrates the expression and purification process of the IH4-IH4-ADA fusion protein. IH4-IH4-ADA was expressed in SHuffle T7 cells and purified using the His- tag via a Ni-NTA resin (a), followed by size exclusion chromatography (b). The collected fractions from the four largest peaks were analysed by SDS-PAGE, followed by Coomassie blue staining. Fractions corresponding to the final 3 peaks were analysed by SDS-PAGE and probed using an anti-His antibody by Western blot evidencing that Peak 3 corresponds to IH4-IH4-ADA (c). An anti-VHH antibody Western blot of the Peak 3 fraction further confirms the presence of IH4-IH4-ADA. The calculated molecular weight of IH4-IH4-ADA is 69 kDa.

[0082] Figure 61 : Figure shows representative deformability index profiles for untreated RBCs (dashed lines), RBCs treated with IH4-ADA (Figure 61A) and IH4-IH4-ADA (grey lines) (Figure 61B) at the indicated concentrations. Data were obtained using an ARCA. Flow cytometry analysis was also performed to confirm the binding of IH4-ADA (Figure 61A) and IH4-IH4-ADA (Figure 61B) to RBCs under the same conditions used for deformability measurements. RBCs were prepared at a concentration of 2 x 106cells and proteins were added at the indicated concentrations. The analysis was conducted on 0.2 x 106cells labelled with AF647-conjugated anti- VHH antibody. Representative flow cytometry histograms are shown, with control RBCs incubated with the anti-VHH antibody alone displayed in grey.

[0083] Figure 62: Figure shows flow cytometry analysis of the IH4-IH4-ADA fusion protein interaction with RBCs. RBCs were incubated with purified IH4-IH4-ADA, and binding activity was detected using an AF647-conjugated anti- VHH antibody, quantified as mean fluorescence intensity (MFI). Representative flow cytometry histograms are shown with control RBCs incubated with the anti-VHH antibody alone displayed in grey. Binding affinity was estimated by fitting the calculated MFI values versus protein concentration to a one-site specific binding model. The estimated Kd values for IH4-IH4-ADA are 30 nM with a corresponding Bmax value of 64 MFI.

[0084] Figure 63: Figure shows enzymatic activity of IH4-IH4-ADA measured in solution, a) IH4- IH4-ADA activity was assessed by spectroscopically monitoring the continuous decrease in absorbance at 265 nm as adenosine was converted to inosine, b) Reaction rates were measured at a fixed protein concentration (10 nM) and varying adenosine concentrations (0-100 pM). The data were analysed using GraphPad Prism.

[0085] Figure 64: Figure shows enzymatic activity of IH4-IH4-ADA measured when bound to RBCs. a) The enzymatic activity of IH4-IH4-ADA bound to RBCs under saturable conditions, by incubating 200 nM IH4-IH4-ADA with 1 x 106RBCs, was analysed across a range of adenosine concentrations (0-100 pM). b) Flow cytometry analysis was performed to confirm the binding of IH4-IH4-ADA to 1 x 106RBCs. Binding was assessed on 0.2 x 106cells using an AF647-conjugated anti-VHH antibody. Representative flow cytometry histograms are shown, with control RBC, incubated with the anti-VHH antibody alone, displayed in grey, c) Enzyme activity of bound IH4-IH4-ADA was measured using the remaining RBCs with varying adenosine concentrations (0-100 pM). The data were analysed using GraphPad Prism. Figure 65: Figure shows a diagram representation of the domains present in an IH4-CCDi- ADA fusion protein dimer with front, side and top views, with ADA shown in white, the coiled coil self dimerisation domain (CCDi) shown in grey, and IH4 in black.

[0086] Figure 66: Figure showing IH4-CCDi-ADA expressed in a soluble form in BL21 (DE3) cells, purified using the His-tag via a Ni-NTA resin, and further purified by size exclusion chromatography, a) The collected fractions from the four largest peaks were analysed by SDS-PAGE, followed by Coomassie blue staining, b) Shows the SEC chromatogram for IH4- CCDi-ADA with the 4 major peaks highlighted, with peak 3 corresponding to the correctly folded protein, c) Concentrated peak 3 protein sample was run on SDS PAGE, transferred to a nitrocellulose membrane and detected using an anti-His antibody. The calculated molecular weight of IH4-CCDi-ADA is 59.1kDa.

[0087] Figure 67: Figure shows particle sizes measured using a dynamic light scattering (DLS) experiment in which both IH4-ADA and IH4-CCDi-ADA were present at 0.3 mg / mL in PBS solution. IH4-CCDi-ADA particle size is approximately double that of IH4-ADA, indicating that IH4-CCDi-ADA has dimerised. Particle sizes from DLS are in line with the dimensions of IH4-ADA and IH4-CCDi-ADA structures predicted by AlphaFold3. All size measurements were performed using PyMOL molecular visualisation software.

[0088] Figure 68: Figure shows flow cytometry analysis of the IH4-CCDi-ADA protein interaction with RBCs. RBCs were incubated with purified IH4-CCDi-ADA, and binding activity was detected using an AF647-conjugated anti-Alpaca IgG VHH antibody, quantified as mean fluorescence intensity (MFI). Representative flow cytometry histograms are shown with control RBCs incubated with the anti-VHH antibody alone shown in grey. Binding affinity was estimated by fitting the calculated MFI values versus protein concentration to a one-site specific binding model. The estimated Kd value for IH4-CCDi-ADA was 156nM with a corresponding Bmax value of 66 MFI.

[0089] Figure 69: This figure shows flow cytometry analysis confirming the specificity of IH4-CCDi- ADA to GPA. Purified IH4-CCDi-ADA was incubated with day 7 differentiated BEL-A cells (solid line) and GPA KO BEL-A cells (dotted line) at the indicated concentrations. Binding activity was detected using an AF647-conjugated anti-Alpaca IgG VHH antibody. Representative flow cytometry histograms are shown, with control BEL-As (no protein) incubated with the anti-VHH antibody alone displayed in grey.

[0090] Figure 70: This figure shows enzymatic activity of IH4-CCDi-ADA bound to the RBC surface. Purified IH4-CCDi-ADA was added to 10 x 106RBCs at the indicated concentrations and 0.2 x 106cells were analysed via flow cytometry with an AF647-conjugated anit-VHH antibody to confirm binding. Representative flow cytometry histograms are shown, with control RBCs, incubated with the anti-VHH antibody alone, shown in grey (a). Enzymatic activity of bound IH4-CCDi-ADA was measured using the remaining RBCs. b) Reaction rates were determined as the initial velocity of the decrease in adenosine concentration and converted to pM / min and plotted against IH4-CCDi-ADA concentration, c) The reaction rates of IH4-CCDi-ADA in solution at various known concentrations combined with 40 pM adenosine were used to calculate the effective bound concentration of IH4-CCDi-ADA on RBCs from the bound activity. Data was analysed in GraphPad Prism and a linear regression fitted: Rate = (1.569 x Concentration) + 0.005804.

[0091] DESCRIPTION OF THE SEQUENCE LISTING

[0092] SEQ ID NOs: 1 and 2: The IH4 DNA sequence (SEQ ID NO: 1) was codon-optimised for efficient expression in E. coli and subsequently cloned into the pET-21b vector, providing a C-terminal His tag. The protein encoded by SEQ ID NO: 1 is shown in SEQ ID NO: 2.

[0093] SEQ ID NOs: 3 and 4: The DNA sequence for TP-IH4 (SEQ ID NO: 3) was codon-optimised for efficient expression in E. coli and subsequently cloned into the pET-28a vector. In this construct, TP is fused to the N-terminal end of the IH4 nanobody, positioning it on the same side as of the CDR loops that form the GPA binding site, and is proceeded by an N-terminal His-tag. The protein encoded by SEQ ID NO: 3 is shown in SEQ ID NO: 4.

[0094] SEQ ID NOs: 5 and 6: The DNA sequence for IH4-TP (SEQ ID NO: 5) was codon-optimised for efficient expression in E. coli and subsequently cloned into the pET-28a vector. In this construct, TP is fused to the C-terminal end of the IH4 nanobody, positioning it on the opposite side of the complementary determining region (CDR) loops that form the GPA- binding site, and is followed by a C-terminal His-tag. The protein encoded by SEQ ID NO: 5 is shown in SEQ ID NO: 6.

[0095] SEQ ID NOs: 7 and 8: The DNA sequence for IH4-GFP (SEQ ID NO: 7) was codon-optimised for efficient expression in E. coli and subsequently cloned into the pET-21b vector, providing a C-terminal His tag. msGFP2 is fused to the C-terminal end of the IH4 nanobody, proceeding the His-tag. The protein encoded by SEQ ID NO: 7 is shown in SEQ ID NO: 8.

[0096] SEQ ID NOs: 9 and 10: The DNA sequence for IH4-SC003 (SEQ ID NO: 9) was codon- optimised for efficient expression in E. coli and subsequently cloned into the pET-21b vector. In this construct, SpyCatcher003 is fused to the C-terminal end of IH4, with an internal His-tag positioned between IH4 and SC003. The protein encoded by SEQ ID NO: 9 is shown in SEQ ID NO: 10.

[0097] SEQ ID NOs: 11 and 12: The DNA sequence for ST003-TID (SEQ ID NO: 11) was codon- optimised for efficient expression in E. coli and subsequently cloned into the pET-21b vector, providing a C-terminal His-tag. In this construct, ST003 is fused to the N-terminal end of TID. The protein encoded by SEQ ID NO: 11 is shown in SEQ ID NO: 12. SEQ ID NOs: 13 and 14: SpyTag003-sfGFP was a gift from Mark Howarth (Addgene plasmid # 133454; http: / / n2t.net / addgene: 133454 ; RRID:Addgene_133454). In this construct, SpyTag003 is fused to the N-terminal end of superfolder GFP, and the entire sequence is cloned into the pET-28a vector, which includes a C-terminal His-tag (SEQ ID NO: 13). The protein encoded by SEQ ID NO: 13 is shown in SEQ ID NO: 14.

[0098] SEQ ID NOs: 15 and 16: The DNA sequence for IH4BS1 (SEQ ID NO: 15) was codon- optimised for efficient expression in E. coli and subsequently cloned into the pET-28a vector. In this construct, IH4BS1 is preceded by an N-terminal His-tag. The protein encoded by SEQ ID NO: 15 is shown in SEQ ID NO: 16.

[0099] SEQ ID NOs: 17 and 18: The DNA sequence for IH4BS1-SPM1 (SEQ ID NO: 17) was codon- optimised for efficient expression in E. coli and subsequently cloned into the pET-28a vector. In this construct, SPM is fused to the C-terminal end of the IH4BS1 nanobody via a GSY-linker, and IH4BS1 is preceded by an N-terminal His-tag. The protein encoded by SEQ ID NO: 17 is shown in SEQ ID NO: 18.

[0100] SEQ ID NOs: 19 and 20: The DNA sequence for IH4BS1-SPM2 (SEQ ID NO: 19) is as described for SEQ ID NO: 17, but with the SPM domain and IH4BS1 nanobody separated by a longer 8-residue linker, SGGGGSGG, proceeding the GSY motif. The protein encoded by SEQ ID NO: 19 is shown in SEQ ID NO: 20.

[0101] SEQ ID NOs: 21 and 22: The DNA sequence for IH4BS1-SPM3 (SEQ ID NO: 1) is as described for SEQ ID NO: 17 and 19, but with the SPM domain and IH4BS1 nanobody separated by a longer 16-residue linker, GGGGSGGGGCGGGGSS, proceeding the GSY motif. The protein encoded by SEQ ID NO: 21 is shown in SEQ ID NO: 22.

[0102] SEQ ID NOs: 23 and 24: The DNA sequence for IH4BS2 (SEQ ID NO: 23) was codon- optimised for efficient expression in E. coli and subsequently cloned into the pET-28a vector. In this construct IH4BS2 is preceded with a N-terminal His-tag. The protein encoded by SEQ ID NO: 23 is shown in SEQ ID NO: 24.

[0103] SEQ ID NOs: 25 and 26: The DNA sequence for IH4BS2-SPM (SEQ ID NO: 25) was codon- optimised for efficient expression in E. coli and cloned into the pET-28a vector. In this construct, SPM is fused to the C-terminal end of the IH4BS2 nanobody via a 8-residue flexible linker (SGGGGSGG) followed by a GSY-linker, and IH4BS2 is preceded by an N- terminal His-tag. The protein encoded by SEQ ID NO: 25 is shown in SEQ ID NO: 26.

[0104] SEQ ID NO: 27 shows the sequence of the IH4 nanobody.

[0105] SEQ ID NO: 28 shows the sequence of IH4BS1.

[0106] SEQ ID NO: 29 shows the sequence of IH4BS2. SEQ ID NO: 30 shows the sequence of CDR.1 of the IH4 nanobody.

[0107] SEQ ID NO: 31 shows the sequence of CDR.2 of the IH4 nanobody.

[0108] SEQ ID NO: 32 shows the sequence of CDR.3 of the IH4 nanobody.

[0109] SEQ ID NO: 33 shows the sequence of thymidine phosphorylase.

[0110] SEQ ID NO: 34-37 show the sequences the linkers used in the Examples.

[0111] SEQ ID NO: 38 shows the sequence of the self-processing module (SPM). The selfprocessing module (SPM) is a protein sequence that enables intermolecular bonds between polypeptides. Here, we use the SPM sequence, derived from the FrpA protein of Neisseria meningitidis and provided by Prof. Mark Howarth (University of Cambridge).

[0112] SEQ ID NOs: 39 and 40: The DNA sequence for ADA (SEQ ID NO: 39) followed by a C- terminal His tag, was codon-optimised for efficient expression in E. coli and subsequently cloned into the pET-21b vector. The protein encoded by SEQ ID NO: 39 is shown in SEQ ID NO: 40.

[0113] SEQ ID NOs: 41 and 42: The DNA sequence for IH4-ADA was codon optimised for efficient expression in E. coli and subsequently cloned into the pET-21b vector. In this construct, ADA is fused to the C-terminal end of the IH4 nanobody, positioning it on the opposite side of the complementary determining region (CDR) loops that form the GPA-binding site, and is followed by a C-terminal His-tag. The protein encoded by SEQ ID NO: 41 is shown in SEQ ID NO: 42.

[0114] SEQ ID NOs: 43 and 44: Includes the kozak sequence, ADAMTS13 signal peptide, DNA sequence for ADAMTS13 MDTCS called here the coding sequence (SEQ ID NO:43) followed by a C-terminal His tag, was codon-optimised for efficient expression mammalian cells and subsequently cloned into the p3.1. The protein encoded by SEQ ID NO: 43 is shown in SEQ ID NO: 44.

[0115] SEQ ID NOs: 45 and 46: The coding DNA sequence for ADAMTS13-IH4 was codon optimised for efficient expression in Homo sapiens and subsequently cloned into pcDNA3.1(+)vector. In this construct ADAMTS13 MDTCS (C-terminal truncation) is fused to the N-terminal end of the IH4 nanobody via a GSS linker, then the IH4 sequence is followed by a GSS linker and C-terminal His-tag. The protein encoded by SEQ ID NO: 45 is shown in SEQ ID NO: 46.

[0116] SEQ ID NOs: 47 and 48: The coding DNA sequence for ADAMTS13 MDTCS (C-terminal truncation) with a His tag and spytag003 was codon optimised for efficient expression in Homo sapiens and subsequently cloned into p3.1 vector. In this construct ADAMTS13 has a C-terminal His tag followed a GSS linker and SpyTag003. The protein encoded by SEQ ID NO: 47 is shown in SEQ ID NO: 48.

[0117] SEQ ID NO: 49 shows a gain-of-function ADAMTS13-IH4 protein.

[0118] SEQ ID NOs: 50 and 51 : The coding DNA sequence for ADAMTS13-CA52 was codon optimised for efficient expression in Homo sapiens and subsequently cloned into pcDNA3.1(+)vector. In this construct ADAMTS13 MDTCS (C-terminal truncation) is fused to the N-terminal end of the CA52 nanobody via a GSS linker, then the CA52 sequence is followed by a GSS linker and C-terminal His-tag. The sequence was codon-optimised for efficient expression in E. coli and subsequently cloned into the pET-21b vector. The protein encoded by SEQ ID NO: 50 is shown in SEQ ID NO: 51.

[0119] SEQ ID NOs: 52 and 53. The coding DNA sequence for the bivalent IH4-IH4-GFP has 2 IH4 nanobody coding sequences separated by a SGGGSG linker between the two IH4 nanobodies, Then a GSS linker between the C-terminal IH4 nanobody and the N-terminus of the msGFP2, which is followed by c-terminal a His-tag. The sequence was codon-optimised for efficient expression in E. coli and subsequently cloned into the pET-21b vector. The protein encoded by SEQ ID NO: 52 is shown in SEQ ID NO: 53.

[0120] SEQ ID NOs: 54 and 55. The coding DNA sequence for the bivalent IH4-IH4-ADA has 2 IH4 nanobody coding sequences separated by a SGGGSG linker between the 2 IH4 nanobodies, Then a GSS linker between the C-terminal IH4 nanobody and the N-terminus of the ADA, which is followed at the C terminus by a His-tag. The sequence was codon-optimised for efficient expression in E. coli and subsequently cloned into the pET-21b vector. The protein encoded by SEQ ID NO: 54 is shown in SEQ ID NO: 55.

[0121] SEQ ID NOs: 56 and 57. The DNA sequence for IH4-CCDi-ADA fusion protein dimer was codon optimised for efficient expression in E. coli and subsequently cloned into the pET-21b vector. In this construct, the C-terminal end of the nanobody sequence is then followed by a linker sequence, then the homodimerization sequence (coil-coil self dimerisation domains), followed by a linker sequence, then ADA which followed by a C-terminal His-tag. The protein encoded by SEQ ID NO: 56 is shown in SEQ ID NO: 57.

[0122] SEQ ID NO: 58 shows the sequence of the CA52 nanobody.

[0123] SEQ ID NO: 59 shows the sequence of CDR.1 of the CA52 nanobody.

[0124] SEQ ID NO: 60 shows the sequence of CDR.2 of the CA52 nanobody.

[0125] SEQ ID NO: 61 shows the sequence of CDR.3 of the CA52 nanobody.

[0126] SEQ ID NO: 62 shows the CDDi self dimerisation domain used in the Examples. DETAILED DESCRIPTION

[0127] General disclosure

[0128] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0129] The present invention is described with respect to particular embodiments and with reference to certain Figures but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein.

[0130] The invention, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying Figures. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to "an / one embodiment", "some embodiments" or "preferred embodiment(s)" means that a particular feature, structure, or characteristic described in connection with the embodiment(s) is included in at least one embodiment of the present invention. Thus, appearances of the embodiment phrases in various places throughout this specification are not necessarily all referring to the same embodiment, but may do so. Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. In addition as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a protein conjugate" includes two or more protein conjugates, reference to "an antibody" includes two or more such antibodies, reference to "a variant" includes two or more such variants, reference to "a polynucleotide" includes two or more polynucleotides, reference to "an enzyme" refers to two or more enzymes, reference to "a method" includes two or more methods, reference to "a subject" includes two or more subjects and the like.

[0131] In all of the discussion herein, the standard one letter codes for amino acids are used. These are as follows: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V).

[0132] Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

[0133] "About" as used herein when referring to a measurable value such as a percentage or an amount and the like, is meant to encompass variations of ± 20 % or ± 10 %, more preferably ± 5 %, even more preferably ± 1 %, and still more preferably ± 0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. Any statement herein including the term "about" includes the same feature without the term. For instance, at least "about" 90% identity includes at least 90% identity.

[0134] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to", and do not exclude other components, integers, or steps. In all instances herein the terms "comprising" and "comprises" are interchangeable with "consisting essentially of" and "consists essentially of" and "consisting of" and "consists of".

[0135] In all instances herein, the term "fragment" encompasses a sequence which is a naturally occurring polymorphic form of the reference sequence as well as synthetic fragment sequences in which one or more amino acids are deleted. In all instances herein, the term "fragment" is interchangeable with "functional fragment". A functional fragment is a sequence which differs from the reference sequence, typically because one or more of amino acids are deleted, and retains at least some functional activity of the reference sequence. In the context of antibodies, a functional fragment is a sequence which differs from the reference sequence, typically because one or more of amino acids are deleted, and retains at least some ability to specifically bind to the antigen on the surface of RBCs. In the context of enzymes, a functional fragment is a sequence which differs from the reference sequence, typically because one or more of amino acids are deleted, and retains at least some catalytic activity of the enzyme.

[0136] In all instances herein, the term "variant" encompasses a sequence which is a naturally occurring polymorphic form of the reference sequence as well as synthetic variant sequences in which one or more amino acids are inserted, deleted, and / or substituted. The term "variant" is interchangeable with "functional variant". A functional variant is a sequence which differs from the reference sequence, typically because one or more of amino acids are inserted, deleted and / or substituted, and retains at least some functional activity of the reference sequence. In the context of antibodies, a functional variant is a sequence which differs from the reference sequence, typically because one or more of amino acids are inserted, deleted and / or substituted, and retains at least some ability to specifically bind to an antigen on the surface of RBCs. In the context of enzymes, a functional variant is a sequence which differs from the reference sequence, typically because one or more of amino acids are inserted, deleted and / or substituted, and retains at least some of the catalytic activity of the enzyme.

[0137] The functional activity of the fragment / functional fragment or the variant / functional variant may be substantially the same or about the same as the reference sequence. The functional activity may be increased. The functional activity may be increased by any amount, such as by at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 90%, at least about a factor of 2, such as least about a factor of 5, at least about a factor of 10, at least about a factor of 50, at least about a factor of 100, at least about a factor of 500, as least about a factor of 1000, at least about a factor of 5000, at least about a factor of 10,000 or more. The functional activity may be decreased. The functional activity may be decreased by any amount, such as by less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2% or less than about 1%.

[0138] In all instances, the term "surface of RBCs" is interchangeable with "surfaces of RBCs".

[0139] Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Within the scope of this application it is expressly intended that the various aspects, embodiments, examples, and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and Figures, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination, unless such features are incompatible.

[0140] Protein conjugates

[0141] The invention provides a protein conjugate. The protein conjugate may also be called a polypeptide conjugate, and the two terms are interchangeable. The protein conjugate may also be called a labelling protein conjugate or labelling polypeptide conjugate. The protein conjugate may also be called an engineered protein conjugate or an engineered polypeptide conjugate. The protein conjugate may also be called a recombinant protein conjugate or a recombinant polypeptide conjugate. The protein conjugate may also be called a recombinant engineered protein conjugate or a recombinant engineered polypeptide conjugate.

[0142] In some embodiments, especially when the components of the protein conjugate are genetically linked, the protein conjugate is a fusion protein or polypeptide. The invention therefore provides a fusion protein comprising (a) one or more antibodies or variants thereof which specifically bind to one or more antigens on the surface of red blood cells (RBCs) and (b) one or more enzymes and / or variants thereof. The invention also provides a fusion protein comprising (a) an antibody or a variant thereof which specifically binds to an antigen on the surface of red blood cells (RBCs) and (b) one or more enzymes and / or variants thereof.

[0143] The protein conjugate comprises or consists of two components (a) and (b). These are defined above. The protein conjugate may comprise at least (a) and (b). The protein conjugate may comprise or consists of any number of components including (a) and (b), such as two, three, four, five, six, seven, eight, nine, ten or more components. The protein conjugate preferably comprises or consists of (a) and (b) as defined above and one or more linkers. Linkers are discussed in more detail below.

[0144] Antibodies or variants thereof

[0145] The protein conjugate comprises (a) one or more antibodies or variants thereof which specifically bind to one or more antigens on the surface of red blood cells (RBCs). The one or more antibodies or variants thereof are functional because they specifically bind to one or more antigens on the surface of RBCs. The protein conjugate preferably comprises (a) two or more antibodies or variants thereof which specifically bind to one or more antigens on the surface of red blood cells (RBCs). The protein conjugate preferably comprises (a) two or more antibodies or variants thereof which specifically bind to two or more antigens on the surface of red blood cells (RBCs). The protein conjugate may comprise any number of antibodies or variants thereof, such as two or more, three or more, four or more or five or more. The protein conjugate preferably comprises two antibodies or variants thereof. The one or more or two or more antibodies or variants thereof may bind to any number of antigens on the surface of RBCs, such as two or more, three or more, four or more or five or more.

