Methods of activating CD32b/c comprising administering an antibody that binds BDCA-2 (CD303)

Abstract

The present invention relates to polypeptides comprising a mutant human IgG.sub.4, which mutant human IgG.sub.4 is capable of increasing the binding to and activation of immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing FcγRIIb/c (CD32b), but not FcγRIIa (CD32a). More specifically, the invention relates to polypeptides comprising at least one human IgG.sub.4 with a lysine at position 409 (409K), using the EU index according to Kabat et al., which IgG.sub.4 is capable of binding to human CD32b/c with a statistically significant (p=0.05) higher binding affinity than a wild-type human IgG.sub.1 and than a wild-type human IgG.sub.4, for use in the prevention and/or treatment of an autoimmune disease or allergy, as further defined in the claims.

Claims

1. A method of activating human CD32b/c on CD32b/c expressing cells in the treatment of an autoimmune disease in a human subject, comprising the step of administering to said human subject a polypeptide comprising at least one human IgG.sub.4 with a lysine at position 409 (409K), using the EU index according to Kabat, wherein the IgG.sub.4 recognizes an epitope of BDCA-2 (CD303) and is capable of binding to human CD32b/c with a statistically significant (p=0.05) higher binding affinity than a wild-type human IgG.sub.1 having the amino acid sequence of SEQ ID NO: 10 and than a wild-type human IgG.sub.4 having the amino acid sequence of SEQ ID NO: 1, when subjecting the polypeptide and the wild type antibodies to an ELISA assay with 1 hour incubation at room temperature in 1×PBS on ELISA plates precoated with recombinant CD32b, wherein the polypeptide comprises the heavy chain CDRs 1-3 shown in SEQ ID NOs: 2-4 and light chain CDRs 1-3 shown in SEQ ID NOs: 5-7.

2. The method of claim 1, wherein the autoimmune disease is an inflammatory autoimmune disease.

3. The method of claim 1, wherein the autoimmune disease is further characterized by increased plasma levels of autoantibodies as compared to healthy subjects.

4. The method of claim 1, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, psoriasis, multiple sclerosis, and rheumatoid arthritis.

5. The method of claim 1, wherein the autoimmune disease is systemic lupus erythematosus or psoriasis.

6. The method of claim 1, wherein the human IgG.sub.4 further comprises a proline at position 241 (241P).

7. The method of claim 1, wherein the human IgG.sub.4 has the amino acid sequence of SEQ ID NO: 1 (hIgG.sub.4) with a lysine at the position corresponding to position 409 (409K) and a proline at the position corresponding to position 241 (241P), using the EU index according to Kabat.

8. The method of claim 1, wherein the human IgG.sub.4 409K shows an increased activation of human CD32b/c as compared to wild type human IgG.sub.4.

9. The method of claim 1, wherein the human IgG.sub.4 409K shows an increased activation of human CD32b/c as compared to wild type human IgG.sub.1.

10. The method of claim 1, wherein said polypeptide is not fused to a bioactive peptide amino acid sequence.

11. The method of claim 1, wherein the polypeptide is a monoclonal antibody.

12. The method of claim 11, wherein the monoclonal antibody does not recognize an epitope of human CD32b/c via its antigen binding region.

13. The method of claim 11, wherein the monoclonal antibody is a monospecific antibody.

14. The method of claim 11, wherein the antibody comprises the heavy chain CDRs 1-3 shown in SEQ ID Nos: 2-4 and light chain CDRs 1-3 shown in SEQ ID Nos: 5-7.

15. The method of claim 11, wherein the antibody comprises the variable heavy chain of SEQ ID NO: 8 and the variable light chain of SEQ ID NO: 9.

16. The method of claim 1, wherein the human subject shows a dysregulation of CD32b expression, as compared to healthy subjects.

17. The method of claim 1, wherein the human subject has a CD32b 695T allele or a 2B.4 haplotype of the CD32b gene, or both; or wherein the human subject has a CD32b 695C allele or a 2B.1 haplotype of the CD32b gene, or both.

18. A method of activating human CD32b/c on CD32b/c expressing cells in the treatment of allergy in a human subject, comprising the step of administering to said human subject a polypeptide comprising at least one human IgG.sub.4 with a lysine at position 409 (409K), using the EU index according to Kabat, wherein the IgG.sub.4 recognizes an epitope of BDCA-2 (CD303) and is capable of binding to human CD32b/c with a statistically significant (p=0.05) higher binding affinity than a wild-type human IgG.sub.1 having the amino acid sequence of SEQ ID NO: 10 and than a wild-type human IgG.sub.4 having the amino acid sequence of SEQ ID NO: 1, when subjecting the polypeptide and the wild type antibodies to an ELISA assay with 1 hour incubation at room temperature in 1×PBS on ELISA plates precoated with recombinant CD32b, wherein the polypeptide comprises the heavy chain CDRs 1-3 shown in SEQ ID NOs: 2-4 and light chain CDRs 1-3 shown in SEQ ID NOs: 5-7.

19. The method of claim 18, wherein the human IgG.sub.4 further comprises a proline at position 241 (241P).

20. The method of claim 18, wherein the human IgG.sub.4 comprises the amino acid sequence of SEQ ID NO: 1 (hIgG.sub.4) with a lysine at the position corresponding to position 409 (409K) and a proline at the position corresponding to position 241 (241P), using the EU index according to Kabat.

21. The method of claim 18, wherein said polypeptide is not fused to a bioactive peptide amino acid sequence.

22. The method of claim 18, wherein the polypeptide is a monoclonal antibody.

23. The method of claim 22, wherein the monoclonal antibody does not recognize an epitope of human CD32b/c via its antigen binding region.

24. The method of claim 22, wherein the monoclonal antibody is a monospecific antibody.

25. The method of claim 22, wherein the antibody comprises the heavy chain CDRs 1-3 shown in SEQ ID Nos: 2-4 and light chain CDRs 1-3 shown in SEQ ID Nos: 5-7.

26. The method of claim 22, wherein the antibody comprises the variable heavy chain of SEQ ID NO: 8 and the variable light chain of SEQ ID NO: 9.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the binding affinity of different IgG.sub.1 mAbs and MB101 to recombinant human FcγRI (CD64) receptor (plate bound). It can be observed, that both IgG.sub.1 variants have a high affinity to CD64, as expected for IgG.sub.1 monoclonal antibodies. MB101 has a significantly lower affinity for CD64, which is also not surprising as MB101 is a humanized IgG.sub.4 molecule for which lower affinities to CD64 are reported, compared to IgG.sub.1 molecules

(2) FIG. 2 shows the binding affinity of a chimeric IgG.sub.1 mAb and MB101.10 to recombinant human FcγRIIa (CD32a) receptor (plate bound). It can be observed that MB101 has a moderately higher affinity for CD32a compared to the chimeric IgG.sub.1 molecule.

