ADAMTS13 PROTEIN VARIANTS AND USES THEREOF
20230242896 · 2023-08-03
Inventors
- Johannes Jacobus VOORBERG (Wormer, NL)
- Nuno Alexandre GOMES GRAÇA (Amsterdam, NL)
- Bogaç ERÇIG (Amsterdam, NL)
Cpc classification
A61P7/02
HUMAN NECESSITIES
International classification
Abstract
The invention relates to ADAMTS13 protein variants comprising residues 1 to 685 of ADAMTS13 and wherein one or more N-linked glycosylation sites are added as compared to wild-type ADAMTS13 and/or one or more existing N-linked glycosylation sites are shifted as compared to wild-type ADAMTS13. The invention further relates to compositions comprising such ADAMTS13 variants, methods for their preparation and uses thereof, in particular as an antithrombotic agent, and in the treatment of thrombotic disease, thrombotic microangiopathy, thrombotic thrombocytopenic purpura (TTP), hemolytic—uremic syndrome (HUS), ischemic stroke, systemic thrombosis, COVID19, antiphospholipid syndrome, pre-eclampsia/HELLP syndrome, sepsis and sickle cell disease.
Claims
1. An ADAMTS13 protein variant comprising residues 1 to 685 of ADAMTS13 and wherein one or more N-linked glycosylation sites are added as compared to wild-type ADAMTS13 and/or one or more existing N-linked glycosylation sites are shifted as compared to wild-type ADAMTS13 in a spacer domain comprising amino acid residues S556 to A685 of ADAMTS13 as shown in
2. The ADAMTS13 protein variant according to claim 1 comprising residues 1 to 1427 of ADAMTS13.
3. The ADAMTS13 protein variant according to claim 1, wherein one or more N-linked glycosylation sites are added as compared to wild-type ADAMTS13 and/or one or more existing N-linked glycosylation sites are shifted in part of the spacer domain comprising residues R568 to R670 of the ADAMTS13 sequence, as shown in
4. The ADAMTS13 protein variant according to claim 1, comprising an N-linked glycosylation site at an amino acid residue of ADAMTS13 as shown in
5. (canceled)
6. The ADAMTS13 protein variant according to claim 1, comprising an N-linked glycosylation site at amino acid residue 608 of ADAMTS13 as shown in
7. The ADAMTS13 protein variant according to claim 1, having proteolytic activity against Von Willebrand Factor (VWF) that is at least 10% of the proteolytic activity against VWF of wildtype ADAMTS13 protein.
8. The ADAMTS13 protein variant according to claim 1 comprising an N-linked glycosylation site comprising an amino acid residue selected from the group consisting of R568, L591, V604, V605, A606, G607, K608, M609, R636, L637, P638, R639, Y665, L668 and combinations thereof, preferably comprising a mutation selected from the group consisting of R568N, L591N, V604N, V605N, A606N, G607N, K608N, M609N, R636N, L637N, P638N, R639N, Y665N, L668N and combinations thereof.
9. The ADAMTS13 protein variant according to claim 1, comprising a mutation selected from the group consisting of 568REY570 to 568NET570 (NGLY1), 591LFT593 to 591NFT593 (NGLY2), 608KMSI611 to 608NMSI611 (NGLY3), 608KMSI611 to 608KNST611 (NGLY4), 636RLPR639 to 636NLSR639 (NGLY5), 636RLPL639 to 636RNAS639 (NGLY6), 665YGNL668 to 665NVTL668 (NGLY7), 667NLTRP671 to 667LNVTA671 (NGLY8), and combinations thereof.
10. (canceled)
11. The ADAMTS13 protein variant according to claim 1, comprising mutation 608KMSI611 to 608NMSI611 (NGLY3).
12. The ADAMTS13 protein variant according to claim 1, further comprising a further mutation at one or more amino acid residues.
13. The ADAMTS13 protein variant according to claim 12, wherein said mutation at one or more amino acid residues is in the spacer domain comprising residues 5556 to A685 of ADAMTS13 as shown in
14. The ADAMTS13 protein variant according to claim 1, further comprising a mutation at an amino acid residue selected from the group consisting of R568, L591, F592, R636, L637, L668, L591, F592, R636, L637, R660, Y661, Y665. L668 and combinations thereof, preferably comprising a mutation selected from the group consisting of R568K, R568A, R568N, L591A, F592Y, F592A, F592N, R636A, L637A, R660K, R660A, R660N, Y661F, Y661A, Y661N, Y665F, Y665A, Y665N, L668A and combinations thereof.
15. (canceled)
16. The ADAMTS13 protein variant according to claim 1, comprising mutations R568A and Y665A or mutations L591A, R636A, L637A, and L668A.
17. The ADAMTS13 protein variant according to claim 1, comprising an N-glycan at said one or more N-linked glycosylation sites that are added and/or wherein said one or more existing N-linked glycosylation sites that are shifted comprise an N-linked glycan.