[0146] The two or more antibodies or variants thereof may bind to the same antigen on the surface of RBCs. The two or more antibodies or variants thereof may bind to different antigens on the surface of RBCs. The two or more antibodies or variants thereof may bind to two or more different antigens on the surface of RBCs. The two or more antibodies or variants thereof may bind to any of the antigens discussed below or any combination of the antigens discussed in more detail below.

[0147] The two or more antibodies or variants thereof may be repeating units of the same antibody or variant thereof. The two or more antibodies or variants thereof may be different antibodies and / or different variants. The protein conjugate may comprise two or more of any of the antibodies and / or variants thereof discussed in more detail below.

[0148] The protein conjugate may comprise (a) an antibody or a variant thereof which specifically binds to an antigen on the surface of red blood cells (RBCs). The antibody or variant thereof is functional because it specifically binds to an antigen on the surface of RBCs.

[0149] The term "antibody" as referred to herein includes whole antibodies. Naturally occurring antibodies typically comprise a tetramer which is usually composed of at least two heavy (H) chains and at least two light (L) chains. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, usually comprised of three domains (CHI, CH2 ad CH3). Heavy chains can be of any isotype, including IgG (IgGl, IgG2, IgG3 and IgG4 subtypes), IgA (IgAl and IgA2 subtypes), IgM and IgE. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). Light chain includes kappa (K) chains and lambda (A) chains. The heavy and light chain variable region is typically responsible for antigen recognition, whilst the heavy and light chain constant region may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0150] The one or more variants thereof may be one or more fragments of one or more intact antibodies. The protein conjugate may comprise (a) one or more antibodies, variants thereof or fragments thereof which specifically bind to one or more antigens on the surface of red blood cells (RBCs), such as two or more antibodies, variants thereof or fragments thereof which specifically bind to one or more antigens or two or more antigens on the surface of RBCs. The variant thereof may be any fragment of an intact antibody. The protein conjugate may comprise (a) an antibody, a variant thereof or a fragment thereof which specifically binds to an antigen on the surface of red blood cells (RBCs).

[0151] Such fragments include, but are not limited to, an antibody heavy chain, the variable region of an antibody heavy chain (VH), an antibody light chain and the variable region of an antibody light chain (VL). The fragment retains the ability to specifically bind to an antigen or target antigen, although in some instances an accessory protein is required for specific binding. For instance, if the fragment is an antibody heavy chain of the variable region of an antibody heavy chain (VH), an antibody light chain or the variable region of an antibody light chain (VL) is needed for specific binding. The antibody light chain or the variable region of an antibody light chain (VL) need not form part of the protein conjugate of the invention and can be supplied or expressed separately so that the protein conjugate forms an antibody or functional fragment thereof capable of specific binding to a target antigen.

[0152] The term "fragment" with reference to an antibody, including a "functional fragment" and "functional antibody fragment", refers to a fragment of an intact antibody that retains the ability to specifically bind to the target antigen. Such fragments include, but are not limited to, Fab fragments, Fab' fragments, monovalent fragments consisting of the VL, VH, CL and CHI domains; F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragment consisting of the VH and CHI domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g. Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "functional fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0153] The one or more, such as two or more, antibodies, variants thereof or fragments thereof each typically comprise one or more, such as 3, 6, 9 or 12, complementarity determining regions (CDRs). The antibody, variant thereof or fragment thereof typically comprises one or more, such as 3, 6, 9 or 12, complementarity determining regions (CDRs). CDRs are defined according to the Kabat definition unless specified that the CDR are defined according to another definition. The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme) and ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) ("IMGT" numbering scheme). For example, for classic formats, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Under IMGT the CDR amino acid residues in the VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3) (numbering according to "Kabat"). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0154] By convention, the CDR regions in the heavy chain are typically referred to as HCDR1, HCDR2 and HCDR3 and in the light chain as LCDR1, LCDR2 and LCDR3. They are numbered sequentially in the direction from the amino terminus to the carboxy terminus.

[0155] The one or more, such as two or more, antibodies may be one or more monoclonal antibodies. The antibody may be a monoclonal antibody. The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. The protein conjugate may comprise a monoclonal antibody, a variant thereof or a fragment thereof.

[0156] The one or more, such as two or more, antibodies may be one or more human antibodies. The antibody may be a human antibody. The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g. human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik, et al., (2000) J Mol Biol; 296:57-86). The protein conjugate may comprise a human antibody, a variant thereof or a fragment thereof.

[0157] The human antibody may include amino acid residues not encoded by human sequences (e.g. mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0158] An antibody can be prepared using an antibody having one or more of the VH and / or VL sequences shown herein as starting material to engineer a modified antibody, which modified antibody may have altered properties from the starting antibody. An antibody can be engineered by modifying one or more residues within one or both variable regions (i.e., VH and / or VL), for example within one or more CDR regions and / or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant region(s), for example to alter the effector function(s) of the antibody.

[0159] One type of variable region engineering that can be performed is antibody binding region / paratope or CDR grafting. Because paratope sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR / paratope sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al., 1998 Nature 332:323-327; Jones, P. et al., 1986 Nature 321:522- 525; Queen, C. et al., 1989 Proc. Natl. Acad. See. U.S.A. 86: 10029-10033; US5,225,539, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0160] Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc- cpe.cam.ac.uk / vbase), as well as in Kabat, E. A., et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al., 1992 J. fol. Biol. 227:776-798; and Cox, J. P. L. et al., 1994 Eur. J Immunol. 24:827-836. An example of framework sequences for use in the antibodies are those that are structurally similar to the framework sequences used by selected antibodies of the disclosure, e.g., consensus sequences and / or framework sequences used by monoclonal antibodies of the disclosure. The VH CDR1, 2 and 3 sequences, and the VL CDR1, 2 and 3 sequences, can be grafted onto framework regions that have the identical sequence as that found in the germline immunoglobulin gene from which the framework sequence derive, or the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences. For example, it has been found that in certain instances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0161] Another type of variable region modification is to mutate amino acid residues within the VH and / or VL CDR.1, CDR2 and / or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest, known as "affinity maturation." Site- directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as described herein and provided in the Examples. Conservative modifications (as discussed above) can be introduced. The mutations may be amino acid substitutions, additions, or deletions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.

[0162] A wide variety of antibody / immunoglobulin frameworks or scaffolds can be employed so long as the resulting polypeptide includes at least one binding region which specifically binds to the target antigen. Such frameworks or scaffolds include the 5 main idiotypes of human immunoglobulins, De fragments thereof (such as those disclosed elsewhere herein), and include immunoglobulins of other animal species, preferably having humanised aspects. Single heavy-chain antibodies such as those identified in camelids are of particular interest in this regard. Novel frameworks, scaffolds and fragments continue to be discovered and developed by those skilled in the art.

[0163] Antibody proteins obtained from members of the camel and dromedary Camelus bactrianus and Camelus dromaderius) family including new world members such as llama species Lama paccos, Lama glama and Lama vicugna) have been characterized with respect to size, structural complexity and antigenicity for human subjects. Certain IgG antibodies from this family of mammals as found in nature lack light chains, and are thus structurally distinct from the typical four chain quaternary structure having two heavy and two light chains, for antibodies from other animals, see WO94 / 04678.

[0164] A region of the camelid antibody which is the small single variable domain identified as VHH can be obtained by genetic engineering to yield a small protein having high affinity for a target, resulting in a low molecular weight antibody-derived protein known as a "camelid nanobody". See US5,759,808; see also Stijlemans, B. et al., 2004 J Biol Chem 279: 1256- 1261; Dumoulin, M. et al., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14: 440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; and Lauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. As with other antibodies of non-human origin, an amino acid sequence of a camelid antibody can be altered recombinantly to obtain a sequence that more closely resembles a human sequence, i.e., the nanobody can be "humanised". Thus the natural low antigenicity of camelid antibodies to humans can be further reduced.

[0165] The camelid nanobody has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e., camelid nanobodies are useful as reagents detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents. Thus yet another consequence of small size is that a camelid nanobody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody.

[0166] The low molecular weight and compact size further result in camelid nanobodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. Another consequence is that camelid nanobodies readily move from the circulatory system into tissues, and even cross the blood-brain barrier and can treat disorders that affect nervous tissue. Nanobodies can further facilitated drug transport across the blood brain barrier, see US2004 / 0161738. These features combined with the low antigenicity to humans indicate great therapeutic potential. Further, these molecules can be fully expressed in prokaryotic cells such as E. coli and are expressed as protein conjugates with bacteriophage and are functional.

[0167] The one or more, such as two or more, antibodies or variants thereof may be one or more camelid antibodies or nanobodies, variants thereof or fragments thereof. The antibody or variant thereof may be a camelid antibody or nanobody, a variant thereof or a fragment thereof. In one embodiment, the camelid antibody, nanobody, variant thereof or fragment thereof is obtained by grafting the CDR sequences of the heavy or light chain of a human antibody into nanobody or single domain antibody framework sequences, as described for example in WO94 / 04678. The one or more, such as two or more, antibodies or variants thereof may be one or more shark antibodies or variants thereof or fragments thereof. The antibody or variant thereof may be a shark antibody or variant thereof or a fragment thereof.

[0168] The one or more, such as two or more, antibodies or variants thereof may comprise nonimmunoglobulin frameworks. The antibody, variant thereof or fragment thereof may comprise non-immunoglobulin frameworks. Known non-immunoglobulin frameworks or scaffolds include, but are not limited to, Adnectins (fibronectin) (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd (Cambridge, MA), now part of GSK) and Ablynx nv (Zwijnaarde, Belgium)), lipocalin (Anticalin) (Pieris Proteolab AG, Freising, Germany), small modular immuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA, now Emergent BioSolutions), maxybodies (Avidia, Inc. (Mountain View, CA)), Protein A (Affibody AB, Sweden) and affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany), protein epitope mimetics (Polyphor Ltd, Allschwil, Switzerland).

[0169] The adnectin scaffolds are based on fibronectin type III domain (e.g., the tenth module of the fibronectin type III (10 Fn3 domain)). The fibronectin type III domain has 7 or 8 beta strands which are distributed between two beta sheets, which themselves pack against each other to form the core of the protein, and further containing loops (analogous to CDRs) which connect the beta strands to each other and are solvent exposed. There are at least three such loops at each edge of the beta sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the beta strands (US6,818,418).

[0170] These fibronectin-based scaffolds are not an immunoglobulin, although the overall fold is closely related to that of the smallest functional antibody fragment, the variable region of the heavy chain, which comprises the entire antigen recognition unit in camel and llama IgG. Because of this structure, the non-immunoglobulin antibody mimics antigen binding properties that are similar in nature and affinity to those of antibodies. These scaffolds can be used in a loop randomization and shuffling strategy in vitro that is similar to the process of affinity maturation of antibodies in vivo. These fibronectin-based molecules can be used as scaffolds where the loop regions of the molecule can be replaced with CDRs of the disclosure using standard cloning techniques.

[0171] Ankyrin technology is based on using proteins with ankyrin derived repeat modules as scaffolds for bearing variable regions which can be used for binding to different targets. The ankyrin repeat module is a 33 amino acid polypeptide consisting of two anti-parallel a- helices and a p-turn. Binding of the variable regions is mostly optimized by using ribosome display. Avimers are derived from natural A-domain containing protein such as LRP-1. These domains are used by nature for protein-protein interactions and in human over 250 proteins are structurally based on A-domains. Avimers consist of a number of different "A-domain" monomers (2-10) linked via amino acid linkers. Avimers can be created that can bind to the target antigen using the methodology described in, for example, US20040175756 US20050053973 US20050048512 and US20060008844.

[0172] Affibody® affinity ligands are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This scaffold domain consists of 58 amino acids, 13 of which are randomized to generate Affibody® libraries with a large number of ligand variants (See e.g., US5,831,012). Affibody® molecules mimic antibodies, they have a molecular weight of 6 kDa, compared to the molecular weight of antibodies, which is 150 kDa. In spite of its small size, the binding site of Affibody® molecules is similar to that of an antibody.

[0173] Anticalins® are products developed by the company Pieris ProteoLab AG. They are derived from lipocalins, a widespread group of small and robust proteins that are usually involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins occur in human tissues or body liquids.

[0174] The protein architecture is reminiscent of immunoglobulins, with hypervariable loops on top of a rigid framework. However, in contrast with antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain with 160 to 180 amino acid residues, being just marginally bigger than a single immunoglobulin domain.

[0175] The set of four loops, which makes up the binding pocket, shows pronounced structural plasticity and tolerates a variety of side chains. The binding site can thus be reshaped in a proprietary process in order to recognize prescribed target molecules of different shape with high affinity and specificity.

[0176] One protein of lipocalin family, the bilin-binding protein (BBP) of Pieris brassicae has been used to develop anticalins by mutagenizing the set of four loops. One example of a patent application describing "anticalins" is WO1999 / 16873.

[0177] Affilin™ molecules are small non-immunoglobulin proteins which are designed for specific affinities towards proteins and small molecules. New Affilin™ molecules can be very quickly selected from two libraries, each of which is based on a different human derived scaffold protein.

[0178] Affilin™ molecules do not show any structural homology to immunoglobulin proteins. Scil Proteins employs two Affilin™ scaffolds, one of which is gamma crystalline, a human structural eye lens protein and the other is "ubiquitin" superfamily proteins. Both human scaffolds are very small, show high temperature stability and are almost resistant to pH changes and denaturing agents. This high stability is mainly due to the expanded beta sheet structure of the proteins. Examples of gamma crystalline derived proteins are described in W02001 / 04144 and examples of "ubiquitin-like" proteins are described in W02004 / 106368.

[0179] PEM are medium-sized, cyclic, peptide-like molecules (MW l-2kDa) mimicking beta-hairpin secondary structures of proteins, the major secondary structure involved in protein-protein interactions. More generally, any polypeptide that mimicks the 3D structure of the epitope of the disclosed antibodies are part of the present disclosure.

[0180] These polypeptides may further be engineered to increase half-life or improve solubility. Especially, fusion constructs of these polypeptides with serum proteins, such as Fc fragments of IgG or human serum albumin can be generated to increase half-life, similarly to Fc engineering described in the following paragraph for antibody fragment molecules of the disclosure.

[0181] Engineered antibodies include those in which modifications have been made to framework residues within VH and / or VL, e.g. to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence by, for example, site- directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the disclosure.

[0182] Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T-cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in US2003 / 0153043.

[0183] In addition or alternative to modifications made within the framework or CDR regions, antibodies may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Furthermore, an antibody of the disclosure may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

[0184] In one embodiment, the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in US5,677,425. The number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0185] In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in US6, 165,745.

[0186] In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in US6,277,375. Alternatively, to increase the biological half-life, the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in US5,869,046 and US6,121,022.

[0187] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigenbinding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in US5,624,821 and US5,648,260. In particular, residues 234 and / or 235 may be mutated, optionally to alanine. Thus, in one embodiment, an antibody according to the disclosure has a mutation in the Fc region at one or both of amino acids 234 and 235. Substitution of both amino acids 234 and 235 results in reduced ADCC activity.

[0188] In another embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered Clq binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in US6,194,551. In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in WO94 / 29351.

[0189] In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids. This approach is described further in WO00 / 42072. Moreover, the binding sites on human IgGl for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R.L. et al., 2001 J. Biol. Chen. 276:6591-6604).

[0190] In still another embodiment, the glycosylation of an antibody is modified. For example, an a glycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for "antigen". Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in US5,714,350 and US6,350,861.

[0191] Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GIcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the disclosure to thereby produce an antibody with altered glycosylation. For example, EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. WO03 / 035835 describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al., 2002 J. Biol. Chem. 277:26733-26740). WO99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(l,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GIcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al., 1999 Nat. Biotech. 17: 176-180). Another modification of the antibodies herein that is contemplated by the disclosure is pegylation. An antibody can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. The pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody.

[0192] Methods for pegylating proteins are known in the art and can be applied to the antibodies of the disclosure. See for example, EP0154316 and EP0401384.

[0193] The one or more, such as two or more, antibodies or variants thereof may be one or more synthetic antibodies, synthetic variants, or synthetic fragments. The antibody or variant thereof may be a synthetic antibody, a synthetic variant, or a synthetic fragment.

[0194] Variants and fragments

[0195] Specific variants and fragments of antibodies are discussed above. The protein conjugate may comprise in (a) any of the variants or fragments discussed above.

[0196] The variant or fragment specifically binds to the antigen on the surface of RBCs. Specific binding is defined below and can be measured using known assays including any of those discussed below.

[0197] The fragment may be any length as long as it is functional. The fragment may comprise at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the antibody. The fragment may be from about 10 to about 200 amino acids in length, such as from about 20 to about 190, from about 30 to about 180, from about 40 to about 170, from about 50 to about 160, from about 60 to about 150, from about 70 to about 140 or from about 80 to about 130 amino acids in length. The fragment is preferably at least about 10 amino acids in length, such as at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190 or at least about 200 amino acids in length. The fragment typically comprises one or more of the regions in the antibody responsible for specific binding to the antigen on the surface of RBCs. The fragment typically comprises one or more of, such as 3, 6, 9 or 12 of, the CDR sequences from the antibody. The fragment may comprise one or more of, such as 3, 6, 9 or 12 of, variants of the CDR sequences from the antibody. The one or more variant CDR sequences, such as 3, 6, 9 or 12 variant CDR sequences, may comprise or consist of sequence(s) having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the one or more CDR sequences in the antibody, such as in a native or wild-type antibody. The one or more variant CDR sequences, such as 3, 6, 9 or 12 variant CDR sequences, may comprise or consist of sequence(s) having any of these levels of homology and / or identity to the native or wild-type CDR sequences. Homology and / or identity is / are typically measured over the entire length of the reference sequence, typically the wild-type or native CDR sequence(s).

[0198] Standard methods in the art may be used to determine homology or identity. For example, the UWGCG Package provides the BESTFIT program which can be used to calculate homology or identity, for example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology and identity or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S.F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0199] The protein conjugate may comprise an antibody variant in (a). The protein conjugate may comprise a variant comprising or consisting of a sequence having at least about 90% homology or identity to the sequence of the antibody or a fragment thereof. The fragment may be any of those described above. In preferred embodiments, the variant has at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence of the antibody, such as a wild-type or native antibody, or the fragment thereof. Homology and / or identity is / are typically measured over the entire length of the reference sequence, typically the antibody or fragment thereof.

[0200] All of the embodiments in this section equally apply to one or more variants and / or one or more fragments.

[0201] Preferred antibody variants The one or more, such as two or more, antibodies or variants thereof may be one or more nanobodies, one or more nanobody fragments, one or more nanobody variants, or one or more humanised nanobodies. The antibody or variant thereof is preferably a nanobody, a nanobody fragment, a nanobody variant, or a humanised nanobody. Humanisation is discussed above.

[0202] Nanobodies are discussed above and any of those may be used in the protein conjugate of the invention. The nanobody may also be called "a single domain-based VHH".

[0203] The nanobody fragment is a functional nanobody fragment. The nanobody variant is a functional nanobody variant. The humanised nanobody is a functional humanised nanobody. The nanobody fragment, nanobody variant or humanised nanobody specifically binds to the antigen on the surface of RBCs. Specific binding is defined below and can be measured using known assays including any of those discussed below.

[0204] The nanobody fragment is preferably a peptide derived from the CDR loop regions of a nanobody. The peptide derived from the CDR loop regions of a nanobody is a functional peptide derived from the CDR loop regions of a nanobody. It specifically binds to the antigen on the surface of RBCs.

[0205] The nanobody fragment or peptide derived from the CDR loop regions of a nanobody may be any length as long as it is functional. The nanobody fragment or peptide may comprise at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the nanobody. The nanobody fragment or peptide may be from about 10 to about 130 amino acids in length, such as from about 20 to about 120, from about 30 to about 110, from about 40 to about 100, or from about 50 to about 90 amino acids in length. The nanobody fragment or peptide is preferably at least about 10 amino acids in length, such as at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, or at least about 130 amino acids in length.

[0206] The nanobody fragment or peptide typically comprises one or more of, such as 1, 2 or 3, of the nanobody CDR sequences. The nanobody fragment or peptide typically comprises one or more of, such as 1, 2 or 3 of, the CDR sequences from the nanobody. The nanobody fragment or peptide may comprise one or more of, such as 1, 2 or 3 of, variants of the CDR sequences from the nanobody. The one or more variant CDR sequences, such as 1, 2 or 3 variant CDR sequences, may comprise or consist of sequence(s) having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the one or more CDR sequences in the nanobody. Homology and / or identity is / are typically measured over the entire length of the reference sequence, typically the wild-type or native CDR sequence(s). Standard methods in the art may be used to determine homology or identity as defined above.

[0207] The nanobody variant preferably comprises or consists of a sequence having at least about 90% homology or identity to the sequence of the nanobody or a fragment thereof. The fragment may be any of those described above. In preferred embodiments, the nanobody variant comprises or consists of a sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence of the nanobody or the nanobody fragment. Homology and / or identity is / are typically measured over the entire length of the reference sequence, typically the nanobody, such as the native or wild-type nanobody, or the nanobody fragment. Standard methods in the art may be used to determine homology or identity as defined above.

[0208] All of the embodiments in this section equally apply to one or more nanobody variants and / or one or more nanobody fragments.

[0209] GPA specific antibodies

[0210] The one or more, such as two or more, antibodies or variants thereof specifically bind to one or more antigens on the surface of red blood cells (RBCs). The antibody or a variant thereof specifically binds to an antigen on the surface of red blood cells (RBCs). The one or more antigens or antigen may be any of those discussed in more detail below, including glycophorin A (GPA). The one or more, such as two or more, antibodies or variants thereof preferably specifically bind to one or more antigens comprising GPA. The antibody or a variant thereof preferably specifically binds to GPA. The protein conjugate may comprise one or more GPA-specific antibodies or variants thereof. The protein conjugate may comprise one or more GPA-specific nanobodies or variants thereof. The protein conjugate may comprise a GPA-specific antibody or a variant thereof. The protein conjugate may comprise a GPA-specific nanobody or a variant thereof. The one or more variants or variant may be any of those described above. The one or more fragments or fragment may be any of those described above. The one or more fragments or fragment is preferably a peptide derived from the CDR loop regions of the GPA-specific antibody or nanobody. IH4 is a nonlimiting example of a GPA-specific nanobody.

[0211] Duffy specific antibodies

[0212] The one or more, such as two or more, antibodies or variants thereof preferably specifically bind to one or more antigens comprising Duffy (also known as DARC or ACKR1). The antibody or a variant thereof preferably specifically binds to Duffy. The protein conjugate may comprise one or more Duffy-specific antibodies or variants thereof. The protein conjugate may comprise one or more Duffy-specific nanobodies or variants thereof. The protein conjugate may comprise a Duffy-specific antibody or a variant thereof. The protein conjugate may comprise a Duffy-specific nanobody or a variant thereof. The one or more variants or variant may be any of those described above. The one or more fragments or fragment may be any of those described above. The one or more fragments or fragment is preferably a peptide derived from the CDR loop regions of the Duffy-specific antibody or nanobody. CA52 is a non-limiting example of a Duffy-specific nanobody.

[0213] IH4

[0214] The one or more antibodies or variants thereof are preferably one or more IH4 nanobodies or variants thereof. The one or more variants may be one or more fragments. The one or more antibodies or variants thereof are preferably one or more IH4 nanobodies, variants thereof or fragments thereof. The one or more variants may be any of those described above. The one or more fragments may be any of those described above. The one or more IH4 fragments are preferably one or more peptides derived from the CDR loop regions of the IH4 nanobody.

[0215] The antibody or a variant thereof is preferably an IH4 nanobody or a variant thereof. The variant may be a fragment. The antibody or a variant thereof is preferably an IH4 nanobody, a variant thereof or fragment thereof. The variant may be any of those described above. The fragment may be any of those described above. The IH4 fragment is preferably a peptide derived from the CDR loop regions of the IH4 nanobody.

[0216] The antibody or a variant thereof may be any of the sequences described in Habib et al. (Analytical Biochemistry, Volume 438, Issue 1, 1 July 2013, Pages 82-89). This study retrieved 208 bona fide VHH sequences from colony PCR of positive clones. The 30 different sequences that were obtained were classified into three distinct families differing in the CDR3 region. The VHH referred to as IH4 was found 140 times and so accounted for 67% of the positively identified sequences. The antibody or a variant thereof may derived from a completely new library or a synthetic library.

[0217] The one or more antibodies or variants thereof preferably comprise or consist of the sequence shown in SEQ ID NO: 27, a variant thereof or a fragment thereof. The one or more fragments of the sequence shown in SEQ ID NO: 27 are one or more functional fragments.