(3) FIG. 3 shows the binding affinity of an chimeric IgG.sub.1 mAb and MB101 to recombinant human FcγRIIb (CD32b) receptor (plate bound). It can be observed that MB101 has a significantly higher affinity for CD32b receptor compared to the chimeric IgG.sub.1 molecule.

(4) FIG. 4 shows the binding affinity of an chimeric IgG.sub.1 mAb and MB101.10 to recombinant human Fcγ RIIIa (CD16a) receptor (plate bound). It can be observed that MB101 has a significantly lower affinity for CD16a compared to the chimeric IgG.sub.1 molecule. A minimal residual binding affinity to CD16a can still be found.

(5) FIG. 5 shows the binding affinity of an chimeric IgG.sub.1 mAb and MB101.10 to recombinant human Fcγ RIIIb/c (CD16b/c) receptor (plate bound). It can be observed that MB101 has no affinity for CD16b/c. In comparison, the chimeric IgG.sub.1 molecule has a medium binding affinity to CD16b/c.

(6) FIG. 6: Interaction of different mAbs with transfectants expressing the human FcγR CD16.sub.V158. BW5147 transfectants expressing the FcγR CD16 were incubated with coated TGN1412 (dot), Tysabri® (square), Mabthera® (triangle), Remicade® (black diamond), or MB101 (blue diamond) at the indicated concentrations and murine IL-2 secretion was measured by an ELISA method (Bender MedSystems). Values are depicted as the mean of duplicate (MB101) or triplicate (TGN1412, Tysabri®, Mabthera®, and Remicade®) measurements±SD.

(7) FIG. 7: Interaction of different mAbs with transfectants expressing the human FcγR CD32A. BW5147 transfectants expressing the FcγR CD32A were incubated with coated TGN1412 (dot), Tysabri® (square), Mabthera® (triangle), Remicade® (black diamond), or MB101 (blue diamond) at the indicated concentrations and murine IL-2 secretion was measured by ELISA (Bender MedSystems). Values are depicted as the mean of duplicate (MB101) or triplicate (TGN1412, Tysabri®, Mabthera®, and Remicade®) measurements±SD.

(8) FIG. 8: Interaction of different mAbs with transfectants expressing the human FcγR CD32b/c. BW5147 transfectants expressing the FcγR CD32b/c were incubated with coated TGN1412 (dot), Tysabri® (square), Mabthera® (triangle), Remicade® (black diamond), or MB101 (blue diamond) at the indicated concentrations and murine IL-2 secretion was measured by ELISA (Bender MedSystems). Values are depicted as the mean of duplicate (MB101) or triplicate (TGN1412, Tysabri®, Mabthera®, and Remicade®) measurements±SD.

(9) FIG. 9: A single amino acid exchange introduced into the constant region of MB101 (Pool 6; R409K) leads to a significantly increased binding to CD32b and CD64. MB101 (anti-CD303) expressed as a wild type IgG.sub.4 and the two single mutation variants Pool 3 (anti-CD303 IgG.sub.4 S241 P) and Pool 6 (anti-CD303 IgG.sub.4 R409K) were coated in a log 3 serial dilution starting with 1 μg to 96 well plates in Na.sub.2HPO.sub.4 binding buffer for 2 h at 37° C. As controls, antibodies from the IgG.sub.4 (TGN1412) and IgG.sub.1 (BT-061) subclass were used. Then transfectants expressing one of the human FcγR were incubated on the coated plates for 18 h. Afterwards, cell-free supernatants were harvested and the mouse IL-2 content was analyzed by an ELISA method. N=5 with two biological duplicates for each value. Statistical analysis was performed using a two-way Anova with Bonferroni's multiple comparison test.

(10) FIG. 10: MB101 (anti-CD303 IgG.sub.4 S241 P/R409K) and Pool 6 (anti-CD303 IgG.sub.4 R409K) induce CD32b expressing transfectants to produce significantly more IL-2 than Pool 3 (anti-CD303 IgG.sub.4 S241 P) and the wt IgG.sub.4. To examine more precisely the CD32b binding of MB101, the wt IgG.sub.4 and the two single mutation variants, linear dilutions of the mAbs were coated to plates starting with 1 μg and incubated with the CD32b expressing transfectants. After 18 h supernatant was harvested and analyzed by an ELISA method. N=3 with two biological duplicates for each value. Statistical analysis was performed using a two-way Anova with Bonferroni's multiple comparison test.

(11) FIG. 11: MB101 (anti-CD303 IgG.sub.4 S241 P/R409K) and Pool 6 (anti-CD303 IgG.sub.4 R409K) show stronger binding to CD32b/c compared to Pool 3 (anti-CD303 IgG.sub.4 S241 P) and the wt IgG.sub.4. To examine more precisely the CD32b/c binding of MB101, the wt IgG.sub.4 and the two single mutation variants, linear dilutions of the mAbs were incubated with ELISA plates previously coated with recombinant CD32b/c. FcR-bound anti-BDCA2 Ab were probed with anti-human kappa-HRP and TMB-substrate.

(12) FIG. 12: MB101 (anti-CD303 IgG.sub.4 S241P/R409K) and Pool 6 (anti-CD303 IgG.sub.4 R409K) induce significantly more GFP expression in BDCA-2 transfectants than Pool 3 (anti-CD303 IgG.sub.4 S241 P) or the wt IgG.sub.4. To examine whether MB101, the wt IgG.sub.4 and the two single mutation variants differ in their binding to BDCA-2, the induction of GFP expression was analyzed in transfectants constitutively expressing BDCA-2. (A) mAbs were added at the indicated concentrations to medium alone and then 1×10.sup.5 BDCA-2 transfectants were added. Percentage of GFP.sup.+ cells was analyzed after 24 h of culture by using an LSRII and FlowJo software. (B) Antibodies were added at the indicated concentrations to 1×10.sup.5 CD99 (upper row) or CD32b (lower row) transfectants before adding 1×10.sup.5 BDCA-2 transfectants. Percentage of GFP.sup.+ cells was analyzed after 24 h of culture. One typical experiment is shown.

