METHOD FOR DETERMINING THE IN VIVO INTERACTION MODE

Abstract

Herein is reported a method for determining the binding interaction with a multimeric antigen of an antibody of the human IgG1 subclass that has at least two binding sites specifically binding to the antigen comprising the steps of 1) determining the binding affinity of the antibody for the multimeric antigen, and 2) incubating a mixture comprising the antibody and a polypeptide that is derived from lysine-gingipain of Porphyromonas gingivalis under conditions and for a time sufficient to cleave the antibody into Fabs and Fc-region, and determining the binding affinity of the Fabs of the antibody for the multimeric, whereby the binding affinity of the antibody to the multimeric antigen to be affinity-driven if the binding affinity determined in both steps are comparable and to be avidity-driven if the binding affinity determined in both steps are different.

Claims

1. A method for determining the binding interaction with a multimeric antigen of an antibody of the human IgG1 subclass comprising the following steps: 1) determining the binding affinity of the antibody for the multimeric antigen, 2) incubating a mixture comprising the antibody, the antigen and lysine-gingipain of Porphyromonas gingivalis or an enzymatically active fragment thereof at a pH of 7.5 to 8.5, in the presence of a reducing agent, at a temperature of 30° C. to 42° C., for a time of 10 minutes to 240 minutes to cleave the antibody into Fabs and a Fc-region, whereby the concentration of the antibody is higher than the concentration of the antigen, and determining the binding affinity of the Fabs of the antibody for the multimeric antigen, and 3) determining the binding affinity of the antibody to the multimeric antigen to be affinity-driven if the binding affinity determined in step 1) and 2) is comparable and to be avidity-driven if the binding affinity determined in step 1) and 2) is different.

2. A method for selecting the assay format for determining the binding interaction of an antibody of the human IgG1 subclass with a multimeric antigen comprising the following steps: 1) determining the binding affinity of the antibody for the multimeric antigen using a surface plasmon resonance method, 2) incubating a mixture comprising the antibody, the antigen and lysine-gingipain of Porphyromonas gingivalis or an enzymatically active fragment thereof at a pH of 7.5 to 8.5, in the presence of a reducing agent, at a temperature of 30° C. to 42° C., for a time of 10 minutes to 240 minutes to cleave the antibody into Fabs and a Fc-region, whereby the concentration of the antibody is higher than the concentration of the antigen, and determining the binding affinity of the Fabs of the antibody for their antigen using surface plasmon resonance by directly applying the incubated reaction mixture obtained in the previous step in the surface plasmon resonance method, whereby the binding affinity of the antibody to the multimeric antigen is i) affinity-driven if the binding affinity determined in step 1) and 2) is comparable, or ii) avidity-driven if the binding affinity determined in step 1) and 2) is different, and selecting i) in case of an affinity-driven interaction with a soluble multimeric antigen a solution assay, ii) in case of an avidity-driven interaction with a soluble multimeric antigen a solution or a surface assay, iii) in case of an affinity-driven interaction with a surface bound antigen a solution assay, or iv) in case of an avidity-driven interaction with a surface bound antigen a surface assay for determining the binding interaction of the antibody of the human IgG1 subclass with the multimeric antigen.

3. The method according to claim 1, wherein the binding affinity is determined in solution using an ELISA or a surface plasmon resonance method.

4. The method according to claim 1, wherein the binding affinity is determined using a cellular assay using FACS or a cellular effect.

5. The method according to claim 3, wherein the binding affinities determined in steps 1) and 2) of the antibody to the multimeric antigen are comparable if the binding affinities determined in both steps differ by a factor of 2 or less, wherein the smaller value is used as basis for the calculation; and is different if the binding affinities determined in both steps differ by more than a factor of 2, wherein the smaller value is set to 100%.

6. The method according to claim 5, wherein the binding affinity is determined with an antibody: multimeric antigen ratio of 10 or more.

7. The method according to claim 1, wherein the polypeptide that is derived from lysine-gingipain of Porphyromonas gingivalis is the lysine-gingipain of Porphyromonas gingivalis.

8. The method according to claim 7, wherein the polypeptide that is derived from lysine-gingipain of Porphyromonas gingivalis comprises the amino acid sequence of SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04 or a functional variant thereof.

9. The method according to claim 7, wherein the polypeptide that is derived from lysine-gingipain of Porphyromonas gingivalis has an amino acid sequence that comprises at least residues 230 to 739 of SEQ ID NO: 01.