[0218] The antibody or a variant thereof preferably comprises or consists of the sequence shown in SEQ ID NO: 27, a variant thereof or a fragment thereof. The fragment of the sequence shown in SEQ ID NO: 27 is a functional fragment. The fragment of the sequence shown in SEQ ID NO: 27 may be any length as it is functional. The fragment may comprise at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the sequence shown in SEQ ID NO: 27.

[0219] The fragment may be from about 10 to about 120 amino acids in length, such as from about 20 to about 110, from about 30 to about 100, from about 40 to about 90, or from about 50 to about 80 amino acids in length. The fragment is preferably at least about 10 amino acids in length, such as at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, or at least about 120 amino acids in length.

[0220] The variant and / or fragment of the sequence shown in SEQ ID NO: 27 typically comprises one or more of, such as 1, 2 or 3, of SEQ ID NO: 27's CDR sequences. These are shown in SEQ ID NOs: 30-32. The variant and / or fragment of the sequence shown in SEQ ID NO: 27 typically comprises one or more of:

[0221] (i) the sequence shown in SEQ ID NO: 30, a variant having at least about 72.7%, at least about 81.8% or at least about 90.9% homology and / or identity to the sequence shown in SEQ ID NO: 30 or a variant having 1, 2 or 3 amino acid mismatches with the sequence shown in SEQ ID NO: 30;

[0222] (ii) the sequence shown in SEQ ID NO: 31, a variant having at least about 82.3%, at least about 88.2% or at least about 94.1% homology and / or identity to the sequence shown in SEQ ID NO: 31 or a variant having 1, 2 or 3 amino acid mismatches with the sequence shown in SEQ ID NO: 31; and

[0223] (iii) the sequence shown in SEQ ID NO: 32, a variant having at least about 80.0%, at least about 86.6% or at least about 93.3% homology and / or identity to the sequence shown in SEQ ID NO: 32 or a variant having 1, 2 or 3 amino acid mismatches with the sequence shown in SEQ ID NO: 32.

[0224] The variant and / or fragment of the sequence shown in SEQ ID NO: 27 may comprise (i), (ii), (iii), (i) and (ii), (0 and (iii), (ii)ar|d (iii) or (i), (ii) and (iii). The variant and / or fragment of the sequence shown in SEQ ID NO: 27 preferably comprises three CDR sequences comprising or consisting of the sequences shown in SEQ ID NOs: 30-32 respectively.

[0225] The variant of the sequence shown in SEQ ID NO: 27 preferably comprises or consists of a sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 27. In preferred embodiments, the variant comprises or consists of a sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 27. Homology and / or identity is / are typically measured over the entire length of the SEQ ID NO: 27. Standard methods in the art may be used to determine homology or identity as defined above.

[0226] The one or more IH4 nanobodies, variants thereof or fragments thereof may have the structure shown in any one of Figures 1, 26, 33, 53, 59 and 67.

[0227] CA52

[0228] The one or more antibodies or variants thereof are preferably one or more CA52 nanobodies or variants thereof. The one or more variants may be one or more fragments. The one or more antibodies or variants thereof are preferably one or more CA52 nanobodies, variants thereof or fragments thereof. The one or more variants may be any of those described above. The one or more fragments may be any of those described above. The one or more CA52 fragments are preferably one or more peptides derived from the CDR loop regions of the CA52 nanobody.

[0229] The antibody or a variant thereof is preferably an CA52 nanobody or a variant thereof. The variant may be a fragment. The antibody or a variant thereof is preferably an CA52 nanobody, a variant thereof or fragment thereof. The variant may be any of those described above. The fragment may be any of those described above. The CA52 fragment is preferably a peptide derived from the CDR loop regions of the CA52 nanobody.

[0230] The one or more antibodies or variants thereof preferably comprise or consist of the sequence shown in SEQ ID NO: 58, a variant thereof or a fragment thereof. The one or more fragments of the sequence shown in SEQ ID NO: 58 are one or more functional fragments.

[0231] The antibody or a variant thereof preferably comprises or consists of the sequence shown in SEQ ID NO: 58, a variant thereof or a fragment thereof. The fragment of the sequence shown in SEQ ID NO: 58 is a functional fragment.

[0232] The one or more fragments or fragment of the sequence shown in SEQ ID NO: 58 may be any length as it is functional. The fragment may comprise at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the sequence shown in SEQ ID NO: 58. The one or more fragments or fragment may be from about 10 to about 120 amino acids in length, such as from about 20 to about 110, from about 30 to about 100, from about 40 to about 90, or from about 50 to about 80 amino acids in length. The one or more fragments or fragment is / are preferably at least about 10 amino acids in length, such as at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, or at least about 120 amino acids in length.

[0233] The variant and / or fragment of the sequence shown in SEQ ID NO: 58 typically comprise(s) one or more of, such as 1, 2 or 3, of SEQ ID NO: 58's CDR sequences. These are shown in SEQ ID NOs: 59-61. The variant and / or fragment of the sequence shown in SEQ ID NO: 58 typically comprises one or more of:

[0234] (i) the sequence shown in SEQ ID NO: 59, a variant having at least about 70%, at least about 80% or at least about 90% homology and / or identity to the sequence shown in SEQ ID NO: 59 or a variant having 1, 2 or 3 amino acid mismatches with the sequence shown in SEQ ID NO: 59;

[0235] (ii) the sequence shown in SEQ ID NO: 60, a variant having at least about 82.3%, at least about 88.2% or at least about 94.1% homology and / or identity to the sequence shown in SEQ ID NO: 60 or a variant having 1, 2 or 3 amino acid mismatches with the sequence shown in SEQ ID NO: 60; and

[0236] (iii) the sequence shown in SEQ ID NO: 61, a variant having at least about 80.0%, at least about 86.6% or at least about 93.3% homology and / or identity to the sequence shown in SEQ ID NO: 61 or a variant having 1, 2 or 3 amino acid mismatches with the sequence shown in SEQ ID NO: 61.

[0237] The variant and / or fragment of the sequence shown in SEQ ID NO: 58 may comprise (i), (ii), (iii), (i) and (ii), (0 and (iii), (ii)ar|d (iii) or (i), (ii) and (iii). The variant and / or fragment of the sequence shown in SEQ ID NO: 58 preferably comprises three CDR sequences comprising or consisting of the sequences shown in SEQ ID NOs: 59-61 respectively.

[0238] The variant of the sequence shown in SEQ ID NO: 58 preferably comprises or consists of a sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 58. In preferred embodiments, the variant comprises or consists of a sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 58. Homology and / or identity is / are typically measured over the entire length of the SEQ ID NO: 58. Standard methods in the art may be used to determine homology or identity as defined above. The one or more CA52 nanobodies, variants thereof or fragments thereof may have the structure shown in Figure 46.

[0239] Beta-strand ( / 3-strand) swapped variants

[0240] The one or more antibody variants or one or more nanobody variants are preferably modified or engineered to alter the position of the N and / or C terminus in the variant(s). The antibody variant or the nanobody variant is preferably modified or engineered to alter the position of the N and / or C terminus in the variant. This allows the position of the one or more enzymes and / or variants thereof in the protein conjugate to be altered. For instance, the position of the C terminus and the one or more enzymes and / or variants thereof conjugated thereto can be altered. An example of this is shown in Figure 20. Antibody variants and nanobody variants modified in this way facilitate covalent binding to the antigen and / or the RBCs. This is discussed in more detail below.

[0241] The position of the N and / or C terminus in the variant may be altered in any way. The N terminus may be moved from the top of the beta-sheet (0-sheet) to the bottom of the 0- sheet or may be moved from the bottom of the 0-sheet to the top of the 0-sheet. The C terminus may be moved from the top of the 0-sheet to the bottom of the 0-sheet or may be moved from the bottom of the 0-sheet to the top of the 0-sheet. The N terminus may be moved from the top of the 0-sheet to the bottom of the 0-sheet and the C terminus may be moved from the bottom of the 0-sheet to the top of the 0-sheet. The N terminus may be moved from the bottom of the 0-sheet to the top of the 0-sheet and the C terminus may be moved from the top of the 0-sheet to the bottom of the 0-sheet. In these embodiments, the

[0242] N and / or C terminus have been "swapped".

[0243] The one or more antibody variants or the one or more nanobody variants are preferably one or more beta-strand (0-strand) swapped variants. The antibody variant or the nanobody variant is preferably a beta-strand (0-strand) swapped variant. Any of the antibodies, nanobodies, variants or fragments discussed above may comprise 0-strand swapped modifications. The 0-strand swapped variant may comprise (a) swapping of existing 0- strands to alter the position of the N and / or C terminus, (b) swapping out existing 0-strands for other sequences that result in swapping the N and / or C terminus and / or (c) modifying the swapped 0-strand to improve the structural stability of the variant. The 0-strand swapped variant may comprise (a), (b), (c), (a) and (b), (b) and (c), (a) and (c) or (a), (b) and (c). Embodiment (a) may involve swapping the N and / or C termini in any of the ways described above.

[0244] The one or more antibodies or variants thereof are preferably one or more beta swapped IH4 nanobodies or variants thereof. The one or more variants may be one or more fragments. The one or more antibodies or variants thereof are preferably one or more beta swapped IH4 nanobodies, variants thereof or fragments thereof. The one or more variants may be any of those described above. The one or more fragments may be any of those described above. The one or more fragments are preferably one or more peptides derived from the CDR loop regions of the beta swapped IH4 nanobody.

[0245] The antibody or variant thereof is preferably a beta swapped IH4 nanobody or a variant thereof. The variant may be a fragment. The antibody or variant thereof is preferably a beta swapped IH4 nanobody, a variant thereof or a fragment thereof. The variant may be any of those described above. The fragment may be any of those described above. The fragment is preferably a peptide derived from the CDR loop regions of the beta swapped IH4 nanobody.

[0246] The one or more beta swapped IH4 nanobodies preferably comprise or consist of the sequence shown in SEQ ID NO: 28 or 29, a variant thereof or a fragment thereof. The one or more fragments of the sequence shown in SEQ ID NO: 28 or 29 are functional fragments.

[0247] The beta swapped IH4 nanobody preferably comprises or consists of the sequence shown in SEQ ID NO: 28 or 29, a variant thereof or a fragment thereof. The fragment of the sequence shown in SEQ ID NO: 28 or 29 is a functional fragment.

[0248] The fragment of the sequence shown in SEQ ID NO: 28 or 29 may be any length as it is functional. The fragment may comprise at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the sequence shown in SEQ ID NO: 28 or 29.

[0249] The fragment may be any of the lengths discussed above with reference to SEQ ID NO: 27 discussed above.

[0250] The variant and / or fragment of the sequence shown in SEQ ID NO: 28 or 29 typically comprises one or more of, such as 1, 2 or 3, of SEQ ID NO: 27's CDR sequences as discussed above. These are shown in SEQ ID NOs: 30-32. The variant and / or fragment of the sequence shown in SEQ ID NO: 28 or 29 may comprise (i), (ii), (iii), (i) and (ii), (i) and (iii), (ii) and (iii) or (i), (ii) and (iii) as defined above. The variant and / or fragment of the sequence shown in SEQ ID NO: 28 or 29 preferably comprises three CDR sequences comprising or consisting of the sequences shown in SEQ ID NOs: 30-32 respectively.

[0251] The beta swapped IH4 nanobody variant preferably comprises or consists of a sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 28 or 29. In preferred embodiments, the beta swapped IH4 nanobody variant comprises or consists of a sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 28 or 29. Homology and / or identity is / are typically measured over the entire length of the SEQ ID NO: 28 or 29. Standard methods in the art may be used to determine homology or identity as defined above.

[0252] The one or more antibodies or variants thereof are preferably one or more beta swapped CA52 nanobodies or variants thereof. The one or more variants may be one or more fragments. The one or more antibodies or variants thereof are preferably one or more beta swapped CA52 nanobodies, variants thereof or fragments thereof. The one or more variants may be any of those described above. The one or more fragments may be any of those described above. The one or more fragments are preferably one or more peptides derived from the CDR loop regions of the beta swapped CA52 nanobody.

[0253] The antibody or variant thereof is preferably a beta swapped CA52 nanobody or a variant thereof. The variant may be a fragment. The antibody or variant thereof is preferably a beta swapped CA52 nanobody, a variant thereof or a fragment thereof. The variant may be any of those described above. The fragment may be any of those described above. The fragment is preferably a peptide derived from the CDR loop regions of the beta swapped CA52 nanobody.

[0254] Tagged nanobodies

[0255] The one or more antibodies or variants thereof may be tagged. The antibody or a variant thereof may be tagged. Suitable tags are discussed in detail below. The one or more tagged antibodies or variants thereof preferably comprise or consist of the sequence shown in SEQ ID NO: 2, 16 or 24, a variant thereof or a fragment thereof. The tagged antibody or variant thereof preferably comprises or consists of the sequence shown in SEQ ID NO: 2, 16 or 24, a variant thereof or a fragment thereof. The fragment of the sequence shown in SEQ ID NO: 2, 16 or 24 is a functional fragment.

[0256] The fragment of the sequence shown in SEQ ID NO: 2, 16 or 24 may be any length as it is functional. The fragment may comprise at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the sequence shown in SEQ ID NO: 2, 16 or 24.

[0257] The fragment may be any of the lengths discussed above with reference to SEQ ID NO: 27 discussed above.

[0258] The variant and / or fragment of the sequence shown in SEQ ID NO: 2, 16 or 24 typically comprises one or more of, such as 1, 2 or 3, of SEQ ID NO: 27's CDR sequences as discussed above. These are shown in SEQ ID NOs: 30-32. The variant and / or fragment of the sequence shown in SEQ ID NO: 2, 16 or 24 may comprise (i), (ii), (iii), (i) and (ii), (i) and (iii), (ii) and (iii) or (i), (ii) and (iii) as defined above. The variant and / or fragment of the sequence shown in SEQ ID NO: 2, 16 or 24 preferably comprises three CDR sequences comprising or consisting of the sequences shown in SEQ ID NOs: 30-32 respectively.

[0259] The tagged variant preferably comprises or consists of a sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 2, 16 or 24. In preferred embodiments, the tagged variant comprises or consists of a sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 2, 16 or 24. Homology and / or identity is / are typically measured over the entire length of the SEQ ID NO: 2, 16 or 24. Standard methods in the art may be used to determine homology or identity as defined above.

[0260] Covalent binding

[0261] In some embodiments, the one or more antibodies or variants thereof are modified to facilitate covalent binding or covalent attachment to the antigen and / or the RBCs. In some embodiments, the antibody or variant thereof is modified to facilitate covalent binding or covalent attachment to the antigen and / or the RBCs. The antibody or variant thereof is preferably modified with a protein or molecule capable of covalent binding to the antigen and / or the RBCs. The protein conjugate preferably comprises a -strand swapped nanobody variant modified at its swapped C-terminus by the covalent attachment of a protein or molecule capable of covalent binding to the antigen and / or the RBCs. The protein conjugate preferably comprises a -strand swapped nanobody variant covalently linked via its swapped C-terminus to a protein or molecule capable of covalent binding to the antigen and / or the RBCs. Any protein or molecule capable of covalent binding to the antigen and / or the RBCs may be used. The protein may be a bacterial protein or a fragment thereof which comprises a self-processing module (SPM). SPMs are capable of generating an anhydride group that allow covalent conjugation. They are discussed in US20230106353A1. The bacterial protein may be from Neisseria meningitis, Alysiella filiformis or Kingella negevensis.

[0262] The protein may be FrpC or FrpA from Neisseria meningitis. The antibody or variant thereof may be modified using NeissLock as described in Scheu, A.H.A., Lim, S.Y.T., Metzner, F.J. et al. NeissLock provides an inducible protein anhydride for covalent targeting of endogenous proteins. Nat Commun 12, 717 (2021) and US20230106353A1.

[0263] The protein comprising a SPM may modified to improve its ability to facilitate covalent binding or covalent attachment to the antigen and / or the RBCs. For instance, the protein may modified to remove one or more reactive amino acids, such as one or more lysines, and / or to reduce non-specific binding or reactions. The one or more reactive amino acids, such as one or more lysines, may be removed by substitution. The protein preferably comprises or consists of a sequence having at least about 90% homology or identity to a SPM-containing protein. In preferred embodiments, the protein comprises or consists of a sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to a SPM-containing protein. Homology and / or identity is / are typically measured over the entire length of the SPM-containing protein. Standard methods in the art may be used to determine homology or identity as defined above.

[0264] The protein is preferably the SPM from FrpA from Neisseria meningitidis. The SPM module from FrpA displays calcium-dependant autoproteolytic activity as an Asp-Pro (D-P) bond, leading to formation of an aspartic anhydride on the Asp of the C-terminus post cleavage of the part of the polypeptide chain that contains the SPM domain. This can then go on to form a intermolecular covalent bond. The protein preferably comprises or consists of the sequence shown in SEQ ID NO: 38, a variant thereof or a fragment thereof. The fragment is a functional fragment. The protein preferably comprises or consists of a sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 38. In preferred embodiments, the protein comprises or consists of a sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 38. Homology and / or identity is / are typically measured over the entire length of the SEQ ID NO: 38. Standard methods in the art may be used to determine homology or identity as defined above.

[0265] The one or more antibodies or variants thereof preferably comprise or consist of the sequence shown in SEQ ID NO: 18, 20, 22 or 26, or a variant or fragment thereof. The antibody or variant thereof preferably comprises or consists of the sequence shown in SEQ ID NO: 18, 20, 22 or 26, or a variant or fragment thereof. The fragment of the sequence shown in SEQ ID NO: 18, 20, 22 or 26 is a functional fragment.

[0266] The fragment of the sequence shown in SEQ ID NO: 18, 20, 22 or 26 may be any length as it is functional. The fragment may comprise at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the sequence shown in SEQ ID NO: 18, 20, 22 or 26. The fragment may be any of the lengths discussed above with reference to SEQ ID NO: 27 discussed above.

[0267] The variant and / or fragment typically comprises one or more of, such as 1, 2 or 3, of SEQ ID NO: 27's CDR sequences as discussed above. These are shown in SEQ ID NOs: 30-32. The variant and / or fragment of the sequence shown in SEQ ID NO: 18, 20, 22 or 26 may comprise (i), (ii), (iii), (i) and (ii), (i) and (iii), (ii) and (iii) or (i), (ii) and (iii) as defined above. The variant and / or fragment of the sequence shown in SEQ ID NO: 18, 20, 22 or 26 preferably comprises three CDR sequences comprising or consisting of the sequences shown in SEQ ID NOs: 30-32 respectively.

[0268] The variant preferably comprises or consists of a sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 18, 20, 22 or 26. In preferred embodiments, the variant comprises or consists of a sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 18, 20, 22 or 26. Homology and / or identity is / are typically measured over the entire length of the SEQ ID NO: 18, 20, 22 or 26. Standard methods in the art may be used to determine homology or identity as defined above.

[0269] Specific binding

[0270] The one or more antibodies, variants, or fragments specifically bind to one or more antigens on the surface of RBCs. The one or more antigens may also be called the one or more target antigens. The antibody, variant, or fragment specifically binds to an antigen on the surface of RBCs. The antigen may also be called the target antigen. The RBCs may be any of those described below.

[0271] The term "specifically binds to" an antigen means binding that is measurably different from a non-specific or non-selective interaction (e.g., with a non-target molecule or antigen). Specific binding can be measured, for example, by measuring binding to a target antigen and comparing it to binding to a non-target molecule or antigen. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target antigen. Such methods are routine in the art. All instances herein of the term "specifically binds to" is interchangeable with "specifically interacts with," "specific for," "selectively binds to" "selectively interacts with" and "selective for".

[0272] The antibody, variant, or fragment binds to the antigen or target antigen if it binds to the antigen or target antigen with preferential or high affinity, but does not bind or binds with only low affinity to other or different molecules or antigen, such as other or different antigens of the surface of RBCs or other cells. Preferably, the antibody, variant, or fragment binds to the antigen or target antigen with an affinity that is at least about 10 times, such as at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 1000 or at least about 10,000 times, greater than its affinity for other molecules or antigen.

[0273] The term "Kassoc" or "Kon", as used herein, is intended to refer to the association rate of a particular binding molecule-target, whereas the term "Kdis" or "Koff," as used herein, is intended to refer to the dissociation rate of a particular binding molecule-target interaction. The term "Kd", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kon to Koff (i.e. Kon / Koff) and is expressed as a molar concentration (M).

[0274] The antibody, variant, or fragment has affinity for the antigen or target antigen. The antibody, variant, or fragment preferably has high affinity for the antigen or target antigen. The antibody, variant, or fragment has high affinity for the antigen or target antigen if it binds with a Kd of about 1 x IO-6M or less, such as about 1 x IO-7M or less, about 5 x IO-8M or less, about 1 x IO-8M or less, or about 5 x IO-9M or less. The antibody, variant, or fragment preferably has a Kd for the antigen or target antigen of about 100 nM or less, such as about 50 nM or less, about 20 nM or less or about 10 nM or less. A molecule or group binds with low affinity if it binds with a Kd of about 1 x IO-6M or more, about 1 x IO-5M or more, about 1 x 10-4M or more, about 1 x IO-3M or more, or about 1 x IO-2M or more.

[0275] Affinity can be measured using known binding assays, such as those that make use of fluorescence and radioisotopes. Competitive binding assays are also known in the art. Other methods include spectroscopic assays, isothermal titration calorimetry (ITC), or optical biosensors such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and grating-coupled interferometry (GCI). Kd values for antibodies and variants thereof can be determined using surface plasmon resonance, such as a Biacore® system, or solution equilibrium titration (SET) (see Friguet, et al., (1985) J. Immunol. Methods, 77(2):305-319, and Hanel et al., (2005) Anal. Biochem., 339(1) : 182-184).

[0276] All of the embodiments in this section equally apply to one or more antibodies, variants thereof and / or fragments thereof.

[0277] Target antigens

[0278] The one or more antibodies, variants, or fragments specifically bind to one or more antigens on the surface of RBCs. The one or more antigens may also be called the one or more target antigens. The one or more antigens or one or more target antigens may be any antigen(s) present on the surface of the RBCs. The one or more antigens or one or more target antigens may be any antigen(s) present on the surface of the RBCs in the subject. The RBCs typically express a detectable level of the one or more antigens or target antigens. All of the embodiments in this section equally apply to one or more antigens or one or more target antigens.

[0279] The antibody, variant, or fragment specifically binds to an antigen on the surface of RBCs. The antigen may also be called the target antigen. The antigen or target antigen may be any antigen present on the surface of the RBCs. The antigen or target antigen may be any antigen present on the surface of the RBCs in the subject. The RBCs typically express a detectable level of the antigen or target antigen.

[0280] The antigen or target antigen may be specific to (or specific for) the RBCs. This typically means the antigen or target antigen is not expressed on the surface of any other cell in the subject or is expressed at a low but detectable level or an undetectable level on the surface of any other cell in the subject.

[0281] In the context of the invention, a "detectable level" may comprise a detectable level of the mRNA encoding the relevant marker, e.g. glycophorin A (GPA) mRNA, and / or the marker protein, e.g. the GPA protein. Preferably, a "detectable level" comprises a detectable level of the protein, e.g. the GPA protein. A "detectable level" may comprise a detectable level of the mRNA encoding the relevant marker, e.g. GPA mRNA, and a detectable level of the marker protein, e.g. the GPA protein. The same applies to a low but detectable level and undetectable level.

[0282] The presence of mRNA and / or protein may be detected using any routine method in the art. Such methods include immunofluorescence, immunohistochemistry, western blotting, quantitative polymerase chain reaction (qPCR), reporter assays, enzyme-linked immunosorbent assay (ELISA), microscopy, flow cytometry, enzymatic staining, dye incorporation, chemiluminescent oxygen detection reagents, fluorometry, fluorescence in situ hybridization (FISH) and an alkaline phosphatase assay. The skilled person is capable of detecting the activity of any particular antigen using known assays. Suitable assays are described in the Examples. The antigen or target antigen is preferably detected using flow cytometry.

