WO2022013304A1 - POLYPEPTIDES PDGFRα EN TANT QUE RÉCEPTEURS LEURRES VIRAUX - Google Patents

POLYPEPTIDES PDGFRα EN TANT QUE RÉCEPTEURS LEURRES VIRAUX Download PDF

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WO2022013304A1
WO2022013304A1 PCT/EP2021/069644 EP2021069644W WO2022013304A1 WO 2022013304 A1 WO2022013304 A1 WO 2022013304A1 EP 2021069644 W EP2021069644 W EP 2021069644W WO 2022013304 A1 WO2022013304 A1 WO 2022013304A1
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pdgfra
polypeptide
pdgf
domain
cell
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PCT/EP2021/069644
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Peter Lischka
Immanuel GRIMM
Cora STEGMANN
Svenja FELDMANN
Christian SINZGER
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Aicuris Gmbh & Co. Kg
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Publication of WO2022013304A1 publication Critical patent/WO2022013304A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to the field of pharmacology and specifically to PDGFRa polypeptides which can be used as viral decoy receptors. It includes fusion proteins comprising these polypeptides as well as nucleic acids, vectors, cells and pharmaceutical compositions.
  • the use in medicine is envisioned specifically for the prevention and treatment of CMV infections.
  • HCMV Human cytomegalovirus
  • PDGFRa-Fc inhibits the infection of various cell types, while many of the highly potent monoclonal antibodies that are currently in development are directed against the viral pentamer complex and inhibit only infection of endothelial and epithelial cells efficiently.
  • Using a PDGFRa-Fc as a decoy receptor against HCMV infection may impair cellular signaling by sequestration of the natural ligands of PDGFRa.
  • the interplay between platelet-derived growth factors (PDGFs) and their receptors PDGFRa and PDGFRp plays an important role in development as well as in the regulation of various physiological processes like cell migration and proliferation in adults.
  • soluble PDGFRa-Fc could cause side effects by sequestration of PDGFs and in consequence reduction of PDGF-dependent signaling.
  • circumvention of PDGF sequestration is desirable. While substitutions or deletions could be an option, it has been shown that PDGFs can outcompete HCMV, indicating that the binding site of PDGFRa for the HCMV glycoprotein complex gH/gL/gO and the PDGFs are located in the same domains and might overlap.
  • the present invention relates to a PDGFRa polypeptide comprising (i) at least a PDGFRa D2 domain and a PDGFRa D3 domain, and (ii) mutations at positions 139 and 206 corresponding to the PDGFRa sequence according to SEQ ID NO: 1.
  • the invention in a second aspect, relates to a fusion protein comprising the PDGFRa polypeptide of the first aspect.
  • the invention in a third aspect, relates to a multimer comprising at least two fusion proteins of the second aspect.
  • the present invention relates to a nucleic acid encoding the PDGFRa polypeptide of the first aspect, the fusion protein according to the second aspect, or one or more fusion proteins forming the multimer of the third aspect.
  • the present invention relates to a vector comprising the nucleic acid of the fourth aspect.
  • the present invention relates to a cell comprising the nucleic acid of the fourth aspect or the vector of the fifth aspect.
  • the present invention relates to a pharmaceutical composition comprising the PDGFRa polypeptide of the first aspect, the fusion protein of the second aspect, the multimer of the third aspect, the nucleic acid of the fourth aspect, the vector of the fifth aspect or the cell of the sixth aspect, and a pharmaceutically acceptable carrier.
  • the invention relates to an in vitro method of treating a device or a composition, comprising the steps of:
  • the present invention relates to the PDGFRa polypeptide of the first aspect, the fusion protein of the second aspect, the multimer of the third aspect, the nucleic acid of the fourth aspect, the vector of the fifth aspect, the cell of the sixth aspect or the pharmaceutical composition of the seventh aspect for use in medicine.
  • the present invention relates to a polypeptide comprising a fragment of PDGFRa, wherein the fragment consists of a PDGFRa D2 domain and a PDGFRa D3 domain, and to the use of this polypeptide for binding to a virus.
  • Figure 1 Screening of deletions in the predicted ligand binding sites of PDGFR- alpha-Fc for their ability to inhibit HCMV infection.
  • A Overview of the relative locations of the predicted ligand binding sites in domains 2 and 3 of PDGFR-alpha-Fc. Position numbers are based on the amino acid sequence of cellular PDGFRa.
  • B to C The effect of PDGFRa-Fc wild type (in the context of a PDGFRa-Fc protein, “wild type” herein refers to the PDGFRa part of the fusion) and deletion mutants on HCMV infection of fibroblasts was tested.
  • soluble receptor variants Different concentrations of the soluble receptor variants were preincubated with a MOI of 1 of HCMV TB40-BAC4-IE-GLuc reporter virus.
  • B Expression of GLuc in samples treated with the different PDGFRa-Fc variants were compared to cells incubated with untreated virus. Dose response curves were generated as an average of 3 independent experiments using 2 different preparations of the luciferase expressing TB40-BAC4-IE-GLuc reporter virus.
  • C Three mutants showed near complete inhibition at high doses (shown in black). For those mutants and the wild type PDGFRa-Fc the half-maximal effective concentration (EC50, shown in grey) was calculated from the neutralization curves shown in B. Error bars indicate the standard error of the mean (SEM) calculated from 3 experiments.
  • Figure 2 Mutation of isoleucine 139 and tyrosine 206 reduces sequestration of PDGF-BB.
  • a + B Quantification of HCMV inhibition by PDGFRa-Fc alanine exchange mutants was assessed by preincubation of the HCMV TB40 GLuc reporter virus with PDGFRa- Fc derivatives before infection of fibroblasts. The degree of infection was measured relative to a virus only condition.
  • A depicts the average dose-response curves as calculated from at least 3 independent experiments.
  • FIG. 3 Replacement of isoleucine 139 with glutamic acid improves the inhibition profile of PDGFRa-Fc.
  • a + B Quantification of HCMV inhibition by the PDGFRa-Fc 1139 mutants.
  • HCMV TB40 at a MOI ⁇ 1 was preincubated with different concentrations of PDGFRa-Fc derivatives before infection of fibroblasts. After 1 day the cells were fixed and stained for the viral immediate early antigens and the percentage of infected cells was determined. Dose-response curves showing the percent of infection relative to a virus only control (A) were used as a basis to calculate the (B). Shown are the means of 5 independent experiments.
  • C to F Biological interference of PDGFRa-Fc 1139 mutants with PDGF- dependent signaling was tested by preincubation of 6 ng/ml PDGF-BB with different concentrations of PDGFRa-Fc before stimulation of fibroblasts.
  • C + D PDGF-dependent signaling was assessed by staining for Phospho-Akt.
  • C shows immunoblot examples and D summarizes the change in phospho-Akt signals after pretreatment of PDGF-BB with 4000g/ml PDGFRa-Fc. 100% interference equals complete inhibition of Akt phosphorylation to the level of cells that not receive PDGF-BB.
  • E + F PDGF-dependent signaling was quantified with a commercial phospho-Akt ELISA.
  • the dose-response curves shown in E depict the mean of 3 independent experiments and F shows the EC50s calculated thereof in grey.
  • the black bars depict the change in Akt phosphorylation when PDGF-BB was pretreated with 562 ng/ml of PDGFRa-Fc wild type or I139A. 100% interference equals complete inhibition of Akt phosphorylation to the level of cells that not receive PDGF-BB. All error bars indicate SEM and all experiments were performed with at least 2 independent protein preparations. Asterisks indicate statistically significant differences as compared to PDGFRa-Fc wild type determined by unpaired t-tests.
  • Figure 4 Dose-response relationship between PDGF-BB and Akt phosphorylation.
  • Serum-starved fibroblasts were incubated for 15 min with various concentrations of PDGF-BB before the cells were lysed.
  • the PDGF-dependent signaling was assessed by immunoblot and staining for phospho-Akt. Actin was included as a loading control.
  • A shows a representative example of such an immunostaining.
  • B depicts the average dose-response in 4 independent experiments. Error bars indicate SEM.
  • FIG. 5 Replacement of tyrosine 206 with serine abolishes PDGF sequestration while maintaining HCMV inhibition.
  • a + B HCMV at a MOI ⁇ 1 was pretreated with various concentrations of PDGFRa-Fc fusion proteins before infection of fibroblasts. Shown is the number of cells expressing the viral immediate early antigens relative to a virus only condition. Dose-response curves, as shown averaged in A, were used as a basis to calculate the half- maximal effective concentrations (B) in 3 independent experiments.
  • C to F Biological interference with PDGF-dependent signaling was tested by preincubation of 6 ng/ml PDGF- BB with various concentrations of PDGFRa-Fc mutants or wild type before stimulation of fibroblasts.
  • C to D Staining of Phospho-Akt served as a measure for PDGF induced signaling.