18. A nucleic acid construct comprising a nucleic acid sequence encoding an ADAMTS13 protein variant according to claim 1.
19. A pharmaceutical composition comprising an ADAMTS13 protein variant according to claim 1 and one or more pharmaceutically acceptable carriers, adjuvants, excipients and/or diluents.
20.-22. (canceled)
23. A method for the treatment of a disorder characterized by aberrant Von Willebrand Factor (VWF) activity and/or VWF processing comprising administering to a subject in need thereof a therapeutically effective amount of an ADAMTS13 protein variant according to claim 1.
24. (canceled)
25. The method according to claim 23, wherein said disorder is a thrombotic disease.
26. The method according to claim 25, wherein said thrombotic disease is a thrombotic microangiopathy or a disorder selected from the group consisting of thrombotic thrombocytopenic purpura (TTP), including immune-mediated TTP (iTTP), hemolytic-uremic syndrome (HUS), ischemic stroke, systemic thrombosis, COVID19, antiphospholipid syndrome, pre-eclampsia/HELLP syndrome, sepsis and sickle cell disease.
27. (canceled)
28. A method for producing an ADAMTS13 protein variant according to claim 1, comprising introducing a nucleic acid molecule comprising a nucleic acid sequence encoding said ADAMTS13 protein variant into a host cell capable of N-linked glycosylation, preferably an eukaryotic host cell, and culturing said host cell under conditions that allow expression of said ADAMTS13 protein variant preferably wherein the host cell is a mammalian cell, preferably selected from the group consisting of CHO cells, NS0 cells, SP2/0 cells, PERC.6 cells and HEK293 cells.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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: galactose;
: fucose;
: sialic acid.
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EXAMPLES
Example 1. Design of N-Glycan Variants of ADAMTS13
[0119] Autoantibodies that develop in patients with immune TTP (iTTP) are frequently directed towards an immunodominant region in the spacer domain that is composed of residues R568, F592, R660, Y661 and Y665 (see
TABLE-US-00001 TABLE 1 List of generated full-length ADAMTS13 NGLY variants in Example 1. Original Mutated Mutation sequence sequence NGLYl 567AREYV571 567ANETV571 NGLY2 590PLFTH594 590PNFTH594 NGLY3 607GKMSI611 607GNMSI611 NGLY4 608KMSIS612 608KNSTS612 NGLY5 635DRLPR639 635DNLSR639 NGLY6 636RLPRL640 636RNASL640
Example 2: Expression and Functional Characterization of N-Glycan Variants
[0120] The N-glycan variants described in Table 1 were expressed in CHO cells employing QMCF technology (described in European Patent EP1851319B1; www.icosagen.com). Full-length wild-type ADAMTS13 (1427 amino acids), and a full-length ADAMTS13 variant which contained the substitutions R568A/F592A/R660A/Y661A/Y665A (designated ADAMTS13-AAAAA) were used as controls. A wild-type ADAMTS13 variant truncated beyond the spacer domain (amino acid sequence 1-685) was used as an additional control; this ADAMTS13 variant was designated MDTCS. An additional MDCTS variant in which substitutions R568A/F592A/R660/Y661A/Y665A were present was also used as a control for our studies; this variant was designated MDTCS-AAAAA. These constructs have been described previously and were all cloned into the plasmid expression vector pQMCF3 (Icosagen Cell Factory OU) (Graca et al., 2019). All cDNAs contained a carboxy-terminal V5 epitope which was followed by a 6xHis-tag (Graça et al., 2019).
[0121] NGLY variants were constructed as follows: synthetic DNA fragments encoding residues M509 to W688 (540 bp) in which NGLY substitutions were introduced were designed and ordered by Genewiz (Leipzig, Germany). The synthetic fragments were flanked by an XmaI site at the 5′ end and a HindIII at the 3′ end of the fragment. The XmaI site is native to the wild-type ADAMTS13 cDNA sequence. The HindIII site was introduced by silent mutations in the nucleotide sequences encoding Q684 (CAG to CAA) and A685 (GCC to GCT) resulting in an overall change from 5′-CAGGCCT-3′ to 5′-CAAGCTT-3′. The plasmid pUC57_mut1.1 was custom-designed and obtained through Genewiz (Leipzig, Germany). In this plasmid was cloned a larger ADAMTS13 fragment coding from F494 to C908 (1245 bp), and flanked by the native sites PagI at 5′ and Esp3I at 3′. In pUC57_mut1.1, an additional artificial XhoI site was introduced by silent mutation of the nucleotide sequence encoding L621 (CTG to CTC), resulting in an overall change from 5′-CTGGAG-3′ to 5′CTCGAG-3′(Graça et al, 2019). The synthetic DNA fragments (540 bp) encoding NGLY1 to NGLY6 were first used to replace the corresponding XmaI-HindIII fragment in pUC57_mut1.1, where they were embedded individually to create pUC57_NGLY1-6. Then, the larger 1245 bp fragment flanked by native PagI-Esp3I in each pUC57_NGLY was used to replace the respective wild-type fragment of ADAMTS13 in pQMCF3. The resulting pQMCF3_ADAMTS13-NGLY1-6 variants were subsequently expressed in CHO cells as described previously (Graça et al., 2019). Supernatants were collected 10-12 days post transfection, cleared by centrifugation and stored at −30° C. until further use.