[0283] The antigen or target antigen may be any antigen on the surface of RBCs. The antigen or target antigen may be selected from Kell, Rh proteins, including RhD, RhCE, and RhAG, Band 3 / AE1, BCAM / Lutheran, Glycophorin A (GPA), Glycophorin B (GPB), Glycophorin C (GPC), CD44, XK, Aquaporin 1, Aquaporin 3, CD47, Complement component Receptor 1 (CR1), CD55 / Daf, Duffy, ICAM4 / LW, Acetylcholinesterase (ACHE), Basigin (CD147), Kidd, Glucose transporter 1 (GLUT1), Monocarboxylate transporter 1 (MCT1), Sodium Dependent multivitamin transporter SMVT (SLC5A6), ERMAP (erythroblast membrane associated protein), XG, Semaphorin 7A (CD108), CD151, ADP-ribosyltransferase, Neutral Amino acid transporter B(0) (SLC1A5), Large neutral amino acids transporter small subunit 1(SLC7A5), Chloride intracellular channel protein 1 (CLIC1), Sodium / potassium-transporting ATPase subunit alpha-1 (ATP1A1), ATPase Na+ / K+ transporting subunit beta 3 (ATP1B3) and CD59. Antibodies which bind these antigens are known in the art.

[0284] The antigen or target antigen may be selected from Kell, Rh proteins, including RhD, RhCE, and RhAG, Band 3 / AE1, BCAM / Lutheran, Glycophorin A (GPA), Glycophorin B (GPB), Glycophorin C (GPC), CD44, XK, Aquaporin 1, Aquaporin 3, CD47, Complement component Receptor 1 (CR1), CD55 / Daf, Duffy, ICAM4 / LW, Acetylcholinesterase (ACHE), Basigin (CD147), Kidd, Glucose transporter 1 (GLUT1), Monocarboxylate transporter 1 (MCT1), Sodium Dependent multivitamin transporter SMVT (SLC5A6), ERMAP (erythroblast membrane associated protein), XG, Semaphorin 7A (CD108), CD151, and ADP- ribosyltransferase. The antigen or target antigen may be selected from Kell, Rh proteins, including RhD, RhCE, and RhAG, Band 3 / AE1, BCAM / Lutheran, Glycophorin A (GPA), Glycophorin B (GPB), Glycophorin C (GPC), XK, Aquaporin 1, Aquaporin 3, Complement component Receptor 1 (CR1), CD55 / Daf, Duffy, ICAM4 / LW, Acetylcholinesterase (ACHE), Kidd, Glucose transporter 1 (GLUT1), Monocarboxylate transporter 1 (MCT1), Sodium Dependent multivitamin transporter SMVT (SLC5A6), ERMAP (erythroblast membrane associated protein), XG, Semaphorin 7A (CD108), CD151, and ADP-ribosyltransferase. Antibodies which bind these antigens are known in the art.

[0285] The antigen or target antigen is preferably specific to (or specific for) the RBCs. The antigen or target antigen is preferably selected from glycophorin A (GPA), Band 3 / AE1, CD47, Duffy, Glucose transporter 1 (Glutl), Basigin (CD147), ICAM4, and Rh proteins, including RhD, RhCE, and RhAG. The one or more antigens or one or more target antigens are preferably selected from this list of antigens. Antibodies which bind these antigens are known in the art.

[0286] The one or more antigens or one or more target antigens preferably comprise glycophorin A (GPA) and / or Duffy. The antigen or target antigen is preferably glycophorin A (GPA) or Duffy.

[0287] The one or more antigens or one or more target antigens preferably comprise glycophorin A (GPA). The antigen or target antigen is preferably glycophorin A (GPA).

[0288] One or more enzymes and / or variants thereof

[0289] The protein conjugate comprises (b) one or more enzymes and / or variants thereof. The protein conjugate may therefore comprise in (b) one or more enzymes and / or one or more enzyme variants. The one or more enzymes and / or variants thereof are functional. The one or more enzymes are functional. The one or more enzyme variants are functional. The invention is used to surface label RBCs with the one or more enzymes and / or variants thereof. The surface labelled RBCs are a delivery system for the one or more enzymes and / or variants thereof. In other words, the RBCs themselves are not the ultimate target for the one or more enzymes and / or variants thereof. The one or more enzymes and / or variants thereof are not being used to correct defects in the RBCs themselves. The RBCs are being used in the invention as a vehicle to deliver the one or more enzymes and / or variants thereof. They may be used to deliver the one or more enzymes to where they are needed, for instance to the site of a disease or disorder.

[0290] The protein conjugate may comprise any number of one or more enzymes and / or variants thereof, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more enzymes and / or variants thereof. The protein conjugate may comprise one or more enzymes, one or more enzyme variants, or one or more enzymes and one or more enzyme variants. The protein conjugate may comprise one or more components of a multi-enzyme complex.

[0291] The one or more enzymes and / or variants thereof may be the same. The one or more enzymes and / or variants thereof may be different. For example, the protein conjugate may comprise two identical enzymes or two identical enzyme variants. Alternatively, the protein conjugate may comprise two different enzymes, two different enzyme variants (including from the same enzyme) or an enzyme and an enzyme variant.

[0292] The one or more enzyme variants are preferably one or more enzyme fragments. The one or more enzyme fragments are functional.

[0293] The one or more enzyme fragments may be any length as long as it is / they are functional. The enzyme fragment may comprise at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the enzyme. The enzyme fragment may be from about 10 to about 500 amino acids in length, such as from about 20 to about 450, from about 30 to about 400, from about 40 to about 350, from about 50 to about 300, from about 60 to about 250, from about 70 to about 200 or from about 80 to about 100 amino acids in length. The fragment is preferably at least about 10 amino acids in length, such as at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 500 or at least about 600 amino acids in length. The one or more enzyme variants preferably comprise or consist of a sequence having at least about 90% homology or identity to the sequence of the enzyme. In preferred embodiments, the enzyme variant comprises or consists of a sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence of the enzyme. Homology and / or identity is / are typically measured over the entire length of the reference sequence, typically the enzyme, such as the native or wild-type enzyme. Standard methods in the art may be used to determine homology or identity as defined above.

[0294] The one or more enzymes may selected from hydrolases, oxidoreductases, lyases, transferases, ligases and isomerases. The one or more enzymes may be selected from transferase, phosphorylases, synthases, kinases, hydroxylase, dehydrogenase, deaminases, proteases, metalloproteinases, lyases, aminotransferases, deiminases, decarboxylase, and oxidases. The one or more enzymes may be selected from transferases, phosphorylases, synthases, kinases, hydroxylases, dehydrogenases, deaminases, proteases, metalloproteinases, lyases, aminotransferases, deiminases, decarboxylases, and oxidases. The one or more enzymes may be selected from transferases, phosphorylases, synthases, kinases, hydroxylases, dehydrogenases, deaminases, proteases, metalloproteinases, lyases, aminotransferases, deiminases, decarboxylases, oxidases, amino acid oxidases, peroxiredoxins, aldehyde dehydrogenases, RNAses, DNAses, glutathione-S-transferases, Organophosphate Hydrolases (OPHs), Phosphotriesterase-like Lactonases (PLLs), alginate lyases, endolysins, N-acetylmuramoyl-L-alanine amidases (peptidoglycan amidases) and lytic transglycosylases. The one or more enzymes may be one or more lysosomal enzymes. The one or more enzymes may be one or more clotting factors.

[0295] The one or more enzymes are preferably selected from branched-chain a-ketoacid dehydrogenase, p-glucocerebrosidase, a-galactosidase a, acid a-glucosidase, a-l- iduronidase, iduronate-2-sulfatase, acid sphingomyelinase, p-hexosaminidase a, cystathionine p-synthase, methylmalonyl-coa mutase, propionyl-coa carboxylase, galactose- 1-phosphate uridylyltransferase, aldolase b, glucose-6-phosphatase, glycogen debranching enzyme, biotinidase, argininosuccinate synthetase, pyruvate dehydrogenase, isovaleryl-coa dehydrogenase, glutaryl-coa dehydrogenase, cholesterol oxidase, cystinosin, thymidine phosphorylase, alanine amino transferase, thymidine phosphorylase, glutamine synthase, hexokinase, glucokinase, phenylalanine hydroxylase, alcohol dehydrogenase, catalase, glucose-6-phosphate dehydrogenase, adenosine deaminase (ADA), asparaginase, uricase, bacterial L-phenylalanine ammonia lyase, alanine aminotransferase, glutamate dehydrogenase, arginine deiminase, arginase, oxalate decarboxylase, oxalate oxidase, a serine protease, tissue plasminogen activator, larondinase, superoxide dismutase, disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), Carboxypeptidase B2 (TAFI), Protein C, Protein S, CD39 (ENTPD1), CD73 (NT5E), Carboxypeptidase N (CPN), Factor Vila (FVIIa), Factor IXa (FIXa), Factor Xa (FXa), Prothrombin / Thrombin (Flla), Factor Xia (FXIa), Factor XHIa (transglutaminase), Urokinase (uPA), Plasmin, Streptokinase, Staphylokinase, Glutathione peroxidase 1 (GPxl), GPx3, Glutathione reductase, Peroxiredoxin 1 (PRDX1), PRDX2, PRDX3, PRDX4, PRDX5, PRDX6, Alkaline phosphatase (ALPL), Acyloxyacyl hydrolase (AOAH), Diamine oxidase (DAO) & Histamine N-methyltransferase (HNMT), Platelet-activating factor acetylhydrolase (PLA2G7), Heme oxygenase-1 (HO-1), Glutathione reductase (GSR), Glutathione-S-transferase Al (GSTA1), GSTA2, GSTA3, GSTA4, GSTA5, GSTK1, GSTM1, GSTM1L (RNAi), GSTM2, GSTM3, GSTM4, GSTM5, GSTO1, GSTO2, GSTP1, GSTT1, GSTT2, GSTT4, GSTZ1 (aka MAAI- Maleylacetoacetate isomerase), Paraoxonase-1 (PON1), Butyrylcholinesterase (BChE) Aldehyde dehydrogenase 2 (ALDH2), ALDH1A1, Phosphotriesterase (PTE), Organophosphorus Hydrolase (OPH), Paraoxonase 1 (PON1), PON2, PON3, Diisopropylfluorophosphatase (DFPase), Methylparathion Hydrolase (MPH), Cocaine esterase (Rhodococcus), Hyaluronidase-2 (HYAL2), a combination of CD39 and CD73, Arylsulfatase B (ARSB), Acid ceramidase (ASAHI), N-acetylgalactosamine-6-sulfatase (GALNS), Galactocerebrosidase (GALC), Arylsulfatase A (ARSA), Aspartylglucosaminidase (AGA), 0- Mannosidase (MANBA), N-acetylglucosamine-6-sulfatase (GNS), Heparan N-sulfatase (SGSH), Alpha-N-acetylglucosaminidase (NAGLU), Acetyl-CoA:alpha-glucosaminide N- acetyltransferase (HGSNAT), Alpha-mannosidase (MAN2B1), Neuraminidase 1 (NEU1), Cathepsin K (CTSK), Cathepsin D (CTSD), Palmitoyl-protein thioesterase 1 (PPT1), Tripeptidyl peptidase 1 (TPP1), DNase I or a variant thereof, DNASE1L3, RNase 1 or a variant thereof, DNASE1L3, Lysozyme (LYZ), AlgL alginate lyase, Al-IV alginate lyase, Al-II alginate lyase, Al-III alginate lyase, alginate lyase Al (AlyAl), AlyA2, AlyPG, AlyM, AlyGC, Algl7c, AlyQ, AlyDRl, AlyDR2, AlyVl, AlyV2, AlyA5, Alg7A, AlySY08, AlyMBl, ALY-1, ALY-2, Dispersin B (bacterial PNAG hydrolase), Secretory phospholipase A2 (SPLA2-IIA), Complement Factor I, LysK, PlyC, Cpl-1, Pal, Lys44, LysH5, LysEF-PIO, LysSA97, Ply511, PlyV12, LysPA26, LysAB2, OBPgp279, KZ144, LysSS, ClyS, Art-175, AmiA, AmiB, AmiC, AtlA, LytA, AtlE, AtlA, CwlC, CwlA, CwlB, CwID, Auto (autolysin with amidase domain), Cpl- 1, PlyC, Pal, Ply511, Plyll8, Slt70, MltA, MltB, MltC, MltD, MltE, MltF, MltG, Sit, MltBl, MltB2, MltF, LtgA, LtgD, RIpA, AmpDh2, AmpDh3, LtgX, Angiotensin converting enzyme 2 (ACE2), Lipoprotein lipase (LPL), Cholesterol esterase, Lecithin-Cholesterol aclytransferasee (LCAT), D-amino acid oxidase and L-amino acid oxidase.

[0296] The one or more enzymes are preferably selected from branched-chain a-ketoacid dehydrogenase, 0-glucocerebrosidase, a-galactosidase a, acid a-glucosidase, a-l- iduronidase, iduronate-2-sulfatase, acid sphingomyelinase, 0-hexosaminidase a, cystathionine 0-synthase, methylmalonyl-coa mutase, propionyl-coa carboxylase, galactose- 1-phosphate uridylyltransferase, aldolase b, glucose-6-phosphatase, glycogen debranching enzyme, biotinidase, argininosuccinate synthetase, pyruvate dehydrogenase, isovaleryl-coa dehydrogenase, glutaryl-coa dehydrogenase, cholesterol oxidase, cystinosin, thymidine phosphorylase, alanine amino transferase, thymidine phosphorylase, glutamine synthase, hexokinase, glucokinase, phenylalanine hydroxylase, alcohol dehydrogenase, catalase, glucose-6-phosphate dehydrogenase, adenosine deaminase (ADA), asparaginase, uricase, bacterial L-phenylalanine ammonia lyase, alanine aminotransferase, glutamate dehydrogenase, arginine deiminase, arginase, oxalate decarboxylase, oxalate oxidase, a serine protease, tissue plasminogen activator, larondinase, superoxide dismutase, and disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13).

[0297] The one or more enzymes are preferably selected from branched-chain a-ketoacid dehydrogenase, p-glucocerebrosidase, a-galactosidase a, acid a-glucosidase, a-l- iduronidase, iduronate-2-sulfatase, acid sphingomyelinase, p-hexosaminidase a, cystathionine p-synthase, methylmalonyl-coa mutase, propionyl-coa carboxylase, galactose- 1-phosphate uridylyltransferase, aldolase b, glucose-6-phosphatase, glycogen debranching enzyme, biotinidase, argininosuccinate synthetase, pyruvate dehydrogenase, isovaleryl-coa dehydrogenase, glutaryl-coa dehydrogenase, cholesterol oxidase, cystinosin, thymidine phosphorylase, alanine amino transferase, thymidine phosphorylase, glutamine synthase, hexokinase, glucokinase, phenylalanine hydroxylase, alcohol dehydrogenase, catalase, glucose-6-phosphate dehydrogenase, adenosine deaminase (ADA), asparaginase, uricase, bacterial L-phenylalanine ammonia lyase, alanine aminotransferase, glutamate dehydrogenase, arginine deiminase, arginase, oxalate decarboxylase, oxalate oxidase, a serine protease, tissue plasminogen activator, larondinase, and disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13).

[0298] The one or more enzymes are preferably selected from thymidine phosphorylase, alanine amino transferase, thymidine phosphorylase, glutamine synthase, hexokinase, glucokinase, phenylalanine hydroxylase, alcohol dehydrogenase, catalase, glucose-6-phosphate dehydrogenase, adenosine deaminase (ADA), asparaginase, uricase, bacterial L- phenylalanine ammonia lyase, alanine aminotransferase, glutamate dehydrogenase, arginine deiminase, arginase, oxalate decarboxylase, oxalate oxidase, a serine protease, tissue plasminogen activator, larondinase, and disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13).

[0299] The one or more enzymes preferably comprise thymidine phosphorylase, adenosine deaminase (ADA) or disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13).

[0300] The one or more enzymes preferably comprise thymidine phosphorylase. The enzyme preferably comprises or consists of the sequence shown in SEQ ID NO: 33, a variant thereof or a fragment thereof. The fragment may be any of those discussed above. The variant preferably has at least about 90% homology or identity to the sequence shown in SEQ ID NO: 33. In preferred embodiments, the protein conjugate comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 33. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 33. Homology and / or identity can be measured as described above. The protein conjugate preferably comprises or consists of the sequence shown in SEQ ID NO: 33. The variant and / or fragment preferably has the structure shown in Figure 1.

[0301] The one or more enzymes preferably comprise adenosine deaminase (ADA). The enzyme preferably comprises or consists of the sequence shown in SEQ ID NO: 40, a variant thereof or a fragment thereof. The fragment may be any of those discussed above. The variant preferably has at least about 90% homology or identity to the sequence shown in SEQ ID NO: 40. In preferred embodiments, the protein conjugate comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 40. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 40. Homology and / or identity can be measured as described above. The variant or fragment of SEQ ID NO: 40 may lack the linker (G residue) and / or C-terminal His tag. The protein conjugate preferably comprises or consists of the sequence shown in SEQ ID NO: 40. The protein conjugate preferably comprises or consists of positions 2-363 of SEQ ID NO: 40. The variant and / or fragment preferably has the structure shown in Figure 26, 59 or 67.

[0302] The one or more enzyme preferably comprise disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13). The ADAMTS13 may be wild-type ADAMTS13. The ADAMTS13 may comprise a gain-of-function mutation. The gain-of-function mutation may be R527K / F551Y / R619K / Y620F / Y624F. An example of a protein conjugate of the invention comprising this gain-of-function mutation is shown in SEQ ID NO: 49. This mutation has been shown to increase ADAMTS13 activity in leaving von Willebrand Factor, as well as decrease autoantibody binding in patient with acquired TTP_£Jian, Cui, Juan Xiao, Lingjie Gong, Christopher G. Skipwith, Sheng-Yu Jin, Hau C. Kwaan, and X. Long Zheng. 'Gain-of-Function ADAMTS13 Variants That Are Resistant to Autoantibodies against ADAMTS13 in Patients with Acquired Thrombotic Thrombocytopenic Purpura'. Blood 119, no. 16 (19 April 2012): 3836-43. https : / / doi.orQ / 10.1182 / blood-2011-12-399501). The variant and / or fragment preferably has the structure shown in Figure 33, 39 or 46. The ADAMTS13 may be a truncated version of ADAMTS13. Previous work has found C- terminal truncation of the TSP1 repeats and CUB domains after the Spacer domain of ADAMTS13 may improve its activity, against a fluorogenic vWF substrate (Gao, W., Anderson, P. J., Majerus, E. M., Tuley, E. A. & Sadler, J. E. Exosite interactions contribute to tension-induced cleavage of von Willebrand factor by the antithrombotic ADAMTS13 metalloprotease. Proc. Natl. Acad. Sci. U.S.A. 103, 19099-19104 (2006)). The ADAMTS13 may be the MDTCS region of ADAMTS13. The MDTCS region consists of a C-terminal truncation (amino acids >A685X), and removal of the pro-peptide (amino acids Q34-R74). The MDTCS region is the minimal structure for ADAMTS13 function, with the C-terminal TSP1 repeats 2-8 and CUB1 / 2 domains acting as auto-inhibitory units (Zhang, P., Pan, W., Rux, A. H., Sachais, B. S. & Zheng, X. L. The cooperative activity between the carboxyl- terminal TSP1 repeats and the CUB domains of ADAMTS13 is crucial for recognition of von Willebrand factor under flow. Blood 110, 1887-1894 (2007)).

[0303] The enzyme preferably comprises or consists of the sequence shown in SEQ ID NO: 44, a variant thereof or a fragment thereof. The fragment may be any of those discussed above. The variant preferably has at least about 90% homology or identity to the sequence shown in SEQ ID NO: 44. In preferred embodiments, the protein conjugate comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 44. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 44. Homology and / or identity can be measured as described above. The variant or fragment of SEQ ID NO: 44 may lack the signal peptide and / or C- terminal His tag. The variant of SEQ ID NO: 44 may comprise R527K, F551Y, R619K, Y620F and Y624F. The protein conjugate preferably comprises or consists of the sequence shown in SEQ ID NO: 44. The protein conjugate preferably comprises or consists of positions 34-644 of SEQ ID NO: 44.

[0304] The one or more enzymes, variants and / or fragments may be derived from any source. They may be derived from humans or bacteria, such as Escherichia coll E. coll). They may be synthetic or recombinant. The one or more variants or one or more fragments include truncated versions of the human enzyme that are structurally more similar to the E. coll enzyme, and variants designed to improve the recombinant expression of the human enzyme in E. coll. Methods for doing this are known in the art (e.g., Karamitros et al., Front Bioeng Biotechnol. 2021 Dec 17:9:793985. doi: 10.3389 / fbioe.2021.793985. eCollection 2021). The one or more enzymes and / or one or more fragments thereof are preferably associated with a disease or disorder and / or may be used for detoxification. The disease or disorder may be any of those discussed below.

[0305] Conjugation

[0306] Components (a) and (b) may be conjugated in any order. The protein conjugate may comprise from N to C terminus (a) and (b). The protein conjugate may comprise from N to C terminus (b) and (a).

[0307] Components (a) and (b) may be conjugated in any way. Components (a) and (b) may be produced separately and conjugated in any way. Components (a) and (b) may be conjugated before the protein conjugate is used to surface label RBCs. Components (a) and (b) may be conjugated on the surface of RBCs.

[0308] Components (a) and (b) are typically covalently attached. The conjugation chemistry between components (a) and (b) is not limited. Any suitable combination of reactive functional groups can be used. Many suitable reactive groups and their chemical targets are known in the art. Some exemplary reactive groups and their corresponding targets include aryl azides which may react with amine, carbodiimides which may react with amines and carboxyl groups, hydrazides which may react with carbohydrates, hydroxmethyl phosphines which may react with amines, imidoesters which may react with amines, isocyanates which may react with hydroxyl groups, carbonyls which may react with hydrazines, maleimides which may react with sulfhydryl groups, NHS-esters which may react with amines, PFP- esters which may react with amines, psoralens which may react with thymine, pyridyl disulfides which may react with sulfhydryl groups, vinyl sulfones which may react with sulfhydryl amines and hydroxyl groups, vinylsulfonamides, and the like. Other suitable chemistry for conjugating the polypeptide to the polynucleotide includes click chemistry. Many suitable click chemistry reagents are known in the art. Suitable examples of click chemistry include, but are not limited to, the following:

[0309] • copper(I)-catalyzed azide-alkyne cycloadditions (azide alkyne Huisgen cycloadditions);

[0310] • strain-promoted azide-alkyne cycloadditions; including alkene and azide [3 + 2] cycloadditions; alkene and tetrazine inverse-demand Diels-Alder reactions; and alkene and tetrazole photoclick reactions;

[0311] • copper-free variant of the 1,3 dipolar cycloaddition reaction, where an azide reacts with an alkyne under strain, for example in a cyclooctane ring such as in bicycle[6.1.0]nonyne (BCN); the reaction of an oxygen nucleophile on one linker with an epoxide or aziridine reactive moiety on the other; and the Staudinger ligation, where the alkyne moiety can be replaced by an aryl phosphine, resulting in a specific reaction with the azide to give an amide bond.

[0312] Any reactive group may be used to form the conjugate. Some suitable reactive groups include [1, 4-Bis[3-(2-pyridyldithio)propionamido]butane; 1,1 1-bis- maleimidotriethyleneglycol; 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester); ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester); 4,4'-diisothiocyanatostilbene- 2,2'-disulfonic acid disodium salt; Bis[2-(4-azidosalicylamido)ethyl] disulphide; 3-(2- pyridyldithio)propionic acid N-hydroxysuccinimide ester; 4-maleimidobutyric acid N- hydroxysuccinimide ester; lodoacetic acid N-hydroxysuccinimide ester; S-acetylthioglycolic acid N-hydroxysuccinimide ester; azide-PEG-maleimide; and alkyne-PEG-maleimide.

[0313] Components (a) and (b) may be conjugated using SpyCatcher-SpyTag (Zakeri, Bijan et al. "Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin." Proceedings of the National Academy of Sciences of the United States of America vol. 109,12 (2012): E690-7. doi: 10.1073 / pnas.1115485109).

[0314] Components (a) and (b) may be expressed separately and then conjugated post expression. Components (a) and (b) are preferably genetically fused. This means the protein conjugate is expressed from a single polynucleotide expressing the protein conjugate. Polynucleotides and their expression are described in more detail below.

[0315] Components (a) and (b) may be fused by one or more linkers. The one or more linkers are preferably one or more amino acid sequences. Suitable amino acid linkers, such as peptide linkers, are known in the art. The length, flexibility and hydrophilicity of the amino acid or peptide linker(s) are typically designed such each region in the protein conjugate can perform its function. Preferred flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16, serine and / or glycine amino acids. More preferred flexible linkers include (SG)i, (SG)2, (SG)3, (SG)4, (SG)5, (SG)8, (SG)IO, (SG)is or (SG)?o wherein S is serine and G is glycine. Preferred rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24, proline amino acids. More preferred rigid linkers include (P)i2 wherein P is proline.

