Quantification of misfolded TNFR2:Fc

Abstract

The present invention is directed to methods for determining the relative amount of wrongly disulphide bridged TNFR2:Fc in a sample of TNFR2:Fc, a fusion protein which is used in a variety of therapeutic applications. In addition, the invention pertains to a method for purifying TNFR2:Fc using said method for determining the percentage of wrongly disulphide bridged TNFR2:Fc, and to TNFR2:Fc compositions obtained thereby.

Claims

1. A method for determining a relative amount of Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc in a sample comprising Cys.sub.74-Cys.sub.88/Cys.sub.78-Cys.sub.96 disulfide bridged TNFR2:Fc and Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc, wherein the method comprises the steps of: (a) providing a sample comprising a mixture of Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc and Cys.sub.74-Cys.sub.88/Cys.sub.78-Cys.sub.96 disulfide bridged TNFR2:Fc; (b) denaturing and alkylating the sample of step (a); (c) subjecting the sample resulting from step (b) to tryptic digestion under non-reducing conditions; (d) determining by HPLC the amount of a fragment indicative of Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc in the sample resulting from step (c); and (e) determining the relative amount of the Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc in the sample based on the amount of the fragment indicative of Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc relative to the amount of a fragment not affected by disulfide bridging of Cys.sub.74, Cys.sub.78, Cys.sub.88 and Cys.sub.96; wherein the amino acid sequence of the TNFR2 part of TNFR2:Fc has at least 97% identity to the amino acids 1-235 of the amino acid sequence of SEQ ID NO: 1, and wherein the fragment indicative of Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc consists of SEQ ID NO:4 (T7).

2. The method of claim 1, wherein the amino acid sequence of the TNFR2:Fc applied to step (a) has at least 97% identity to the amino acid sequence of SEQ ID NO: 3 (etanercept).

3. The method of claim 1, wherein the peak not affected by disulfide bridging of Cys.sub.74, Cys.sub.78, Cys.sub.88 and Cys.sub.96 is not affected by disulfide bridging at all and is indicative of the total TNFR2:Fc in the sample.

4. The method of claim 3, wherein the fragment indicative of total TNFR2:Fc comprises the amino acid sequence shown in SEQ ID NO: 5 (T27).

5. The method of claim 4, wherein the fragment indicative of total TNFR2:Fc consists of the amino acid sequence shown in SEQ ID NO: 5 (T27).

6. The method of claim 4, wherein the relative amount of Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc is determined by (i) determining the peak areas in the HPLC chromatogram indicative of Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc (T7 area) and indicative of total TNFR2:Fc (T27 area); and (ii) calculating the relative amount according to formula (1) $\begin{array}{cc}x\left(in\%\right)=\frac{\left[T7\mathrm{area}\right]}{\left[T7\mathrm{area}\right]+\left[T27\mathrm{area}\right]}100.& \left(1\right)\end{array}$

7. The method of claim 1, wherein step (b) is carried out in a buffer having a pH in the range of 7 to 9, and wherein the buffer comprises at least one of: 10-100 mM TRIS, 0.5-1.5 M iodoacetamide, and 0.02%-0.5% of a cleavable surfactant.

8. The method of claim 7, wherein step (b) is carried out in a buffer having a pH in the range of 7.5 to 8.5, and wherein the buffer comprises at least one of: 20-80 mM TRIS, 0.9-1.2 M iodoacetamide, and 0.1%-0.2% of a cleavable surfactant.

9. The method of claim 8, wherein the buffer comprises a cleavable surfactant which is selected from the group consisting of: sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate, sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate, and sodium 3-(4-(1,1-bis(hexyloxy)ethyl)pyridinium-1-yl)propane-1-sulfonate.

10. The method of claim 9, wherein the cleavable surfactant is sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate.

11. The method of claim 1, wherein step (b) is carried out at 40 to 70 C. for 30 to 60 min.

12. The method of claim 1, wherein step (b) is carried out at 50 to 60 C. for 30 to 45 min.

13. The method of claim 1, wherein step (c) is carried out in a digestion buffer having a pH in the range of 5 to 7; and wherein the digestion buffer comprises: MES as the buffering agent; or 0.02%-0.5% of a cleavable surfactant; or MES as the buffering agent and 0.02%-0.5% of a cleavable surfactant.

14. The method of claim 13, wherein step (c) is carried out in a digestion buffer having a pH in the range of 5.5 to 6.5; and wherein the digestion buffer comprises: 10-100 mM MES as the buffering agent; or 0.1%-0.2% of a cleavable surfactant; or 10-100 mM MES as the buffering agent and 0.1%-0.2% of a cleavable surfactant.