10. The method according to claim 1, wherein the reducing agent is selected from the group consisting of 2-mercaptoethanol, cysteine, and dithiothreitol.

11. The method according to claim 10, wherein the reducing agent is cysteine.

12. The method according to claim 10, wherein the reducing agent is cysteine at a concentration of from 0.5 mM to 10 mM.

13. The method according to claim 1, wherein the pH value is about pH 8.

14. The method according to claim 1, wherein the temperature is of from 35° C. to 38° C.

15. The method according to claim 1, wherein the incubating is for a time of about 60 minutes.

16. The method according to claim 1, wherein the incubated mixture is used for the determination of the binding affinity without intermediate purification.

17. The method according to claim 1, wherein the determining of the binding affinity is by surface plasmon resonance.

18. The method according to claim 6, wherein the incubated mixture is used for the determination of the binding affinity without intermediate purification.

19. The method according to claim 18, wherein the determining of the binding affinity is by surface plasmon resonance.

20. The method according to claim 2, wherein the incubated mixture is used for the determination of the binding affinity without intermediate purification.

21. The method according to claim 20, wherein the determining of the binding affinity is by surface plasmon resonance.

22. The method according to claim 21, wherein the binding affinities determined in steps 1) and 2) of the antibody to the multimeric antigen are comparable if the binding affinities determined in both steps differ by a factor of 2 or less, wherein the smaller value is used as basis for the calculation, and is different if the binding affinities determined in both steps differ by more than a factor of 2, wherein the smaller value is set to 100%.

23. The method according to claim 19, wherein the binding affinity is determined with an antibody: multimeric antigen ratio of 10 or more.

24. The method according to claim 22, wherein the binding affinity is determined with an antibody: multimeric antigen ratio of 10 or more.

25. The method according to claim 23, wherein the polypeptide that is derived from lysine-gingipain of Porphyromonas gingivalis is the lysine-gingipain of Porphyromonas gingivalis.

26. The method according to claim 24, wherein the polypeptide that is derived from lysine-gingipain of Porphyromonas gingivalis is the lysine-gingipain of Porphyromonas gingivalis.

27. The method according to claim 25, wherein the polypeptide that is derived from lysine-gingipain of Porphyromonas gingivalis comprises the amino acid sequence of SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04 or a functional variant thereof.

28. The method according to claim 26, wherein the polypeptide that is derived from lysine-gingipain of Porphyromonas gingivalis comprises the amino acid sequence of SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04 or a functional variant thereof.

29. The method according to claim 8, wherein the reducing agent is selected from the group consisting of 2-mercaptoethanol, cysteine, and dithiothreitol.

30. The method according to claim 27, wherein the reducing agent is selected from the group consisting of 2-mercaptoethanol, cysteine, and dithiothreitol.

31. The method according to claim 28, wherein the reducing agent is selected from the group consisting of 2-mercaptoethanol, cysteine, and dithiothreitol.

32. The method according to claim 29, wherein the reducing agent is cysteine at a concentration of from 0.5 mM to 10 mM.

33. The method according to claim 30, wherein the reducing agent is cysteine at a concentration of from 0.5 mM to 10 mM.

34. The method according to claim 31, wherein the reducing agent is cysteine at a concentration of from 0.5 mM to 10 mM.

Description

DESCRIPTION OF THE FIGURES

[0233] FIG. 1 Decision tree.

[0234] FIG. 2 UHR ESI-QTOF mass spectrometry of bevacizumab digested with lysine-gingipain of Porphyromonas gingivalis for one hour at 37° C. Only Fab and Fc fragments were detected.

[0235] FIG. 3 UHR ESI-QTOF mass spectrometry of bevacizumab digested with papain for 1.5 h at 37° C. Beside Fab and Fc fragments, several Fab- and antibody fragments were detected.

[0236] FIG. 4 UHR ESI-QTOF mass spectrometry of bevacizumab digested with papain for 2 h at 37° C. Several antibody fragments were detected. Fab and Fc fragment could not be identified.

[0237] FIG. 5A Surface plasmon resonance sensorgrams of bevacizumab.

[0238] FIG. 5B Surface plasmon resonance sensorgrams of a recombinant bevacizumab Fab.

[0239] FIG. 5C Surface plasmon resonance sensorgrams of bevacizumab digested with lysine-gingipain of Porphyromonas gingivalis.

[0240] FIG. 5D Surface plasmon resonance sensorgrams of bevacizumab, bevacizumab digested with papain without termination of the digest, and bevacizumab digested with papain with termination of the digest.