  • C shows immunoblot examples and D summarizes the interference of different PDGFRa-Fc variants with Akt phosphorylation. 100% interference equals complete inhibition of Akt phosphorylation and negative values indicate enhanced Akt phosphorylation as compared to cells that were stimulated with untreated PDGF-BB.
  • E +F PDGF-dependent signaling was quantified using a phospho-Akt ELISA.
  • E depicts the mean of 3 independent experiments and F shows the EC50s calculated thereof (grey bars) as well as the interference of 562 ng/ml of PDGFRa-Fc variants with PDGF-dependent Akt phosphorylation (black bars).
  • N.d. stands for not determined and indicates that in this case no EC50 could be calculated due to lack of response.
  • Figure 6 Combination of multiple amino acid exchanges in PDGFRa further reduce inhibition of PDGF dependent signaling.
  • a + B Quantification of HCMV inhibition by PDGFRa-Fc double amino acid exchange mutants. To measure the inhibition of infection, the rate of immediate early positive cells after infection with HCMV untreated or pretreated with PDGFRa-Fc was measured relative to a virus only condition. An average of 3 independent dose-response experiments is shown in A. The corresponding EC50 values are shown in B.
  • C + D PDGF-dependent signaling after preincubation of 6 ng/ml PDGF-BB with various concentrations of PDGFRa-Fc mutants was quantified with a commercial phospho-Akt ELIS A.
  • the dose-response curves shown in C depict the mean of 3 independent experiments and D compares the interference of 562 ng/ml of the different PDGFRa-Fcs with PDGF-dependent cellular signaling. 100% interference equals complete inhibition of Akt phosphorylation to level of unstimulated cells whereas negative values indicate enhanced Akt phosphorylation as compared to cells that were stimulated with untreated PDGF-BB.
  • FIG. 8 MST traces for PDGF-BB bound to PDGFRa-Fc variants.
  • Microscale thermophoresis was performed with 0.1 nM (2.5 ng/ml) fluorescently labelled (NT-647) PDGF-BB which was mixed with different concentrations of PDGFRa-Fc wild type and mutants (2fold dilution series starting from A + E: 5 nM, B: 500 nM, C: 650 nM, D: 550 nM or F: 500 nM).
  • the darkest line represents the highest concentration of soluble receptor and lightest grey depicts the lowest concentration. Each concentration was tested three times, shown is one example. All experiments were performed with medium MST power and 60% excitation power.
  • a to D Direct fluorescence analysis measuring binding-induced fluorescence quenching. After 5 sec of initial photobleaching the change in fluorescence was monitored over the indicated time (shaded in grey). The rate of change in fluorescence per second was used to generate dose-response curves shown in Figure 6 A-C. E + F: After 5 sec of initial photobleaching the change in fluorescence was monitored for 6 seconds. For the dose response curves shown in Figure 7, the fluorescence intensity after infrared laser activation (FI, second grey bar) over the initial fluorescence (F0, first grey bar) was calculated from the data shown here.
  • FI infrared laser activation
  • FIG. 9 Serum induced cellular signaling is unaffected by treatment with the PDGFRa-Fc I139E Y206S mutant.
  • PDGFRa-Fc I139E Y206S mutant To test for cellular signaling under more physiological conditions, the effect of PDGFRa-Fc wild type and I139E+Y206S double mutant on serum- induced phosphorylation of Akt was measured. A pool of sera from 3 donors was preincubated with 562 ng/ml of soluble receptors before stimulation of fibroblasts. The absolute amount of Akt phosphorylation was measured using a commercial ELISA. Shown are the mean values for 3 experiments testing 2 independent protein preparations. The error bars indicate SEM. The asterisk indicates that only PDGFRa-Fc wild type but not the I139E + Y206S mutant significantly interfered with serum induced signaling as determined by unpaired t-tests comparing the response of untreated serum to the treated sera.
  • FIG. 10 Mutation of V242K abolishes PDGF sequestration without impact on HCMV inhibition.
  • a + B Quantification of HCMV inhibition by PDGFRa-Fc V242K mutants. To measure the inhibition of infection, the rate of immediate early antigen-positive cells after infection with HCMV untreated or pretreated with PDGFRa-Fc was measured. A shows the average of 3 independent dose-response experiments and B shows the corresponding EC50 values.
  • C + D PDGF-dependent signaling after preincubation of 6 ng/ml PDGF-BB with various concentrations of PDGFRa-Fc mutants was quantified with a phospho-Akt ELIS A.
  • the dose-response curves shown in C depict the mean of 3 independent experiments and D compares the interference of 500 ng/ml of the different PDGFRa-Fcs with PDGF-dependent cellular signaling. 100% interference equals complete inhibition of Akt phosphorylation to the level of unstimulated cells whereas negative values indicate enhanced Akt phosphorylation as compared to cells that were stimulated with untreated PDGF-BB. All error bars indicate standard error of the mean.
  • FIG. 11 Combination of multiple mutations affecting PDGF binding reduces the affinity of PDGFRa-Fc for PDGF beyond detection. Quantification of biochemical binding affinity of PDGFRa-Fc variants for PDGF-BB was assessed by microscale thermophoresis.
  • Various concentrations of PDGFRa-Fc wildtype, PDGFRa-Fc V242K (A), PDGFRa-Fc I139E + V242K (B), PDGFRa-Fc Y206S + V242K (C) or PDGFRa-Fc I139E + Y206S + V242K (D) were mixed with 0.1 nmol/1 (A) or 1 nmol/1 (B to D) fluorescently labeled PDGF-BB.
  • Binding curves were generated by analysis of the ratio (D F n0rm ) of fluorescence at MST-on time (1.5- 2.5) seconds over the steady-state fluorescence (F0) for each concentration. Measurements were performed at 60% excitation power (A) or 20% excitation power (B). Error bars indicate standard deviation from 3 replicate measurements.
  • the present invention relates to a PDGFRa polypeptide comprising (i) at least a PDGFRa D2 domain and a PDGFRa D3 domain, and (ii) mutations at positions 139 and 206 corresponding to the PDGFRa sequence according to SEQ ID NO: 1 (also referred to herein as “139 mutation” and “206 mutation”).
  • PDGFRa polypeptide herein refers to a polypeptide derived from PDGFRa, in particular according to SEQ ID NO: 1.
  • PDGFRa comprises several domains, an extracellular domain (ECD), a transmembrane domain (TM) and an intracellular domain (ICD).
  • ECD comprises domains 1, 2, 3, 4 and 5 (Dl, D2, D3, D4 and D5) as well as a signal peptide (SP) prior to maturation.
  • the SP extends from amino acid position 1 to 23
  • the ECD extends from amino acid position 24 to 528
  • the TM extends from amino acid position 529 to 549
  • the ICD extends from amino acid position 550 to 1089.
  • Dl, D2, D3, D4 and D5 extend from amino acid position 24 to 113, 117 to 201, 202 to 306, 319-410 and 414 to 517, respectively.
  • PDGFRa also comprises a PDGF binding domain and a virus binding domain within the ECD. While both may involve some residues outside of D2 and D3, the inventors found that D2 and D3 are sufficient for PDGF and virus binding. Therefore, when referring to “the PDGF binding domain” and “the virus binding domain” herein, in particular the PDGF binding domain and the virus binding domain herein as comprised in D2 and D3 are meant.
  • the virus is a virus binding to (usually mature) PDGFRa, preferably a virus binding to mature PDGFRa according to amino acids 24-1089 of SEQ ID NO: 1, in particular to the ECD or only D2 and D3 thereof. More preferably, the virus is CMV, with respect to all references to “a virus” or “viral” herein.
  • the CMV is preferably HCMV, with respect to all references to CMV herein.
  • the PDGFRa from which the PDGFRa polypeptide of the first aspect is derived can be any PDGFRa, specifically from any species (although preferably it is from a mammal, more preferably human), or a variant thereof.
  • the positions and domains boundaries of homologous PDGFRa or of variants can be determined by the skilled person without undue burden by sequence alignment.
  • it is PDGFRa according to SEQ ID NO: 1 or a variant thereof.
  • the PDGFRa polypeptide is derived from mature PDGFRa or a variant thereof, i.e. PDGFRa or a variant thereof lacking the SP (or any other signal peptide).
  • mature PDGFRa corresponds to amino acids 24-1089 of SEQ ID NO: 1.
  • a nucleic acid of the invention encodes for the PDGFRa polypeptide comprising a (any) signal peptide (preferably one which directs the PDGFRa polypeptide into the endoplasmic reticulum of a cell such that it is secreted, such as a signal peptide comprising amino acids 1-20 of SEQ ID NO: 2 or a variant thereof), preferably the PDGFRa SP (e.g. comprising amino acids 1-23 of SEQ ID NO: 1 or a variant thereof).
  • any PDGFRa domain recited herein (SP, ECD, TD, ICD, Dl- D5), i.e. these domains can be from any PDGFRa (any species, preferably mammalian, more preferably of SEQ ID NO: 1), and it can be a variant of domains of that PDGFRa.