15. The method of claim 13, wherein the buffer comprises a cleavable surfactant which is selected from the group consisting of: sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate, sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate, and sodium 3-(4-(1,1-bis(hexyloxy)ethyl)pyridinium-1-yl)propane-1-sulfonate.

16. The method of claim 15, wherein the cleavable surfactant is sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate.

17. The method of claim 1, wherein step (c) is carried out using an effective amount of trypsin for 1-24 h; and at 32-38 C.

18. The method of claim 17, wherein step (c) is carried out using an effective amount of trypsin for 6-18 h; and at 36-37 C.

19. The method of claim 1, wherein step (d) is carried out in a mobile phase comprising 0.05%-0.5% TFA in water.

20. The method of claim 19, wherein step (d) is carried out in a mobile phase comprising 0.1%-0.2% TFA in water.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Schematic illustration of TNFR2:Fc.

(2) FIG. 2: TNFR2:Fc (left), TNF-alpha (right).

(3) FIG. 3: Complex of TNFR2:Fc and TNF-alpha.

(4) FIG. 4: Structural representation of the TNFR2:Fc N-terminal TNF alpha receptor domain (see also SEQ ID NO: 1). Amino acids are indicated by single letter code. Asparagine linked N-glycans and serine or threonine linked O-glycans are indicated graphically. Correct disulphide bridging is shown by light grey bars between specific cysteine residues.

(5) FIG. 5: Structural representation of incorrectly disulphide bridged peptide T7 (see also SEQ ID NO: 4) and the internal reference peptide T27 (see also SEQ ID NO: 5). Amino acids are indicated by single letter code. The T7 peptide exhibits an aberrant disulphide bridge between cysteins 78 and 88 and its abundance correlates negatively with bioactivity. The reference peptide T27 is not involved in disulphide bridging.

(6) FIG. 6: Disulphide bridges of the receptor (X-ray structure taken from Solution of the Structure of the TNF-TNFR2 Complex. Mukai et al., Sci Signal 3(148), ra83, November 2010; labeling of bridges and text added).

(7) FIG. 7: Representative data showing the relative amount of T7 determined according to the method using peak integration of T7 and T27 as described above in different samples with varying levels of bioactivity. The potency on the y-axis was determined using a reporter gene assay, the values are arbitrary values. Samples of different quality were analyzed and the correlation was determined based on all data points shown.

DESCRIPTION OF THE SEQUENCES

(8) TABLE-US-00003 (humanTNFreceptortype2;CD120b;p75/80;RefSeq(protein):NP_001057) SEQIDNO:1 MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPG60 QHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTC120 RPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICR180 PHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTS240 FLLPMGPSPPAEGSTGDFALPVGLIVGVTALGLLIIGVVNCVIMTQVKKKPLCLQREAKV300 PHLPADKARGTQGPEQQHLLITAPSSSSSSLESSASALDRRAPTRNQPQAPGVEASGAGE360 ARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDTDSSPSESPKDEQ420 VPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS461 (humanIgG1classheavychainconstantdomain) SEQIDNO:2 AlaSerThrLysGlyProSerValPheProLeuAlaProSerSerLysSerThr SerGlyGlyThrAlaAlaLeuGlyCysLeuValLysAspTyrPheProGluPro ValThrValSerTrpAsnSerGlyAlaLeuThrSerGlyValHisThrPhePro AlaValLeuGlnSerSerGlyLeuTyrSerLeuSerSerValValThrValPro SerSerSerLeuGlyThrGlnThrTyrIleCysAsnValAsnHisLysProSer AsnThrLysValAspLysLysValGluProLysSerCysAspLysThrHisThr CysProProCysProAlaProGluLeuLeuGlyGlyProSerValPheLeuPhe ProProLysProLysAspThrLeuMetIleSerArgThrProGluValThrCys ValValValAspValSerHisGluAspProGluValLysPheAsnTrpTyrVal AspGlyValGluValHisAsnAlaLysThrLysProArgGluGluGlnTyrAsn SerThrTyrArgValValSerValLeuThrValLeuHisGlnAspTrpLeuAsn GlyLysGluTyrLysCysLysValSerAsnLysAlaLeuProAlaProIleGlu LysThrIleSerLysAlaLysGlyGlnProArgGluProGlnValTyrThrLeu ProProSerArgGluGluMetThrLysAsnGlnValSerLeuThrCysLeuVal LysGlyPheTyrProSerAspIleAlaValGluTrpGluSerAsnGlyGlnPro GluAsnAsnTyrLysThrThrProProValLeuAspSerAspGlySerPhePhe LeuTyrSerLysLeuThrValAspLysSerArgTrpGlnGlnGlyAsnValPhe SerCysSerValMetHisGluAlaLeuHisAsnHisTyrThrGlnLysSerLeu SerLeuSerProGlyLys (Etanercept) SEQIDNO:3 LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDST60 YTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRK120 CRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTS180 TSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSC240 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD300 GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK360 GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS420 DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK467