[0241] FIG. 6 Results of the analysis of the participation of a second binding site to target binding for the in vivo interaction mode.

[0242] FIG. 7 Results of the analysis of the participation of a second binding site to target binding for the in vivo interaction mode.

EXAMPLES

[0243] Bevacizumab was obtained from Roche Diagnostics GmbH (Mannheim, Germany). Papain was obtained as suspension with a concentration of 10 mg/mL from Sigma-Aldrich/Roche Diagnostics GmbH. Lysine-gingipain of Porphyromonas gingivalis was obtained under the trade name GingisKHAN from Genovis (Lund, Sweden). GingisKHAN was reconstituted in 200 μL double distilled water (ddH2O) resulting in 2000 U/200 μL, and the 10× reducing agent was freshly prepared in 50 μL ddH2O (final concentration: 20 mM cysteine) prior to each digestion.

Example 1

Transient Fab Expression and Purification

[0244] The antibody light chain and heavy chain Fd-fragments were ordered as gene syntheses and cloned via unique restriction sites using standard cloning procedures into separate expression vectors for each chain enabling secretory expression in HEK cells growing in suspension. Transfection (1:1 plasmid ratios) into HEK293-F cells (Invitrogen, Cat. No. 510029) was performed according to the cell supplier's instructions using Maxiprep (Qiagen, Cat. No. 12163) preparations of the antibody vectors, Opti-MEM I medium (Invitrogen, Cat. No. 31985) 293fectin (Invitrogen, Cat. No. 31985070), and an initial cell density of 1-2×10E+06 viable cells/mL in serum-free FreeStyle 293 expression medium (Invitrogen, Cat. No. 12338018). Antibody containing cell culture supernatants were harvested after 7 days of cultivation in shake flasks by centrifugation at 14,000×g for 30 min. and filtered through a 0.22 μm sterile filter (Thermo Scientific, Cat. No. 566-0020). The antibodies were purified directly from the supernatant, or the supernatant was stored at −80° C. until purification. The quality of the purified Fab was analyzed by SEC and BioAnalyzer.

Example 2

[0245] Enzymatic Cleavage of Bevacizumab with Papain

Without Purification:

[0246] The antibody was diluted in 20 mM Histidine, 140 mM NaCl, pH 6.0 to a final concentration of 1 mg/mL, added 2 μL 250 mM L-cysteine (Sigma-Aldrich, Schnelldorf, Germany) and 10.9 μL diluted papain (7.34 U/mL in 20 mM Histidine, 140 mM NaCl, pH 6.0), and incubated 1 h at 37° C.

With Purification:

[0247] The antibody was incubated with Papain (0.8 U/mg mAb; Sigma-Aldrich/Roche) in presence of 5 mM Cystein for 170 minutes at 37° C. To isolate the Fab from non-cleaved antibodies, Fc-fragments and Papain, the mixture was applied to a CaptureSelect IgG-CH1 and MabSelectSuRe affinity chromatography (GE Healthcare) according to manufacturer protocol. Finally, a size exclusion chromatography using a Superdex 75 10/300 GL column (GE Healthcare) was performed using 140 mM NaCl, 20 mM histidine (pH 6.0) as running buffer. Protein concentration of the Fab was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. The purity was analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining with Coomassie brilliant blue.

Example 3

[0248] Enzymatic Cleavage of Bevacizumab with Lysine-Gingipain of Porphyromonas gingivalis

[0249] GingisKHAN was reconstituted in 200 μL ddH2O resulting in 2000 U/200 μL, and the 10× reducing agent was freshly prepared in 50 μL ddH2O (final concentration: 20 mM Cysteine) prior to each digestion. 100 μg antibody was diluted to a final concentration of 1 mg/mL in 100 mM Tris, pH 8.0 and subsequently digested with 10 μL GingisKHAN and 11 μL of freshly prepared 10× reducing agent at 37° C. for 1 hour.

Example 4

UHR-ESI-QTOF Mass Spectrometry

[0250] Samples were desalted by HPLC on a Sephadex G25 column (Kronlab, 5×250 mm, TAC05/250G0-SR) using 40% acetonitrile with 2% formic acid (v/v). The total mass was determined via ESI-QTOF MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion). Calibration was performed with sodium iodide (Waters ToF G2-Sample Kit 2 Part: 700008892-1). For the recombinant and purified Fabs, data acquisition was done at 900-2600 m/z (ISCID: 0.0 eV), for the hIgG1s or digested hIgG1s, data acquisition was done at 900-4000 m/z (ISCID: 0.0 eV). The raw mass spectra were evaluated and transformed into individual relative molar masses using an in-house developed Roche software tool. For visualization of the results, the same in-house developed software was used to generate deconvoluted mass spectra.