(13) FIG. 13: Incubation of BDCA-2 transfectants with MB101 (anti-CD303 IgG.sub.4 S241 P/R409K) and Pool 6 (anti-CD303 IgG.sub.4 R409K) induce significantly more GFP expression than Pool 3 (anti-CD303 IgG.sub.4 S241P) and the wt IgG.sub.4. To examine whether MB101, the wt IgG.sub.4 and the two single mutation variants differ in their binding to BDCA-2, GFP induction of BDCA-2 transfectants was analyzed. (A) Antibodies were added at the indicated concentrations to 1×10.sup.5 CD32b transfectants and then 1×10.sup.5 BDCA-2 transfectants were added. Percentage of GFP.sup.+ BDCA-2 transfectants was analyzed after 24 h of culture. Percentage of GFP.sup.+ cells was analyzed after 24 h of culture by using an LSRII and FlowJo software. (B) like (A) but the mean fluorescence intensity (MFI) was analyzed. Data from five independent experiments are shown. Statistical analysis was performed using a two-way Anova with Tukey's multiple comparison test.

(14) TABLE-US-00002 DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1-Human IgG.sub.4 heavy chain constant region (CH1-hinge-CH2-CH3; accession number UNIPROT P01861-1; S241 and R409 are emphasized)         10         20         30         40         50 ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV         60         70         80         90        100 HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES        110        120        130        140        150 KYGPPCPSCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED        160        170        180        190        200 PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK        210        220        230        240        250 CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK        260        270        280        290        300 GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG        310        320 NVFSCSVMHE ALHNHYTQKS LSLSLGK SEQ ID NO: 2-MB101 heavy chain CDR1 SGFSLSTSGMGVG SEQ ID NO: 3-MB101 heavy chain CDR2 HIWWEDDKYYNPSLKS SEQ ID NO: 4-MB101 heavy chain CDR3 TRNWDYYTMDY SEQ ID NO: 5-MB101 light chain CDR1 RASQEISGYLS SEQ ID NO: 6-MB101 light chain CDR2 YAASTLDS SEQ ID NO: 7-MB101 light chain CDR3 LQYASYPPT SEQ ID NO: 8-MB101 variable region heavy chain (CDRs emphasized) QVTLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWEDDKYY NPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCTRNWDYYTMDYWGQGTTVTVSSAST KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP SEQ ID NO: 9-MB101 variable region light chain (CDRs emphasized) DIQMTQSPSSVSASVGDRVTITCRASQEISGYLSWYQQKPGKAIKRLIYAASTLDSGVPSR FSGSRSGTDFTLTISSLQSEDFATYYCLQYASYPPTFGGGTKLEIKGTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 10-Human IgG.sub.1 heavy chain constant region (CH1-hinge-CH2-CH3; GenBank accession number AAC82527.1) STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK

EXAMPLES

Example 1—Fc Receptor Binding Characteristics of MB101

(15) In this example, different mAb were analyzed with regard to their ability to interact with five human Fcγ receptors CD16a CD16b/c, CD32a, CD32b and CD64. Two mutations (S241 P and R409K) were introduced into the human IgG.sub.4 antibody MB101 for stability purposes. MB101 is a recombinant humanized therapeutic monoclonal IgG.sub.4 antibody directed against CD303. MB101 was originally designed as a IgG.sub.4 therapeutic antibody in order to exhibit no or significantly reduced effector functions. In order to evaluate the ADCC (antibody dependent cell cytotoxicity) and CDC (complement derived cytotoxicity)-potential via CD16 and CD64, MB101 was characterized for its binding to these receptors.

(16) Despite not relevant for ADCC and CDC, MB101 (aCD303-hIgG.sub.4-S241P/R409K) was also tested for its binding to human Fcγ receptors CD32a and CD32b/c. As MB101 was intended and designed as humanized IgG.sub.4 molecule with stabilization, it was expected that MB101 behaves as typical IgG.sub.4 molecule and shows no affinity or functional interactions with human Fcγ receptors like CD16A, CD32a, CD32b/c and CD64.

(17) Fc receptor binding characteristics were analyzed in vitro in comparison to chimeric IgG.sub.1 antibody variants of MB101 by evaluating the binding profile of to recombinant plate bound human recombinant Fcγ receptors (FcγR) CD16a, CD32a, CD32b/c and CD64.

Abbreviations

(18) ADCC: antibody dependent cell cytotoxicity

(19) ELISA: enzyme-linked immunosorbent assay

(20) Fc: fragment crystallizable (of an Ab)

(21) HRP: Horse-radish peroxidase

(22) ITAM: immunoreceptor tyrosine-based activation motif

(23) ITIM: immunoreceptor tyrosine-based inhibition motif

(24) mAb: monoclonal antibody

(25) PBS: phosphate buffered saline

(26) RT: room temperature

(27) SD: standard deviation

(28) TMB: 3,3′,5,5′-Tetramethylbenzidine

(29) Material/Equipment

(30) MB101.10 IgG.sub.4 antibody (SAP 320-002-807, L/N A303P022) recombinant chimeric anti-CD303 (BDCA-2) IgG.sub.1 wildtype (Cor 6.10.11) chimeric anti-BDCA2 IgG.sub.1 (FE 060620.02)

(31) MB101.10 (L/N A303P022) is a humanized IgG.sub.4 antibody directed against BDCA-2 (CD303) and intended for clinical applications. Beside this version of MB101, a chimeric anti-BDCA-2 IgG.sub.1 antibody generated in chimeric mice (hybridoma) and a recombinant chimeric anti-CD303 (BDCA-2) IgG1 antibody (AC144 binding region with human Fc region) were used. Mb101.10 was manufactured in CHO DG44 cells, recombinant chimeric anti-CD303 (BDCA-2) IgG.sub.1 was manufactured in HEK293.EBNA cells and chimeric anti-BDCA-2 IgG.sub.1 antibody was derived from hybridoma cells.