EXAMPLES

Example 1

Determination of Relative Amount of T7

(9) TNFR2:Fc is a fusion protein composed of a C-terminal Fc antibody domain and an N-terminal TNF alpha receptor 2 domain. The structure of the TNF alpha receptor domain 2 is critical for bioactivity of this biopharmaceutical and is highly complex containing multiple O-glycans, two N-glycans and eleven disulphide bridges (see FIGS. 4 and 6). It could be shown that at least one variant form of the molecule exists in the final TNFR2:Fc drug substance as a result of different disulphide bridging.

(10) By digesting TNFR2:Fc samples with trypsin under non-reducing conditions, the protein can be cleaved into smaller components, while the disulphide bridge structures remain intact. Elucidation of the peptides using RP-HPLC-MS analysis verified the presence of the expected correctly bridged peptides as well as a peptide termed T7, which was shown to contain an aberrant disulphide bridge between cysteins 78 and 88 (see FIGS. 5 and 6, and Table 2 above). While the abundance of some correctly bridged structures were found to correlate with increased bioactivity, their diversity and complex elution profiles precluded them from use as stable indicators of bioactivity using an LC-UV/Vis approach. However, the incorrectly bridged peptide T7 exhibited a stable correlation with reduced bioactivity. Representative data is shown in FIG. 7, demonstrating the strong correlation between bioactivity and the relative amount of T7 determined according to the method using peak integration of T7 and T27 as described above.

(11) The relative T7 amount can be determined as follows.

(12) All samples are thawed at room temperature. All centrifugation steps are carried out on a refrigerated centrifuge (e.g., Eppendorf Centrifuge 5804R; Eppendorf, Hamburg, Germany). About 80-300 g, preferably 100-200 g of protein are typically used per sample. In order to adapt the buffer it may be necessary to concentrate the samples to an appropriate protein concentration, e.g. by using a concentration device such as Vivaspin 500, 10 kDa, Sartorius Art. Nr.: VS0102. To the samples or their concentrates, wash buffer (50 mM TRIS pH 8) is added to a final volume of about 200 l of wash buffer. 100 l of denaturation solution are added. The denaturation solution is prepared by mixing 950 l of 0.1% RapiGest (Waters, no. 186001861) in 50 mM TRIS pH 8 with 50 l M iodoacetamide (Sigma, no. 11149) in 50 mM TRIS pH 8. The reagent is prepared directly before use, and covered e.g. with aluminum foil for protection against light. Bubbles are removed by light tapping and the sample is incubated for 40 min at 50 C. (e.g. by using Thermomixer Comfort; Eppendorf Hamburg, Germany).

(13) The samples are allowed to cool to room temperature and then buffer exchanged to a final volume of 20-40 l of digestion buffer (50 mM MES (Sigma, M5287) in HPLC water pH 6). Then the samples are each transferred into a safe lock reaction tube, and 25 l of 50 mM digestion buffer (50 mM MES in HPLC water pH)+25 l of digestion buffer with surfactant (0.1% RapiGest (Waters, no. 186001861) in 50 mM MES pH 6 buffer) are added. 12 l of the freshly reconstituted 1 g/l trypsin are added (Promega, Trypsin Gold, Mass Spec Grade, reconstituted with 50 mM MES pH 6.0 buffer directly before use). The sample is carefully agitated by gentle flicking, then spinned down shortly, and incubated for 17 h at 37 C. in a heating block (e.g. Thermomixer Comfort; Eppendorf Hamburg, Germany).

(14) Following incubation, the samples are removed from the thermomixer, and 49 l of termination solution (1% formic acid (HPLC grade, e.g., ThermoScientific, no. 40967) in 10% acetonitrile (acetonitrile of HPLC grade 99.9%, e.g. Merck no. (1.00030.2500)) are added. It is gently mixed by lightly flicking. The samples are centrifuged for approx. 10 min at 16,000 g and 6 C. A slight opaque pellet may be barely visible after centrifugation. If the sample is still opaque following this first centriguation, the centrifugation is repeated. Then the supernatant is transferred into a 300 l autosampler glass vial, water is added to an overall volume of approximately 236 l, and the samples are placed in a cooled autosampler.