[0122] ADAMTS13 levels present in culture supernatant were quantified by ELISA using a previously established assay (Alwan et al., 2017; Graça et al., 2019). ADAMTS13 antigen levels measured for the different proteins ranged approximately from 1.0-2.5 μg/ml (see Table 2) and were similar to levels of wild-type ADAMTS13 in culture supernatants. In agreement with previous findings MDCTS and MDCTS-AAAAA were expressed at higher levels (Table 2). These data show that ADAMTS13-NGLY1-6 are secreted from transfected CHO cells at levels similar to that of wild type ADAMTS13.
TABLE-US-00002 TABLE 2 Antigen levels of ADAMTS13 NGLY variants and controls Variant Antigen level (μg/mL) Full-length ADAMTS13 wild-type 1.44 Full-length AAAAA 1.22 MDTCS (wild-type) 12.94 MDTCS-AAAAA 10.78 NGLY1 1.83 NGLY2 2.36 NGLY3 2.33 NGLY4 2.21 NGLY5 2.31 NGLY6 1.88
[0123] We also tested the ability of the different ADAMTS13-NGLY variants to process a small fluorogenic substrate designated FRETS-VWF73, the minimum peptide representative of the A2 domain of VWF harboring the Tyr1605-Met1606 scissile bond and to be used for ADAMTS13 activity assessment (Kokame et al., 2005). Diluted culture supernatants containing the ADAMTS13 NGLY variants at a concentration of 1.05 nM (0.2 μg/ml) were used for these assays. ADAMTS13 was first diluted in an activity buffer composed of 20 mM HEPES, 20 mM Bis-Tris, 20 mM Tris-HCl, 25 mM CaCl.sub.2) (pH 6.0) supplemented with 0.005% Tween20 to 2.10 nM in 100 μl volume. Then, the FRETS-VWF73 substrate was added (100 μl, 4 μM), diluting further ADAMTS13 to 1.05 nM, and the reaction started. In parallel, a calibration curve was done in a similar manner using the wild-type ADAMTS13 diluted in a concentration range of 0.13125-2.10 nM. The activity-levels were interpolated and compared to that of wild-type ADAMTS13 with final concentration of 1.05 nM, which was set at 100%. The results of this analysis are shown in
[0124] Subsequently, we tested the activity of the ADAMTS13-NGLY1-6 variants in a more physiological VWF multimer assay essentially as described previously (Graça et al., 2019). ADAMTS13 variants were incubated for 30 mins at 37° C. at a concentration of 0.2 μg in an activation buffer composed of 20 mM Bis-Tris, 20 mM Tris-HCl, 20 mM HEPES, 25 mM CaCl2 (pH 7.5), 0.005% Tween20 and supplemented with 2% Bovine Serum Albumin fraction V (Merck) (ADAMTS13=3.8 nM). In parallel, human recombinant VWF produced in HEK293 cells was incubated with 3 M urea for 30 min at 37° C. at a final concentration of 80 nM. Denatured recombinant VWF multimers were then added to the ADAMTS13 containing mixture at a 1 to 1 ratio (final ADAMTS13=1.9 nM; final VWF=40 nM). Capacity to cleave VWF in these conditions is visualized by the disappearance of High-Molecular Weight (HMW) multimers from the top of the gel, and accumulation of cleavage products seen through higher intensity of bands in the lower part of the gel, as well as the appearance of satellite bands. Samples were collected and quenched with 4× loading buffer (composition: urea 9.6 M, 4% SDS m/v, Tris-base 0.035 M, EDTA 25 mM, Bromophenol blue 7.5 μM, no pH adjustment) at 0 and 30 minutes and 24 hours to assess the ability of the different ADAMTS13-NGLY1-6 variants to process VWF. Results are shown in
Example 3. Binding of Pathogenic Autoantibodies from TTP Patients to ADAMTS13 NGLY Variants
[0125] Binding of autoantibodies present in a collection of samples from patients with iTTP. A previously developed ELISA was used to assess the binding of a panel of 13 samples of iTTP patients (kindly provided by Prof. Paul Coppo and Prof. Agnés Veyradier (Centre de Reference des Microangiopathies Thrombotiques—CNR-MAT, AP-HP, Paris France). The protocol for the assessment of patient-derived autoantibodies was published previously (Graça et al., 2019). Plates were coated overnight with 100 μl monoclonal antibody 3H9 (kindly provided by Prof Vanhoorelbeke, KU Leuven, Belgium) at a concentration of 1 μg/ml. Plates were then blocked with phosphate buffered saline (PBS) supplemented with 2% BSA. Plates were then incubated with 1.05 nmol/well of ADAMTS13 (200 ng/well for full-length ADAMTS13 and 78.75 ng/well for MDTCS variants). Subsequently, 100 μl of the different dilutions of each patient