  • SP, ECD, TD, ICD, or D1-D5 as referred to herein can be any PDGFRa SP, ECD, TD, ICD, or D1-D5, respectively, and in particular can be a variant of the PDGFRa SP, ECD, TD, ICD, or D1-D5, respectively according to SEQ ID NO: 1.
  • the PDGFRa polypeptide of the first aspect differs from the PDGFRa from which it is derived by at least the 139 and 206 mutations.
  • the PDGFRa polypeptide may further differ from the PDGFRa from which it is derived by deletion of one or more domains or parts thereof, wherein the deleted domains (or domains with deletion of at least a part) are preferably the TM and/or the ICD, and optionally further D4 and/or D5, and optionally also Dl.
  • the PDGFRa polypeptide of the first aspect comprises (i) D2 and (ii) D3, in a preferred embodiment it further comprises (iii) Dl, and optionally (iv) D4 and/or D5.
  • the PDGFRa polypeptide of the first aspect consists of the ECD and lacks the SP, TM and ICD.
  • any of these domains can be from any PDGFRa (any species, preferably mammalian, more preferably of SEQ ID NO: 1), and it can be a variant of corresponding domains of that PDGFRa.
  • a PDGFRa polypeptide lacking SP may comprise a different signal peptide, but preferably does not comprise any signal peptide.
  • the above variants (of PDGFRa or of any of its domains) independently have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (higher identities being preferred over lower ones) to the sequence of polypeptide they vary (e.g. SEQ ID NO: 1 or domains thereof).
  • the ECD has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the ECD of SEQ ID NO: 1 (rather than the ECD of SEQ ID NO: 1 or of a variant of SEQ ID NO: 1).
  • the results of the examples provide evidence that mutations of distinct sites within the PDGF binding domain can act synergistically against binding of PDGFs (the 139 and 206 mutations render the PDGF binding domain largely non-functional), so it is reasonable to assume that also mutations without a strong individual impact on PDGF sequestration by themselves may lead to a synergistic reduction when replaced e.g. with amino acids other than alanine and in combination with the 139 and 206 mutations.
  • the PDGFRa polypeptide may further differ from the PDGFRa (or variant) from which it is derived by one or more further mutations (e.g.
  • Preferred amino acids to be mutated are those that are predicted by in silico models to be relevant for PDGF binding.
  • substitutions are preferably non-conservative and change one or more properties of the substituted amino acid selected from the group consisting of from polarity, charge and size.
  • Exemplary substitutions of e.g. V242 are V242H, V242K and V242R, specifically V242K. While the V242 mutation may be a deletion or substitution, a substitution is preferred. Most preferred is a point mutation.
  • the PDGFRa polypeptide comprises a functional virus binding domain.
  • Functional in this respect means that the PDGFRa polypeptide has a substantial affinity for the virus.
  • Substantial in this respect preferably means at least 5%, at least 10%, at least 15%, or at least 20% of the virus affinity of a corresponding PDGFRa according to SEQ ID NO: 1, e.g. at least at least 5%, 10%, at least 15%, or at least 20% of the virus affinity of the ECD of SEQ ID NO: 1.
  • functional may mean that the PDGFRa polypeptide has a substantial inhibitory effect on virus cell-entry.
  • Substantial in this respect preferably means at least 5%, at least 10%, at least 15%, or at least 20% of the inhibitory effect of a corresponding PDGFRa according to SEQ ID NO: 1, e.g. at least at least 5%, 10%, at least 15%, or at least 20% of the virus affinity of the ECD of SEQ ID NO: 1. Higher percentages are preferred over lower ones.
  • a “corresponding PDGFRa according to SEQ ID NO: 1” above and below has the same PDGFRa domains (or parts thereof) as the PDGFRa polypeptide, but of exactly SEQ ID NO: 1.
  • the 139 and 206 mutations alter the PDGF binding domain, i.e. the PDGFRa polypeptide comprises an altered PDGF binding domain.
  • the altered PDGF binding domain is characterized functionally as a result of these mutations by a reduced affinity for PDGF (at least PDGF-BB) compared to the corresponding PDGFRa according to SEQ ID NO: 1.
  • Reduced affinity in this respect preferably means that the altered PDGF binding domain has 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.01% or less, 0.005% or less, or 0.001% or less of the PDGF affinity of the corresponding PDGFRa according to SEQ ID NO: 1. Lower percentages are preferred over higher ones. The preferred minimal affinity is, if zero cannot be achieved, 0.001% or 0.0001% of the corresponding PDGFRa according to SEQ ID NO: 1.
  • Mutations referred to herein, specifically the 139 and 206 mutations may independently be selected from the group consisting of a deletion and a substitution, preferably a non conservative substitution.
  • the mutation is a point mutation. This means that at least the one adjacent position upstream and the one adjacent position downstream are not mutated. This applies in particular to the mutation being a deletion, i.e. at least the one adjacent position upstream and the one adjacent position downstream are not deleted.
  • the 139 substitution preferably is to a polar, more preferably polar and charged, most preferably polar and negatively charged amino acid and/or the 206 substitution is to a non-polar amino acid or preferably to a smaller amino acid.
  • the smaller amino acid is preferably polar, and more preferably also uncharged.
  • Polar amino acids have either an OH or N3 ⁇ 4 group (when in an aqueous environment) and can therefore form hydrogen bonds with other suitable groups. Due to the hydrophilic properties of their side chain, they have a tendency to be on the outside of a protein and to affect the overall protein structure. Polar amino acids are D and E (negatively charged); R, K and H (positively charged); and N, Q, S, T and Y (uncharged). Non-polar amino acids have side chains consisting of hydrocarbons and optionally other side chain atoms such as sulphur which do not confer polar properties. Non-polar amino acids are G, A, P, V, L, I, M, W and F.
  • the expression “smaller” relates to the Tyrosine at position 206 in SEQ ID NO: 1 and preferably means that the amino acid has a lower molecular weight than Tyrosine. Alternatively or in addition, it means that is has a shorter side chain than Tyrosine, e.g. a side chain comprising less than 6, less than 5, less than 4, less than 3 C, N, O, S and Se atoms in total (lower numbers preferred over higher ones).
  • the 139 substitution is selected from the group consisting of I139E and II 39 A, more preferably it is I139E, and/or the 206 substitution is selected from the group consisting of Y206S, Y206T, Y206N, Y206Q, Y206C, Y206U, Y206G, Y206A and Y206F, preferably from the group consisting of Y206S, Y206T, Y206N, Y206Q, Y206C, Y206U, Y206A and Y206G, more preferably from the group consisting of Y206S, Y206T, Y206N, Y206A and Y206Q, even more preferably from the group consisting of Y206S and Y206T. Most preferably it is Y206S. In the most preferred embodiment, the 139 substitution is I139E and the 206 substitution is Y206S.
  • the PDGFRa polypeptide comprises or consists of a fragment consisting of amino acids 60-206, 60-243, 60-262, 60-273, 60-294, 60-528, 30-206, 30-243, 30-262, 30-273, 30-294, 30-528, 24-206, 24-243, 24-262, 24-273, or 24-528 (the latter being preferred) of SEQ ID NO: 1 or a variant of any of the fragments, wherein the fragments and variants have the 139 and 206 mutations.
  • the PDGFRa polypeptide is a decoy receptor.
  • decoy receptor refers to viral decoy receptor, i.e. to a receptor that binds to a virus, but is not capable of mediating cell entry of the virus. In order to exert its function, the decoy receptor is usually not integrated into or attached to the plasma membrane of a cell.
  • the decoy receptor is soluble.
  • soluble indicates that the PDGFRa polypeptide is not bound to a cellular membrane, and it is usually characterized by the functional disruption of the TM (preferably by deletion of at least part of it, e.g.
  • the soluble PDGFRa polypeptide is devoid of any membrane anchoring function. It is therefore generally secreted by the cell producing it. At least part of the ICD may also be absent.
  • the decoy receptor e.g. if not soluble, can be linked to a carrier.
  • the carrier can be any suitable biocompatible substance, but is not a host cell.
  • the decoy receptor may be covalently bound to a particle, e.g. a nanobead, or may be integrated in an artificial lipid membrane of e.g. a liposome.
  • the present invention relates to a PDGFRa polypeptide comprising a mutation at (a) a position selected from the group consisting of M133, VI 84, G185, N204, N240, E241, V242, V243, D244, L245, T259, M260, L261, E262, K270, T273, Q294, A295, T296 and E298 and (b) position 206 and/or 139 corresponding to the PDGFRa sequence according to SEQ ID NO: 1.
  • the mutation can be at one position (e.g. V242) or at more than one of the positions.
  • the mutation of (a) is preferably a point mutation. It may be a deletion or substitution. Preferably it is a substitution, more preferably a non-conservative substitution, changing one or more properties of the substituted amino acid selected from the group consisting of from polarity, charge and size.
  • Exemplary substitutions of e.g. V242 are V242H, V242K and V242R, specifically V242K.