[0316] The one or more linkers may be selected from G, GSS, SSG and GSY. The one or more linker preferably comprise or consist of the sequence shown in SEQ ID NO: 34, 35, 36 or 37 or a variant having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 34, 35, 36 or 37, such as at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 34, 35, 36 or 37. Homology and / or identity is / are typically measured over the entire length of the reference sequence. Homology and / or identity may be measured as described above.

[0317] The protein conjugate can have any conformation. The protein conjugate preferably has the conformation from N to C terminus or from C to N terminus: component (b) - component (a) - component (b), wherein the two instances of component (b) are the same or different; component (b) - component (b) - component (a), wherein the two instances of component (b); component (a) - component (b) - component (b), wherein the two instances of component (b) are the same or different; component (a) - one or more linkers - component (b); component (b) - one or more linkers - component (a); component (b) - one or more linkers - component (a) - one or more linkers - component (b), wherein the two instances of component (b) are the same or different; component (b) - one or more linkers - component (b) - one or more linkers - component (a), wherein the two instances of component (b) are the same or different; or component (a) - one or more linkers - component (b) - one or more linkers - component (b), wherein the two instances of component (b) are the same or different.

[0318] Oligomer formation

[0319] The protein conjugate may be a monomer. The protein conjugate is preferably capable of forming an oligomer. Advantages of forming an oligomer are discussed in more detail below. The protein conjugate may be capable of forming any oligomer, such as a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamers, decamer, undecamer, or dodecamer. The protein conjugate is preferably capable of forming a dimer. The ability of a protein conjugate can be measured using routine methods and as disclosed in the Examples.

[0320] The protein conjugate may be capable of forming an oligomer because the one or more enzymes and / or fragments thereof may be capable of forming an oligomer. For instance, thymidine phosphorylase is capable of forming a dimer. The ability of the one or more enzymes and / or fragments thereof to form an oligomer may be measured using routine methods. Some enzymes and their fragments are known in the art to form oligomers.

[0321] The protein conjugate may further one or more modifications which increases its ability to form an oligomer. The protein conjugate may comprise any number of such modifications, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. Modifications capable of increase the ability of a protein to oligomerise are known the art. For example, the protein conjugate can be modified using biotin. This allows the protein conjugate to be oligomerised using streptavidin. The protein conjugate can also be modified using alpha-helical coiled-coils (e.g., Swainsbury et al., Biochim Biophys Acta 2016 Dec; 1857(12): 1829-1839. doi: 10.1016 / j.bbabio.2016.09.002). The protein conjugate may comprise the sequence shown in SEQ ID NO: 62. This sequence is disclosed in Fletcher et al., ACS Synthetic Biology 2012 1 (6), 240-250. This sequence may be located at any position in the protein conjugate, such as between (a) and (b). These can be used to achieve homo- but also heterooligomerisation. The protein conjugate can also be modified by the inclusion of other protein regions that are known to modulate oligomerisation, such as the Cartilage Oligomeric Matrix Protein (COMP) Pentamerisation Domain (e.g., Bushell et al., Genome Res. 2008 Apr; 18(4):622-30. doi: 10.1101 / gr.7187808. Epub 2008 Feb

[0322] Preferred protein conjugate

[0323] The protein conjugate preferably comprises or consists of the sequence shown in SEQ ID NO: 4 or 6 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 4 or 6. In preferred embodiments, the protein conjugate comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 4 or 6. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 4 or 6. Homology and / or identity can be measured as described above. The protein conjugate preferably comprises or consists of the sequence shown in SEQ ID NO: 4 or 6.

[0324] The protein conjugate preferably comprises or consists of the sequence shown in SEQ ID NO: 42 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 42. In preferred embodiments, the protein conjugate comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 42. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 42. Homology and / or identity can be measured as described above. The protein conjugate preferably comprises or consists of the sequence shown in SEQ ID NO: 42.

[0325] The protein conjugate preferably comprises or consists of the sequence shown in SEQ ID NO: 46 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 46. In preferred embodiments, the protein conjugate comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 46. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 46. Homology and / or identity can be measured as described above. The protein conjugate preferably comprises or consists of the sequence shown in SEQ ID NO: 46.

[0326] The protein conjugate preferably comprises or consists of the sequence shown in SEQ ID NO: 49 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 49. In preferred embodiments, the protein conjugate comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 49. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 49. Homology and / or identity can be measured as described above. The protein conjugate preferably comprises or consists of the sequence shown in SEQ ID NO: 49.

[0327] The protein conjugate preferably comprises or consists of the sequence shown in SEQ ID NO: 51 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 51. In preferred embodiments, the protein conjugate comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 51. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 51. Homology and / or identity can be measured as described above. The protein conjugate preferably comprises or consists of the sequence shown in SEQ ID NO: 51.

[0328] The protein conjugate preferably comprises or consists of the sequence shown in SEQ ID NO: 55 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 55. In preferred embodiments, the protein conjugate comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 55. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 55. Homology and / or identity can be measured as described above. The protein conjugate preferably comprises or consists of the sequence shown in SEQ ID NO: 55.

[0329] The protein conjugate preferably comprises or consists of the sequence shown in SEQ ID NO: 57 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 57. In preferred embodiments, the protein conjugate comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 57. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 57. Homology and / or identity can be measured as described above. The protein conjugate preferably comprises or consists of the sequence shown in SEQ ID NO: 57.

[0330] The protein conjugate preferably has the structure shown in Figure 1, 26, 33, 46, 59 or 67.

[0331] Production of protein conjugates

[0332] The protein conjugate of the invention may be modified in any way. Methods for introducing or substituting non-naturally occurring amino acids in proteins are also well known in the art. The protein conjugate may also be produced using D-amino acids or a mixture of L- amino acids and D-amino acids. This is conventional in the art for producing such proteins or peptides.

[0333] Any protein conjugate of the invention may be chemically modified. The protein conjugate can be chemically modified in any way and at any site. The protein conjugate may be chemically modified by attachment of a molecule to one or more cysteines (cysteine linkage), attachment of a molecule to one or more lysines, attachment of a molecule to one or more non-natural amino acids, enzyme modification of an epitope or modification of a terminus. Suitable methods for carrying out such modifications are well-known in the art. The protein may be chemically modified by the attachment of any molecule, such as a dye or a fluorophore.

[0334] Any of the protein conjugates may be modified to assist their identification or purification, for example by the addition of histidine residues (a his tag), aspartic acid residues (an asp tag), a streptavidin tag, a flag tag, a SUMO tag, a GST tag or a MBP tag, or by the addition of a signal sequence to promote their secretion from a cell where the protein does not naturally contain such a sequence. The his tag typically comprises or consists of from 2 to 10 histidine residues, such as 6 histidine residues. An alternative to introducing a genetic tag is to chemically react a tag onto a native or engineered position on the protein. An example of this would be to react a gel-shift reagent to a cysteine engineered on the outside of the protein.

[0335] Any of the protein conjugates may be labelled with a revealing label. The revealing label may be any suitable label which allows the protein conjugate to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g., 1251, 35S, enzymes, antibodies, antigens, polynucleotides, and ligands such as biotin.

[0336] The protein conjugate may also contain other non-specific modifications as long as they do not adversely interfere with the function of the protein. Some mutations may also improve functionality. A number of non-specific side chain modifications are known in the art and may be made to the side chains of the protein(s). Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH4, amidation with methylacetimidate or acylation with acetic anhydride.

[0337] Any of the protein conjugates can be produced using standard methods known in the art. Polynucleotide sequences encoding a protein may be derived and replicated using standard methods in the art. Polynucleotide sequences encoding a protein may be expressed in a bacterial host cell using standard techniques in the art. The protein may be produced in a cell by in situ expression of the polypeptide from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control the expression of the polypeptide. These methods are described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0338] Proteins may be produced in large scale following purification by any protein liquid chromatography system from protein producing organisms or after recombinant expression. Typical protein liquid chromatography systems include FPLC, AKTA systems, the Bio-Cad system, the Bio-Rad BioLogic system, and the Gilson HPLC system.

[0339] The protein conjugate of the invention may be isolated, substantially isolated, purified or substantially purified. A protein conjugate is isolated or purified if it is completely free of any other components, such as free from RBCs or other cells or free from components used to produce the conjugate. A protein conjugate is substantially isolated if it is mixed with carriers or diluents which will not interfere with its intended use. This may include RBCs as discussed in more detail below.

[0340] Oligomeric construct

[0341] The invention also provides an oligomeric construct comprising two or more protein conjugates of the invention. The two or more protein conjugates may be any of those discussed above, especially in relation to conjugates which form oligomers. The two or more protein conjugates may oligomerise naturally, for instance via the oligomeric nature of the one or enzymes present in the protein conjugates, or artificially following the incorporation of one or more oligomerisation motifs as discussed above.

[0342] The oligomeric construct may be any of the oligomers discussed above, such a monomeric construct, dimeric construct, trimeric construct, tetrameric construct, pentameric construct, hexameric construct, heptameric construct, octameric construct, nonameric constructs, decameric construct, undecameric construct, or dodecameric construct. The construct is preferably a dimeric construct. The construct may comprise any number of two or more protein conjugates of the invention, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more or 12 or more.

[0343] The oligomeric constructs of the invention have several advantages. The oligomerised antibodies or variants thereof may increase the avidity of the construct. The oligomerised enzymes and / or variants thereof may increase the enzymatic activity of the constructs. The oligomeric constructs may also be used to bring the components of a multi-enzyme complex together, for instance if different protein conjugates comprise different enzymes and / or variants thereof.

[0344] The two or more protein conjugates may be the same. The two or more protein conjugates may be different. The two or more protein conjugates may differ based on their antibodies or variants thereof. The two or more protein conjugates may differ based on the one or more enzymes and / or variants thereof. The two or more protein conjugates may differ based on their antibodies or variants thereof and the one or more enzymes and / or variants thereof.

[0345] The two or more protein conjugates in the oligomeric constructs may be conjugated, attached or covalently attached. Any of the methods discussed above for conjugating, attaching or covalently attaching the components of the protein conjugates of the invention may be used. The two or more protein conjugates in the oligomeric constructs may be genetically fused. The two or more protein conjugates may be conjugated, attached, covalently attached or genetically fused using one or more linkers. The one or more linkers may be any of those described above.

[0346] The two or more protein conjugates in the oligomeric conjugate may be expressed from a single polynucleotide or separate polynucleotides. This is discussed in more detail below.

[0347] The invention preferably provides a dimeric construct comprising two protein conjugates, wherein each protein conjugate comprises (a) an antibody that specifically binds GPA, a variant thereof or a fragment thereof or a GPA-specific nanobody, a variant thereof or a fragment thereof and (b) thymidine phosphorylase or a variant thereof. The invention preferably provides a dimeric construct comprising two protein conjugates, wherein each protein conjugate comprises (a) an IH4 nanobody, a variant thereof or a fragment thereof and (b) thymidine phosphorylase or a variant thereof. Any of the embodiments discussed above equally apply to this dimeric construct of the invention. Component (b) preferably comprises a variant of human thymidine phosphorylase designed to improve its recombinant expression in E. coli. Component (b) preferably comprises or consists of the sequence shown in SEQ ID NO: 33 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 33. Each protein conjugate preferably comprises in (a) the sequence shown in SEQ ID NO: 27, 28, 29, 2, 16, 24, 18, 20, 22 or 26 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 27, 28, 29, 2, 16, 24, 18, 20, 22 or 26. In preferred embodiments, each protein conjugate comprises in (a) a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 27, 28, 29, 2, 16, 24, 18, 20, 22 or 26. Each protein conjugate preferably comprises in (b) the sequence shown in SEQ ID NO: 33 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 33. In preferred embodiments, each protein conjugate comprises in (b) a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 33. Homology and / or identity is / are typically measured over the entire length of the reference sequence. Homology and / or identity can be measured as described above. The protein conjugate preferably comprises (a) the sequence shown in SEQ ID NO: 27, 28, 29, 2, 16, 24, 18, 20, 22 or 26 and (b) the sequence shown in SEQ ID NO: 33.

[0348] Each protein conjugate preferably comprises in the sequence shown in SEQ ID NO: 4 or 6 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 4 or 6. In preferred embodiments, each protein conjugate comprises a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 4 or 6. Homology and / or identity is / are typically measured over the entire length of the reference sequence. Homology and / or identity can be measured as described above.

[0349] The invention preferably provides a dimeric construct comprising two protein conjugates, wherein each protein conjugate comprises (a) an antibody that specifically binds GPA, a variant thereof or a fragment thereof or a GPA-specific nanobody, a variant thereof or a fragment thereof, (b) ADA or a variant thereof and (c) a self dimerisation domain. The invention preferably provides a dimeric construct comprising two protein conjugates, wherein each protein conjugate comprises (a) an IH4 nanobody, a variant thereof or a fragment thereof, (b) ADA or a variant thereof and (c) a self dimerisation domain. Any of the embodiments discussed above equally apply to this dimeric construct of the invention. Component (b) preferably comprises a variant of human thymidine phosphorylase designed to improve its recombinant expression in E. coli. Component (b) preferably comprises or consists of the sequence shown in SEQ ID NO: 40 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 40. Each protein conjugate preferably comprises in (a) the sequence shown in SEQ ID NO: 27, 28, 29, 2, 16, 24, 18, 20, 22 or 26 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 27, 28, 29, 2, 16, 24, 18, 20, 22 or 26. In preferred embodiments, each protein conjugate comprises in (a) a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 27, 28, 29, 2, 16, 24, 18, 20, 22 or 26. Each protein conjugate preferably comprises in (b) the sequence shown in SEQ ID NO: 40 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 40. In preferred embodiments, each protein conjugate comprises in (b) a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 40. Homology and / or identity is / are typically measured over the entire length of the reference sequence. Homology and / or identity can be measured as described above. Each protein conjugate preferably comprises (a) the sequence shown in SEQ ID NO: 27, 28, 29, 2, 16, 24, 18, 20, 22 or 26 and (b) the sequence shown in SEQ ID NO: 33. Each protein conjugate preferably comprises (c) the sequence shown in SEQ ID NO: 62.

[0350] Each protein conjugate preferably comprises in the sequence shown in SEQ ID NO: 57 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 57. In preferred embodiments, each protein conjugate comprises a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 57. Homology and / or identity is / are typically measured over the entire length of the reference sequence. Homology and / or identity can be measured as described above.

[0351] The oligomeric construct of the invention may be isolated, substantially isolated, purified or substantially purified. These terms are defined above.

[0352] Polynucleotides The invention also provides one or more polynucleotides, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more or 12 or more encoding a protein conjugate of the invention or an oligomeric construct of the invention. The invention also provides a polynucleotide encoding a protein conjugate of the invention or an oligomeric construct of the invention. The protein conjugate or oligomeric construct may be any of those described above.

[0353] The invention also provides two or more polynucleotides, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more or 12 or more polynucleotides, encoding an oligomeric construct of the invention. The protein conjugate or the oligomeric construct may be any of those described above.

[0354] The one or more polynucleotides or two or more polynucleotides preferably encodes a protein conjugate which comprises or consists of the sequence shown in SEQ ID NO: 4 or 6 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 4 or 6. In preferred embodiments, the one or more polynucleotides or two or more polynucleotides preferably encode a protein conjugate which comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 4 or 6. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 4 or 6. Homology and / or identity can be measured as described above. The one or more polynucleotides or two or more polynucleotides preferably encode a protein conjugate which comprises or consists of the sequence shown in SEQ ID NO: 4 or 6.

[0355] The one or more polynucleotides or two or more polynucleotides preferably comprise or consist of the sequence shown in SEQ ID NO: 3 or 5 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 3 or 5. In preferred embodiments, the one or more polynucleotides or two or more polynucleotides preferably comprise or consist of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 3 or 5. Homology and / or identity can be measured as described above. The one or more polynucleotides or two or more polynucleotides preferably comprise or consist of the sequence shown in SEQ ID NO: 3 or 5.

[0356] The one or more polynucleotides or two or more polynucleotides preferably encodes a protein conjugate which comprises or consists of the sequence shown in SEQ ID NO: 42 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 42. In preferred embodiments, the one or more polynucleotides or two or more polynucleotides preferably encode a protein conjugate which comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 42. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 42. Homology and / or identity can be measured as described above. The one or more polynucleotides or two or more polynucleotides preferably encode a protein conjugate which comprises or consists of the sequence shown in SEQ ID NO: 42.

[0357] The one or more polynucleotides or two or more polynucleotides preferably comprise or consist of the sequence shown in SEQ ID NO: 41 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 41. In preferred embodiments, the one or more polynucleotides or two or more polynucleotides preferably comprise or consist of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 41. Homology and / or identity can be measured as described above. The one or more polynucleotides or two or more polynucleotides preferably comprise or consist of the sequence shown in SEQ ID NO: 41.

[0358] The one or more polynucleotides or two or more polynucleotides preferably encodes a protein conjugate which comprises or consists of the sequence shown in SEQ ID NO: 46 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 46. In preferred embodiments, the one or more polynucleotides or two or more polynucleotides preferably encode a protein conjugate which comprises or consists of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 46. Homology and / or identity is / are typically measured over the entire length of the sequence shown in SEQ ID NO: 46. Homology and / or identity can be measured as described above. The one or more polynucleotides or two or more polynucleotides preferably encode a protein conjugate which comprises or consists of the sequence shown in SEQ ID NO: 46.

[0359] The one or more polynucleotides or two or more polynucleotides preferably comprise or consist of the sequence shown in SEQ ID NO: 45 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 45. In preferred embodiments, the one or more polynucleotides or two or more polynucleotides preferably comprise or consist of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 45. Homology and / or identity can be measured as described above. The one or more polynucleotides or two or more polynucleotides preferably comprise or consist of the sequence shown in SEQ ID NO: 45.

[0360] The one or more polynucleotides or two or more polynucleotides preferably comprise or consist of the sequence shown in SEQ ID NO: 50 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 50. In preferred embodiments, the one or more polynucleotides or two or more polynucleotides preferably comprise or consist of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 50. Homology and / or identity can be measured as described above. The one or more polynucleotides or two or more polynucleotides preferably comprise or consist of the sequence shown in SEQ ID NO: 50.

[0361] The one or more polynucleotides or two or more polynucleotides preferably comprise or consist of the sequence shown in SEQ ID NO: 54 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 54. In preferred embodiments, the one or more polynucleotides or two or more polynucleotides preferably comprise or consist of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 54. Homology and / or identity can be measured as described above. The one or more polynucleotides or two or more polynucleotides preferably comprise or consist of the sequence shown in SEQ ID NO: 54.

[0362] The one or more polynucleotides or two or more polynucleotides preferably comprise or consist of the sequence shown in SEQ ID NO: 56 or a variant sequence having at least about 90% homology or identity to the sequence shown in SEQ ID NO: 56. In preferred embodiments, the one or more polynucleotides or two or more polynucleotides preferably comprise or consist of a variant sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the sequence shown in SEQ ID NO: 56. Homology and / or identity can be measured as described above. The one or more polynucleotides or two or more polynucleotides preferably comprise or consist of the sequence shown in SEQ ID NO: 56.

[0363] A polynucleotide, such as a nucleic acid, is a polymer comprising two or more nucleotides. The nucleotides can be naturally occurring or artificial. A nucleotide typically contains a nucleobase, a sugar and at least one linking group, such as a phosphate, 2'0-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate group. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar and the nucleobase together form a nucleoside. Preferred nucleosides include, but are not limited to, adenosine, guanosine, 5-methyluridine, uridine, cytidine, deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. The nucleosides may be adenosine, guanosine, uridine and cytidine.

[0364] The nucleotides are typically ribonucleotides or deoxyribonucleotides. The nucleotides may be deoxyribonucleotides. The nucleotides typically contain a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5' or 3' side of a nucleotide.

[0365] Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5- hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5- hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP), 5-methyl-2'-deoxycytidine monophosphate, 5-methyl-2'-deoxycytidine diphosphate, 5-methyl-2'-deoxycytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine diphosphate and 5-hydroxymethyl-2'- deoxycytidine triphosphate. The nucleotides may be selected from AMP, UMP, GMP, CMP, dAMP, dTMP, dGMP or dCMP. In some embodiments, the nucleotides are selected from dAMP, dTMP, dGMP or dCMP.

[0366] The nucleotides may contain additional modifications. In particular, suitable modified nucleotides include, but are not limited to, 2'amino pyrimidines (such as 2'-amino cytidine and 2'-amino uridine), 2'-hyrdroxyl purines (such as , 2'-fluoro pyrimidines (such as 2'- fluorocytidine and 2'fluoro uridine), hydroxyl pyrimidines (such as 5'-a-P-borano uridine), 2'-0-methyl nucleotides (such as 2'-O-methyl adenosine, 2'-O-methyl guanosine, 2'-O- methyl cytidine and 2'-O-methyl uridine), 4'-thio pyrimidines (such as 4'-thio uridine and 4'- thio cytidine) and nucleotides have modifications of the nucleobase (such as 5-pentynyl-2'- deoxy uridine, 5-(3-aminopropyl)-uridine and l,6-diaminohexyl-N-5-carbamoylmethyl uridine).

[0367] One or more nucleotides in the polynucleotides may be modified, for instance with a label or a tag. The label may be any suitable label which allows the polynucleotides to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g.125I,35S, enzymes, antibodies, antigens, other polynucleotides and ligands such as biotin.

[0368] The nucleotides in the polynucleotides may be attached to each other in any manner. The nucleotides may be linked by phosphate, 2'0-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate linkages. The nucleotides are typically attached by their sugar and phosphate groups. The nucleotides may be connected via their nucleobases as in pyrimidine dimers.

[0369] The polynucleotide may comprise a deoxyribonucleic acid (DNA) or a ribonucleic acid (RIMA). The polynucleotide may be any synthetic polynucleotide known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), morpholino nucleic acid or other synthetic polymers with nucleotide side chains. The polynucleotide may comprise any of the nucleotides discussed above, including the modified nucleotides.

[0370] Unless otherwise indicated, a particular polynucleotide also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a different base, mixed- base and / or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al., 1985, J. Biol. Chem. 260:2605-2608; and Rossolini et al., 1994, Mol. Cell. Probes 8:91-98).

[0371] Substitutions may be used for the practices of codon optimisation and codon wobble, both of which are known to those skilled in the art. Thus, it will be appreciated that codon- optimised and codon-wobbled polynucleotides are also envisaged. In an embodiment, the polynucleotides are codon-optimised for human expression.

[0372] The polynucleotides can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence. Direct chemical synthesis of polynucleotides can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., 1979, Meth. Enzymol. 68: 109; the diethylphosphoramidite method of Beaucage et al., 1981, Tetra. Lett., 22: 1859; and the solid support method of U.S. Pat. No. 4,458,066. Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H. A. Erlich (Ed.), Freeman Press, NY, N.Y., 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif, 1990; Mattila et al., 1991, Nucleic Acids Res. 19:967; and Eckert et al., 1991, PCR Methods and Applications 1: 17.

[0373] Vectors

[0374] The invention also provides a vector comprising the one or more polynucleotides of the invention, the polynucleotide of the invention or the two or more polynucleotides of the invention. The polynucleotide(s) may be any of those described above.

[0375] The vector is typically an expression vector. Various expression vectors can be employed. Both viral-based and non-viral expression vectors can be used in the invention. Non-viral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., 1997, Nat Genet. 15:345). For example, non-viral vectors useful for expression in mammalian (e.g., human) cells include pThioHis A, B and C, pcDNA3.1 / His, pEBVHis A, B and C, (Invitrogen, San Diego, Calif.), MPS V vectors, and numerous other vectors known in the art for expressing other proteins and / or nucleotide sequences. The non-viral expression vector may be a pET E. coli expression vector. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al., supra; Smith, 1995, Annu. Rev. Microbiol. 49:807; and Rosenfeld et al., 1992, Cell 68: 143.

[0376] Preferably, the vector is a retroviral, lentiviral, adenoviral, or adeno-associated viral vector. The vector is preferably a retroviral or lentiviral vector. The vector is preferably a lentiviral vector.

[0377] Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen, et al., 1986, Immunol. Rev. 89:49-68), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell typespecific, stage-specific, and / or modulatable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPS V promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, the SSFV promoter, the EFl alpha promoter, the phosphoglycerate kinase (PGK) promoter and promoter-enhancer combinations known in the art.

