ETANERCEPT VARIANTS WITH IMPROVED THERAPEUTICAL EFFECT

Abstract

The present invention provides improved Etanercept variants which comprise one (A105E), preferably two (A105E/L106F), amino acid substitutions regarding the Etanercept original amino acid sequence of SEQ ID NO: 3. These variants inhibit TNF activity but fail to neutralize human LTα (hLTα). Thus, they are proposed herein as a great alternative to be used in the clinic for the treatment of autoimmune or inflammatory diseases in which an exacerbated TNF activity is involved, since they prevent the side effects associated to the use of the original Etanercept molecule while retaining the TNF blocking activity.

Claims

1. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1, wherein said amino acid sequence of SEQ ID NO: 1 comprises the amino acid substitution A105E.

2. The isolated polypeptide according to claim 1, that further comprises the amino acid substitution L106F.

3. The isolated polypeptide according to claim 2, that comprises the amino acid substitutions A105E and L106F.

4. The isolated polypeptide according to any one of claims 1 to 3, linked to the Fc fragment of a human IgG1.

5. The isolated polypeptide according to any of claim 3 or 4, comprising the amino acid sequence of SEQ ID NO: 4.

6. An isolated polynucleotide comprising a nucleic acid sequence encoding the isolated polypeptide according to any one of claims 1 to 5.

7. A gene construct comprising the isolated polynucleotide according to claim 6, preferably wherein said gene construct is a viral vector.

8. A host cell comprising the isolated polypeptide according to any one of claims 1 to 5, the isolated polynucleotide according to claim 6, or the gene construct according to claim 7.

9. A pharmaceutical composition comprising the isolated polypeptide according to any one of claims 1 to 5.

10. The pharmaceutical composition according to claim 9, formulated for subcutaneous administration.

11. The isolated polypeptide according to any one of claims 1 to 5 for use as a medicament.

12. The isolated polypeptide according to any one of claims 1 to 5 for use in the treatment of an autoimmune or inflammatory disease.

13. The isolated polypeptide according to any one of claims 1 to 5 for use according to claim 12, wherein the disease is a TNF-dependent inflammatory disease.

14. The isolated polypeptide according to any one of claims 1 to 5 for use according to any of claim 12 or 13, wherein the disease is a rheumatic disorder.

15. The isolated polypeptide according to any one of claims 1 to 5 for use according to any one of claims 12 to 14, wherein the disease is selected from the list consisting of: rheumatoid arthritis, Crohn's disease, psoriasis, ankylosing spondylitis, multiple sclerosis or inflammatory bowel disease.

Description

DESCRIPTION OF THE DRAWINGS

[0099] FIG. 1. An EFE motif in the 90s loop of CrmD abrogates its anti-hLTα activity. A cytotoxic dose of hTNF and hLTα was incubated with L929 cells in the absence or presence of CrmD wild-type (WT) or the indicated mutants at increasing cytokine:protein molar ratios (legend above each graph). After 18 h cell survival was assessed as the A490 determined using Cell Titer Aqueous One Solution Kit. Data are represented as the % relative to the A490 recorded for cells incubated without cytokine (Media, 100% viability). The corresponding effector cytokine is indicated above each graph. Results are shown as mean±SD of triplicates of three representative experiments. Asterisks indicate mutants that display significantly different viability values compared to CrmD WT at the same protein dose (*p<0.05, ANOVA with Bonferroni multiple comparison test).