  • PDGFRa polypeptide of the first aspect applies also to this PDGFRa polypeptide, and corresponding fusion proteins, multimers, nucleic acids, vectors, cells, pharmaceutical compositions and methods uses as described below (but corresponding to the (a) and (b) mutations instead) are also intended.
  • the invention in a second aspect relates to a fusion protein comprising the PDGFRa polypeptide of the first aspect.
  • the fusion protein further comprises one or more moieties selected from the group consisting of a moiety facilitating the crossing of the blood-brain- barrier (BBB moiety); a moiety targeting the brain; a moiety facilitating the crossing of the placenta from the mother towards the fetus (placenta moiety); a moiety preventing the crossing of the placenta from the mother towards the fetus; a multimerization moiety, preferably a dimerization moiety; a half-life extending moiety; and an extracellular anti-viral agent (EAA).
  • the moieties are preferably polypeptide moieties.
  • An example for a BBB moiety is an insulin receptor binding moiety, e.g. an antibody or an antigen-binding fragment thereof binding to the insulin receptor.
  • An example for a moiety targeting the brain is a scopine moiety (lR,2R,4S,5S,7S)-9-Methyl-3-oxa-9-azatricyclononan- 7-ol).
  • An example for a placenta moiety is an Fc domain of IgG.
  • An example for a moiety preventing the crossing of the placenta from the mother towards the fetus is an Fc domain of IgM.
  • the multimerization moiety can be a heterodimerization moiety or preferably a homodimerization moiety.
  • the homodimerization domain is selected from the group consisting of an Fc domain, a CH3 domain, a CH2-CH3 domain, and a domain where homodimerization is mediated by an Ig-like fold, a rossmann- or rossmann-like alpha-beta-alpha sandwich fold, an alpha-sandwich fold, a continuous-beta-sheet fold, a beta-sandwich fold, a mixed beta-sheet fold, a 2-helix orientation, an antiparallel alpha-helix-orientation, a parallel alpha-helix orientation, a 4-helix bundle motif, a leucine zipper or a coiled-coil domain.
  • it is an Fc domain, more preferably of IgG (specifically IgGl) or IgM, most preferably
  • the heterodimerization domain is selected from the group consisting of a knob or a hole CH3 (or CH2-CH3) domain of a pair of knob-into-hole CH3 (or CH2-CH3) domains; an Fc-domain with mutations forcing heterodimerization (e.g.
  • a domain of a pair of interchanged domains such as Fc-one/kappa heterodimerization domain, CL and CH domains
  • an Ig-like fold with introduced mutations to force heterodimerization and a domain mediating heterodimerization containing a rossmann- or rossmann-like alpha-beta-alpha sandwich fold, an alpha-sandwich fold, a continuous-beta- sheet fold, a beta-sandwich fold, a mixed beta-sheet fold, a 2-helix orientation, an antiparallel alpha-helix-orientation, a parallel alpha-helix orientation, a 4-helix bundle motif, a leucine zipper or a coiled-coil domain.
  • a rossmann- or rossmann-like alpha-beta-alpha sandwich fold an alpha-sandwich fold, a continuous-beta- sheet fold, a beta-sandwich fold, a mixed beta-sheet fold,
  • the half-life extending moiety can be a moiety selected from the group consisting of an Fc domain, albumin, an elastin-like polypeptide (ELP), an XTEN polypeptide, a PAS polypeptide, a polyethylene glycol (PEG) moiety and a lipid moiety.
  • EAA e.g. an agent, like an antibody, disrupting the virus capsid
  • PEG polyethylene glycol
  • the fusion protein comprises a multimerization moiety or a half-life extending moiety. More preferably, it comprises both. In especially preferred embodiments, the fusion protein comprises a half-life extending moiety which is a multimerization moiety. Most preferably, the half-life extending moiety which is a multimerization moiety is an Fc domain (e.g. of IgM or preferably IgG, specifically IgGl). The Fc domain increases the plasma half-life of fusion proteins. This is caused by binding of the Fc domain to the receptor FcRn, which enables recycling during circulation. It also is a dimerization moiety.
  • Fc domain e.g. of IgM or preferably IgG, specifically IgGl
  • dimerization domain One of the advantages of the use of a dimerization domain considered by the inventors is that it increases the binding efficiency which suggests that the Fc-mediated dimerization of PDGFRa-Fc further enhances virus inhibition by increasing the avidity. Furthermore, it has placenta-crossing properties as described above.
  • SEQ ID NO: 2 represents the fusion protein of the examples. It comprises (i) a signal peptide (which is not the PDGFRa SP) at positions 1-20, (ii) the PDGFRa ECD at positions 26-530 with the 139 and 206 mutations (numbering of the mutations according to SEQ ID NO: 1 as in the entire disclosure), (iii) the Fc domain of human IgGl at positions 533-759, and (iv) non-functional (with respect to effects described herein) residues which are remnants of the recombination process at positions 21-25 and 531-532.
  • the fusion protein comprises or consists of an amino acid sequence according to positions 26-530 of SEQ ID NO: 2 and an amino acid sequence according to positions 533-759 of SEQ ID NO: 2, or variants of these amino acid sequences.
  • it comprises or consists of an amino acid sequence according to positions 26-530 of SEQ ID NO: 2 and an amino acid sequence according to positions 533-759 of SEQ ID NO: 2, or variants of these amino acids sequences, as well as any signal peptide (preferably any that directs the fusion protein into the endoplasmic reticulum of a cell, e.g.
  • Linking sequences are up to 20, preferably up to 15 or 10 or more preferably up to 5 amino acids long.
  • the fusion protein comprises or consists of an amino acid sequence according to SEQ ID NO: 2 or a variant thereof.
  • Variants have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (higher identities being preferred over lower ones) to the sequence of polypeptide they vary (here SEQ ID NO: 2 or parts thereof). Variants maintain the 139 and 206 mutations and all other mutation that may be intended for the PDGFRa polypeptide of the first aspect, in as far as they include the corresponding positions.
  • the invention relates to a multimer comprising at least two fusion proteins of the second aspect. This is preferably afforded by the fusion protein comprising a multimerization domain as described above.
  • the multimer is a dimer.
  • the dimer preferably is a homodimer, but it may also be a heterodimer.
  • the present invention relates to a nucleic acid encoding the PDGFRa polypeptide of the first aspect, the fusion protein according to the second aspect, or one or more fusion proteins forming the multimer of the third aspect. It is preferred that the nucleic acid encoding the PDGFRa polypeptide of the first aspect encodes also a (any) signal peptide preferably one directing to the endoplasmic reticulum of a cell, more preferably the PDGFRa SP or the signal peptide of SEQ ID NO: 2 or a variant thereof with at least 80%, at least 90% or preferably at least 95% sequence identity.
  • the present invention relates to a vector comprising the nucleic acid of the fourth aspect.
  • Any suitable vector known in the art can be used, preferably it is a DNA vector e.g. an expression vector like a plasmid. Examples are given further below.
  • the present invention relates to a cell comprising the nucleic acid of the fourth aspect or the vector of the fifth aspect.
  • the cell may be any prokaryotic or eukaryotic cell.
  • the cell secretes the PDGFRa polypeptide of the first aspect, the fusion protein of the second aspect or the multimer of the third aspect.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the PDGFRa polypeptide of the first aspect, the fusion protein of the second aspect, the multimer of the third aspect, the nucleic acid of the fourth aspect, the vector of the fifth aspect or the cell of the sixth aspect, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may further comprise an anti- viral agent, e.g. an agent selected from the group consisting of Letermovir, Formiviren, Foscarnet, Cidofovir, Ganciclovir, Valganciclovir, a direct antiviral (DAA), a viral antigen, and an antibody to a viral antigen.
  • an anti- viral agent e.g. an agent selected from the group consisting of Letermovir, Formiviren, Foscarnet, Cidofovir, Ganciclovir, Valganciclovir, a direct antiviral (DAA), a viral antigen, and an antibody to a viral antigen.
  • an anti- viral agent e.g. an agent selected from the group consisting of Letermovir, Formiviren, Foscarnet, Cidofovir, Ganciclovir, Valganciclovir, a direct antiviral (DAA), a viral antigen, and an antibody to a viral antigen.
  • DAA direct antiviral
  • Preferred viral antigens of CMV are CMV complexes comprising gH and gL, such as the gH/gL/gO trimer or the gIT/gL/pUL128/pUL130/pUL131A pentamer, or one or more of the CMV proteins gH, gL, gO, pUL128, pUL130 and/or pUL131A.
  • the therapeutically effective amount results in a therapeutically effective concentration in blood of a subject that is suitable for neutralizing a virus, but does not detectably influence PDGF cellular signaling, i.e. it does not have any unspecific effects on cells.
  • Wide ranges of concentrations are possible since the PDGFRa polypeptide of the first aspect is engineered to maintain virus neutralization but to have a diminished PDGF binding capability.