(32) Fcγ Receptors:

(33) Recombinant Human Fc gamma RI/CD64, R&D Systems, 1257-FC-050

(34) Recombinant Human Fc gamma RIIa/CD32a, R&D Systems, 1330-CD-050

(35) Recombinant Human Fc gamma RIIb/CD32b) R&D Systems, 1875-CD-050

(36) Recombinant Human Fc gamma RIIIa/CD16a, R&D Systems, 4325-FC-050

(37) Recombinant Human Fc gamma RIIIb/CD16b, R&D Systems, 1597-FC-050

(38) Method ELISA plates were coated with Fc gamma receptors I, IIa, IIb, IIIa and IIIb with 5 μg/mL each in coating buffer overnight at 4° C. Coated plates were washed 3-fold with 1×PBS 0.05% Tween 20 and blocked with 5% BSA in 1×PBS. Antibodies were applied to ELISA plates with 50 μL/well at serial 2-fold dilution of antibody solution and incubated at room temperature for 1 h. As blank controls, respective antibody concentrations were also applied to wells lacking coated Fc gamma receptors, in order to substract unspecific binding from resulting signals. After washing (see above), the detection antibody anti-human kappa-HRP (FE 110316.07) 1:4000 in 1×PBS was applied with 50 μL/well and incubated again at RT for 1 h. After additional washing (see above), for assay development 50 μL/well TMB (CH111027.37) were applied and stopped after approx. 5 minutes with 50 μL/well 10% H.sub.2SO.sub.4. The developed plate was then analyzed via ELISA reader at 450 nm wavelength.
Results

(39) With regard to the binding to Fcγ RI (CD64, High Affinity Fc gamma Receptor), FIG. 1 shows that both IgG.sub.1 variants have a high affinity to CD64, as expected for IgG.sub.1 monoclonal antibodies. MB101 has a significantly lower affinity for CD64, which is also not surprising as MB101 is a humanized IgG.sub.4 molecule for which lower affinities to CD64 are reported, compared to IgG.sub.1 molecules. It has to be noted that MB101 still has a minimal affinity to CD64.

(40) With regard to the binding to Fcγ RIIa (CD31a, Low Affinity Fc gamma Receptor), FIG. 2 shows that MB101 has a moderately higher affinity for CD32a compared to the chimeric IgG.sub.1 molecule.

(41) With regard to the binding to Fcγ RIIb (CD31b, Low Affinity Fc gamma Receptor), FIG. 3 shows that MB101 has a significantly higher affinity for CD32b receptor compared to the chimeric IgG.sub.1 molecule.

(42) With regard to the binding to Fcγ RIIIa (CD16a, Low Affinity Fc gamma Receptor), FIG. 4 shows that MB101 has a significantly lower affinity for CD16a compared to the chimeric IgG.sub.1 molecule. A minimal residual binding affinity to CD16a can still be found.

(43) With regard to the binding to Fcγ RIIIb/c (CD16b/c), FIG. 5 shows that MB101 has no affinity for CD16b/c. In comparison, the chimeric IgG1 molecule has a medium binding affinity to CD16b/c.

Conclusions

(44) Clear differences in FcγR interactions could be observed for the different tested receptors. The interactions of MB101 with CD16a, CD16b/c and CD64 were significantly lower than the compared chimeric anti-BDCA-2 IgG.sub.1. The significantly lower affinity of MB101 for CD16a, CD16b/c and CD64 was an expected design feature of MB101 by the choice of using a human IgG.sub.4 backbone without effector function. This design feature should reduce antibody interactions with effector cells (e.g. macrophages, monocytes and natural killer cells). As an effect of this low affinity and activation potential, a low or absent ADCC activity potential of MB101 is assumed and the intended design goal was achieved.

(45) Interestingly, MB101 shows a notable affinity to CD32a and CD32b, which is moderately to significantly higher than the binding affinity of the chimeric anti-BDCA-2 IgG.sub.1 and could not be expected from the choice of an human IgG.sub.4 molecule or from the introduced stabilization mutations that change the IgG.sub.4 molecule to a slightly IgG.sub.1-ish human monoclonal antibody backbone. Surprisingly, it was found that the affinity of MB101 for the CD32a and CD32b receptors is even higher than for the IgG.sub.1 control.

(46) The Fcγ receptor FcγIIa (CD32a) has an activating ITAM (immunoreceptor tyrosine-based activation motif) and is found on monocytes, neutrophils, platelets and dendritic cells. Binding to this receptor may lead to activated cells via the ITAM. The affinity of MB101 to this receptor may therefore induce activation of B cells and dendritic cells. The Fcγ receptor FcγIIb (CD32b) has an inhibiting ITIM (immunoreceptor tyrosine-based inhibition motif) and is found on B cells and myeloid dendritic cells. Binding to this receptor may lead to inhibited cells via the ITIM. The affinity of MB101 to this receptor may therefore inhibit the antibody production of B cells and inhibit the maturation and cell activation of certain dendritic cells.

Example 2—Cell-Based In Vitro Assays for Functionality on CD32a and CD32b

(47) The purpose of this set of experiments is to evaluate the activation profile of antibody MB101 on low-affinity human Fcγ receptors (FcγR) CD16A, CD32a and CD32b/c on recombinant stable transfected reporter cell lines in in vitro assays. The aim of this study is to evaluate the potency of MB101 to activate different FcγR expressing cells upon Fc-part mediated binding. A particular interest was to analyze potential interactions of MB101 with Fc receptors regarding binding affinity and receptor activity upon binding. Both wild-type IgG.sub.1 and IgG.sub.4 antibodies are used as controls in these assays.

(48) In the human population several different allelic forms of low-affinity FcγRs are expressed, albeit at comparably low frequency. We tested IgG interactions with the following allelic forms of Fcγ receptors, CD16aV158 (FcγRIIIAV158), CD32aR131 (FcγRIIAR131) and CD32b/c (FcγRIIB/C). In case of CD16 two important allelic forms are found, CD16aF158 and CD16aV158 (amino acid sequence numbering according to Nimmerjahn, F., and Ravetch, J. V. 2008. Fc gamma receptors as regulators of immune responses. Nature Reviews Immunology 8: 34). We studied the more abundant allelic form of CD16, CD16aV158. In case of CD32 three different receptors are found that are encoded by three separate gene loci, CD32a, b and c. Although CD32b and CD32c exhibit different biological functions, they express identical extracellular amino acid sequences in amino-acid positions 43-217. Thus, to determine Fcγ interactions of CD32b and CD32c we used one transfectant expressing the extracellular domains of CD32b. In the following this clone is referred to as CD32b/c. In case of CD32a we expressed the allelic form CD32aR131, which is broadly found in the human population.

(49) For the generation of transfectants the murine thymoma cell line BW5147 was used. For surface expression of the different Fcγ receptors fusion proteins were engineered consisting of the extracellular domain of the respective human FcγR portion linked with the transmembrane and intracellular ζ-chain of the murine T cell receptor. As BW5147 cells carry an intracellular response element that leads to the production of murine Interleukin (IL)-2 once a signal via the ζ-chain of the T cell receptor is conveyed, transfectants are expected to secrete murine IL-2 upon interaction of the expressed FcγRs with IgG. The amount of murine IL-2 production is supposed to be directly linked with the intensity of the interaction of IgG with the respective FcγR.