sample were tested for reactivity with each ADAMTS13 variant. Next, 100 μl of a pool of monoclonal antibodies directed towards human IgG1, IgG2, IgG3 and IgG4, each conjugated with horseradish peroxidase and diluted 1:10 000 (Sanquin, The Netherlands) was incubated to assess the binding of patient IgG to the immobilized ADAMTS13 variants, essentially as described previously (Graça et al., 2019). Dilutions used for the different patient samples ranged from 30× to 400× depending on the amount and affinity of anti-ADAMTS13 antibodies present in the patient sample. Dilutions of patient samples were adjusted to meet an optimal target optical density at 450 nm (using the 540 nm as a reference) of 1.6. To correct for potential inter-assay variation, a dilution curve of human monoclonal anti-ADAMTS13 antibody II-1 was included in all experiments as outlined previously for data interpolation (Graça et al., 2019). Reactivity of autoantibodies in each patient sample with the ADAMTS13 NGLY-variants was compared to that of the observed reactivity with wild-type ADAMTS13. Binding of patient autoantibodies was expressed as a percentage of the binding of patient autoantibodies to wild-type ADAMTS13. The results of this analysis are presented in
Example 4: N-Glycan Variants of ADAMTS13 Retain Activity in the Presence of Pathogenic Autoantibodies from Patients with iTTP
[0126] In Example 2 we showed that NGLY2, NGLY3, NGLY4 and NGLY5 were capable of proteolytic processing of VWF multimers as well as the small peptide substrate FRETS-VWF73. In Example 3 we showed that NGLY3 was poorly recognized by pathogenic autoantibodies present in samples of patients with iTTP. This prompted us to assess whether NGLY3 was still capable of processing FRETS-VWF73 in the presence of plasma samples from patients with iTTP. In order to test this we selected two patient samples based on the data presented in
[0127] We further analyzed the ability of NGLY3 to process VWF in different flow or shear rate assays. First we assessed the ability of NGLY3 to process VWF strings under flow on the surface of endothelial cells. Endothelial cells were grown in Ibidi ti-Slide VI channels coated in 1% gelatin prior to seeding. HUVEC's (Promocell, passage 3) were seeded 50.000 cells per channel. Channel medium was refreshed twice per day with EGM-18 medium (Promocell) with Supplement Mix (2% v/v) (Promocell) and penicillin/streptomycin (1% v/v) (Sigma). Measurements were made at the fourth day of confluency. The flow experiments were performed using a flow rate of 2 mL/min, which corresponds to a shear stress of approximately 2.5 dynes/cm2. Before measurements, cells were starved using M199 medium (Gibco) supplemented with 0.2% BSA for 5 minutes. Subsequently, cells were stimulated with 100 μM histamine in M199 medium supplemented with 0.2% BSA for 10 minutes. Next, the VWF strings were stained using anti-VWF polyclonal antibody (DAKO) labeled with AlexaFluor-488, at a dilution 1:2000 for 5 minutes. The ADAMTS13 variants were diluted to a final concentration of 0.1 μg/mL in M199 medium supplemented with 0.2% BSA. ADAMTS13 containing medium was flown over the cells for 10 minutes, during which 3 separate positions were imaged at 10 seconds intervals using a Zeiss Axio Observer Z1 microscope. The first and last image of each position were analyzed with ImageJ. The length of each VWF string was measured manually, and the difference between the total length before and after ADAMTS13 incubation was used to determine the activity of the protein. For the control, we used the medium which was in contact with ExpiCHO cells not producing any ADAMTS13 (
[0128] We also determined the ability of NGLY3 to process VWF multimers under turbulent flow employing a so-called vortex assay (Zhang et al., 2007). Incubation of 40 nM VWF with 1.0 ug/ml recADAMTS13 under turbulent flow of 3000 rpm for 30 minutes in a reaction buffer composed of 25 mM CaCl.sub.2); 20 mM Bis-Tris; 20 mM HEPES; 20 mM Tris-HCl; Tween20 0.005% v/v; pH 7.5 (final volume of reaction=200 μL) resulted in loss of the high molecular weight multimers from the sample (
[0129] These results show that the NGLY3 variant retains significant proteolytic activity in the presence of autoantibodies directed towards ADAMTS13.