(15) HPLC is carried out using a liquid chromatograph with a UV detector (e.g. 1200SL Series LC system with online degasser (G1322A), binary pump module (G1312), thermostatted autosampler (G1329A/G1330A), thermostatted column department (G1316A), VWD detector (G1314A); all Agilent Technologies, Waldbronn, Germany) and a suitable column (e.g., Ascentis Express Peptide ES-C18, 2.1 mm ID15 cm L Cat. No. 53307-U; Supelco). The following parameters are used:

(16) TABLE-US-00004 Run time: 45 min Flow rate: 0.8 mL/min CompressibilityLeftPump 46 CompressibilityRightPump 115 Column temperature: 60 C. (=setpoint) Injection volume: 50 L Autosampler temperature: 2-10 C. UV detector: Wavelength: 215 nm PeakWidth: 0.025 min MWD/DAD detector: Wavelength: 215 nm PeakWidth: 0.03 min Bandwidth: 4 nm No Reference SlitWidth: 4 nm Mobile phase A 0.1% TFA (HPLC grade, Fluka no. 40967) in HPLC water Mobile phase B 0.1% TFA in 90% Acetonitrile and 10% HPLC water Gradient

(17) TABLE-US-00005 TABLE 3 Gradient Flow Rate Time [min] % B [ml/min] 0.0 0 0.8 2.5 0 0.8 25 16 0.8 28 18 0.8 33 100 0.8 37 100 0.8 40 0 0.8 45 0 0.8

(18) To check for carryover, blank samples (mobile phase A) can be injected every e.g. 10th injection.

(19) Integration of the chromatograms is performed using a suitable chromatography data system, e.g. Chromeleon (Dionex, Sunnyvale, Calif., USA). The relative amount of T7 peptide is calculated according to the following equation (formula (1)):

(20) $\begin{array}{cc}\mathrm{rel}.\%\left(T7\right)=\frac{\mathrm{area}\left(T7\right)}{\mathrm{area}\left(T7\right)+\mathrm{area}\left(T27\right)}100& \left(1\right)\end{array}$ Area(T7): peak area of T7 Area(T27): peak area of T27

(21) To guarantee that proper amount of sample was applied onto the column, the peak area T27 in a given samples is compared to the average peak area of a reference substance injection. Calculations are performed according to following equation:

(22) $\mathrm{Applied}\mathrm{sample}\mathrm{amount}\left[\%\right]=\frac{a_{\mathrm{sample}}}{a_{\mathrm{reference}}}*100$ a.sub.sample T27 peak area of TNFR:Fc sample a.sub.reference average T27 peak area of TNFR:Fc reference substance injections

(23) All used substances were Ph. Eur. Grade or of comparable quality. The buffers were prepared with purified and de-ionized water. The suppliers and order numbers for instruments, materials and reagents indicated are given as examples. These products can be considered interchangeable with comparable products of the same or better quality.

(24) Most notably, the relative amounts of T7 found in all US and EU batches of Enbrel examined showed values of 2.3% or higher when analysed and calculated according to the determination method of the present invention using the T27 peptide's signal as reference peak, cf. Table 5.

(25) TABLE-US-00006 TABLE 4 Relative amounts of T7 in the reference product Enbrel Batch T7 [%] #1026663 (US) 2.4 #F36988 (EU) 2.4 #F76195 (EU) 2.8 #1028435 (US) 2.3 #1026662 (US) 2.3 #F69006 (EU) 2.8

(26) This demonstrated that the methods of production and purification presented herein are capable of producing TNFR2:Fc and in particular etanercept at an unprecedented level of reduced T7 amount.

Example 2

Producing TNFR2:Fc with Varying Amounts of T7

(27) It is known that wrongly bridged variants can already be formed in the upstream process (USP) for the manufacturing of TNFR2:Fc. By analyzing samples of DoE (Design of Experiments) process characterization studies it could be shown that the amount of wrongly bridged variants can be influenced on the USP level (see Table 4). The provided values are obtained from a statistical model. The TNFR2:Fc samples taken to establish this model were subjected only to Protein A affinity chromatography and not purified via hydrophobic interaction chromatography. The relative amount of T7 was determined according to the method using peak integration of T7 and T27 as described below.