Example 5

Surface Plasmon Resonance

[0251] Binding affinities and kinetics were investigated by surface plasmon resonance using a BIAcore T200 instrument (GE Healthcare). All experiments were performed at 25° C. using PBS-T (10 mM Na2HPO4, 140 mM NaCl, 0.05% Tween 20, pH 7.4) as running and dilution buffer. An anti-His-tag (GE Healthcare, #28995056) or an anti-human Fab antibody (GE Healthcare, #28958325) was immobilized on a Series S CM5 Sensor Chip (GE Healthcare, #29104988) using standard amine coupling chemistry. Histidine-tagged human VEGF or full length IgG/Fabs were captured on the surface leading to a response between 10 and 50 RU. The analytes were injected for 180 s at concentrations from 2.2 nM up to 1800 nM onto the surface (association phase) at a flow rate of 30 μL/min. The dissociation phase was monitored for up to 3600 sec. by washing with running buffer. The surface was regenerated by injecting 10 mM Glycine pH 1.5 for 60 sec. at a flow rate of 5 μL/min. Bulk refractive index differences were corrected by subtracting the response obtained from a mock surface and by subtracting blank injections (double referencing). The derived curves were fitted to a 1:1 Langmuir binding model using the BIAevaluation software.

Example 6

VEGF-A Specific Reporter Gene Assay

[0252] A reporter gene cell line GloResponse™ NFAT-RE-luc2P/KDR HEK293 expressing KDR (KDR=VEGF receptor 2) and a NFAT responsive element in front of the firefly luciferase was purchased from Promega Corporation, Madison, USA. Upon binding of VEGF-A to the KDR a signal transduction via Calcineurin results in activation of NFAT, translocation to the nucleus, binding to the NFAT responsive element and subsequently expression of the luciferase gene. VEGF121 (10.8 nM 40 μL/well) was incubated with the anti-VEGF antibody, said antibody digested with lysine-gingipain of Porphyromonas gingivalis, a bispecific anti-VEGF-A/second, non-related antigen CrossMab, and a negative lysine-gingipain of Porphyromonas gingivalis control (diluted in DMEM, 1% FBS, 40 μL/well) for approximately 30 min. at room temperature. 5×10.sup.4 GloResponse™ HEK293 cells (Promega Coop., cultured in FreeStyle™ 293 Expression Medium, 100 μg/mL Hygromycin B, 250 μg/mL Geneticin (Thermo Fischer Scientific, Sigma-Aldrich, Calbiochem) in 40 μL DMEM, supplemented with 1% FBS, were added as suspension and incubated for 5 hours at 37° C., 5% CO.sub.2. The plate was equilibrated at room temperature for approximately 15 min. before the luminescence substrate (Promega Coop., ONE-Glo™ EX, 60 μL/well) was added. The contents were mixed on an orbital shaker for about 1-3 min. at 600 rpm. The luminescence intensity was measured with a luminescence reader.

Example 7

CEACAM5 Cell Surface Binding Assay

[0253] 1×10.sup.5 gastric adenocarcinoma cells cultured in RPMI1640, 20% fetal bovine serum (FBS), 1× GIBCO GlutaMax (Thermo Fischer Scientific, Dreieich, Germany) were washed twice with PBS, 5% FBS, resuspended in PBS, 5% FBS and incubated with the anti-CEA antibody, a purified Fab fragment obtained by papain digestion of said anti-CEA antibody, said antibody digested with lysine-gingipain of Porphyromonas gingivalis and negative controls (an antibody binding to a non-related target, lysine-gingipain of Porphyromonas gingivalis only) for one hour at 4° C. Bound antibodies/Fab fragments were detected using a mouse anti-human kappa light chain antibody (150 μg/mL) labeled using the Alexa Fluor 647 Protein Labeling Kit according to the instructions of the manufacturer (Molecular Probes, Thermo Fischer Scientific). The mixture was incubated in the dark at 4° C. for 30 min. and analyzed by flow cytometry using a BD FACSCanto II and the FACSDiva Software (BD Biosciences, Heidelberg, Germany). The specificity was verified with an isotype control (Alexa Fluor 647-labelled mouse IgG2a, BD Biosciences). Gating of viable cells was done using forward and sideward scatter based on size and granularity, and the bound antibody/Fab fragment was detected by measuring the fluorescence signal.