  • Suitable steady state concentrations in blood are for example from about 0.3 nM to about 12 nM, from about 0.6 nM to about 10 nM, from about 1.5 nM to about 6 nM, or from about 3 nM to about 3.6 nM.
  • Such concentrations can be achieved, for example, by administering from about 0.03 mM to about 0.5 mM, from about 0.1 mM to about 0.3 mM, or from about 0.12 mM to about 0.2 mM per week in total, in one or more doses (e.g. daily or weekly doses).
  • the pharmaceutically acceptable carrier is not limited, definitions and examples can be found further below.
  • the invention relates to an in vitro method of treating a device or a composition, comprising the steps of:
  • PDGFRa agent refers to a PDGFRa polypeptide of the first aspect, a fusion protein of the second aspect, a multimer of the third aspect, or a pharmaceutical composition of the seventh aspect.
  • the device can be e.g. a medical device including an intravaginal device, and the composition, which is preferably liquid and more preferably aqueous, can be a body liquid such as blood or breast milk.
  • the in vitro method can be one of the following methods:
  • An in vitro method of coating a medical device, in particular an intravaginal device comprising the steps of:
  • Adherence is such that it is maintained during washing steps. More preferably, it is such that it is maintained within an animal, preferably human, body. Methods for conferring adherence are well-known in the art and are not limited herein.
  • An in vitro method of treating a body liquid comprising virus comprising the steps of (i) providing the body liquid, and (ii) contacting the body liquid with a PDGFRa agent such that vims comprised in the body liquid binds to the PDGFRa agent, and
  • vims preferably blood, more preferably human blood; e.g. from a donor
  • At least potentially comprising vims means that only the body liquid comprising vims is treated, or that body liquid for which it is unknown whether it comprises vims is treated for the event that it does comprise it. The latter would be a routine treatment applied to any body liquid (specifically blood) without ascertaining that it comprises vims. Any step of optionally removing the PDGFRa agent from the body liquid also removes all vims from the body liquid, which can be afforded by contacting the body liquid with an excess amount of PDGFRa agent (compared to vims comprised or at least potentially comprised, e.g. at least lOx, at least 50x, at least lOOx or at least lOOOx; larger x preferred over lower ones).
  • the treated medical device can be used to treat a patient that is infected with vims or to prevent infection of a patient or a child to be born, the treated blood can be used to prevent infection of a patient in need of blood, and the treated breast milk can be used to prevent infection of an infant in need of the breast milk.
  • the present invention relates to the PDGFRa polypeptide of the first aspect, the fusion protein of the second aspect, the multimer of the third aspect, the nucleic acid of the fourth aspect, the vector of the fifth aspect, the cell of the sixth aspect or the pharmaceutical composition of the seventh aspect for use in medicine.
  • the use in medicine includes various uses, such as the use of a medical device coated with a PDGFRa in particular an intravaginal device, e.g. produced with the method of the eighth aspect, or the use of a body liquid (preferably human breast milk or blood, e.g. of a donor, i.e. of a subject who will not receive the blood) treated with a PDGFRa agent to capture (decoy) vims, e.g. as described in the eighth aspect.
  • a body liquid preferably human breast milk or blood, e.g. of a donor, i.e. of a subject who will not receive the blood
  • a PDGFRa agent to capture (decoy) vims e.g. as described in the eighth aspect.
  • breast milk expressed from the adult body
  • this can be advantageous compared to methods like shortterm pasteurization, since there are concerns that short term pasteurization may destroy valuable components comprised in the milk.
  • the use as a medicament is in an amount or concentration that is therapeutically effective as described above.
  • the subject is preferably a mammal, more preferably human. In some embodiments, it is female and optionally pregnant. In other embodiments, the subject has a malfunctioning organ and is going to have transplant surgery, or the subject had transplant surgery (preferably within 3 months of the start administration of the medicament), or the subject is characterized by having recurring acute transplant rejections.
  • the use as a medicament is the use as a virus-decoying medicament.
  • the terms “virus-trapping”, “virus-sinking” and “virus-capturing” may be used instead to describe this function.
  • this use is a use as a viral entry inhibitor, since the decoying prevents cell-entry of decoyed viral particles.
  • Preferred specific uses may be one or more of the following:
  • infection refers to a viral infection, i.e. to the entry of a virus into at least one cell of a host and its replication within the at least one cell.
  • An infection may be acute (i.e. active) or, as e.g. in the case of CMV, also latent (i.e. inactive, hidden, dormant).
  • latent infection the virus is replicating, infects cells and potentially causes symptoms, whereas in a latent infection, the virus does not replicate independent from the host cell genome and infect further cells, it rather “hides” in a cell.
  • a latent infection can be interrupted by acute infections in which the hidden virus starts replicating and infecting further cells.
  • the use in preventing of an infection preferably relates to preventing an acute infection by preventing the hidden virus from infecting further cells, i.e. from spreading.
  • this can be described as a treatment of a latent infection, wherein the treatment is not curative (but keeps the virus in check).
  • infectious disease refers to a disease resulting from an infection.
  • the infectious disease is a viral infectious disease, i.e. a disease resulting from a viral infection (primary viral disease) or a “virus-associated diseases”, which is a related (secondary) disease that is caused or contributed to by a viral infection.
  • primary viral disease primary viral disease
  • virus-associated diseases which is a related (secondary) disease that is caused or contributed to by a viral infection.
  • virus-associated disease e.g.
  • a CMV-associated disease is selected from the group consisting of retinitis, encephalitis, ventriculitis, hepatitis, nephritis, cystitis, myocarditis, pancreatitis, esophagitis, colitis, pneumonia, atherosclerosis, neonatal infection sequelae, transplant rejection, mucoepidermoid carcinoma, glioblastoma, prostate, breast and colon cancer, a cardiovascular disease, a gastrointestinal disease, an acute or chronic inflammatory disease (including e.g. rheumatoid arthritis, and an age-related disease (including e.g. diabetes, particularly of type 2, immunosenescence and cognitive impairment).
  • Transmission of a virus to a fetus may occur when the mother is infected (or has an acute infection) with the virus during pregnancy and can be prevented.
  • Infections can be through the vagina, the cervix, the fallopian tubes, invasive procedures such as amniocentesis, or through the placenta (transplacental infection).
  • Prevention or treatment of virus infection can also prevent maternal pregnancy complications including preeclampsia.
  • Adverse pregnancy outcomes that can be due to preeclampsia include stillbirth, neonatal death, intrauterine growth restriction and preterm birth, and can therefore also be prevented.
  • Children bom with a viral infection may have one or more of the following secondary disorders (“neonatal sequelae”): hepatomegaly, splenomegaly, jaundice, pneumonitis, fetal growth retardation, petechiae, purpura, thrombocytopenia and/or major neurological sequelae including microcephaly, intracranial calcifications, sensorineural hearing loss (SNHL), vision loss, optic atrophy, strabismus, chorioretinitis, intellectual disabilities, motor disabilities, and/or seizure disorders.
  • SNHL sensorineural hearing loss
  • vision loss optic atrophy
  • strabismus chorioretinitis
  • intellectual disabilities motor disabilities, and/or seizure disorders.
  • the cancer is generally one which is adversely oncomodulated by the viral infection, with respect to CMV infection, e.g. glioblastoma or other cancers including but not limited to colon cancer, prostate cancer and breast cancer. This is based on known oncomodulatory and malignancy increasing effects of viral infection, including CMV infection, of tumour cells.
  • the transplant rejection can be an acute or chronic rejection. It is known that infection with pathogens including vimses, specifically CMV, increases morbidity and decreases transplant survival, and that control of the infection can control the transplant rejection.
  • An acute rejection usually occurs any time from the first week to 3 months after the infection, and in a preferred embodiment, the medical use according to the invention prevents an acute rejection.
  • a chronic rejection can take place over several years, during which the transplant is slowly damaged.
  • a chronic rejection usually comprises several episodes of acute rejections, and it is preferred that the medical use according to the invention treats a chronic rejection.
  • the route of administration is not particularly limited and can for example be oral, intravenous or subcutaneous.
  • the present invention relates to a polypeptide comprising a fragment of PDGFRa, wherein the fragment consists of a PDGFRa D2 domain and a PDGFRa D3 domain.
  • the polypeptide comprising the fragment may further comprise other polypeptides which are not domains of PDGFRa.
  • the fragment comprises the 139 and 206 mutations.
  • the polypeptide is a decoy receptor.
  • Parts of this aspect of the invention are further a fusion protein comprising the polypeptide, a multimer comprising the fusion protein, a nucleic acid encoding for the polypeptide, a vector comprising the nucleic acid, a cell comprising the nucleic acid or the vector, a pharmaceutical composition comprising the polypeptide, fusion protein, multimer, nucleic acid, vector or cell, and their (and of the polypeptide of the tenth aspect) use for binding a virus, specifically for use in medicine.