[0378] Methods of producing conjugates or constructs

[0379] The invention also provides a method of producing a protein conjugate of the invention. The method may comprise conjugating (a) one or more antibodies or variants thereof which specifically bind to one or more antigens on the surface of red blood cells (RBCs) to (b) one or more enzymes and / or variants thereof. The method may comprise conjugating (a) an antibody or a variant thereof which specifically binds to an antigen on the surface of red blood cells (RBCs) to (b) one or more enzymes and / or variants thereof. Any of the discussion above relating to components (a) and (b) and their conjugation equally apply to the method of the invention. The method may comprise producing components (a) and (b) separately by in vitro translation and transcription (IVTT) and then conjugating them to form the protein conjugate of the invention. The method may comprise producing the protein conjugate of the invention by IVTT. The method may comprise expressing components (a) and (b) separately and then conjugating them to form the protein conjugate of the invention. The method preferably comprises expressing the protein conjugate of the invention. The method preferably comprises expressing a polynucleotide encoding the protein conjugate of the invention or a vector comprising such a polynucleotide. The polynucleotide or vector are preferably expressed in a host cell. Suitable host cells are known in the art. The host cell may be mammalian, such as human, fungal, such as Saccharomyces cerevisiae, or bacterial, such as E. coli.

[0380] The invention also provides a method of producing an oligomeric construct of the invention. The method may comprise contacting two or more conjugates of the invention under conditions which allow them to oligomerise. Any of the discussion above relating to protein conjugates and oligomeric constructs of the invention equally apply to the method of the invention. The skilled person will recognise suitable conditions allowing oligomerisation. The method may comprise producing the two or more protein conjugates separately by in vitro translation and transcription (IVTT) and then contacting them under conditions which allow them to form the oligomeric construct of the invention. The method may comprise expressing the two or more protein conjugates separately and then contacting them under conditions which allow them to form the oligomeric construct of the invention. The method preferably comprises expressing one or more polynucleotides encoding the oligomeric construct of the invention or a vector comprising such one or more polynucleotides. The polynucleotide or vector are preferably expressed in a host cell. Suitable host cells are known in the art. The host cell may be mammalian, such as human, fungal, such as Saccharomyces cerevisiae, or bacterial, such as E. coli.

[0381] Population of RBCs

[0382] The invention also provides a population of red blood cells (RBCs) surface labelled with a protein conjugate of the invention and / or an oligomeric construct of the invention. The protein conjugate and / or the oligomeric construct may be any of those described above.

[0383] The RBCs are typically surface labelled by specific binding of the one or more antibodies or variants thereof to the one or more antigens on the surface of the RBCs of the antibody or a variant thereof to the antigen on the surface of the RBCs. The one or more antigens or antigen may be any of those described above.

[0384] The protein conjugate(s) and / or the oligomer conjugate is / are preferably non-covalently attached to the RBCs. The non-covalent attachment is typically via normal antibody / antigen interactions. One advantage of using an IH4 nanobody or a variant thereof is its affinity provides longer lasting stability than other antibody / antigen interactions even via the non- covalent approach.

[0385] The protein conjugate(s) and / or the oligomer conjugate is / are preferably covalently attached to the RBCs. The covalent attachment may be achieved using any of the approaches discussed above.

[0386] The one or more enzymes and / or variants thereof in the protein conjugate(s) and / or oligomeric conjugate typically remain active on the surface of the RBCs. This can be measured using routine methods including those described in the Examples. The activity of the one or more enzymes and / or variants thereof may be substantially the same or about the same as in protein conjugate(s) and / or oligomeric construct in the absence of the RBCs. The functional activity may be increased. The activity on the surface of the RBCs may be increased by any amount, such as by at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 90%, at least about a factor of 2, such as least about a factor of 5, at least about a factor of 10, at least about a factor of 50, at least about a factor of 100, at least about a factor of 500, as least about a factor of 1000, at least about a factor of 5000, at least about a factor of 10,000 or more. The activity may be decreased. The activity on the surface of the RBCs may be decreased by any amount, such as by less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2% or less than about 1%.

[0387] The protein conjugate(s) and / or the oligomer conjugate may affect the RBC deformability distribution (RBC-DD) of the population. The protein conjugate(s) and / or the oligomer conjugate may increase the RBC deformability distribution (RBC-DD) of the population. The protein conjugate(s) and / or the oligomer conjugate may decrease the RBC deformability distribution (RBC-DD) of the population. The protein conjugate(s) and / or the oligomer conjugate may not significantly affect the RBC deformability distribution (RBC-DD) of the population. The skilled person can identify circumstances when is advantageous to change the deformability of the RBCs or not. The RBC-DD can be measured using routine methods including as described in the Examples. The effect of the protein conjugate(s) and / or the oligomer conjugate can be determined by measuring RBC-DD of the RBCs before and after they are surface labelled.

[0388] The population preferably comprises at least about lxlO5, at least about lxlO6, at least about lxlO7, at least about lxlO8, at least about lxlO9, or at least about lxlO10RBCs. The population preferably comprises at least about lxlO8, at least about lxlO9, at least about lxlO10, at least about lxlO11, at least about lxlO12, at least about lxlO13, or at least about lxlO14RBCs.

[0389] The population may comprise other cell types. The identity of the other cell types is not particularly limited. They may be a single cell type or can be a mixture of different cell types. They are typically one or more of the types of cells used to produce or engineer the RBCs.

[0390] The population typically comprises or consists of mammalian RBCs. The RBCs may be human, dog, cat, primate, horse, murine, rat, rodent, bovine, murine, porcine, or ovine RBCs. The population is preferably a population of human RBCs. The RBCs may be wildtype. The RBCs may be genetically modified.

[0391] The population may be a fraction of whole blood. A skilled person is able to tell whether or not a population is a fraction of whole blood using standard means in the art. For instance, the skilled person can perform assays on the population to detect markers of whole blood, such as compounds found in whole blood. The RBCs may be isolated from whole blood. The population may still comprise other cells present in whole blood.

[0392] The population may be autologous. In other words, the population may be derived from the subject into which the cell will be administered. The population is preferably allogeneic. In other words, the population is preferably derived from a subject that is immunologically compatible with the subject into which the population will be administered. The population is preferably matched to the subject. The administration of autologous or allogeneic cells to subjects is well documented.

[0393] The population is preferably produced by cell culture. The population is preferably derived from a population of erythroid progenitor cells. The erythroid progenitor cells can comprise any type of cells that are capable of differentiated and maturing into RBCs. The term erythroid progenitor cell" or "erythroid progenitor" can be used to refer to cells at different stages along the differentiation / maturation pathway. The erythroid progenitor cells can be stem cells, haematopoietic stem cells, induced pluripotent stem cells (iPSCs), erythroid immortalized cell lines, erythroblast cells or reticulocyte cells. Preferably the erythroid progenitors are CD34+ cells, and / or BEL-A cells. For instance, the erythroid progenitor cells can be CD34+ cells derived from stem cells or iPSCs. Where the erythroid progenitor cells are non-CD34+ stem cells, iPSCs or immortalised cells, the cell type or lineage can produce erythroblasts which then differentiate to RBCs.

[0394] The population typically comprises or consists of intact RBCs. "Intact cells" are generally cells that are viable. In other words, they are cells that have not been ruptured. This can be measured using routine methods. For example, the cells can be assessed on a flow cytometer, for instance as described in the Examples.

[0395] The RBCs preferably further comprise, contain or display a therapeutic protein or polypeptide. The therapeutic protein or polypeptide is not typically present in wild-type RBCs. A "therapeutic protein or polypeptide" is a protein or polypeptide with a useful therapeutic function. In other words, the RBCs can be used as a carrier of therapeutic proteins or polypeptides. An advantage of this is that, once inserted into the circulatory system of the body, the RBCs can transport the therapeutic protein or polypeptide around the entire circulatory system providing the therapeutic protein or polypeptide with rapid and systemic distribution around the body of the subject. In addition, the RBCs can protect the therapeutic protein or polypeptide from the body's metabolic or defensive processes, preventing the therapeutic protein or polypeptide from being degraded. In addition, for a therapeutic protein or polypeptide that is intracellular within the RBCs, the reticulocyte can protect against the body launching an immune response.

[0396] The RBCs may comprise, contain or display a therapeutic protein or polypeptide comprising a sequence that is not naturally present in wild-type RBCs. The therapeutic protein or polypeptide may an exogenous protein or polypeptide. "Exogenous" refers proteins or polypeptides which are not naturally expressed in the RBCs. This includes proteins or polypeptides which comprise one or more amino acid mutations with respect to an endogenous protein. Exogenous can also cover isoforms from other cells or species, or non- naturally occurring proteins or polypeptides, such as hybrid proteins, chimeric proteins, fusion proteins or de novo protein or polypeptide sequences.

[0397] The therapeutic protein or polypeptide can be a variant of an endogenous protein or polypeptide. The therapeutic protein or polypeptide can be an exogenous protein or polypeptide or a variant of an endogenous protein or polypeptide. The RBCs may overexpress the therapeutic protein or polypeptide. The therapeutic protein or polypeptide is typically able to exhibit a benefit, such as a therapeutic benefit, in a subject. The therapeutic protein or polypeptide is typically not a haem-containing protein, such as haemoglobin or methaemoglobin. The therapeutic protein or polypeptide is typically intracellular within the RBCs. The therapeutic protein or polypeptide may be displayed on the surfaces of the RBCs.

[0398] One challenge faced with expression of a therapeutic protein or polypeptide is that the therapeutic protein or polypeptide is expelled or degraded during the enucleation process that leads to the RBCs. Methods for suppressing this reduction in therapeutic protein or polypeptide are known in the art and have been outlined in WO 2021 / 053243, the entirety of which is incorporated by reference herein. In brief, ubiquitination was identified as an important method by which protein and polypeptide concentrations are reduced during enucleation. In some embodiments, ubiquitination can be inhibited through use of a ubiquitinase inhibitor. Ubiquitinase inhibitors are non-specific and can have deleterious effects beyond simply retaining the therapeutic protein or polypeptide. It has been reported that ubiquitination is an essential part of the erythroid cell maturation process (Nguyen, A.T., et al., Science, 2017, 357(6350)). As such, global inhibition of ubiquitination (using ubiquitinase inhibitors such as MG132) means many non-erythroid proteins would be retained in the developing reticulocyte or erythrocyte. As a result, many aspects of cell function would be disrupted which can adversely impact important aspects of the cell such as lifetime and immune system compatibility. In certain cases, broad ubiquitination inhibition may prevent differentiation altogether and / or lead to cell death.

[0399] In some embodiments, the therapeutic protein or polypeptide is configured such that ubiquitination of the therapeutic protein or polypeptide is hindered or prevented. As such, the inhibition occurs locally at the therapeutic protein or polypeptide rather than by blanket inhibition of ubiquitination. This is a much more targeted technique for preventing or hindering ubiquitin-mediated degradation of the therapeutic protein or polypeptide.

[0400] The population of RBCs of the invention may be present in any of the pharmaceutically or physiologically acceptable diluents and / or carriers discussed below.

[0401] Making surface labelled RBCs

[0402] The invention also provides a method of producing a population of RBCs of the invention. The method comprises contacting the RBCs with a protein conjugate of the invention and / or an oligomeric construct of the invention such that the RBCs are surface labelled with the protein conjugate of the invention and / or oligomeric construct of the invention. Any of the embodiments discussed above with reference to the population of the invention, the protein conjugate of the invention, and the oligomeric construct of the invention equally apply to the method of the invention. The surface labelling may occur instantaneously when the RBCs are contacted with a protein conjugate of the invention and / or an oligomeric construct of the invention. The method may comprise contacting the components for less than about 5 minutes.

[0403] The method may comprise contacting the RBCs with a protein conjugate of the invention and / or an oligomeric construct of the invention and incubating them such that the RBCs are surface labelled with the protein conjugate of the invention and / or oligomeric construct of the invention. Incubating the components may increase the surface labelling. Incubation may be for at least about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours or about 4 hours. Incubation may be for longer, for example up to about 12 hours, about 16 hours, about 24 hours, about 48 hours or about 72 hours.

[0404] The total incubation may be for at least about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 12 hours, about 16 hours, about 24 hours, about 48 hours or about 72 hours.

[0405] The contacting and / or incubation may be at a temperature of at least about 5°C, about 10°C, about 15°C, about 30°C, about 35°C, about 37°C, about 40°C, or at least about 45°C. Incubation may be at a temperature of about 37°C.

[0406] The contacting and / or incubation is typically in a buffered aqueous solution, for example a salt solution such as phosphate buffered saline (PBS). Incubation may be at a pH of about pH 6-9, for example about pH 6, 6.5, 7, 7.5, 8, 8.5 or about 9. In some embodiments, the contacting and / or incubation is at a pH of from about pH 5 to about pH 9. In some embodiments, the contacting and / or incubation is at a pH of about 7.

[0407] The method may be an in vitro method. The method may be an ex vivo method. For instance, the RBCs may be isolated from a subject, surface labelled in accordance with the invention and then reintroduced into the subject. The method may be an in vivo method. For instance, administration of the protein conjugate of the invention and / or oligomeric construct of the invention to a subject may surface label the RBCs in the subject's circulatory system.

[0408] Pharmaceutical compositions

[0409] The invention also provides a pharmaceutical composition comprising one or more protein conjugates of the invention, an oligomeric construct of the invention, a population of RBCs of the invention, one or more polynucleotides of the invention or a vector of the invention. The pharmaceutical composition also comprises a pharmaceutically acceptable diluent and / or carrier. The one or more protein conjugates of the invention, the oligomeric construct of the invention, the population of RBCs of the invention, the one or more polynucleotides of the invention or the vector of the invention may be any of those discussed above. The pharmaceutical composition may comprise any number of one or more protein conjugates of the invention, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more or 12 or more. If there are two or more protein conjugates of the invention, they are typically different.

[0410] The carrier and / or diluent is generally selected to be suitable for the intended mode of administration and can include agents for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, colour, isotonicity, odour, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Typically, these carriers and / or diluents include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and / or buffered media.

[0411] Suitable further agents for inclusion in the pharmaceutical compositions include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulphite, or sodium hydrogensulphite), buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as free serum albumin, gelatin, or immunoglobulins), colouring, flavouring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as Polysorbate 20 or Polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides, such as sodium or potassium chloride, or mannitol sorbitol), delivery vehicles, excipients and / or pharmaceutical adjuvants.

[0412] The carrier and / or diluent may be a parenteral, optionally intravenous vehicle. Suitable parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates may be included. Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. In some cases, one might include agents to adjust tonicity of the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in a pharmaceutical composition. For example, in many cases it is desirable that the composition is substantially isotonic. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, may also be present. The precise formulation will depend on the route of administration. Additional relevant principle, methods and components for pharmaceutical formulations are well known (see, e.g., Allen, Loyd V. Ed, (2012) Remington's Pharmaceutical Sciences, 22ndEdition).

[0413] A pharmaceutical composition of the invention can be administered by 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 person, the route and / or mode of administration will vary depending upon the desired results. Routes of administration for pharmaceutical compositions of the invention include intravenous, intramuscular, intradermal, intraperitoneal, intrapleural, subcutaneous, spinal, or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means 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, intrapleural and intra-sternal injection and infusion. In some embodiments, the pharmaceutical composition is administered intratumourally. In other embodiments, administration is intrapleural or intraperitoneal. When parenteral administration is contemplated, the pharmaceutical compositions are usually in the form of a sterile, pyrogen-free, parenterally acceptable composition. A particularly suitable vehicle for parenteral injection is a sterile, isotonic solution, properly preserved. The pharmaceutical composition can be in the form of a lyophilizate, such as a lyophilized cake.

[0414] Alternatively, the pharmaceutical composition of the invention can be administered by a nonparenteral route, such as a topical, epidermal, or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.

[0415] In some embodiments, the pharmaceutical composition is for subcutaneous administration. Typically, the pharmaceutical compositions for subcutaneous administration contain suitable stabilizers (e.g., amino acids, such as methionine, and or saccharides such as sucrose), buffering agents and tonicifying agents.

[0416] Alternatively, the pharmaceutical composition may be for intravenous administration.

[0417] Delivery methods

[0418] The invention also provides a method of delivering one or more enzymes and / or variants thereof to a target cell or tissue. The one or more enzymes and / or variants may be any of those discussed above. The method comprises contacting the cell or tissue with one or more protein conjugates of the invention, an oligomeric construct of the invention, a population of RBCs of the invention, one or more polynucleotides of the invention, a vector of the invention or a pharmaceutical composition of the invention. The one or more protein conjugates of the invention, the oligomeric construct of the invention, the population of RBCs of the invention, the one or more polynucleotides of the invention, the vector of the invention or the pharmaceutical composition of the invention may be any of those discussed above. The method may comprise contacting the cell or tissue with any number of one or more protein conjugates of the invention, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more or 12 or more. If there are two or more protein conjugates of the invention, they are typically different.

[0419] The cell or tissue may be any cell or tissue. The cell or tissue may be present in a subject. The cell or tissue may be associated with a disease or disorder. The disease or disorder may be any of those discussed below.

[0420] Therapeutic methods

[0421] The invention also provides a method of treating or preventing a disease or disorder in a subject. The subject is typically in need thereof. The subject may be a mammal. Optionally, the subject is a human, horse, dog, or cat. The subject is preferably human.

[0422] The method comprises administering to the subject one or more protein conjugates of the invention, an oligomeric construct of the invention, a population of RBCs of the invention, one or more polynucleotides of the invention, a vector of the invention or a pharmaceutical composition of the invention.

[0423] The invention also provides one or more protein conjugates of the invention, an oligomeric construct of the invention, a population of RBCs of the invention, one or more polynucleotides of the invention, a vector of the invention or a pharmaceutical composition of the invention for use in therapy. The invention also provides one or more protein conjugates of the invention, an oligomeric construct of the invention, a population of RBCs of the invention, one or more polynucleotides of the invention, a vector of the invention or a pharmaceutical composition of the invention for use in a method of treating or preventing a disease or disorder in a subject.

[0424] The invention also provides the use of one or more protein conjugates of the invention, an oligomeric construct of the invention, a population of RBCs of the invention, one or more polynucleotides of the invention, a vector of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for treating or preventing a disease or disorder.

[0425] The one or more protein conjugates of the invention, the oligomeric construct of the invention, the population of RBCs of the invention, the one or more polynucleotides of the invention, the vector of the invention or the pharmaceutical composition of the invention may be any of those discussed above. The therapy may comprise any number of one or more protein conjugates of the invention, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more or 12 or more. If there are two or more protein conjugates of the invention, they are typically different.

[0426] As explained above, one key advantage of the invention is the one or more protein conjugates of the invention or oligomeric construct of the invention can be administered to the subject such that the subject's RBCs are surface labelled.

[0427] The invention may be used to treat or prevent any disease or disorder. The disease or disorder is preferably associated with a reduced function or absence of the enzyme. In this context, the "enzyme" is one of the one or more enzymes and / or variants thereof in the one or more protein conjugates or the oligomeric construct. The reduced function may be a reduced activity and / or reduced amount of the enzyme. The skilled person can determine using routine techniques if a disease or disorder is associated with a reduced function or absence of the enzyme. Diseases or disorders associated with reduced enzyme function are also known in the art.

[0428] The disease or disorder may be a metabolic disease or disorder, an infectious disease or disorder, an autoimmune disease or disorder, an immune deficiency disease or disorder, a cardiovascular disease or disorder or cancer.

[0429] The metabolic disease or disorder may be mitochondrial neurogastrointestinal encephalopathy (MNGIE), Hypercholesterolemia (familial hypercholesterolemia), Gaucher disease, Fabry disease, Pompe disease (Glycogen Storage Disease Type II), Hurler syndrome (Mucopolysaccharidosis Type I), Hunter syndrome (Mucopolysaccharidosis Type II), Krabbe disease, maple syrup urine disease (MSUD), metachromatic leukodystrophy, mitochondrial encephalopathy lactic acidosis stroke-like episodes (MELAS), Niemann-Pick disease, phenylketonuria (PKU), porphyria, Tay-Sachs disease, Homocystinuria, Methylmalonic Acidemia, Propionic Acidemia, Galactosemia, Fructose Intolerance, Glycogen Storage Disease Type I (Von Gierke Disease), Glycogen Storage Disease Type III (Cori Disease), Biotinidase Deficiency, Citrullinemia Type I, Pyruvate Dehydrogenase Complex Deficiency, Isovaleric Acidemia, Glutaric Acidemia Type I, Cystinosis, or Wilson's disease. The Niemann-Pick disease may be type A and / or B.

[0430] The infectious disease or disorder may be caused by any pathogenic agent. The pathogenic agent may be a bacterium, an archaeon, a fungus, or a virus.

[0431] The bacterium may be Gram negative or Gram positive. The Gram-positive bacterium is preferably from the genus Bacillus, Clostridium, Enterococcus, Mycobacterium, Staphylococcus or Streptococcus. The Gram-positive bacterium may be from the genus Pasteurella or Nocardia.

[0432] The Gram-negative bacterium is preferably from the genus Aggregatibacter, Bacteroides, Bartonella, Brucella, Campylobacter, Chylamidia, Enterbacter, Francisella, Haemophilus, Heliobacter, Klebsiella, Legionella, Moraxella, Neisseria, Porphyromonas, Pseudomonas, Salmonella, Serratia, Stenotrophomonas, Vibrio or Yersinia. The Gram-negative bacterium may be from the genus Escherichia or Pseudomonas.

[0433] The bacterium may be from the genus Borrelia, Chlamydophila, Listeria, Mycoplasma, Proteus, or Treponema. The bacterium is preferably Aggregatibacter actinomycetemcomitans, Bacillus anthracis, Bacillus licheniformis, Bacteroides fragilis, Bartonella henselae, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Campylobacter jejuni, Chlamydia trachomatis, Chlamydophila pneumoniae, Clostridium difficile, Clostridium perfringens, Enterobacter aerogenes, Enterococcus faecalis, Enterococcus faecium, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella oxytoca, Legionella pneumophila, Listeria monocytogenes, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium bovis, Mycoplasma genitalium, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Porphyromonas gingivalis, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enter ica, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Stenotrophomonas maltophilia, Streptococcus mutans, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Treponema pallidum, Vibrio cholera, Vibrio parahaemolyticus or Yersinia enterocolitica.

[0434] Other specific examples of bacteria include, but are not limited, to Mycobacterium tuberculosis, Mycobacterium intracellilare, Mycobacterium kansaii, Mycobacterium gordonae, Streptococcus agalactiae, Streptococcus viridans group, Streptococcus faecalis, Streptococcus bovis, Streptococcus pneumoniae, Corynebacterium diptheriae, Erysipelothrix rhusiopathie, Clostridium tetani, Klebsiella pneumoniae, Pasteurella multocida, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pertenue and Actinomyces israelii.

[0435] The fungus is preferably from the genus Absidia, Acremonium, Aspergillus, Aureobasidium, Basidiobolus, Blastomyces, Blastoschizomyces, Candida, Cladosporium, Coccidioides, Cryptococcus, Cunninghamella, Curvularia, Debaryomyces, Exophiala, Exserohilum, Fonsecea, Fusarium, Geotrichum, Histoplasma, Issatchenkia, Kluyveromyces, Malezzesia, Mucor, Paracoccidioides, Paecilomyces, Penicillium, Pichia, Pneumocystis, Rhizomucor, Rhizopus, Rhodotorula, Saccharomyces, Scedosporium, Schizophyllum, Scopulariopsis, Sporothrix, Trichoderma, Trichophyton or Trichosporon. The fungus is preferably Aspergillus fumigatus, Aspergillus flavus, Aspergillus lentulus, Aspergillus terreus, Aspergillus nidulans, Aspergillus oryzae, Aspergillus niger, Candida albicans, Candida caribbica ( Candida fermentati), Candida dubliniensis, Candida famata (Debaryomyces hansenii), Candida fukuyamaensis (Candida xestobii or Candida carpophila), Candida guilliermondii, Candida kefyr (Kluyveromyces marxianus), Candida krusei (Issatchenkia orientalis), Candida metapsilosis, Candida orthopsilosis, Candida parapsilosis, Candida parapsilosis, Candida pelliculosa, Candida psychrophila, Candida rugosa, Candida smithsonii, Candida tropicalis, Candida utilis, Coccidioides immitis , Cryptococcus bacillisporus, Cryptococcus gattii, Cryptococcus grubii, Cryptococcus neoformans, Debaryomyces coudertii, Debaryomyces maramus, Debaryomyces nepalensis, Debaryomyces prosopidis, Debaryomyces robertsiae, Debaryomyces udenii, Histoplasma capsulatum, Kluyveromyces lactis, Pichia cecembensis, Rhodotorula araucariae, Rhodotorula babjevae, Rhodotorula dairensis, Rhodotorula diobovatum, Rhodotorula glutinis, Rhodotorula kratochvilovae, Rhodotorula paludigenum, Rhodotorula sphaerocarpum, Rhodotorula toruloides, Rhodotorula mucliaginosa, Saccharomyces 'sensu stricto', Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces cariocanus, Saccharomyces kudiavzevii, Saccharomyces mikatae, Saccharomyces paradioxus, Saccharomyces pastorianus, Saccharomyces uvarum, Saccharomyces cerevisiae or Tsuchiyaea wingfieldii.