(50) To test the ability of different mAbs to bind to and to cross-link the individual FcγRs, mAbs were coated to plastics and the transfectants were added. If a mAb interacted with a tested FcγR, murine IL-2 production was induced. Absolute IL-2 levels are taken as readout of Fcγ-FcγR-interaction. Tests with control mAbs allow comparative evaluation of interaction strength. This experimental setting is suited to study conditions of Fc-FcR-interactions that are also relevant in vivo, when a mAb is bound to a cell-surface target and interacts with Fc-receptors on different immune cells.

Abbreviations

(51) ADCC: antibody dependent cell-mediated cytotoxicity CD: cluster of differentiation ELISA: enzyme-linked immunosorbent assay Fc: fragment crystallizable (of an Ab) FCS: fetal calf serum IL: Interleukin mAb: monoclonal antibody PBS: phosphate buffered saline RT: room temperature SD: standard deviation
FcγR Transfectants

(52) BW5147-transfectants were used expressing fusion proteins consisting of the mouse ζ-transmembrane and cytoplasmic domains and the extracellular domains of CD16a, CD32a, or CD32b/c.

(53) TABLE-US-00003 Cell line Referred to as Plasmid BW5147 CD16A.sub.V158-ζ CD16a pcDNAZ-CD16zeta BW5147CD32A.sub.R131-ζ CD32a pcDNAZ-CD32Azeta BW5147 CD32B-ζ CD32b/c pcDNAZ-CD32Bzeta
Cell Culture Material for FcγR-Expressing Transfectants

(54) TABLE-US-00004 RPMI Biochrom AG # F1215 500 ml FCS (100%) Sigma # F7524 10%  Glutamax (100x) Invitrogen # 35050-038 1% Sodium Pyruvate (100 mM) Biochrom # AG L0473 1% Zeocin (for selection) Invitrogen # ant-zn-5b 0.5 mg/ml end concentration

(55) Cells were passaged every 2-3 days in medium enriched with 0.5 mg/ml Zeocin.

(56) Coating of Plastic with Antibody and Assay Procedure

(57) mAbs were serially 3-fold diluted in 1:10 (total log 3 dilution) in binding buffer (10 mM Bis-Tris, pH 6) starting with a concentration of 10 μg/ml. 100 μl of each dilution were transferred to a single well and the coating was performed over night at 4° C. After removal of the coating reagent, 200 μl per well blocking buffer (PBS+FCS 10%) were added and incubated for 1 h at RT. After blocking the wells were washed 3 times with 300 μl PBS. FcγR-expressing transfectants were transferred into the wells at a concentration of 2×10.sup.5 cells per 200 μl cell culture medium. Cells were incubated for 24 h at 37° C., 5% CO.sub.2. Cell-free supernatant was then collected and analyzed for murine IL-2 using a mouse IL-2 ELISA kit (Bender MedSystems, #BMS601) following the manufacturers' instructions.

(58) Results

(59) In order to quantify the interaction of different mAbs with human FcγRs, the antibodies TGN1412, Tysabri®, Mabthera®, Remicade®, and MB101 were coated in duplicates (MB101) or triplicates (TGN1412, Tysabri®, Mabthera®, and Remicade®) in log 3 dilutions ranging from 10 to 0.1 μg/ml. TGN1412, Tysabri®, and MB101 are IgG.sub.4 mAb, whereas Mabthera® and Remicade® are IgG.sub.1 mAb. As shown in FIG. 6, MB101 showed minor interactions with the low affinity FcγR CD16. The interaction of MB101 is comparable with the interaction of the other IgG.sub.4 mAb Tysabri® and the IgG.sub.1 mAb Mabthera®. Furthermore, interaction of MB101 with CD16-expressing transfectants is lower than those observed with the IgG.sub.4 mAb TGN1412.

(60) The mAb MB101 showed very minor interactions with FcγR CD32a expressing transfectants (FIG. 7) and are similar to those found with Tysabri® (IgG.sub.4), Remicade® (IgG.sub.1), TGN1412 (IgG.sub.4) or Mabthera® (IgG.sub.1). Activation profile data of MB101 on recombinant CD32a reporter cells shows that MB101 has no activation potential on CD32a (but binding potential, see above) and is judged only as binding, but not activating molecule on CD32a.

(61) However, MB101 shows enhanced interaction with CD32b/c-expressing transfectants when compared with the other IgG.sub.4 mAbs (TGN1412 or Tysabri®) and IgG.sub.1 mAbs (Mabthera® or Remicade®) (see FIG. 8). Cells were highly activated to secrete IL-2 as indicator for receptor mediated effects. Activation profile data of MB101 on recombinant CD32b reporter cells shows that MB101 has not only a significant binding activity, which is much higher than for IgG.sub.4 and IgG.sub.1 molecules, but also a significant activation potential on CD32b that is not found at this level for other tested IgG.sub.4 and IgG.sub.1 molecules. This activation capacity on the inhibitory ITIM receptor CD32b is expected to result in B cell and dendritic cell inhibition.

Conclusions

(62) In this study, different mAb were analyzed with regard to their ability to interact with the low-affinity human FcγR CD16 (FcγRIIIA.sub.V158), CD32a (FcγRIIA.sub.R131) and CD32b/c (FcγRIIB). The typical high affinity CD64 (FcγRI) was not analyzed, as it would deliver not-comparable results due to the different nature (low vs. high-affinity receptor). Clear differences in FcγR interactions could be observed for the different tested mAb. The interactions of MB101 with CD16- and CD32a-expressing transfectants seemed to be lower than the interactions of TGN1412 and Remicade® with CD16 as well as TGN1412 and Mabthera® with CD32a. Compared with IgG.sub.1 and IgG.sub.4 mAbs, MB101 showed a highly enhanced interaction and activation on CD32b/c-expressing transfectants. This activation capacity on the inhibitory ITIM receptor CD32b is expected to result in B cell and dendritic cell inhibition.