  • All definitions and embodiments described above with regard to the first to ninth aspect apply here, not necessarily limited to the 206 and 139 mutations, in as far as they are applicable.
  • PDGFRa platelet-derived growth factor receptor a
  • CD 140a The human PDGFRA gene, which is located on the long arm of chromosome 4 (4ql2), encodes a 1089 amino acid protein precursor (platelet-derived growth factor receptor alpha isoform 1 precursor, SEQ ID NO: 1), which is processed into a mature polypeptide consisting of amino acids 24-1089.
  • the extracellular portion of PDGFRa interacts with ligands of the platelet-derived growth factor (PDGF) family, in particular with the homo- or heterodimers PDGF-AA, -AB, -BB, and -CC.
  • fusion protein relates to a protein comprising two or more polypeptides, preferably functional domains, derived from different proteins.
  • the two or more polypeptides are linked directly or indirectly by peptide bonds.
  • a fusion protein is generated by joining two or more nucleic acid sequences. This can be done recombinantly and also via nucleic acid synthesis. Translation of this fusion construct results in a single protein with the functional properties derived from the two or more polypeptides.
  • Fc domain refers to the crystallisable fragment of the constant region of an antibody. Suitable Fc domains may be derived from IgG, IgA, IgD or IgM antibody isotypes.
  • Knob-into-hole mutations are amino acid substitutions in order to create a “knob” on one CH3 domain and a “hole” on the other CH3 domain.
  • Various knob-into-hole mutations are known in the art, for instance the knob is represented by a tyrosine (Y), whereas the hole is represented by a threonine (T).
  • knob-into-hole mutations are T366Y in one CH3 domain and Y407T in the other, wherein the two CH3 domains are IgGl constant domains, and optionally wherein the Fc region comprising the T366Y mutation ("knob" chain) further comprises the mutations S354 and T166W and the Fc region comprising the Y407T mutation ("hole” chain) further comprises the mutations Y349C, T366S, L368A and Y407V.
  • variant is, with respect to polypeptides, to be understood as a polypeptide which differs in comparison to the polypeptide from which it is derived by one or more changes in the amino acid sequence.
  • the polypeptide from which a protein variant is derived is also known as the parent polypeptide.
  • the changes in the amino acid sequence may be amino acid exchanges, insertions, deletions, N-terminal truncations, or C-terminal truncations, or any combination of these changes, which may occur at one or several sites. Amino acid exchanges may be conservative (preferred) or non-conservative.
  • a “variant” herein is characterized by a certain degree of sequence identity (as provided above) to the parent polypeptide from which it is derived.
  • identity refers to the number of residues in the two sequences that are identical when aligned for maximum correspondence. Specifically, the percent sequence identity of two sequences is the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. Alignment tools that can be used to align two sequences are well known to the person skilled in the art and can, for example, be obtained on the World Wide Web, e.g. Needle (EMBOSS) (https://www.ebi.ac.uk/Tools/psa/emboss_needle/), MUSCLE
  • the alignments between two sequences may be carried out using default parameters settings, e.g.
  • MATRIX BLOSUM62, Gap Open: 10.0, Gap Extend: 0.5
  • MAFFT Matrix: Blosum62, Gap Open 1.53, Gap Extend 0.123
  • WATER polynucleotides preferably: MATRIX: DNAFULL, Gap Open: 10.0, Gap Extend 0.5 and for WATER polypeptides preferably MATRIX: BLOSUM62, Gap Open: 10.0, Gap Extend: 0.5.
  • the "best sequence alignment" is defined as the alignment that produces the largest number of aligned identical residues while having a minimal number of gaps. Preferably, it is a global alignment, which includes every residue in every sequence in the alignment.
  • nucleic acid includes RNA and DNA, but preferably refers to DNA.
  • vector includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus (Ad) vectors), adeno-associated virus (AAV) vectors, alphavirus vectors (e.g., Venezuelan equine encephalitis virus (VEE), Sindbis virus (SIN), semliki forest virus (SFV), and VEE-SIN chimeras), herpes virus vectors, measles virus vectors, pox virus vectors (e.g., vaccinia virus, modified vaccinia virus Ankara (MV A), NYVAC (derived from the Copenhagen strain of vaccinia), and avipox vectors: canarypox (ALVAC) and fowlpox (FPV) vectors), and vesicular stomatitis virus vectors, or virus like particles.
  • Ad adenovirus
  • AAV adeno-
  • virus-like particle refers to a non-replicating, empty viral shell.
  • VLPs are generally composed of one or more viral proteins, such as, but not limited to those proteins referred to as capsid, coat, shell, surface and/or envelope proteins. They contain functional viral proteins responsible for cell penetration by the virus, which ensures efficient cell entry. Methods for producing particular VLPs are known in the art.
  • prokaryotic cell refers to any kind of bacterial organism suitable for application in recombinant DNA technology such as cloning or protein expression includes both Gram-negative and Gram-positive microorganisms.
  • Preferred is Escherichia (in particular A. coli).
  • a eukaryotic cell is in particular a yeast or an animal cell.
  • a yeast cell can be, in the broadest sense, any cell of a yeast organism.
  • the yeast cell is a Saccharomyces cell, in particular a Saccharomyces cerevisiae cell.
  • An animal cell may be a cell of a primate, mouse, rat, rabbit, dog, cat, hamster, cow, insect (e.g. Sf9 or Sf21) etc., preferably a human. Also, it may be a suspension or an adherent cell. In one embodiment, the adherent cell is a monolayer cell.
  • the cell is selected from the group consisting of a hybridoma cell, a primary epithelial cell, an endothelial cell, a keratinocyte, a monocyte/macrophage, a lymphocyte, a hematopoietic stem cell, a fibroblast, a chondrocyte and a hepatocyte.
  • a CHO-K1 SV cell a CHO DG44 cell, a CHO DP- 12 cell, a CHO DHFR cell, a CHO-GS cell, a BHK-21 cell, a HEK-293 embryonic kidney cell, a HeLa cervical epithelial cell, a PER- C6 retinal cell, an MDCK cell, an HDMEC cell, a HepG2 cell, an HL-60 cell, an HMEC-1 cell, a HUVEC cell, an HT1080 cell, a Jurkat cell, an MRC5 cell, a K562 cell, a HeLa cell, an NS0 cell, an Sp20 cell, a COS cell, and a VERO cell.
  • pharmaceutically acceptable carrier refers to any substrate which serves to improve the selectivity, effectiveness, and/or safety of drug administration.
  • Such carriers can be used to control the release of a drug into systemic circulation. This can be accomplished either by slow release of the drug over a long period of time (typically diffusion) or by triggered release at the drug's target by some stimulus, such as changes in pH, application of heat, and activation by light.
  • Carriers can also be used to improve the pharmacokinetic properties, specifically the bioavailability, of many drugs with poor water solubility and/or membrane permeability.
  • a wide variety of drug carrier systems have been developed and studied.
  • Examples include liposomes, polymeric micelles, microspheres, nanoparticles, nanofibers, protein-drug conjugates, erythrocytes, virosomes and dendrimers.
  • Different methods of attaching the drug to the carrier can be used, including adsorption, integration into the bulk structure, encapsulation, and covalent bonding.
  • HFFs Primary human foreskin fibroblasts
  • HEK 293 F suspension cells were propagated in 293 Freestyle medium (Thermo) supplemented with 100 pg/ml gentamicin under 8% C02 and agitation at 130 rpm to a maximal density of 3> ⁇ 10 6 cells/ml. Human sera were obtained from adult healthy seronegative donors.
  • HCMV TB40-BAC4-IE-GLuc a BAC-cloned reporter virus based on strain TB40/E that expresses Gaussia luciferase under control of the major immediate early (IE) promotor/enhancer (Falk et al. 2016, Journal of Virological Methods 235 (September): 182-89).
  • IE immediate early
  • Virus stocks were harvested 5 to 7 days after infection of HFFs. The virus was cleared from cells and cell debris by centrifugation for 10 min at 2,700 x g. To additionally remove the luciferase released by the producer cells, the virus particles were ultra-centrifuged for 70min at 100, 000 /g. The virus pellet was immediately resuspended in medium and stored at -80°C until used.
  • the extracellular domain (corresponding to aa 24 to 528) of PDGFRa (CD140a) has been cloned previously into pFuse-hIgGl-Fc2 as described previously (Stegmann et al. 2019, Journal of Virology 93 (11)).
  • Mutagenesis primers were designed using the Agilent primer design tool, because the mutagenesis was performed using the QuikChange Lightning site- directed mutagenesis kit (Agilent).
  • the template plasmid was amplified with primers containing the desired mutation, then the template DNA was digested with Dpn I and transformed into XL 10 chemically competent cells. Sanger sequencing of the PDGFR sequence was performed to exclude unwanted secondary changes. Expression and purification of soluble PDGFRa-Fc
  • Soluble PDGFR-alpha-Fc fusion proteins were transiently expressed from HEK 293 cells.