[0130] Next we tested the NGLY3 variant against an extended panel of 28 patient's plasmas (
[0131] We performed a subset-analysis of plasma samples containing high titer inhibitors. High titer inhibitors were defined as the level of inhibitors that gave rise to at least 50% inhibition of the wild type ADAMTS13 (
[0132] These observations suggest that therapeutic administration of a “glycan-shielded” ADAMTS13 variant may comprise a more efficient treatment option for treatment of patients with iTTP when compared to the administration of a wild-type ADAMTS13, either in a recombinant form or as plasma-derived ADAMTS13 in either purified form or as being administered through plasma-exchange.
Example 5: Autoantibody-Resistance of an Extended Panel of N-Glycan Variants
[0133] As evident from the heatmap shown in
[0134] N-glycan shielded ADAMTS13 variant containing at least one newly introduced or shifted N-glycan can also be combined with individual amino acid substitutions that diminish binding of pathogenic autoantibodies. Therefore, we are aiming to make additional combinations of NGLY3 with alanine mutations (mostly outside exosite-3), and other NGLY modifications within the vicinity of exosite-3 (Table 3). Due to lack of other potential N-glycosylation sequons within this region, we are seeking the strategy of shifting the natural existing N-glycans in the spacer domain of ADAMTS13 1-2 residues in either direction (N- or C-terminus), namely the glycan present at N667.
TABLE-US-00003 TABLE 3 ADAMTS13 NGLY3 variants with additional mutations Mutation Original sequence Mutated sequence NGLY3 607GKMSI611 607GNMSI611 NGLY7 664EYGNLT669 664ENVTLT669 (glycan shift from N667 to Y665) NGLY8 667NLTRP671 667LNVTA671 Glycan shift from N667 to L668) NGLY3 + NGLY7 607GKMSI611/ 607GNMSI611/ 664EYGNLT669 664ENVTLT669 NGLY3 + NGLY8 607GKMSI611/ 607GNMSI611/ 667NLTRP671 667LNVTA671 NGLY3 plus 607GKMSI611 plus 607GNMSI611 plus L591A/R636A/L637A/ L591, R636, L637,L668 591A, 636A, 637A, L668A 668A NGLY3 + R568A/Y665A 607GKMSI611 607GNMSI611 plus plus R568, Y665 568A, 665A NGLY3 + R568A/ 607GKMSI611 R568, 607GNMSI611 plus Y665A + L591A/R636A/ L591, R636, L637, 568A, 591A, 636A, L637A/L668A Y665, L668 637A, 665A, 668A
[0135] These variants are designed as outlined in Example 1. Synthetic DNA fragments encoding residues M509 to W688 (540 bp) in which the novel NGLY variants were introduced were designed and ordered by Genewiz (Leipzig, Germany). The synthetic fragments were flanked by an XmaI site at the 5′ end and a HindIII site at the 3′ end of the fragment. The synthetic DNA's were first cloned into the XmaI-HindIII site of plasmid pUC57_mut1.1 (see example I), embedded in a larger fragment flanked by PagI-Esp3I. This larger fragment was then used to replace the corresponding fragment in wild type ADAMTS13 as present in pcDNA3.1ADAMTS13.
[0136] The resulting NGLY-variants were expressed in Expi-CHO cells according to the instructions of the manufacturer (Thermo Fisher Scientific). Supernatants were harvested after 4 days post transfection, cleared by centrifugation, supplemented with 10 mM benzamidine and stored at −30° C. until further use. ADAMTS13 levels present in culture supernatants were quantified by ELISA using a previously established assay (Alwan et al., 2017; Graça et al., 2019). ADAMTS13 antigen levels ranged from 1.43 to 5.27 μg/ml (see table 4).
[0137] Subsequently we measured the activity of the novel NGLY variants employing the FRETS-VWF73 fluorogenic substrate (Table 4). NGLY7 was 125% active when compared to the wild-type, NGLY8 was 75% active when compared to wild-type ADAMTS13; NGLY3 plus NGLY7 was 125% active when compared to the wild type; NGLY3 plus NGLY8 was 75% active when compared to wild type ADAMTS13. R568A/Y665A was 120% active when compared to wild type; L591A/R636A/L637A/L668A was 90% active when compared to wild type. NGLY3 plus R568A/Y665A was 120% active when compared to wild type, NGLY3 plus L591A/R636A/L637A/L668A was 95% active when compared to wild type ADAMTS13, NGLY3 plus R568A/Y665A plus L591A/R636A/L637A/L668A was 40% active when compared to wild type ADAMTS13.
[0138] Overall these results show that combinations of NGLY3 and NGLY7, NGLY3 and NGLY8 as well as NGLY7 and NGLY8 retain the ability to convert the FRETS-VWF73 substrate. Combinations of NGLY3 with R568A/Y665A and/or L591A/R636A/L637A/L668A also retain their ability to convert the FRETS-VWF73 substrate.