(28) TABLE-US-00007 TABLE 5 95% Confidence 95% Confidence interval interval T7 low high Temperature T7 low high pH [%] limit limit [ C.] [%] limit limit 6.65 3.86 3.59 4.14 30.5 2.21 1.92 2.5 6.70 3.59 3.42 3.76 31.5 2.4 2.24 2.55 6.80 3.25 3.12 3.38 33.0 3.19 3.05 3.32 6.90 3.2 3.04 3.35 34.5 4.59 4.3 4.88 6.95 3.27 3.07 3.48 35.5 5.87 5.31 6.42

Example 3

Purification of TNFR2:Fc

(29) During downstream processing (DSP), wrongly bridged variants are mainly depleted on the HIC purification step, while small amounts may already be depleted by a previous anion exchange purification step, such as a MMC purification step in flow-through mode.

(30) Affinity Chromatography (Protein A)

(31) The purification process starts from cell free culture supernatants. The material was 0.2 m filtered. Utilizing the Fc part of the fusion protein, TNFR:Fc was captured by affinity chromatography on Protein A resin. The Protein A interaction with the Fc part is very specific. Therefore, the capture chromatography very efficiently separates host cell proteins (HCPs), DNA and virus from the product.

(32) Process temperature was 21 C. The cell culture supernatant was loaded onto MabSelect SuRe resin (GE Healthcare), equilibrated with sodium phosphate buffer of pH 7.0 further comprising 150 mM sodium chloride. Then, the column was washed with the same buffer until UV.sub.280 returns to signal close to baseline (about 2 to 6 column volumes).

(33) To increase the HCP removal capacity of the capture step, an additional wash step was introduced. This wash buffer contained sodium acetate and 0-500 mM sodium chloride. It was followed by product elution with an acidic buffer having a pH of 3.2. The eluates were combined and processed to the next purification step.

(34) Anion Exchange Chromatography (AEX)

(35) The intermediate resulting from the affinity chromatography step was adjusted to pH 7.5 and loaded onto a Fractogel TMAE HiCap (M) resin (Merck). Subsequently, the column was rinsed with sodium phosphate buffer and finally the product was eluted with sodium phosphate buffer containing 150 mM sodium chloride. The eluates were combined and processed to the next purification step.

(36) MM Chromatography (MMC)

(37) The combined eluates from the anion exchange chromatography step were adjusted in conductivity using 4M sodium chloride, and the pH was adjusted to pH 6.0 using a phosphoric acid solution of pH 2. Then the intermediate was loaded onto Capto adhere resin (GE Healthcare), equilibrated with 20 mM sodium phosphate, 450 mM sodium chloride pH 6.0 and the column was then washed with 20 mM sodium phosphate, 450 mM sodium chloride pH 6.0. The flow through and the early wash comprising the product were collected and pooled.

(38) Hydrophobic Interaction chromatography (HIC)

(39) The pooled fractions from the MM chromatography were diluted with sodium citrate buffer pH 6.0 comprising 1.4M sodium sulfate. The conductivity of the solution was about 80 mS/cm. Then, the solution was loaded onto Toyopearl Phenyl 650 (M) and equilibrated with sodium citrate buffer pH 6.0 comprising sodium sulfate. The column is then rinsed with the same buffer. Finally, the column is eluted using a 0-100% gradient from the equilibration buffer to elution buffer (25 mM sodium citrate pH 6.0).

(40) The purity of the product was determined using size exclusion chromatography (SEC), and by determining the amount of DNA, host cell proteins (HCP), Protein A, and endotoxin. Further, the step yield and total yield was calculated for each purification step. The following Table 8 shows data obtained with the above described method for at least three runs.

(41) TABLE-US-00008 TABLE 6 Depletion of peptide T7 during downstream processing Purification Potency Batch steps T7 [%] [arbitrary units] 1 Prot A 3.63 71 1 +AEX 3.05 74 +MMC 1 +HIC 1.54 91 2 Prot A 3.44 72 2 +AEX 3.16 75 +MMC 2 +HIC 1.23 93

Example 4

Stability Under Stress Conditions

(42) Applying the methods disclosed herein allows obtaining TNFR:Fc preparations with a decreased relative T7 amount as compared to TNFR:Fc preparations in the state of the art. The low amount of T7 is also maintained upon stress treatment.

(43) TABLE-US-00009 TABLE 7 T7 relative to T27 Sample No. (% T7) 3 Final formulation 1.2 3 1 month @ 40 C. 1.8 4 1 month @ 40 C. 1.9

LIST OF REFERENCES

(44) U.S. Pat. No. 7,294,481 U.S. Pat. No. 6,048,728 WO 2011/134920 WO 2011/134921 Mukai et al. (2010) Solution of the Structure of the TNF-TNFR2 Complex., Sci Signal 3 (148), ra83