[0100] FIG. 2. Etanercept A105E/L106F mutant displays enhanced TNF neutralizing specificity. A) Localization of the side chains of the EFE motif in the 90s loop of CrmD. CrmD three-dimensional folding was modeled using I-TASSER (Yang J., Yan T., Roy A. et al., 2015, Nat. Methods, 12, 7-8) and aligned with the crystallographic structure of the TNFR2-TNF structure (PDB: 3ALQ) (left panel) in Chimera. In the right panel, magnification of the 90s loop region (dashed frame) showing the side chains of the overlapping EFE and ALS motifs of CrmD and TNFR2, respectively. B) hTNF- and hLTα-mediated cytotoxicity assays. L929 cells were incubated with 1.2 nM of the corresponding cytokine, as labeled above each graph, with increasing concentrations of wild type (WT), L106F or Al 05E/L106F Etanercept. After 18h, cell viability was assessed as the A490 detected using Cell Titer Aqueous One Solution Kit. Data are represented as the % relative to the A490 recorded for cells incubated without cytokine (100% cell viability). Mean ±SD of triplicates from two independent experiments are shown. The horizontal line above the hTNF graph indicates the Etanercept concentrations in which statistically significant differences were detected between the anti-hTNF activity of the WT and the A105E/L106F mutant (*p<0.05, two-tailed t test).

EXAMPLES

Example 1. Transfer of the EFE Motif of CrmD into the 90s Loop of Etanercept Specifically Impair its Anti-hLTα Activity

[0101] The inflammatory diseases currently treated with Etanercept are predominantly TNF-driven. Therefore, the anti-hLTα activity of Etanercept not only appears to be clinically unnecessary but it could also pose a source of unwanted complications. It has been identified in this invention that a 90s loop EFE motif in the soluble viral TNF decoy receptor termed CrmD specifically hinders its anti-hLTα activity. Unlike CrmD wild-type, a CrmD E96A/F97A/E98A mutant is able to inhibit the cytotoxic activity of hLTα (FIG. 1). Then, it was here hypothesized that by transferring the EFE motif of CrmD into the 90s loop of Etanercept, we could disrupt its anti-hLTα activity while keeping it active against hTNF.

[0102] An amino acid sequence alignment showed that the EFE motif (Glu.sup.96-Phe.sup.97-Glu.sup.98) in the CRD3 of CrmD aligns with an ALS motif (Ala105-Leu.sup.106-Ser.sup.107) in the 90s loop of human TNFR2, which corresponds to the TNF-binding moiety of Etanercept. Furthermore, it was observed that in the crystal structure of the TNFR2:TNF complex (PDB:3ALQ), the TNFR2 Ser.sup.107 was not facing to the ligand (FIG. 2A). Similarly, the structural superimposition of a CrmD model with the structure of TNFR2 suggested that the third amino acid of this motif in CrmD, Glu.sup.98, would also be far from the ligand interface. Thus, to introduce the lowest number of modifications into the original sequence of Etanercept, we mutated only the TNFR2 Ala.sup.105 and Leu.sup.106 to their equivalent amino acids in the 90s loop of CrmD (Glu (E) and Phe (F)). The WT (non-mutant or original Etanercept) and the L106F and A105E/L106F forms of Etanercept were expressed in a baculovirus system and purified by affinity chromatography.

[0103] hTNF and hLTα binding affinity of wild type (WT), L106F and A105E/L106F Etanercept was calculated by SPR. The kinetic affinity constants, association (Ka), dissociation (Kd) and binding affinity (K.sub.D), and their standard errors (SE), are shown for each interaction. SPR analysis revealed that the hTNF and hLTα binding affinities of Etanercept were not significantly affected in the mutants (Table 1).

TABLE-US-00001 TABLE 1 (Ka ± SE) × (Kd ± SE) × K.sub.D Ligand Etanercept 10.sup.+5 (1/Ms) 10.sup.−3 (1/s) (nM) hTNF WT 28.00 ± 0.67 0.79 ± 0.01 0.28 A105E/L106F 30.07 ± 0.56 2.10 ± 0.03 0.69 L106F 30.00 ± 0.85 2.01 ± 0.05 0.67 hTLα WT  8.85 ± 0.54 3.49 ± 0.21 3.95 A105E/L106F  2.37 ± 0.05 1.69 ± 0.07 7.13 L106F  4.64 ± 0.03 1.20 ± 0.01 2.59