[0139] We also assessed the ability of the new variants to process VWF multimers under shear stress employing a vortex assay. Under these conditions NGLY3 was fully active; also NGLY7 was clearly capable of processing the large VWF multimers (
[0140] We also assessed the ability of the new variants to process VWF multimers employing denaturing conditions. Under these conditions NGLY3 was fully active whereas NGLY7 and NGLY8 also displayed a reduced activity (Table 4). Combinations of NGLY3/NGLY7 and NGLY3/NGLY8 also revealed a reduced ability to process VWF multimers under these experimental conditions. Combinations of NGLY3 plus L591A/R636A/L637A/L668A and NGLY3 plus R568A/Y665A also were less efficient in processing VWF multimers employing these specific condition (Table 4).
TABLE-US-00004 TABLE 4 Antigen levels and activity of ADAMTS13 NGLY variants and controls Ability to Activity level process VWF (% relative to multimers Antigen wild type under level ADAMTS13) denaturing Variant (μg/ml) VWFFRETS73 conditions Full length ADAMTS13 wild 5.27 100 ++++++ type NGLY3 4.60 125 ++++++ NGLY7 3.45 125 +++++ NGLY8 1.43 76 ++ NGLY3 plus NGLY7 4.26 135 +++++ NGLY3 plus NGLY8 2.49 120 ++ NGLY3 plus 4.42 95 ++ L591A/R636A/L637A/L668A NGLY3 plus R568A/Y665A 4.36 120 ++ NGLY3 plus R568A/Y665A 2.32 40 Not tested plus L591A/R636A/L637A/L668A L591A/R636A/L637A/L668A 4.77 90 ++ R568A/Y665A 3.78 120 ++
[0141] Next we assessed whether the newly designed ADAMTS13 variants were capable of neutralizing pathogenic autoantibodies that develop or originate from patients with immune TTP.
[0142] We first assessed the antibody-resistance properties of NGLY7, NGLY8 as well as the combinations of NGLY3 plus NGLY7 and NGLY3 plus NGLY8 (
[0143] We subsequently tested combinations of NGLY3 plus L591A/R636A/L637A/L668A and NGLY3 plus R568A/Y665A for their efficiency of autoantibody resistance in patient samples. In 8 out of 8 samples NGLY3 plus R568A/Y665A retained slightly more activity when compared to wild type ADAMTS13 (
Example 6. Further N-Glycan Variants of ADAMTS13
[0144] The previous examples focus on the spacer domain which contains a major binding site for pathogenic autoantibodies that develop in patients with immune TTP. It is well-known that autoantibodies can also target other domains on ADAMTS13 (Klaus et al., 2004; Thomas et al., 2015; Pos et al., 2011). Similar to the methods described in Example 1-4 N-glycan shielded ADAMTS13 variants can be designed that prevent the binding of autoantibodies targeting antibody binding sites present in the metallo-protease, disintegrin domain, TSP-1 repeat, the Cys-rich domain, epitopes outside R568, F592, R660, Y661 and R665 in the spacer domain, the TSP2-8 repeats and the CUB1/2 domains. 5. Additionally, NGLY3 and/or other N-glycan shielded ADAMTS13 variants can be combined with individual or multiple amino acid substitutions in the above-mentioned domains which results in a decline on auto-antibody binding while retaining at least partial proteolytic activity.
[0145] The 3D structure of ADAMTS13 was used to select the surface residues of ADAMTS13 to select potential N-glycosylation sites. ADAMTS13 crystal structure (PDB: 6qig) was used for following domains: Metalloprotease, Disintegrin-like domain, thrombospondin type 1 repeat 1 (TSP1-1), cysteine-rich and spacer domain. The rest of the structure was built by homology modeling from TSP1-2 to CUB2 domains as described previously (Ercig et al., 2018b). SwissPDB Viewer was used to investigate the 3D structure of ADAMTS13 to select the surface residues manually.