[0436] The virus may belong to the family Retroviridae, such as human deficiency viruses, such as HIV-I (also referred to as HTLV- III), HIV-II, LAC, IDLV-III / LAV, HIV-III or other isolates such as HIV-LP, the family Picornaviridae, such as poliovirus, hepatitis A, enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses, the family Calciviridae, such as viruses that cause gastroenteritis, the family Togaviridae, such as equine encephalitis viruses and rubella viruses, the family Flaviviridae, such as dengue viruses, encephalitis viruses and yellow fever viruses, the family Coronaviridae, such as coronaviruses, including SARS-Cov-2 (COVID-19), the family Rhabdoviridae, such as vesicular stomata viruses and rabies viruses, the family Filoviridae, such as Ebola viruses, the family Paramyxoviridae, such as parainfluenza viruses, mumps viruses, measles virus and respiratory syncytial virus, the family Orthomyxoviridae, such as influenza viruses, the family Bungaviridae, such as Hataan viruses, bunga viruses, phleoboviruses and Nairo viruses, the family Arena viridae, such as hemorrhagic fever viruses, the family Reoviridae, such as reoviruses, orbiviruses and rotaviruses, the family Bimaviridae, the family Hepadnaviridae, such as hepatitis B virus, the family Parvoviridae, such as parvoviruses, the Papovaviridae, such as papilloma viruses and polyoma viruses, the family Adenoviridae, such as adenoviruses, the family Herpesviridae, such as herpes simplex virus (HSV) I and II, varicella zoster virus and pox viruses, or the family Iridoviridae, such as African swine fever virus). The virus may be an unclassified virus, such as the etiologic agents of Spongiform encephalopathies, the agent of delta hepatitis, the agents of non-A, non-B hepatitis (class 1 enterally transmitted; class 2 parenterally transmitted such as Hepatitis C), Norwalk and related viruses and astroviruses. The autoimmune disease or disorder may be psoriatic arthritis, rheumatoid arthritis (RA), Sjogren's syndrome, systemic lupus erythematosus (Lupus, SLE), Crohn's disease, celiac disease, ulcerative colitis, Graves' disease, Hashimoto's thyroiditis, Addison's disease, dermatomyositis, psoriasis, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome, multiple sclerosis (MS), myasthenia gravis, autoimmune vasculitis, type 1 diabetes, pernicious anaemia or vasculitis.

[0437] The immunodeficiency disease or disorder may be Autoimmune Lymphoproliferative Syndrome (ALPS), APS-1 (APECED), CARD9, Chronic Granulomatous Disease (CGD), congenital neutropenia syndromes, Common Variable Immunodeficiency (CVID), CTLA4 deficiency, DOCK8 deficiency, glycosylation disorders with immunodeficiency, Hyper- Immunoglobulin E Syndromes (HIES), PI3 kinase disease, PLAID, Severe Combined Immunodeficiency (SCID), STAT3 Dominant-Negative Disease, WHIM Syndrome, X-Linked Agammaglobulinemia (XLA) or X-Linked Lymphoproliferative Disease (XLP).

[0438] The cardiovascular disease or disorder mat coronary artery disease, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, thromboembolic disease, venous thrombosis, including deep vein thrombosis, thrombotic microangiopathies, such as thrombocytopenic purpura (TPP), and other conditions that might benefit from the addition of ADAMT13.

[0439] The cancer may include, but not necessarily be limited to, a solid tumour cancer, a soft tissue tumour, a metastatic lesion, and a haematological cancer. For example, the cancer can be liver cancer, lung cancer, breast cancer, prostate cancer, lymphoid cancer, colon cancer, renal cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, such as squamous cell carcinoma of the head and neck (SCCHN), cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the oesophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukaemias including acute myeloid leukaemia, chronic myeloid leukaemia, acute lymphoblastic leukaemia, chronic lymphocytic leukaemia, solid tumours of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumour angiogenesis, spinal axis tumour, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, myelodysplastic syndrome (MDS), chronic myelogenous leukaemia-chronic phase (CMLCP), diffuse large B-cell lymphoma (DLBCL), cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), hepatocellular carcinoma (HCC), gastrointestinal stromal tumours (GIST), non-small cell lung carcinoma (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), environmentally induced cancers including those induced by asbestos, and combinations of said cancers. In embodiments, the cancer is selected from the above group.

[0440] The cancer may be a solid tumour cancer.

[0441] In some embodiments, the cancer is selected from the group consisting of cancer of the head and / or neck, ovarian cancer, malignant mesothelioma, breast cancer, pancreatic cancer, colorectal cancer, lung cancer, gastric cancer, bladder cancer, prostate cancer, oesophageal cancer, endometrial cancer, hepatobiliary cancer, duodenal carcinoma, thyroid carcinoma, cancer of the central nervous system or renal cell carcinoma.

[0442] In some embodiments, the cancer is selected from ovarian cancer, breast cancer, optionally triple-negative breast cancer, pancreatic cancer, malignant mesothelioma, and combinations of said cancers.

[0443] In some embodiments the cancer is breast cancer, ovarian cancer, melanoma or mesothelioma.

[0444] In some embodiments the cancer is ovarian cancer, breast cancer or combinations thereof.

[0445] The breast cancer may be triple negative breast cancer.

[0446] The subject may have been pre-treated with a chemotherapeutic agent.

[0447] The following table shows the one or more enzymes and / or variants thereof that may be used in specific diseases or disorders. The disease or disorder may be any of the diseases or disorders in the first column. For each disease or disorder, the one or more enzymes and / or variants thereof is preferably the enzyme in the same row or a variant thereof.

[0448] The disease or disorder may be (1) a hemostasis or thrombosis disease or disorder, (2) an oxidative stress, ischemia-reperfusion, or inflammation disease or disorder, (3) poisoning with xenobiotics or toxins, (4) cancer, including any of the cancers listed above, (5) a rare metabolic disease or disorder or a lysosomal disease or disorder, (6) a pulmonary disease or disorder, a neuroendocrine tumour or a mucus clearance disease or disorder, (7) an infectious disease or disorder or sepsis or (8) a cardiometabolic or vascular disease or disorder.

[0449] General therapeutic methods If a population of RBCs of the invention are administered, the RBCs may be autologous to the subject. The RBCs may be allogeneic to the subject or may be produced by cell culture from erythroid progenitor cells. The RBCs are preferably matched with the subject. The administration of the one or more protein conjugates of the invention, the oligomeric construct of the invention, the population of RBCs of the invention, the one or more polynucleotides of the invention, the vector of the invention or the pharmaceutical composition of the invention may reduce symptoms by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or even about 100%, when compared to an untreated subject. The administration may result in a decrease in tumour size of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or even about 100%, when compared to an untreated tumour.

[0450] The amount and / or number of the one or more protein conjugates, the oligomeric construct, the population of RBCs, the one or more polynucleotides, the vector or the pharmaceutical composition administered to the subject should take into account the route of administration, the disease or disorder being treated, the weight of the subject and / or the age of the subject. From about 1 mg / ml to about 100 mg / ml may be administered to the subject. In some embodiments, from about 1 mg / ml to about 10 mg / ml are administered to the subject. For RBCs, from about 1 x 106to about 1 x 1012RBCs may be administered to the subject. In one embodiment, from about 1 x 107to about 1 x 1010RBCs, or from about 1 x 108to about 1 x 109RBCs are administered to the subject.

[0451] The administration route may be any of those discussed above. In preferred embodiments, administration is conducted by intravenous injection.

[0452] The therapy or method typically comprises administering a therapeutically effective amount or a prophylactically effective amount of the one or more protein conjugates, the oligomeric construct, the population of RBCs, the one or more polynucleotides, the vector or the pharmaceutical composition. A therapeutically effective amount is an amount which ameliorates one or more symptoms, such as all the symptoms, of the disease or disorder and / or abolishes one or more symptoms, such as all the symptoms, of the disease or disorder. The therapeutically effective amount preferably cures the disease or disorder. A prophylactically effective amount is an amount which prevents the onset of the disease or disorder and / or prevents the onset of one or more symptoms, such as all the symptoms, of the disease or disorder. The prophylactically effective amount preferably prevents the subject from developing the disease or disorder. Suitable amounts are discussed in more detail below.

[0453] Any of the one or more protein conjugates, oligomeric constructs, populations of RBCs, one or more polynucleotides, vectors or pharmaceutical compositions may be administered to a subject that displays symptoms of disease or disorder. Any of the one or more protein conjugates, oligomeric constructs, populations of RBCs, one or more polynucleotides, vectors or pharmaceutical compositions may be administered to a subject that is asymptomatic, i.e., does not display symptoms of disease or disorder. Any of the one or more protein conjugates, oligomeric constructs, populations of RBCs, one or more polynucleotides, vectors or pharmaceutical compositions may be administered when the subject's disease status is unknown, or the subject is expected not to have a disease or disorder. Any of the one or more protein conjugates, oligomeric constructs, populations of RBCs, one or more polynucleotides, vectors or pharmaceutical compositions may be administered to a subject that is predisposed, such as genetically predisposed, to developing the disease or disorder.

[0454] The method or therapy may comprise one or more diagnostic tests to establish the presence or absence of the disease or disorder and / or for use in determining treatment options. Examples of suitable diagnostic tests include detection of specific mutations in cancer cells, detection of specific mutations associated with particular diseases, detection of viruses, and so forth.

[0455] In embodiments where the subject is human, the subject may be a human adult or child. In the context of the present invention, an adult will be understood to be an at least 18-year- old human. A child will be understood to be a human less than 18 years old. In some embodiments, the adult is at least 60 years old.

[0456] The invention may be used in combination with other means of, and substances for, treating or preventing the disease or disorder. In some cases, any of the one or more protein conjugates, oligomeric constructs, populations of RBCs, one or more polynucleotides, vectors or pharmaceutical compositions may be administered simultaneously, sequentially, or separately with other substances which are intended for treating or preventing the disease or disorder or ameliorating the symptoms of the disease or disorder, or for providing pain relief. Any of the one or more protein conjugates, oligomeric constructs, populations of RBCs, one or more polynucleotides, vectors or pharmaceutical compositions may be used in combination with existing treatments for disease or disorder and may, for example, be simply mixed with such treatments. Thus the invention may be used to increase the efficacy of existing treatments for disease.

[0457] Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Within the scope of this application it is expressly intended that the various aspects, embodiments, examples, and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination, unless such features are incompatible. SEQUENCE LISTING

[0458]

[0459]

[0460]

[0461]

[0462]

[0463] Ill

[0464] EXAMPLES

[0465] Example 1 : IH4-containincj fusion proteins, including dimeric therapeutic enzymes, can be expressed and purified from bacterial, for assembly on human RBCs.

[0466] The anti-GPA binding nanobody, IH4, was originally described in the US patent No. 9879090 and the following paper: Anal Biochem. 2013 Jul l;438(l):82-9. doi: 10.1016 / j.ab.2013.03.020. Epub 2013 Mar 26. IH4 specifically recognises human glycophorin A (GPA) on the surface of RBCs. This nanobody was isolated from a human blood-immunised dromedary library and binds to a conserved epitope, Y52PPE55, on the extracellular domain of GPA independently of blood group antigens, reacting with RBCs of all humans except possibly very rare individuals who do not express GPA. GPA is a major sialoglycoprotein on the surface of RBCs, present in approximately 1 million copies per cell, characterised by its heavily glycosylated extracellular domain and a single transmembrane domain.

[0467] The version of IH4 utilised in this invention was derived from the following paper: ACS Synth Biol. 2020 Feb 21;9(2): 191-197. doi: 10.1021 / acssynbio.9b00273. Epub 2019 Dec 19. It was modified to include a point mutation (Phe80Tyr) and an additional amino acid (Thrll8) in framework regions 3 and 4, respectively. These modifications were to correct potential unintentional changes from the germline sequence or transcriptional errors. Its high affinity and specificity make IH4 an ideal candidate for use in fusion proteins to attach therapeutic enzymes to the RBC surface, ensuring prolonged circulation time of therapeutic enzymes in the bloodstream. Furthermore, previous research has demonstrated that although certain anti-GPA antibodies can induce RBC membrane rigidification, IH4 does not significantly affect red blood cell deformability.

[0468] IH4-containing fusion proteins, notably featuring the novel aspect of fusing dimeric therapeutic enzymes to both sides of the nanobody, can be expressed and purified from bacteria. This capability allows for the attachment of multiple enzymes at both termini, facilitating synergistic effects and combinations of enzymes for broader therapeutic applications.

[0469] As proof-of-concept for creating IH4 fusion proteins in this Example, five different IH4- containing proteins were constructed for use in this invention :

[0470] • IH4

[0471] • TP-IH4

[0472] • IH4-TP

[0473] • IH4-GFP

[0474] • IH4-SC003

[0475] IH4 alone was made for characterisation comparison of IH4-containing fusion proteins. IH4 was also prepared fused to dimeric thymidine phosphorylase (TP), monomeric superfolder GFP (GFP) and SpyCatcher003 (SC003).

[0476] IH4 was fused to TP to serve as a model dimeric therapeutic enzyme aimed at treating mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a rare and fatal metabolic disorder, characterised by severe neurological and gastrointestinal issues. Current enzyme replacement therapy for MNGIE utilise E. coli TP (ecTP) encapsulated inside RBCs (EE-ecTP) to ensure prolonged plasma persistence and minimise immunogenicity. However, the use of bacterial enzymes in human therapies poses safety and efficacy concerns due to their recognition by the patient's immune system as foreign, potentially eliciting adverse immune responses and the production of clearing anti-drug antibodies. To address these issues, we aimed to use the human ortholog, expected to be a safer and more advantageous alternative.

[0477] While expressing therapeutic enzymes in E. coli is desirable due to low costs and high protein yields, several reports indicate that full-length hsTP expresses very poorly in E. coli. The DNA sequence for hsTP used in this invention was derived from the hsTP218construct as described in the following paper: Front Bioeng Biotechnol. 2021 Dec 17:9:793985. doi: 10.3389 / fbioe.2021.793985. eCollection 2021. The hsTP218construct omits the entire unstructured N-terminal region, comprising the first thirty-three amino acid residues of the full-length hsTP. Structurally, this truncated version resembles the respective N-terminus of ecTP. The paper demonstrated that N-terminal truncations of the human enzyme significantly improve its recombinant expression. Among the various truncations tested, the hsTP218construct was found to yield large amounts of soluble protein when expressed at low temperatures (16 and 22°C) and low ITPG concentrations (0.1 mM). Given these findings, we chose to utilise the hsTP218construct in this invention, leveraging its improved expression and solubility characteristics to enhance the safety and efficacy of the therapeutic enzyme.

[0478] Two different versions of IH4 fused to TP were made with TP attached to either the N or C- terminal ends of IH4. In IH4-TP, TP is fused to the C-terminal end of IH4 and in TP-IH4, TP is fused to the N-terminal end of IH4. Figure 1 illustrates the predicted protein structure of dimeric IH4-TP (Figure 1A) and TP-IH4 (Figure IB) generated by AlphaFold3. The N- terminal end is close to the complementary determining (CDR) loop region and GPA binding site of the nanobody, while the C-terminal end is on the opposite site. This was to test how amenable each end of the nanobody was to fusion in terms of binding and affinity, giving scope for the generation of more complex fusion proteins in the future. TP is also a dimeric protein, and we wanted to test how well enzyme-nanobody fusion proteins worked with more complicated enzymes than a monomer, assessing if dimeric enzymes are still feasible and functioning.

[0479] IH4-GFP was designed to test IH4 fusions with an alternative protein to TP. GFP is monomeric, making it a suitable alternative to dimeric TP, and serves as an easy visual reporter to detect binding to RBCs. msGFP2, used in this invention, is a green fluorescent protein derived from Aequorea victoria and published in 2019. It retains the monomeric superfolder properties of its predecessor, msGFP, but with improved photostability comparable to EGFP. Modifications in the N- and C-terminal peptides of msGFP2 reduce nonspecific interactions, minimising disturbances to partner protein functions.

[0480] IH4-SC003 was used to test the modular assembly of nanobody-enzyme fusion proteins using the SpyCatcher003-SpyTag003 system. In this system SpyTag003 is fused to partner protein, in this case two test proteins: turboID (TID) and GFP. This approach addresses scenarios where fusion proteins of nanobody-enzyme are too large for efficient expression or when the enzyme requires an alternative expression system, such as mammalian cells instead of f. coll. Individual components can be expressed either as smaller fractions and in their optimal expression systems, and the final fusion protein can be assembled in vitro through SpyCatcher-SpyTag interaction. Method: The IH4-containing proteins were expressed as soluble proteins in commercially available SHuffle T7 Express cells, an engineered E. coil strain optimised for the efficient cytoplasmic expression of disulphide-bonded proteins. Expression involved inoculating lL of LB medium in a 2L flask with a 1 / 100 dilution of an overnight culture, followed by incubation at 30°C with shaking at 250 rpm. When OD600nm reached 0.6 to 0.8, protein production was induced by adding 0.1 mM IPTG, and the culture was incubated overnight at 20°C with shaking at 225rpm. Cell pellets were harvested by centrifugation at 6000xg for 20 mins at 4°C and resuspended in 30 ml of PBS containing 300 mM NaCI (PBS-NaCI) with an EDTA-free protease inhibitor cocktail tablet and a trace amount of DNAse. The mixture was stirred until homogenous. Cells were lysed by two passages through a French pressure cell at 1000 psi, followed by clarification by centrifugation at 39 OOOxg for 30 mins at 4°C. The supernatant, containing the cytoplasmic extract, was purified using a HisTrap HP nickel affinity column (5 ml bed volume) pre-equilibrated with 5 column volumes (CV) of PBS- NaCI. After loading the sample, the column was washed with 10 CV of PBS-NaCI and then with 5 CV of PBS-NaCI containing 10 mM imidazole. The protein was eluted with PBS-NaCI containing 500 mM imidazole. The eluted protein was further purified by size exclusion chromatography on a Superdex 200 column (high load 16 / 60; 120 ml bed volume) equilibrated with PBS using an AKTApurifier system. Fractions corresponding to correctly folded protein were pooled and concentrated using a Vivaspin 20 spin concentrator with a 10 kDa molecular weight cut-off. The final protein concentration was determined by absorbance spectroscopy. To date, every IH4-containing protein produced in SHuffle cells was expressed as soluble protein and in high yields of tens of mg / L.

[0481] Figures 2-4 follow the expression and purification of the various IH4-containing constructs described above.

[0482] Example 2: SpyTaQ-containincj fusion proteins can be expressed and purified in bacteria, for modular assembly of IH4-enzvme fusion proteins, via SpyTacj003-SpyCatcher003. SpyTag-containing fusion proteins can be expressed and purified in bacteria for the modular assembly of IH4-containing fusion proteins using IH4-SC003. To test this assembly, two exemplar proteins, GFP and TurboID (TID), were fused with ST003. SpyTag003 and SpyCatcher003 are engineered variants of the original SpyCatcher and SpyTag systems, designed to improve upon the properties of their predecessors Keeble AH, Turkki P, Stokes S, Khairil Anuar INA, Rahikainen R, Hytonen VP, Howarth M. Approaching infinite affinity through engineering of peptide-protein interaction. Proc Natl Acad Sci U S A. 2019 Dec 26;116(52):26523-26533. doi:10.1073 / pnas.1909653116. Epub 2019 Dec 10. PMID: 31822621; PMCID: PMC6936558.

[0483] ST003-sfGFP, available on Addgene (plasmid #133454) from the Mark Howarth lab, uses superfolder GFP, which serves as a visual reporter to detect binding to RBCs. TID is an engineered biotin ligase that rapidly and efficiently biotinylates proximal proteins in living cells, facilitating the identification and study of protein-protein interactions by tagging neighbouring proteins with biotin, allowing for their subsequent isolation and analysis. In the context of creating an IH4-TID fusion protein, TID serves as an alternative monomeric enzyme to TP, providing a tool to target and label proteins in close proximity to IH4, offering insights into the protein environment around GPA on RBCs.

[0484] Method: The ST003-containing proteins were expressed as soluble proteins in commercially available BL21(DE3) cells. For ST-GFP, expression involved inoculating IL of LB medium in a 2L flask with a 1 / 100 dilution of an overnight culture, followed by incubation at 37°C with shaking at 250 rpm. When OD600nm reached approximately 0.8, protein production was induced by adding 0.5 mM IPTG, and the culture was incubated at 37°C for 3-4 hours with shaking at 250 rpm. Cell pellets were harvested by centrifugation at 6000xg for 20 minutes at 4°C and resuspended in 30 ml of lysis buffer (50 mM Na2HPO4, 300 mM NaCI, and 10 mM imidazole, pH 8) with an EDTA-free protease inhibitor cocktail tablet and a trace amount of DNAse. The mixture was stirred until homogenous. Cells were lysed by two passages through a French pressure cell at 1000 psi, followed by clarification by centrifugation at 39,000xg for 30 minutes at 4°C. The supernatant, containing the cytoplasmic extract, was purified using a HisTrap HP nickel affinity column (5 ml bed volume) pre-equilibrated with lysis buffer. After loading the sample, the column was washed with 10 column volumes of 50 mM Na2HPO4, 300 mM NaCI, and 20 mM imidazole. The protein was eluted with 50 mM Na2HPO4, 300 mM NaCI, and 300 mM imidazole, pH 8. The eluted protein was further purified by size exclusion chromatography on a Superdex 200 column (high load 16 / 60; 120 ml bed volume) equilibrated with 20 mM Tris, pH 8, and 150 mM NaCI using an AKTApurifier system. Fractions corresponding to correctly folded protein were pooled and concentrated using a Vivaspin 20 spin concentrator with a 10 kDa molecular weight cut-off. The final protein concentration was determined by absorbance spectroscopy.

[0485] The expression and purification of the ST-TID construct were carried out similarly to the IH4-containing fusion proteins, with growth and expression at 37°C.

[0486] Figures 5 and 6 illustrate the purification of ST003-containing constructs.

[0487] Example 3: IH4-TP fusions retain binding capability to RBCs and specificity to GPA.

[0488] The IH4 nanobody is known to bind with high affinity to the extracellular domain of GPA on the surface of human RBCs. In these experiments, we aimed to determine whether this binding capability is retained when IH4 is fused with dimeric TP at either the N- or C- terminus. The binding of two different IH4-TP fusion proteins, IH4-TP and TP-IH4, to human RBCs was compared to that of IH4 alone using flow cytometry. Both fusion proteins demonstrated retention of binding to RBCs and maintained binding affinity, with estimated Kd values of 203nM for IH4-TP and 284 nM for TP-IH4, within the same range as IH4 alone (139 nM). The fusion proteins showed increased binding levels compared to IH4 alone, attributed to the dimeric nature of TP increasing avidity. Results are shown in Figure 7.

[0489] In all cases, saturating binding was achieved at protein concentrations between 0.5 and 1 pM.

[0490] Further experiments were conducted to confirm the specificity of binding to GPA, through using GPA knockout (KO) BEL-A (Bristol Erythroid Line-Adult) cell lines previously generated through CRISPR-Cas9-mediated gene editing. The flow cytometry histograms shown in Figure 8 illustrate that IH4-TP and TP-IH4 retain their binding specificity to the normal unedited BEL-A cells that express GPA, but there was no binding observed to GPA KO BEL- As.

[0491] Method: For flow cytometry, 0.2 xlO6cells were washed in PBSAG (PBS + 1 mg / ml BSA, 2 mg / ml glucose) and resuspended with purified IH4, IH4-TP or TP-IH4 at indicated dilutions in PBSAG. The suspension was incubated for 1 hour at RT, washed in PBSAG, and incubated for 30 minutes at 4°C with APC-conjugated anti-His antibody, then washed again. Data were acquired on a Miltenyi MACSQuant 10 flow cytometer. Controls with cells incubated with anti-His antibody alone were prepared similarly. For experiments using BEL-As, cells were maintained in expansion medium [StemSpan SFEM (Stem Cell Technologies) supplemented with 50 ng / ml SCF, 3 U / ml EPO, 1 pM dexamethasone and 1 pg / ml doxycycline] at 1-3 x 105cells / ml, with complete medium changes every 48 hours prior to the experiment.