Example 3—FcγR Interactions of MB101 and its Single Mutation Variants

(63) Murine transfectants expressing the extracellular domains of human FcγRs were used to assess the multivalent interaction of mAb variants with different FcγRs. In the used system the interaction is measured by the release of murine IL-2 that is secreted into the supernatant after FcγR engagement of the transfectants. mAbs were coated to maxisorb 96 well plates at concentrations of 1 μg, 0.3 μg, 0.1 μg and 0.03 μg. In addition to MB101 (aCD303-hIgG4-S241P/R409K), two single mutation variants, Pool 3 (aCD303-hIgG4-S241P) and Pool 6 (aCD303-hIgG4-R409K), the wild type IgG.sub.4 antibody (aCD303-hIgG.sub.4), and other IgG.sub.4 and IgG.sub.1 control mAbs were tested. The antibodies were coated in binding buffer for 2 h at 37° C. and then blocked with 1% BSA for one hour at room temperature. 2×10.sup.5 transfectants were added and incubated for 18 h at 37° C. before the supernatant was harvested and tested for murine IL-2 content by an ELISA method.

(64) The results are shown in FIG. 9. IL-2 expression levels detected after incubation with the CD16 expressing transfectants revealed that the classical IgG.sub.1 control antibody induced significant IL-2 expression, whereas the IgG.sub.4 control, the wt IgG.sub.4 antibody, and Pool 3 (aCD303-hIgG.sub.4-S241 P) induce only minor amounts of IL-2 that further decreased with increased mAb dilution. The data obtained for Pool 6 (aCD303-hIgG.sub.4-R409K) and MB101 (aCD303-hIgG.sub.4-S241 P/R409K) suggest that there was a trend towards increased IL-2 expression, however, the data were not significant.

(65) The data obtained with the CD32a expressing transfectants were overall rather similar for MB101 (aCD303-hIgG.sub.4-S241P/R409K), the two variants and the wt antibody. The detected IL-2 levels were generally slightly higher than those of the IgG.sub.4 control but clearly weaker than those of the IgG.sub.1 control. Analyzing the induction of IL-2 after addition of the CD32b transfectant revealed a significant difference between MB101 (aCD303-hIgG.sub.4-S241P/R409K) and Pool 6 (aCD303-hIgG.sub.4-R409K) compared to Pool 3 (aCD303-hIgG.sub.4-S241P) and the wt IgG.sub.4. This difference was particularly evident at the coating concentration of 0.3 μg. This argues that the inclusion of a single mutation in the hinge region (Pool 6; aCD303-hIgG.sub.4-R409K) leads to enhanced interaction with CD32b and therefore increased IL-2 production, shifting the IgG.sub.4 FcγR binding profile towards that of an IgG.sub.1. The enhanced CD32B interaction of Pool 6 (aCD303-hIgG.sub.4-R409K) and MB101 (aCD303-hIgG.sub.4-S241P/R409K) was similarly observed in experiments with the CD64 transfectant.

(66) To more closely analyze the interaction of MB101 (aCD303-hIgG.sub.4-S241P/R409K) with CD32B, the two MB101 variants and the wt antibody were coated at concentrations of 1 μg, 0.8 μg, 0.6 μg, 0.4 μg and 0.2 μg. The result is shown in FIG. 10. Also under such conditions MB101 (aCD303-hIgG.sub.4-S241P/R409K) and Pool 6 (aCD303-hIgG.sub.4-R409K) induced significantly more IL-2 production at concentration of 0.8 and 0.6 μg than Pool 3 (aCD303-hIgG.sub.4-S241P) and the wt IgG.sub.4. This further verified that a single mutation in the hinge region of the original IgG.sub.4 antibody does indeed change the FcγR binding profile.

(67) These data was further confirmed using a CD32a/b binding ELISA. To this end ELISA plates were coated with recombinant human FcγRIlb (CD32b) protein (R&D Reagents, Cat:1875-CD; Lot: GJZ0814031) and binding of MB101 (aCD303-hIgG.sub.4-S241P/R409K), the wt MB101 and the two mutated variants Pool 3 (aCD303-hIgG.sub.4-S241P) and Pool 6 (aCD303-hIgG.sub.4-R409K) was probed. Similar to the data obtained from the previous experiment MB101 (aCD303-hIgG.sub.4-S241 P/R409K) and Pool 6 variant (aCD303-hIgG.sub.4-R409K) showed stronger binding to the recombinant human FcγRIIB/C than WT IgG.sub.4 mAb and Pool 3 (aCD303-hIgG.sub.4-S241P) as evident from the higher OD.sub.450 values (FIG. 11).

(68) To next validate whether the change in the FcγR binding profile of Pool 6 (aCD303-hIgG.sub.4-R409K) also influenced the specific target binding of MB101 (aCD303-hIgG.sub.4-S241P/R409K), we made use of a transfectant showing induced GFP expression upon engagement of BDCA-2 (kindly provided by Miltenyi). This allows testing whether the increased binding of MB101 (aCD303-hIgG.sub.4-S241 P/R409K) and Pool 6 (aCD303-hIgG.sub.4-R409K) to CD32B also leads to more abundant BDCA-2 triggering as measured by a stronger GFP expression in the transfectants compared with the induction of Pool 3 (aCD303-hIgG.sub.4-S241P) and the wt antibody.

(69) Therefore, 1×10.sup.5 CD32b or the control CD99 transfectant were seeded in a 96 well plate in 100 μl medium. MB101 (aCD303-hIgG.sub.4-S241P/R409K), Pool 3 (aCD303-hIgG.sub.4-S241P), Pool 6 (aCD303-hIgG.sub.4-R409K) and the wt antibody were added at concentrations of 0.5, 0.1, 0.05, 0.04, 0.03, 0.02 and 0.01 μg. Then 1×10.sup.5 BDCA-2 transfectants were added and the cells were co-cultured for 24 h.

(70) As shown in FIG. 12A, addition of the soluble antibodies to the BDCA-2 transfectants resulted in GFP induction when high concentrations of MB101 were used, while the different antibody variants behaved overall similarly. When additionally the CD99 control transfectant was added BDCA-2 transfectants showed only background GFP expression, whereas co-culture with CD32b transfectants lead to massive GFP induction (FIG. 12B lower row). This expression varied between the antibody variants, especially when concentrations of 0.05 to 0.01 μg mAb were used. There one can see that MB101 (aCD303-hIgG.sub.4-S241P/R409K) and Pool 6 (aCD303-hIgG.sub.4-R409K) induce higher GFP levels than Pool 3 (aCD303-hIgG.sub.4-S241P) or the wt antibody.