TABLE-US-00005 TABLE 5 Exposed regions on ADAMTS13 that allow for insertion or shifting of N- glycans to prevent the binding of pathogenic autoantibodies. Residues in bold are (part of) natural glycosylation sites of ADAMTS13. Bold and underlined residues have been shown to contain O-glycans. Residues indicated in bold and italics are modified by O-fucosylation of Ser (S) residues or C-mannosylation of Trp (W) residues. Numbering of residues (as shown in No. Domain Amino acid sequence FIG. 1) 1 Metalloprotease (80-286) DVFQAHQEDTER 91-102 2 Metalloprotease (80-286) ELLRDPSLGAQFR 113-125 3 Metalloprotease (80-286) KMVILTEPEGAPNITANLTSSLL 130-152 4 Metalloprotease (80-286) QTINPEDDTDP 159-169 5 Metalloprotease (80-286) RFDLELPDGNRQ 180-191 6 Metalloprotease (80-286) QLGGACSPTW 197-206 7 Metalloprotease (80-286) EHDGAPGSGCGPS 233-245 8 Metalloprotease (80-286) SDGAAPRAGL 251-260 9 Metalloprotease (80- PCSRRQLLSLLSAGRARCVWDPPRPQPGSAG 264-300 286)/Disintegrin-like HPPDAQ Domain (287-383) 10 Disintegrin-like Domain RVAFGPKAVACTFAREHLDMCQ 312-333 (287-383) 11 Disintegrin-like Domain TDPLDQSSCSRLL 339-351 (287-383) 12 Disintegrin-like Domain DGTECGVEK 356-364 (287-383) 13 Disintegrin-like Domain KGRCRSLVELTPIAAVHGR SSWGPRSPCSR
368-399 (287-383)/region between Disintegrin-like Domain and TSP type-1 1 (383-394)/TSP type-1 1 (394-439) 14 TSP type-1 1 (384-439) RRRQ 407-410 15 TSP type-1 1 (384-439) GGRACVGADLQAE 419-431 16 TSP type-1 1 (384-439)/ NTQACEKTQLE 434-444 Cysteine-rich (440-556) 17 Cysteine-rich (440-556) QQCARTDGQPLRSSPGGA 448-465 18 Cysteine-rich (440-556) FYHWGAAVPHSQGDALCR 467-484 19 Cysteine-rich (440-556) RAIGESFIMKRGDSFL 488-502 20 Cysteine-rich (440-556) SGPRE 511-515 21 Cysteine-rich (440-556) SGSCR 524-528 22 Cysteine-rich (440-556) DGRMDSQQVWDR 533-544 23 Cysteine-rich (440-556)/ VCGGDNSTCSPRKGSFTAGRARE 547-569 Spacer (556-685) 24 Spacer (556-685) TFLTVTPN 572-579 25 Spacer (556-685) YIANHRPLF 584-592 26 Spacer (556-685) GGRYVVAGKMSISPN 600-614 27 Spacer (556-685) YPSLLED 617-623 28 Spacer (556-685) RVALTEDRLPR 629-639 29 Spacer (556-685) RIWGPLQED 644-652 30 Spacer (556-685) RRYGEEYGNLTR 659-670 31 Spacer (556-685) TFTYFQPK 674-681 32 TSP type-1 2 (682-730) PRQAWVWAAVRGPCS 682-696 33 TSP type-1 2 (682-730)/ AGLRWVNYSCLDQARKELVE 701-720 region between TSP type-1 2 and TSP type-1 3 (730-742) 34 TSP type-1 2 (682-730)/ QGSQQPPAWPEACVLEP 725-741 region between TSP type-1 2 and TSP type-1 3 (730-742) 35 TSP type-1 3 (742-805) PPYWAVGDFGPCSA
CG 743-759 36 TSP type-1 3 (742-805) LRERPVRCVEAQGSLL 762-777 37 TSP type-1 3 (742-805)/ PPARCRAGAQQPAVALETCNPQPCPAR 781-807 region between TSP type-1 3 and TSP type-1 4 (806-807) 38 TSP type-1 4(808-859) WEVSEPSSCTSAGGAGL 808-824 39 TSP type-1 4(808-859) NETCVP 828-833 40 TSP type-1 4(808-859) LEAPVTEGPGSVDEK 838-852 41 TSP type-1 4(808-859) APEPCVGMSCPPG 855-867 /region between TSP type-1 4 and TSP type-1 5 (859-896) 42 region between TSP LDATSAGEKAP 871-881 type-1 4 and TSP type-1 5 (859-896) 43 region between TSP SP
GSIRTGAQAAHVW 882-897 type-1 4 and TSP type-1 5 (859-896)/TSP type-1 5 (896-950) 44 TSP type-1 5 (896-950) V
CGR 906-910 45 TSP type-1 5 (896-950) ELRFLCMDSALRVPVQEELCGL 915-936 46 TSP type-1 5 (896-950/ KPGSRRE CPARWQYKLAACSV
CGR 939-968 TSP typel 6(951-1011) 47 TSP type-1 6(951-1011) RRILYCARAHGED 972-984 48 TSP type-1 6(951-1011) EEILLDTQCQGLPRPEPQEACSLEP 987-1011 49 TSP type-1 7 (1012-1068) CPPR
1012-1016 50 TSP type-1 7 (1012-1068) PCSA
CGLGTAR 1023-1034 51 TSP type-1 7 (1012-1068) VQLDQGQDVEVDEAA 1040-1054 52 TSP type-1 7 (1012- LVRPEASVPCLIAD 1058-1071 1068)/region between TSP type-1 7 and TSP type-1 8 (1069-1071) 53 TSP type-1 8 (1071-1131) RWHVGTWMECSV