  • 293 cells were diluted to 5x 10 5 cells per ml in gentamycin-free medium.
  • 1 pg of sterile DNA and 3 pg of polyethylenimine (PEI, 25K linear, Polysciences #23966) per 10 6 cells were mixed in serum-free Opti-MEM (Thermo) and incubated for 15min before the mixture was added to the cells. Incubation for 6 hours, then a medium exchange was performed.
  • the PDGFRa-Fc containing medium was harvested at day 5 or 6 after transfection by pelleting the cells at 2,700 g for 10 min.
  • the supernatants were filtered through a 0.22 pm filter prior to concentrating the proteins with centrifugal filters pore size lOOkDa (Amicon).
  • the PDGFR-Fc fusion proteins were then purified using Protein A beads.
  • the proteins were eluted from the beads with an elution buffer of pH 2.8 (Thermo #21004) and stored in the same buffer supplemented with 0.1 M Tris pH 8.0 at 4°C.
  • the purified proteins were quantified photometrically and for additional quality control, all protein preparations were checked by gel electrophoresis followed by visualization using TCE (2,2,2-trichloroethanol).
  • HFFs were seeded on 96well plates at a density of 15,000 cells per well.
  • HCMV TB40-BAC4-IE-GLuc virus stocks were diluted to obtain a final infection rate of about 50% in the untreated sample.
  • These virus dilutions were then mixed with either only maintenance medium (untreated control) or with serial dilutions of PDGFRa-Fc proteins.
  • the virus-inhibitor mixtures were incubated for 2 h at 37°C, before they were added to the cells for infection.
  • the percentage of inhibition was determined either by quantification of Gaussia-luciferase activity in the supernatants or by immunofluorescence staining of the cells.
  • Gaussia luciferase for quantification of infection has been described previously (Falk et al. 2016, Journal of Virological Methods 235 (September): 182-89). Briefly, the Gaussia luciferase-containing cell culture supernatants were either stored at -20°C or mixed immediately with the luciferase substrate coelenterazine (PjK GmbH). Coelenterazine was diluted to 0.2 pg/ml in phosphate buffered saline with 5 mM NaCl. The substrate was added to the cell culture supernatants automatically in a plate reader (Hidex Chameleon) to ensure timely measurement of the luminescence signal.
  • the percentage of infection was calculated from the resulting luminescence signals by normalization to the untreated virus control.
  • the cells were fixed with 80% acetone for 5 min at room temperature, before sequential incubation with an antibody against the viral immediate early antigens (E13, Argene) and secondary antibody goat anti-mouse IgG F(ab') 2 -Cy3 (Jackson ImmunoRe search). Cell nuclei were stained with 4',6-Diamidin-2- phenylindol (DAPI). Quantification was performed using Zen software (Zeiss).
  • HFFs Stimulation of cellular signaling: HFFs were seeded at a density of 100,000 cells per well in maintenance medium onto 24well plates. About 4 hours after seeding, when the cells had adhered to the plates, a medium exchange was performed and the FBS-containing medium was replaced by MEM without any additives. The cells were kept in serum- and growth factor- free medium for 1 day. Prior to stimulation of the cells, PDGF-BB (R&D # 220-BB) was diluted in MEM to 12 ng/ml and then mixed 1 : 1 with different concentrations of PDGFRa-Fc, resulting in a final PDGF concentration of 6 ng/ml. For stimulation with human sera, the sera were diluted to a final concentration of 5%.
  • PDGF-BB R&D # 220-BB
  • Ligand/Serum and receptor were preincubated at 37°C for 2h. Then the mixture was added to the serum starved HFFs and incubated for 15min at 37°C to allow signaling, cumulating in Akt phosphorylation. The stimulation was terminated by lysis of the cells for either immunoblot analysis or ELISA. Please note that the protocol described here was optimized for increased sensitivity. The first experiments using alanine exchange mutants (Fig. 2) had been performed under slightly different conditions, using 50 ng/ml of PDGF-BB and stimulating the cells for 2h.
  • Akt phosphorylation by immunoblot The cells were lysed in laemmli lysis buffer with b-mercaptoethanol on ice, then scraped off the plate and boiled for 10 min at 95°C. An equivalent of 28,500 cells was loaded onto 10% polyacrylamide gels and electrophoresis was performed in tris glycine SDS buffer. The proteins were transferred onto PVDF membranes in tris-glycine buffer with 15% methanol. Membranes were blocked with TBS plus 0.1 % Tween and 5 % milk powder before staining with rabbit anti-phospho-Akt (Cell Signaling #4060) and rabbit anti-actin (Sigma # A 5060).
  • the cells of each well were lysed with 150 m ⁇ of ice-cold cell lysis buffer containing 1 mM phenylmethylsulfonyl fluoride for 5 min on ice. Then the lysate was homogenized using a syringe before the insoluble parts were removed by centrifugation for 5 min at 18,800xg. The samples were stored at -80°C until the assay was performed. 100 m ⁇ of undiluted sample per well was added to a microwell plate coated with phospho-Akt (Ser473) rabbit antibodies. After washing off unbound lysate, the samples were incubated subsequently with Aktl mouse antibody and HRP conjugated anti-mouse antibody. Detection was performed at 450 nm in a plate reader (Hidex Chamaeleon) after incubation of the sample with TMB substrate for 10 min.
  • Binding affinities were quantified using Monolith NT.115 (NanoTemper) instrumentation and analysis was performed using inbuild MO. Affinity Analysis Software. To allow for fluorescence detection, PDGF-BB (R&D # 220-BB) was labelled with NT-647 using the amine reactive protein labelling kit RED-NHS (NanoTemper) according to the manufacturer’s instructions. For all measurements, PDGF-BB was used at a constant concentration of 0.1 nmol/1. To allow for proper curve fitting, the PDGFRa-Fc variants were added in 16 different concentrations spanning 51og steps.
  • a standard microscale thermophoresis (MST) measurement was used to calculate the K D for PDGF binding. For this, after 5 seconds of photobleaching, the sample was heated and the change of fluorescence was recorded for 7 seconds. To generate dose response curves, the normalized difference between the steady state (4 sec after heating (FI)) and the initial fluorescence (F0) was plotted against the respective PDGFRa-Fc concentration to calculate the K D . All experiments were performed at 25°C, medium MST power and with 60% excitation power.
  • Example 1 Deletions of predicted PDGF binding sites in PDGFRa-Fc differentially affect HCMV inhibition
  • the capacity of the PDGFRa-Fc mutants to inhibit HCMV infection was assessed with a standard neutralization assay utilizing a Gaussia luciferase reporter virus (TB40-BAC4-IE-GLuc, Falk et al. 2016, Journal of Virological Methods 235 (September): 182-89).
  • Cell- and luciferase-cleared preparations of the virus were diluted to a MOI of ⁇ 1 (resulting in less than 68% infection) and preincubated with various concentrations (4,000 to 1 ng/ml) of purified PDGFRa-Fc. After an incubation period of 2 hours at 37°C, the virus was incubated with the fibroblasts for 2 hours. One day later, the luciferase- containing medium was collected and the Gaussia activity therein was measured. The extent of infection initiated by the pretreated virus was quantified relative to untreated control samples.
  • Wild type PDGFRa-Fc inhibited HCMV at a halfmaximal effective concentration (EC50) of ⁇ 20 ng/ml and fully blocked HCMV inhibition already at 100 ng/ml. All deletions reduced the inhibitory effect of PDGFRa-Fc to some extent, as indicated by a shift of the dose- response towards higher concentrations (Fig. IB). With four out of seven mutations a concentration of 4,000 ng/ml was insufficient to achieve full inhibition. Yet, PDGFRa-Fc deletion mutants delM133-I139, delN204-Y206 and delQ294-E298 completely blocked HCMV entry at 2000 ng/ml (Fig. 1C).
  • Example 2 Mutations of isoleucine 139 and tyrosine 206 in PDGFRa-Fc reduce sequestration of PDGF-BB
  • the next step was to test whether any of those mutations would reduce interference with cellular signaling.
  • a biological assay was preferred over biochemical measurements of protein-protein interaction.
  • the effect of soluble PDGFRa-Fcs on PDGF-dependent signaling in cells was measured directly.
  • PDGFRa can bind to a variety of different growth factors including PDGF-A, PDGF-B and PDGF-C, which will start signaling cascades that include phosphorylation of Akt. Therefore, the effect of soluble PDGFRa-Fc on Akt phosphorylation was chosen as a downstream measure of changes in binding to growth factors.
  • PDGF-BB Dimeric PDGF-B at a final concentration of 50 ng/ml was preincubated with different concentrations of PDGFRa-Fc wild type and mutants for 2 hours at 37°C before stimulation of fibroblasts. To reduce background phosphorylation, the cells were serum-starved prior to addition of the PDGF-B/PDGFRa-Fc mixtures. After stimulation for 90 min, the cells were lysed and Akt phosphorylation was determined in immunoblots (Fig. 2C). It was observed that those mutations that reduced inhibition of HCMV also reduced sequestration of PDGF-B.