(71) To verify this data and to perform statistical analysis, experiments were as shown in FIG. 13 repeated. Addition of the highest concentration of the antibodies (0.5 μg) in soluble form and without subsequent crosslinking to the BDCA-2 transfectants already sufficed to trigger GFP expression of the transfectants (data not shown). Nevertheless, the extent of GFP expression wanes with lower antibody concentrations markedly. In the co-culture experiments of the CD32b transfectants and the BDCA-2 transfectant incubated with the highest antibody concentrations no significant differences of GFP induction were detected. Nevertheless, there seems to be a trend that MB101 (aCD303-hIgG.sub.4-S241P/R409K) and Pool 6 (aCD303-hIgG.sub.4-R409K) induce slightly more GFP expression than the other antibodies. This trend gets significant starting at concentrations of 0.05 μg of antibody used. This phenomenon can be detected by analyzing the percentage of GFP.sup.+ cells (FIG. 13A) as well as the mean fluorescence intensity (MFI) of the GFP signal (FIG. 13B). These results clearly indicate that introducing stabilizing mutation R409K into the hinge region of an IgG.sub.4 antibody does not only change the FcγR binding profile of the antibody but may also enhances the specific antigen binding capacity by immobilizing the Ab via cell surface FcγR thereby providing functional advantage against WT IgG.sub.4 variants.

Example 4— S241P and R409K Stabilize IgG.SUB.4

(72) Formation of half-molecules and Fab arm exchange are inherent phenomena considering IgG.sub.4. Mutations in the hinge region, i.e. S241P, and/or the CH3-domain, i.e. R409K, are analyzed in the present example for their capacity to eliminate dissociation of aCD303-IgG.sub.4 antibodies into half-molecules and occurrence of Fab arm exchange intermediates.

(73) Antibodies

(74) TABLE-US-00005 Specifity Lot aCD303-hIgG.sub.4-WT Miltenyi Biotec, aCD303-hIgG.sub.4-S241P Miltenyi Biotec aCD303-hIgG.sub.4-R409K Miltenyi Biotec aCD303-hIgG.sub.4-S241P/R409K Miltenyi Biotec Tysabri ® CH080808.03 Remicade ® Klon: CA2, Charge: 5040102008 AC144 (aCD303-mIgG.sub.1) Lot 008 human IgG.sub.4-λ CH 091202.24 Mouse anti-human-κ-HRP Southern Biotech, Clone HP6062, Lot: D2703-5716B; CPD090429.01; FE100112.04
Buffers and Dilutions

(75) TABLE-US-00006 Buffer/dilution composition 10x PBS 400.3 g sodium chloride 57.5 g disodium hydrogen phosphate 9.7 g potassium chloride 9.55 g potassium dihydrogen phosphate ad 5000 mL with H.sub.2O 1x PBS 100 mL 10xPBS ad 1000 mL with H.sub.2O DTT 1M 0.154 g DTT ad 1 mL with H.sub.2O GSH 0.1M 15.5 mg GSH ad 1 mL with H2O

Abbreviations

(76) TABLE-US-00007 ADCC antibody-dependent cell-mediated cytotoxicity BCA bicinchoninic acid BSA bovine serum albumin DMSO dimethyl sulfoxide DTT dithiotreitol EDTA 2,2′,2″,2′″-(ethane-1,2-diyldinitrilo)tetraacetic acid ELISA enzyme-linked immunosorbent assay Fab fragment antigen-binding Fc fragment crystallizable region GSH glutathione, reduced H.sub.2SO.sub.4 sulfuric acid HRP horseradish peroxidase IgG immunoglobulin G MSH mercaptoethanol PBS phosphate buffered saline SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis TMB 3,3′,5,5′-tetramethylbenzidine Tris-HCl 2-Amino-2-hydroxymethyl-propane-1,3-diol buffered hydrochloric acid
SDS-PAGE Analysis

(77) First, samples were applied non-boiled and boiled on non-reducing gels. 3.5 μg aCD303-IgG.sub.4-wt, 2.5 μg Tysabri®, 2.5 μg Remicade® and 2.5 μg AC144 were applied. From all other antibodies, 5 μg were put on each gel. Antibodies were loaded native or boiled for 5 minutes at 94° C. As control, aCD303-IgG.sub.4-wt was incubated with β-mercaptoethanol to show the completely dissociated heavy and light chains. Samples were put on 4-12% Tris/Glycin-SDS-gel. Each gel was run at 30 mA, 5 W, 200 V, 1 h 15 min and stained Coomassie.

(78) For non-boiled samples, all antibody bands could be seen around the 170 kDa marker band, corresponding to the whole antibody, which is approx. 150 kDa. aCD303-hIgG.sub.4-R409K showed also a high molecular weight contaminating band. This could be due to cultivation of antibody-producing cells in serum-containing media.

(79) After boiling of samples, all antibody bands run above the 170 kDA marker band. Half-molecule formation, characterized by the band between the 72 kDa and 95 kDa marker bands, could only be seen for Tysabri® and aCD303-IgG.sub.4-wt, comprising an IgG.sub.4-wild type hinge-region. Half-molecule formation was significantly decreased for all three IgG.sub.4 variants (faint bands). AC144 and Remicade® showed no half-molecule band, representing a stable IgG.sub.1-phenotype. The high molecular weight contaminating band of aCD303-hIgG.sub.4-R409K could be detected again. In addition, a band slightly above the 55 kDa marker band could be detected. This was suspected to be a co-purified protein contamination from the serum-containing cell culture.

(80) In a second set of experiments, the antibodies were incubated in the presence of 1 mM DTT. Antibodies were pre-incubated with 1 mM or 1 M DTT for 1 hour at 37° C. Samples were loaded on 4-12% Tris/Glycin-SDS-gel. Each gel was run at 30 mA, 5 W, 200 V, 1 h 15 min and stained Coomassie. 3.5 μg aCD303-IgG.sub.4-wt, 2.5 μg Tysabri®, 2.5 μg Remicade® and 2.5 μg AC144 were applied. From all other antibodies, 5 μg were put on each gel. As a control, aCD303-IgG.sub.4-wt was incubated with β-mercaptoethanol to show the completely dissociated heavy and light chains.

(81) After the incubation of samples with 1 mM DTT, AC144 and Remicade® dissociated into H2 and light chains. In contrast, aCD303-IgG.sub.4-wt and aCD303-hIgG.sub.4-S241P dissociated completely into light and heavy chains. Tysabri® also showed a residual half-molecules band (upper band). IgG.sub.4 variants R409K and S241 P/R409K showed the same pattern of H2 and light chain bands as the IgG.sub.1 isotype antibodies AC144 and Remicade®, demonstrating the desired stabilized phenotype. These results demonstrated, that IgG.sub.1 stability could be restored for the IgG.sub.4 variants R409K and S241 P/R409K with mutation R409K having the most profound effect.