CGD 1075-1090 54 TSP type-1 8 (1071-1131) T 1098 55 TSP type-1 8 (1071-1131) AQAPVPADFCQHLP 1104-1117 56 TSP type-1 8 (1071- RGCWAGPCVGQGTPSLVPHEEAAAPGR 1123-1149 1131)/region between TSP type-1 8 and CUB1 domain (1132-1191) 57 region between TSP PAGASLEW 1154-1161 type-1 8 and CUB1 domain (1132-1191) 58 region between TSP RGLLFSPAPQPRRLLPGPQENS 1165-1186 type-1 8 and CUB1 domain (1132-1191) 59 CUB1 (1192-1298) CGRQHLEPTGT 1192-1202 60 CUB1 (1192-1298) DMRGPGQAD 1204-1212 61 CUB1 (1192-1298) GRPLGE 1218-1223 62 CUB1 (1192-1298) PGQAD 1208-1212 63 CUB1 (1192-1298) PLG 1220-1222 64 CUB1 (1192-1298) R 1228 65 CUB1 (1192-1298) SSLNCSAGDMLLLWGRL 12332-1248 66 CUB1 (1192-1298) NCSAGDMLL 1235-1243 67 CUB1 (1192-1298) WGRLTWRKMCRKLLDM 1245-1260 68 CUB1 (1192-1298) WRKMCRKLLDM 1250-1260 69 CUB1 (1192-1298) TFSSKTNT 1261-1268 70 CUB1 (1192-1298) KTNT 1265-1268 71 CUB1 (1192-1298) RQRSGRPGGGV 1272-1282 72 CUB1 (1192-1298) RCGRPG 1274-1279 73 CUB1 (1192-1298) RYGSQLAPETFYRE 1285-1298 74 CUB1 (1192-1298) QLAPETFYRE 1289-1298 75 CUB2 (1299-1427) DMQLFGPWG 1300-1308 76 CUB2 (1299-1427) DMQLFGPWGEIVSPSLSPATSNA 1300-1322 77 CUB2 (1299-1427) SPSLSPATSNAGG 1312-1324 78 CUB2 (1299-1427) RLFINVAPHARI 1326-1337 79 CUB2 (1299-1427) APHAR 1332-1336 80 CUB2 (1299-1427) LA 1342-1343 81 CUB2 (1299-1427) TNMGAGTEGANASYIL 1344-1359 82 CUB2 (1299-1427) AGTEGAN 1348-1354 83 CUB2 (1299-1427) IRDTHSLRT 1360-1368 84 CUB2 (1299-1427) RDTHSLRTTAF 1361-1371 85 CUB2 (1299-1427) QQVLYWESESSQ 1374-1385 86 CUB2 (1299-1427) WESESSQAE 1379-1387 87 CUB2 (1299-1427) EFSEGFLKAQAS 1389-1400 88 CUB2 (1299-1427) SEGFLKAQASLRGQY 1391-1405 89 CUB2 (1299-1427) LQSWVPEMQDPQSWKGKEGT 1408-1427
Example 7. Mass Spectrometry Based Identification of N-Glycan Modified ADAMTS13
[0146] In this example we employed mass spectrometry to provide proof of principle for the successful N-glycosylation of newly engineered consensus-sites within ADAMTS13. We selected the NGLY3 variant for this analysis. As outlined in a previous example K608 is replaced by N608 in NGLY3 thereby introducing a consensus site for the addition of N-glycan (see
[0147] First we analyzed which peptide-sequences were retrieved following mass spectrometry analysis of wild type ADAMTS13. Overall coverage of trypsin-digested purified wild-type ADAMTS13 was 66%. Only peptides corresponding to amino acid sequence 599-629 are displayed in
[0148] Next we analyzed NGLY3 in a similar manner. Following trypsin-digestion no peptides corresponding to region I599-R629 were recovered. This observation indicates that one or more N-glycans may be present in this part of NGLY3. Upon treatment with PGNaseF one peptide Y603-R629 was identified (peptide 3:
[0149] As an additional control we analyzed an ADAMTS13 variant in which K608 was replaced by an alanine at this position. Identification of trypsin-cleaved peptides corresponding to this region were not observed in the absence of PGNaseF treatment consistent with the presence of a N-linked glycan in this area. Following digestion with PGNaseF a single Y603-R629 peptide was identified which contained a deamidated N at position 614 (indicated by box in
[0150] Overall, the approach outlined in this proposal shows that introduction of a consensus-site for the addition of N-glycan at amino acid position 608 in NGLY3 results in attachment of an N-glycan at this position. Similarly, the presence of other N-linked glycans in NGLY1,2,4,5,6,7,8 and other glycan-modified ADAMTS13 variants including those listed in Example 7 can be successfully determined using the protocol outlined in this example.
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