[0104] However, binding affinity does not always correlate with neutralizing potency. Therefore, the capacity of A105E/L106F, L106F and WT Etanercept to block the cytotoxic activity of hTNF and hLTα on L929 cells was compared. As shown in FIG. 2B, 20 nM of Etanercept WT was enough to fully neutralize both hTNF and hLTα (FIG. 2B), reaching 50% cell viability at 10-20 nM and 5 nM (EC50), respectively. The anti-hTNF and anti-hLTα activities of the mutant L106F were comparable to those of WT Etanercept. On the other hand, the A105E/L106F mutant protected 50% of the cells from hTNF at 30-60 nM and reached full protection at 90 nM (FIG. 2B). In contrast, this double mutant showed a very low anti-hLTα activity and required a high 300 nM dose to protect only 35% of the cells from this cytokine (FIG. 2B), positioning its anti-hLTα EC50 at even higher concentrations. Therefore, the A105E/L106F variant was at least 60× weaker as hLTα inhibitor.

[0105] It has been demonstrated herein that the anti-hLTα activity of Etanercept can be vastly hampered by making its 90s loop look more like that of CrmD in a A105E/L106F Etanercept mutant. The slight defect observed in the anti-hTNF activity of this mutant could be potentially overcome in the clinic by a small dose increase without compromising hLTα-mediated immune functions.

[0106] Therefore, this A105E/L106F variant could set the foundation for a safer second generation of Etanercept featuring the benefits of a soluble decoy receptor and the high TNF specificity of the antibody therapy.

Example 2. Materials and Methods

[0107] Cells and Reagents

[0108] L929 cells (ATCC, Manassas, Va.) were grown in DMEM supplemented with 10% FCS.

[0109] Recombinant baculoviruses were generated and amplified in adherent Hi5 insect cells cultured in TC-100 medium supplemented with 10% FCS and 1× non-essential amino acids. Suspension Hi5 cells maintained in Express Five (Life Technologies, Carlsbad, Calif.) medium supplemented with 8 mM L-glutamine were used for the expression of the recombinant protein.

[0110] Recombinant cytokines were purchased from R&D Systems (Minneapolis, Minn.) and reconstituted and stored following the manufacturer's recommendations.

[0111] Construction of Recombinant Baculovirus

[0112] All the proteins described in this study were expressed by recombinant baculoviruses. The ECTV strain Hampstead CrmD coding sequence was extracted by PCR from a pBAC1 (Life Technologies) derived plasmid termed pMS1 (Saraiva M., Alcami A., 2001, J. Viral., 75, 226-233) using the primers Crm34 (5′-gcgggatccgatgttccgtatacacccattaatggg-3′, SEQ ID NO: 5) and CrmD33 (5′-gcgctcgaggcatctctttcacaatcatttgg-3′, SEQ ID NO: 6). The CrmD gene lacking the signal peptide (residues 21-320) was cloned into pAL7 (Montanuy I., Alejo A., Alcami A., 2011, FASEB J., 25, 1960-1971), a modified pFastBac1 vector, in frame with a N-terminal honeybee melittin signal peptide and a C-terminal V5-6×His tag. The resulting plasmid was termed pSP3.

[0113] The CrmD point mutants were generated using the QuikChange II site-directed mutagenesis kit (Agilent Technologies, Santa Clara, Calif.). For this, pSP3 was used as template for PCR reactions with the corresponding pair of primers for each mutation. The Etanercept L106F and A105E/L106F mutants were generated using the primer pairs, RM6mut2F (5′-gctggtactgcgcgttcagcaagcaggaggg-3′, SEQ ID NO: 7) and RM6mut2R (5′-ccctcctgcttgctgaacgcgcagtaccagc-3′, SEQ ID NO: 8), and RM6mut3F (5′-cggctggtactgcgagttcagcaagcaggaggg-3′, SEQ ID NO: 9) and RM6mut3R (5′-ccctcctgcttgctgaactcgcagtaccagccg-3′, SEQ ID NO: 10), respectively.