  • Example 3 Selected permutations at positions 139 and 206 of PDGFRa improve the interaction profile of the soluble receptor
  • Isoleucine to Valine exchange should maintain the non-polar character of the site, yet due to the smaller side chain might preclude hydrophobic interactions that are regarded important for PDGFRa-PDGF binding.
  • Isoleucine to glutamic acid exchange in contrast does not change the size much but alters it ' s biochemical characteristic to polar and preferentially surface exposed, potentially disrupting the structural integrity.
  • isoleucine with leucine which is a very mild change, only affecting the stereoisomerism of the side chain.
  • HCMV inhibition For measurement of HCMV inhibition the virus was preincubated with the soluble receptors before incubation with the cells for 1 day. To obtain even more precise data, infection was measured by immunofluorescence staining for the viral immediate early antigens instead of by luciferase activity.
  • Replacement of isoleucine 139 with leucine or valine had no or only a marginal effect as compared with wild type PDGFRa-Fc (Fig. 3 A and B). The replacement with glutamic acid decreased the efficiency of HCMV inhibition 5fold, comparable to the alanine exchange mutant.
  • the protocol of the assay was also adjusted to obtain higher sensitivity which was important because the alanine exchange mutants had already reduced PDGF sequestration close to the limits of detection.
  • the dose-response relationship of cellular Akt phosphorylation after treatment of cells with different concentrations of PDGF-B was determined (Fig. 4).
  • a concentration of 6 ng/ml PDGF-B (about its concentration in human blood) was found to result in strong Akt phosphorylation below saturation, increasing the assays sensitivity by limiting the amount of stimulant.
  • tyrosine 206 was identified as another important site for interaction with PDGF-B. Therefore, analogous to the permutation strategy employed for isoleucine 139, the influence of size and charge at position 206 of PDGFRa-Fc on its interactions with HCMV and PDGFs was investigated. Tyrosines have polar, aromatic side chains that are often involved in stacking interactions. Phenylalanine similarly has an aromatic side chain that is however non-polar, whereas serine is polar but much smaller without an aromatic ring. As described for the 1139 permutations, the Y206F and Y206S PDGFRa-Fc mutants were analyzed regarding inhibition of HCMV and interference with PDGF-dependent signaling.
  • Y206F did not improve interference as compared to the alanine exchange mutant, in contrast Y206S significantly decreased the inhibition of PDGF-dependent cellular signaling as compared to wild type and the alanine mutant. No inhibition of Akt phosphorylation at all was observed even at 36,000 ng/ml. The same was true when Akt phosphorylation was quantified by ELISA. It is noteworthy that PDGFRa-Fc Y206S, when applied at very high concentrations (greater than 4,000 ng/ml), slightly enhanced cellular signaling. Comparison at lower concentrations, that are more likely to be used against HCMV (i.e. 562 ng/ml), however allowed to quantify the difference between PDGFRa-Fc wild type and mutant.
  • PDGFRa-Fc Y206S caused almost no reduction in Akt phosphorylation whereas wild type led to near complete inhibition of cellular signaling.
  • no EC50s could be determined for this PDGF sequestration by PDGFRa-Fc.
  • PDGFRa-Fc already inhibits HCMV potently at low concentrations it is not necessary to use high concentrations that can lead to unspecific effects on the cells and the same is true for PDGFRa-Fc I139E + Y206S. Furthermore, the inventors observed that this increase in signaling at high concentrations (about 50fold above the EC50 for HCMV inhibition) can be avoided also by combining the Y206 substitution or the I139+Y206 substitutions with a V242 substitution such as V242K, see Example 6.
  • PDGF-BB was fluorescently labelled with the dye NT-647.
  • a dose-dependent decrease of fluorescence was observed (Fig. 7 A to C and Fig. 8 A to D), which can be explained by quenching of the ligand’s fluorescence upon binding of the receptor.
  • the single amino acid exchange mutants I139E and Y206S as well as the double alanine exchange mutant decreased the affinity for PDGF-BB by 30 to 50fold to 4.0, 7.3 and 4.6 nmol/1, respectively (Fig. 7 A to D).
  • the combination of I139E and Y206S however did not cause any measurable change in PDGF-BB fluorescence, indicating loss of binding or strongly reduced affinity.
  • thermophoresis was performed to calculate the affinity of I139E + Y206S mutant for PDGF- BB (Fig. 7 E, F and Fig. 8 E, F).
  • Example 5 Serum-induced cellular signaling is unaffected by treatment with the modified PDGFRa-Fc
  • the improved PDGFRa-Fc presented here is a promising molecule for neutralization of HCMV because it is can be expected to have desirable pharmacokinetics, it is most likely non-immunogenic, probably unlikely to induce resistance mutations, and it neutralizes various strains of HCMV and different cell types efficiently. It is conceivable that PDGFRa-Fc alone or in combination with anti-pentamer antibodies may be favorable as compared to known antibody combinations. The identification of mutations which prevent interference with PDGFs now pave the way for further development of this decoy receptor against HCMV infection.
  • a set of PDGFRa-Fc mutants was generated: a V242K single mutant, as well as combinations of V242K with I139E and Y206S. Like with the previous PDGFRa-Fc mutants, all of them were expressed as efficient as wild type and did not show any signs of degradation or aggregation. After purification, these mutants were first tested regarding their ability to inhibit HCMV infection.
  • V242K mutation did not affect HCMV inhibition ( Figure 10).
  • Introduction of V242K into PDGFRa-Fc I139E or Y206S did also not significantly alter the efficiency of HCMV inhibition.
  • the I139E + V242K mutant had an EC50 of 27 ng/ml as compared to 33 ng/ml for I139E, and Y206S + V242K caused half-maximal inhibition of HCMV at 47 ng/ml as compared to 31 ng/ml with Y206S.
  • PDGF- BB was pretreated with different concentrations of the PDGFRa-Fc mutants (2-500 ng/ml) and the effect on PDGF-dependent signaling was assessed with a p-Akt ELISA.
  • PDGFRa-Fc V242K mutations caused any reduction in Akt phosphorylation.

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Abstract

La présente invention relève du domaine de la pharmacologie et concerne spécifiquement des polypeptides PDGFRα qui peuvent être utilisés en tant que récepteurs leurres viraux. Elle comprend des protéines de fusion comprenant ces polypeptides ainsi que des acides nucléiques, des vecteurs, des cellules et des compositions pharmaceutiques. L'utilisation en médecine est spécifiquement envisagée pour la prévention et le traitement d'infections à CMV.
PCT/EP2021/069644 2020-07-14 2021-07-14 POLYPEPTIDES PDGFRα EN TANT QUE RÉCEPTEURS LEURRES VIRAUX WO2022013304A1 (fr)

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WO2018002081A1 (fr) 2016-06-27 2018-01-04 Aicuris Anti-Infective Cures Gmbh Inhibiteurs d'entrée de hcmv.

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Publication number Priority date Publication date Assignee Title
WO2018002081A1 (fr) 2016-06-27 2018-01-04 Aicuris Anti-Infective Cures Gmbh Inhibiteurs d'entrée de hcmv.

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
"Helvetica Chimica Acta", 1995, article "A multilingual glossary of biotechnological terms: (IUPAC Recommendations"
CORA STEGMANN ET AL: "A derivative of platelet-derived growth factor receptor alpha binds to the trimer of human cytomegalovirus and inhibits entry into fibroblasts and endothelial cells", PLOS PATHOGENS, vol. 13, no. 4, 15 May 2019 (2019-05-15), pages e1006273, XP055755207, DOI: 10.1371/journal.ppat.1006273 *
DATABASE UniProt [online] 26 November 2014 (2014-11-26), "RecName: Full=Platelet-derived growth factor receptor alpha {ECO:0000256|PIRNR:PIRNR500950}; Short=PDGF-R-alpha {ECO:0000256|PIRNR:PIRNR500950}; Short=PDGFR-alpha {ECO:0000256|PIRNR:PIRNR500950}; EC=2.7.10.1 {ECO:0000256|PIRNR:PIRNR500950}; AltName: Full=Alpha platelet-derived growth factor receptor", XP002801281, retrieved from EBI accession no. UNIPROT:A0A091D875 Database accession no. A0A091D875 *
FALK ET AL., JOURNAL OF VIROLOGICAL METHODS, vol. 235, September 2016 (2016-09-01), pages 182 - 89
JIHYE PARK ET AL: "Engineered receptors for human cytomegalovirus that are orthogonal to normal human biology", PLOS PATHOGENS, vol. 16, no. 6, 19 June 2020 (2020-06-19), pages e1008647, XP055755225, DOI: 10.1371/journal.ppat.1008647 *
STEGMANN ET AL., JOURNAL OF VIROLOGY, vol. 93, no. 11, 2019
WU ET AL., PNAS, vol. 115, no. 42, 2018, pages E9889 - 98

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