[0492] Preliminary experiments were also conducted to evaluate the stability and attachment duration of TP-nanobody fusion proteins on RBCs using flow cytometry (Figure 9). These studies aimed to gauge whether nanobodies alone are sufficient for achieving long-lasting therapeutic attachment, potentially eliminating the need for further optimisation such as covalent attachment. We monitored the interaction of TP-IH4 with RBCs at a concentration of 1 pM, following previous binding protocols, and periodically assessed the presence of bound TP-IH4 by sampling, spinning down the cells, and detecting with an APC-conjugated anti-His antibody. These experiments were carried out in PBSAG at 4°C, room temperature, and 37°C over a period of up to 8 days.

[0493] Remarkably, TP-IH4 remained bound to RBCs for the entire 8-day period at 4°C, far exceeding our expectations and demonstrating potential for long-lasting non-covalent attachment. Even under more physiological temperatures, TP-IH4 showed a stable interaction for up to 3 days. While storage conditions in PBSAG are not ideal for RBC preservation, these results are highly encouraging as they suggest that nanobody-enzyme fusions can provide sustained therapeutic action without the need for covalent modification. Example 4: IH4-TP can be loaded onto RBC surfaces without affecting their deformability. Previous studies have demonstrated that the binding of the IH4 nanobody does not significantly affect RBC deformability, unlike several other anti-GPA antibodies, such as the mouse monoclonal antibodies R-10 and Brie 256, which are known to induce erythrocyte membrane rigidification. Therefore, in these experiments, we aimed to evaluate whether the binding of the dimeric IH4-TP fusion protein, which is significantly larger than IH4 alone and has the potential to bind multiple copies of GPA, alters RBC membrane rigidity. The goal was to determine and optimise the loading concentrations of dimeric constructs to achieve a balance between enhanced binding through dimerisation and the potential trade-off with cellular deformability.

[0494] To determine whether IH4-TP perturbs membrane rigidity, ARCA deformability measurements were performed on RBCs treated with either IH4 alone or IH4-TP, using the anti-GPA antibody Brie 256 as a positive control for negative effects on cell deformability. As shown in Figure 10, the addition of IH4 alone did not significantly affect red cell deformability at tested concentrations up to 50 pM, whereas the monoclonal antibody Brie 256 caused a notable reduction in cell deformability, consistent with previous reports.

[0495] Remarkably, and contrary to expectations, there is a certain amount of IH4-TP that can be loaded and tolerated by RBCs without affecting the deformability index, specifically at tested concentrations up to 100 nM. To confirm that IH4-TP was indeed binding to treated RBCs under these conditions, binding was verified by flow cytometry using an anti-His antibody (Figure 11). However, at concentrations of 500 nM and higher, binding of IH4-TP to RBCs resulted in a gradual and detectable reduction in the deformability index profile, indicating there is an optimal range at which the dimeric construct can be loaded onto RBCs without altering membrane rigidity.

[0496] Method: For these experiments, 2 xlO6cells were washed in PBSAG and resuspended with purified IH4 or IH4-TP at indicated dilutions in PBSAG. The suspension was incubated for 1 hour at RT and then washed in PBSAG. To confirm IH4 and IH4-TP had bound to treated RBCs under these conditions, 0.2 xlO6cells were incubated for 30 minutes at 4°C with APC- conjugated anti-His antibody and then washed again. Data were acquired on a Miltenyi MACSQuant 10 flow cytometer. Controls with cells incubated with anti-His antibody alone were prepared similarly. A total of 1.8 xlO6cells were resuspended in 200 pl of a polyvinylpyrrolidone solution (PVP viscosity 28.1) for assessment of cell deformability distributions. Samples were loaded onto an Automated Rheoscope and Cell Analyzer (ARCA) consisting of a plate-plate optical shearing stage mounted on a Linkam imaging station assembly and temperature controlled using Linksys32 software. The microscope was equipped with an LMPIanFL 50X with a 10.6 mm working distance objective illuminated by an X-1500 stroboscope through a band-pass interference filter. Images were acquired using a uEye camera. At least 2000 valid cells per sample were analysed using bespoke ARCA analysis software. Incubation with the monoclonal anti-GPA antibody, Brie 256, was used as a positive control for negative effects on cell deformability.

[0497] Example 5: Enzyme activity of IH4-TP is retained in solution and at the RBC surface.

[0498] In these experiments, we aimed to test the activity of our TP constructs fused with IH4 to ensure the enzyme retains its catalytically ability. IH4 was fused to TP to serve as a model dimeric therapeutic enzyme for treating MNGIE. TP is a homodimer that plays a crucial role in cellular nucleotide pool homeostasis by catalysing the phosphorolysis of thymidine to thymine and 2-deoxy-D-ribose 1-phosphate. In MNGIE patients, TP activity is drastically reduced due to mutations in the TYMP gene, leading to thymidine accumulation and severe metabolic consequences. Ensuring that our TP-IH4 fusion proteins retain catalytic activity is critical for their potential therapeutic application in correcting thymidine imbalances in MNGIE patients. Given that TP functions as a homodimer, it was essential to verify that the ability to assemble into the active dimeric form remained in the fusion proteins.

[0499] Endpoint thymidine phosphorylase activity assays were performed for TP alone and our two IH4-TP fusion constructs, and the conversion of thymidine to thymine was monitored spectroscopically, as shown in Figure 12. Proteins were added to reaction buffer, thymidine was added, and following incubation and termination, absorbance measurements at 300 nm were used to determine the amount of thymine formed. This assay allowed us to compare the activity of our TP-IH4 fusion proteins to TP alone, ensuring that the fusion constructs maintained their intended catalytic function.

[0500] Notably, at the lower protein concentrations tested (0.1 pM and less), there was a reduction in activity compared to TP alone (Figure 13). However, this activity was matched at higher protein concentrations of 0.2 pM and above. This observation suggests that the IH4 fusion may initially hinder the catalytic efficiency at lower concentrations, possibly due to steric effects or suboptimal dimerization. Nonetheless, at higher concentrations, these effects are overcome, and the catalytic activity is fully restored. This indicates that our TP-IH4 fusion construct is capable of achieving comparable enzymatic activity to TP alone when appropriately dosed, demonstrating its potential viability as a therapeutic agent.

[0501] To assess the catalytic activity of surface-bound IH4-TP, with TP fused to the C-terminal end of the nanobody, we conducted experiments to ensure its activity was maintained following binding to RBCs, comparing results to untreated RBCs (no protein) and RBCs treated with TP alone, which should not bind to RBCs.

[0502] As shown in Figure 14, IH4-TP attached to the surface of RBCs remains catalytically active, converting thymidine to thymine. This is evidenced by the absorbance spectra obtained after endpoint activity assays which are characteristic of thymine. In contrast, untreated RBCs (with no protein) or RBCs treated with TP alone then washed in the same way, did not show this activity, indicating that the observed activity is not due to residual TP in the reactions but instead due to surface bound IH4-TP. Flow cytometry analysis in Figure 15 also confirmed that IH4-TP indeed binds to RBCs under these conditions, validating the experimental setup prior to the activity assay.

[0503] Method: Thymidine phosphorylase activity assays. Endpoint thymidine phosphorylase activity assays were performed by monitoring the conversion of thymidine to thymine spectroscopically between 200-360 nm. Enzyme concentrations ranging from of 0 - 1 pM were used, with reactions carried out in a reaction buffer consisting of 25 mM HEPES and 50 mM KH2PO4 pH 7.5, in a final volume of 0.1 ml. After the addition 1 mM thymidine, the reactions were incubated for 1 hour at 37°C. The reactions were terminated by the addition of 1 ml of 0.3 M NaOH. Absorbance measurements were taken using a spectrophotometer with UV cuvettes having a pathlength of 1 cm. To compare the activity of TP-IH4 fusion proteins to that of TP alone, the absorbance at 300 nm was measured for both the reaction and blank mixtures. The blank absorbance was subtracted from the reaction absorbance to obtain the corrected absorbance. The amount of thymine formed was calculated using the difference in the molar extinction coefficient between thymidine and thymine at alkaline pH, which is 3.4 x 103L / mol / cm. Enzyme activity was expressed as the amount of thymine (nanomoles) formed per hour per mg of protein. This activity was then normalised and expressed as a percentage relative to the activity of TP alone.

[0504] Cell-surface bound IH4-TP activity. For experiments measuring the activity of IH4-TP bound at the cell surface, TP alone or IH4-TP was first incubated with increasing numbers of RBCs, ranging from 2.5 x 106cells to 50 x 106cells, in PBSAG, with protein added at a ~22- fold molar excess relative to the calculated GPA concentration per cell number (assuming 800 000 copies of GPA per RBC; 2.5, 5, 10, 25 and 50 pM protein added to 2.5, 5, 10, 25 and 50 x 106cells, respectively). The suspension was incubated for 1 hour at RT and then washed twice in PBSAG. Following binding and washing, 0.2 x 106cells were taken and incubated for 30 minutes at 4°C with APC-conjugated anti-His antibody, followed by washing to confirm IH4-TP binding to RBCs under these conditions by flow cytometry analysis. The remaining RBCs were pelleted and resuspended in 10 pl PBSAG and used in the endpoint thymidine phosphorylase activity assay described above. Briefly, the cells were added to reaction buffer in a final volume of 0.1 ml. After the addition of 1 mM thymidine, reactions were incubated for 1 hour at 37°C and terminated by adding 1 ml of 0.3 M NaOH before taking absorbance spectra. TP alone, which was not expected to bind to RBCs, was used as a control to determine the residual activity of non-specifically bound protein.

[0505] Example 6: Modular assembly of IH4 fusion proteins using the SpyCatcher-SpyTao system. Building on work described in previous sections where IH4-containing fusion proteins, including those fused to dimeric therapeutic enzymes, were expressed and purified from bacteria as a single polypeptide chain, we explored a more modular approach to creating nanobody-enzyme constructs. This approach addresses potential challenges in the expression and purification of such constructs as single entities or the need for enzyme expression in mammalian cells. Specifically, we employed the SpyCatcher (SC)-SpyTag (ST) system to facilitate the modular assembly of IH4 fusion proteins. The SC-ST system, developed by Mark Howarth (e.g., EP3615556B1), is a well-known tool for protein conjugation that utilises the formation of an irreversible isopeptide bond between SC, a 138-residue incomplete immunoglobulin-like domain (15 kDa) and ST, a shorter 13-residue peptide.

[0506] In our approach, the smaller ST moieties are fused to enzymes, while the larger SC component is attached to the IH4 nanobody. This configuration leverages the nanobody's ability to tolerate the binding of proteins larger than SC at both termini. The primary goal is to assemble the final fusion proteins through SC-ST interaction, either in vitro prior to RBC attachment or on the RBC surface.

[0507] To validate the functionality of the IH4-SC fusion protein, we first assessed its ability to bind to RBCs and its specificity to GPA using flow cytometry. As shown in Figure 16, IH4-SC retains its binding capability and specificity, similar to IH4 alone. We confirmed this by incubating purified IH4 or IH4-SC with RBCs, BEL-A cells, and GPA KO BEL-A cells, and detecting binding activity with an APC-conjugated anti-His antibody. The representative flow cytometry histograms demonstrate that IH4-SC specifically binds to GPA on RBCs and BEL- A cells, but not to GPA KO BEL-A cells.

[0508] Next, we verified the functionality of the SC component in IH4-SC by assessing its ability to form an isopeptide bond with ST in the form of ST-GFP. IH4-SC and ST-GFP were mixed at a molar ratio of 5: 10 pM in PBS or PBSAG for 90 minutes at room temperature. Samples were periodically analysed by SDS-PAGE with Coomassie staining after quenching the reaction with SDS-PAGE sample buffer and boiling. Figure 17, confirmed the formation of the SC-IH4-ST-GFP construct, indicating by the appearance of a higher molecular weight band corresponding to the assembled complex.

[0509] There are two potential methodologies for attaching these fusion proteins to RBCs. The first involves assembling the fusion protein construct before RBC attachment. Alternatively, the second method involves assembling the fusion protein directly on the cell surface by first binding IH4-SC to the RBCs, followed by the addition of the ST-tagged component.

[0510] In this example, we employed the first method, using two distinct constructs: ST-GFP and ST-TID, with the latter serving as an exemplar enzyme. While TID is not intended for therapeutic use, it is a useful research tool for mapping protein interactions and its application in this study serves only as a proof-of-concept for the broader potential of this technology. The assembly of the complete fusion proteins was first confirmed via SDS-PAGE analysis (Figure 18). Specifically, IH4-SC was mixed with ST-GFP or ST-TID in a 1:2 molar ratio at specified concentrations and incubated for 1 hour at room temperature in PBSAG. The SDS-PAGE results demonstrated the successful formation of full-length fusion proteins comprising IH4 and either GFP or TID.

[0511] Subsequently, the binding efficiency of these pre-assembled fusion proteins to RBCs was evaluated through flow cytometry (Figure 19). The assembled fusion proteins, once incubated with RBCs, showed significant binding. This was detected using an APC- conjugated anti-His antibody or intrinsic GFP fluorescence for GFP-containing reactions. Control experiments confirmed that IH4-SC alone could bind to RBCs, while ST-GFP or ST- TID alone did not. However, pre-assembled IH4-SC with either ST-GFP or ST-TID successfully bound to the RBCs, confirming the necessity of the complete fusion protein assembly for effective RBC coupling of the GFP or TID proteins.

[0512] Method: IH4-SC and ST-GFP or ST-TID were mixed in molar ratios of 1:2, 10:20, and 100:200 pM, maintaining a two-fold molar excess of ST-GFP or ST-TID, in PBSAG. Control experiments were also conducted using o...

Claims

1. CLAIMS1. A protein conjugate comprising (a) one or more antibodies or variants thereof which specifically bind to one or more antigens on the surface of red blood cells (RBCs) and (b) one or more enzymes and / or variants thereof.

2. A protein conjugate according to claim 1, wherein the protein conjugate comprises (a) an antibody or a variant thereof which specifically binds to an antigen on the surface of red blood cells (RBCs).

3. A protein conjugate according to claim 1 or 2, wherein (i) the one or more antibodies or variants thereof are one or more nanobodies, one or more nanobody fragments, one or more nanobody variants, or one or more humanised nanobodies or (ii) the antibody or variant thereof is a nanobody, a nanobody fragment, a nanobody variant, or a humanised nanobody.

4. A protein conjugate according to any one of the preceding claims, wherein the one or more antigens are or the antigen is selected from the group containing Kell, Rh proteins, including RhD, RhCE, and RhAG, Band 3 / AE1, BCAM / Lutheran, Glycophorin A (GPA), Glycophorin B (GPB), Glycophorin C (GPC), CD44, XK, Aquaporin 1, Aquaporin 3, CD47, Complement component Receptor 1 (CR1), CD55 / Daf, Duffy, ICAM4 / LW, Acetylcholinesterase (ACHE), Basigin (CD147), Kidd, Glucose transporter 1 (GLUT1), Monocarboxylate transporter 1 (MCT1), Sodium Dependent multivitamin transporter SMVT (SLC5A6), ERMAP (erythroblast membrane associated protein), XG, Semaphorin 7A (CD108), CD151, ADP-ribosyltransferase, Neutral Amino acid transporter B(0) (SLC1A5), Large neutral amino acids transporter small subunit 1(SLC7A5), Chloride intracellular channel protein 1 (CLIC1), Sodium / potassium-transporting ATPase subunit alpha-1 (ATP1A1), ATPase Na+ / K+ transporting subunit beta 3 (ATP1B3) and CD59.

5. A protein conjugate according to any one of the preceding claims, wherein the one or more antigens are or the antigen is specific to RBCs.

6. A protein conjugate according to any one of the preceding claims, wherein (i) the one or more antibodies or variants thereof are one or more IH4 nanobodies or variants thereof or one or more CA52 nanobodies or variants thereof or (ii) the antibody or a variant thereof is an IH4 nanobody or a variant thereof or a CA52 nanobody or variant thereof.

7. A protein conjugate according to any one of the preceding claims, wherein the variant is a beta-strand ( -strand) swapped variant.

8. A protein conjugate according to any one of the preceding claims, wherein the one or more antibodies or variants thereof are or the antibody or a variant thereof is modified to facilitate covalent binding to the antigen and / or the RBCs.

9. A protein conjugate according to any one of the preceding claims, wherein the protein conjugate is capable of forming an oligomer.

10. A protein conjugate according to claim 9, wherein the protein conjugate is capable of forming a dimer.

11. A protein conjugate according to any one of the preceding claims, wherein the one or more enzymes are selected from branched-chain a-ketoacid dehydrogenase, 0- glucocerebrosidase, a-galactosidase a, acid a-glucosidase, a-l-iduronidase, iduronate-2- sulfatase, acid sphingomyelinase, 0-hexosaminidase a, cystathionine 0-synthase, methylmalonyl-coa mutase, propionyl-coa carboxylase, galactose-l-phosphate uridylyltransferase, aldolase b, glucose-6-phosphatase, glycogen debranching enzyme, biotinidase, argininosuccinate synthetase, pyruvate dehydrogenase, isovaleryl-coa dehydrogenase, glutaryl-coa dehydrogenase, cholesterol oxidase, cystinosin, thymidine phosphorylase, alanine amino transferase, thymidine phosphorylase, glutamine synthase, hexokinase, glucokinase, phenylalanine hydroxylase, alcohol dehydrogenase, catalase, glucose-6-phosphate dehydrogenase, adenosine deaminase (ADA), asparaginase, uricase, bacterial L-phenylalanine ammonia lyase, alanine aminotransferase, glutamate dehydrogenase, arginine deiminase, arginase, oxalate decarboxylase, oxalate oxidase, a serine protease, tissue plasminogen activator, larondinase, superoxide dismutase, disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), Carboxypeptidase B2 (TAFI), Protein C, Protein S, CD39 (ENTPD1), CD73 (NT5E), Carboxypeptidase N (CPN), Factor Vila (FVIIa), Factor IXa (FIXa), Factor Xa (FXa), Prothrombin / Thrombin (Flla), Factor Xia (FXIa), Factor XHIa (transglutaminase), Urokinase (uPA), Plasmin, Streptokinase, Staphylokinase, Glutathione peroxidase 1 (GPxl), GPx3, Glutathione reductase, Peroxiredoxin 1 (PRDX1), PRDX2, PRDX3, PRDX4, PRDX5, PRDX6, Alkaline phosphatase (ALPL), Acyloxyacyl hydrolase (AOAH), Diamine oxidase (DAO) & Histamine N-methyltransferase (HNMT), Platelet-activating factor acetylhydrolase (PLA2G7), Heme oxygenase-1 (HO-1), Glutathione reductase (GSR), Glutathione-S-transferase Al (GSTA1), GSTA2, GSTA3, GSTA4, GSTA5, GSTK1, GSTM1, GSTM1L (RNAi), GSTM2, GSTM3, GSTM4, GSTM5, GSTO1, GSTO2, GSTP1, GSTT1, GSTT2, GSTT4, GSTZ1 (aka MAAI-Maleylacetoacetate isomerase), Paraoxonase-1 (PON1), Butyrylcholinesterase (BChE) Aldehyde dehydrogenase 2 (ALDH2), ALDH1A1, Phosphotriesterase (PTE), Organophosphorus Hydrolase (OPH), Paraoxonase 1 (PON1), PON2, PON3, Diisopropylfluorophosphatase (DFPase), Methylparathion Hydrolase (MPH), Cocaine esterase (Rhodococcus), Hyaluronidase-2 (HYAL2), a combination of CD39 and CD73, Arylsulfatase B (ARSB), Acid ceramidase (ASAHI), N-acetylgalactosamine-6- sulfatase (GALNS), Galactocerebrosidase (GALC), Arylsulfatase A (ARSA), Aspartylglucosaminidase (AGA), 0-Mannosidase (MANBA), N-acetylglucosamine-6- sulfatase (GNS), Heparan N-sulfatase (SGSH), Alpha-N-acetylglucosaminidase (NAGLU),Acetyl-CoA:alpha-glucosaminide N-acetyltransferase (HGSNAT), Alpha-mannosidase (MAN2B1), Neuraminidase 1 (NEU1), Cathepsin K (CTSK), Cathepsin D (CTSD), Palmitoyl-protein thioesterase 1 (PPT1), Tripeptidyl peptidase 1 (TPP1), DNase I or a variant thereof, DNASE1L3, RNase 1 or a variant thereof, DNASE1L3, Lysozyme (LYZ), AlgL alginate lyase, Al-IV alginate lyase, Al-II alginate lyase, Al-III alginate lyase, alginate lyase Al (AlyAl), AlyA2, AlyPG, AlyM, AlyGC, Algl7c, AlyQ, AlyDRl, AlyDR2, AlyVl, AlyV2, AlyA5, Alg7A, AlySY08, AlyMBl, ALY-1, ALY-2, Dispersin B (bacterial PNAG hydrolase), Secretory phospholipase A2 (SPLA2-IIA), Complement Factor I, LysK, PlyC, Cpl-1, Pal, Lys44, LysH5, LysEF-PIO, LysSA97, Ply511, PlyV12, LysPA26, LysAB2, OBPgp279, KZ144, LysSS, ClyS, Art-175, AmiA, AmiB, AmiC, AtlA, LytA, AtlE, AtlA, CwlC, CwlA, CwlB, CwID, Auto (autolysin with amidase domain), Cpl-1, PlyC, Pal, Ply511, Plyll8, Slt70, MltA, MltB, MltC, MltD, MltE, MltF, MltG, Sit, MltBl, MltB2, MltF, LtgA, LtgD, RIpA, AmpDh2, AmpDh3, LtgX, Angiotensin converting enzyme 2 (ACE2), Lipoprotein lipase (LPL), Cholesterol esterase, Lecithin-Cholesterol aclytransferasee (LCAT), D-amino acid oxidase and L-amino acid oxidase.

12. A protein conjugate according to any one of the preceding claims, wherein the one or more enzymes comprise thymidine phosphorylase, adenosine deaminase (ADA) or disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13).

13. A protein conjugate according to any one of the preceding claims, wherein the protein conjugate further comprises one or more linkers.

14. A protein conjugate according to any one of the preceding claims, wherein (a) and (b) are genetically linked to form a fusion protein.

15. A protein conjugate according to any one of the preceding claims, wherein the RBCs are human RBCs.

16. An oligomeric construct comprising two or more protein conjugates according to any one of the preceding claims.

17. An oligomeric construct according to claim 16, wherein the oligomeric construct is a dimeric conjugate comprising two protein conjugates.

18. A population of red blood cells (RBCs) surface labelled with a protein conjugate according to any one of claims 1-15 and / or an oligomeric construct according to claim 16 or 17.

19. A population according to claim 18, wherein the RBCs are surface labelled by specific binding of the one or more antibodies or variants thereof to the one or more antigens onthe surface of the RBCs or the antibody or a variant thereof to the antigen on the surface of the RBCs.

20. A population according to claim 18 or 19, wherein the protein conjugate and / or the oligomer conjugate is / are covalently attached to the RBCs.

21. A population according to any one or claims 18-20, wherein the one or more enzymes and / or variants thereof remain active on the surface of the RBCs.

22. A pharmaceutical composition comprising one or more protein conjugates according to any one of claims 1-15, an oligomeric construct according to claim 16 or 17 or a population of RBCs according to any one of claims 18-21 and a pharmaceutically or physiologically acceptable diluent and / or carrier.

23. A method of treating or preventing disease or disorder in a subject, comprising administering to the subject one or more protein conjugates according to any one of claims 1-15, an oligomeric construct according to claim 16 or 17, a population of RBCs according to any one of claims 18-21 or a pharmaceutical composition according to claim 22.

24. A method according to claim 23, wherein the disease or disorder is associated with a reduced function or absence of the enzyme.

25. A method according to claim 23 or 24, wherein the disease or disorder is a metabolic disease or disorder, an infectious disease or disorder, an autoimmune disease or disorder, an immunodeficiency disease or disorder, a cardiovascular disease or disorder, or cancer.

26. One or more protein conjugates according to any one of claims 1-15, an oligomeric construct according to claim 16 or 17, a population of RBCs according to any one of claims 18-21 or a pharmaceutical composition according to claim 22 for use in a method of treating or preventing a disease or disorder in a subject.