[0114] pRM6 is a pFastBacl-based plasmid containing the wild-type form of a Fc fusion protein of the TNF binding domain of TNFR2, known in the clinic as Etanercept (Pontejo, S. M., Alejo, A., Alcami, A., 2015, J Biol Chem 290, 15973-15984). Mutagenesis was confirmed by sequencing.

[0115] The plasmids described above were used to generate recombinant baculoviruses using the Bac-to-Bac system (Life Technologies) following the manufacturer's instructions.

[0116] Subsequently, viral stocks were amplified by infecting adherent Hi5 cells at low multiplicity of infection (0.1-0.01 pfu/cell).

[0117] Protein Expression and Purification

[0118] Hi5 suspension cells were infected with the corresponding recombinant baculovirus at high multiplicity of infection (2 pfu/cell). Supernatants were harvested 3 days after infection, clarified at 6,000×g for 40 min and then concentrated to 2.5 ml in a Stirred Ultrafiltration Cell 8200 (Millipore, Burlington, Mass.). The concentrate was desalted and buffer was exchanged to 0.1 M phosphate buffer containing 300 mM NaCl and 10 mM imidazole using PD-10 desalting columns (GE Healthcare, Chicago, Ill.).

[0119] His-tagged CrmD proteins were purified by metal chelate affinity chromatography (Ni-NTA resin, Qiagen, Germantown Md.). Etanercept (TNFR2-Fc) proteins were purified using protein A-coupled sepharose columns (Sigma, St. Louis, Mo.). Protein containing fractions were pooled, concentrated and dialyzed in PBS. Final protein concentration was calculated by gel densitometry.

[0120] Cytotoxicity Assays

[0121] The ability of CrmD, Etanercept and their mutants to inhibit TNF superfamily ligands (TNFSF) was tested by cytotoxicity assays on L929 cells as previously described (Pontejo, S. M., Alejo, A., Alcami, A., 2015, J Biol Chem 290, 15973-15984). Briefly, 20 ng/ml of hTNF and hLTα were incubated for 1h at 37° C. in the presence of increasing amounts of recombinant protein. Subsequently, the cytokine-protein mixtures were added to L929 cells seeded at 12,000 cells/well in 96-well plates in the presence of 4 μg/ml of actynomicyn D (Sigma). Cell viability was assessed after 18 h using Cell Titer Aqueous One Solution Kit (Promega, Madison, Wis.) following the manufacturer's instructions and the absorbance at 490 nm (A490) was determined in a Sunrise microplate reader (Tecan, Mannedorf, Switzerland). The A490 of all samples was normalized with the A490 of cells incubated only with the cytokine (0% viability). Cell viability for each sample was calculated in reference to the A490 obtained in wells where cells were incubated without cytokine (“media”, 100% viability).

[0122] Surface Plasmon Resonance (SPR) Assays

[0123] The ligand binding properties of recombinant proteins were characterized by SPR using a Biacore X biosensor (GE Healthcare). For determination of kinetic affinity constants, recombinant proteins were immobilized on CM4 chips at low density (≈500 RU).

[0124] Increasing concentrations of TNFSF cytokines were injected in HBS-EP buffer at 30 pl/min during 2 min and a 5-min dissociation was recorded. A 0.1-1000 nM concentration range of analyte was typically used. Between analyte injections, the chip surface was regenerated with 10 mM glycine-HCl pH 2.0. Kinetic data were globally fitted to a 1:1 Langmuir model using the Biaevaluation 3.2 software. Bulk refractive index changes were removed by subtracting the responses recorded in the reference flow cell, and the response of a buffer injection was subtracted from all sensorgrams to remove systematic artifacts. The average KD of 10 fittings containing sensorgrams for at least 6 different analyte concentrations from at least two independent experiments was calculated. The fitting providing the closest KD to the average KD was chosen to represent the kinetic affinity constants of each interaction.