(82) For all antibodies, DTT concentration was set to 1 M DTT. The pattern of bands was comparable to the treatment with 1 mM DTT for all antibodies except for Tysabri®. Tysabri® did not show any half-molecules but dissociated completely into heavy and light chains. Altogether, results for the reduction with 1 mM DTT were verified in 4 experiments; results for the reduction with 1 M DTT were verified in 2 experiments.

(83) In a last experimental set-up, antibodies were incubated with 1 mM GSH, according to physiological GSH concentration (Michelet et al., 1995). Hereby, Tysabri® and aCD303-hIgG.sub.4-wt dissociated into half-molecules, whereas all the other antibodies were stable. 3.5 μg aCD303-IgG.sub.4-wt, 2.5 μg Tysabri®, 2.5 μg Remicade® and 2.5 μg AC144 were applied. From all other antibodies, 5 μg were put on each gel. Antibodies were put on pre-incubated with 1 mM GSH for 1 hour at 37° C. Samples were put on 4-12% Tris/Glycin-SDS-gel and gel was run at 30 mA, 5 W, 200 V, 1 h 15 min. afterwards, gel was stained Coomassie. As a control, aCD303-IgG.sub.4-wt was incubated with β-mercaptoethanol to show the completely dissociated heavy and light chains.

(84) This also showed, that stability of aCD303-hIgG.sub.4-R409K and aCD303-hIgG.sub.4-S241 P/R409K was comparable to IgG.sub.1 antibodies AC144 and Remicade® under physiological conditions in vitro.

(85) Fc-Fc-Interaction-Assay with Coated Antibodies

(86) To detect, whether antibodies are engaged in Fab arm exchange, assays in ELISA-format are commonly used. Van der Neut Kolfschoten et al. (2007) established an assay, in which plates are coated with one antigen and bispecific antibodies, binding to two antigens, are detected using the other antigen. Rispens et al. (2009) coupled IgG.sub.1 and IgG.sub.4 to a solid phase and determined binding of either IgG.sub.1 or IgG.sub.4. Thereby, they found that Fc-Fc-interactions take place when CH3-domains of IgG.sub.4 or IgG.sub.1 coupled to a solid phase as well as IgG.sub.4 in the fluid phase dissociated slightly before binding. Binding of IgG.sub.4 to immobilized IgG.sub.4 is interpreted as an intermediate state of fab arm exchange, which cannot succeed, because one IgG.sub.4 partner is bound to a solid phase.

(87) For the present example, for each ELISA-plate, 12 mL of coating solution was prepared by diluting an antibody, in general AC144 antibody, to a concentration of 5 μg/mL in 1×PBS. Microplates were coated with 100 μL/well of coating solution and sealed with sealing tape on top of microplate to prevent evaporation. Incubation was performed overnight at 4° C. Plates were washed three times with 350 μL/well washing buffer with 10 seconds soaking between washes. To block potential nonspecific binding, 200 μL/well assay buffer were added and sealed plates were incubated for 2 h at room temperature. Plates could be stored at 4° C. for up to one week with blocking solution when they were tightly sealed with sealing tape.

(88) Antibodies were diluted to 100 ng/mL using assay buffer. If samples were reduced with DTT or GSH, aliquots were pipetted into microcentrifuge tubes and appropriate volumes of reducing stock solutions were added. Reduction was performed at 37° C. for 1 h either in the incubator or in a thermomixer. For the assay, 100 μL/well of sample were applied and sealed plates were incubated for 2 h at room temperature. In general, samples were put on as duplicates and assay buffer was used as blank. When incubation was finished, plates were washed again three times with 350 μL/well washing buffer. Mouse anti-human-K-HRP, diluted 1:1,000 to 1:2,000 in assay buffer, was applied and plates were incubated with 100 μL/well for 1 h at room temperature. Afterwards, plates were washed three times with 350 μL/well washing buffer. For detection, 100 μL/well TMB were used and color reaction was stopped after approx. 5 minutes with 100 μL/well 10 sulfuric acid, resulting in a color change from blue to yellow. Absorbance was measured at 450 nm in a microtiter plate reader.

(89) To find out, whether the aCD303-IgG.sub.4 variants could be engaged in Fc-Fc-interactions, ELISA-plates were coated with AC144, and antibodies were incubated with 1-20 mM DTT in 1 mM-steps. Remicade® was also incubated with DTT to include a stable hIgG.sub.1-antibody as control. Staining for non-stabilized Tysabri® was the brightest with highest OD (450 nm) values, followed by humanized CD303-IgG.sub.4-wt and IgG.sub.4 variant S241 P, resulting from binding of applied antibodies to the Fc-parts of coated antibodies. IgG.sub.4 variants R409K and S241 P/R409K showed lowest staining comparable to Remicade®, demonstrating the desired stabilized phenotype. Results were verified in three experiments.

(90) In the next experimental set-up, human IgG.sub.4-λ was coated on ELISA-plates. Again, antibodies were incubated with 1-10 mM DTT and triplicates were applied. Again, non-stabilized Tysabri® showed brightest staining, followed by humanized CD303-IgG.sub.4-wt and IgG.sub.4 variant S241 P. IgG.sub.4 variants R409K and S241 P/R409K showed lowest staining comparable to Remicade® and AC144. OD (450 nm) of these four antibodies was not much higher than blank measurements. Assay buffer, consisting of PBS and BSA, was used as blank. These results demonstrated again, that IgG.sub.1 stability could be restored for the IgG.sub.4 variants R409K and S241 P/R409K with mutation R409K having the most profound effect. In two orthogonal assays, non-reducing SDS-PAGE analyses and Fc-Fc-interaction-assays, aCD303-IgG.sub.4 variants R409K and S241 P/R409K exhibited the most stabilized phenotype comparable to the IgG.sub.1 isotype antibodies AC144 and Remicade®.

Example 5—Phase I Clinical Trial of MB101

(91) MB101 is a humanized anti-BDCA-2 (anti-CD303) monoclonal antibody derived from the murine antibody AC144 using a germ line-based humanization strategy of the variable regions of the light and heavy chain. MB101 is a humanized IgG.sub.4 with substitutions 409K and 241 P. Binding of BDCA-2 receptor by MB101 suppresses induction of interferon (IFN)-alpha production in plasmacytoid dendritic cells (PDC). Production of IFN-alpha by PDCs is considered to be a major pathophysiological factor in autoimmune diseases and triggering of BDCA-2 is presumed to be a potential therapeutic strategy for blocking production of IFN-alpha in patients.

(92) The production of IFN-alpha by PDCs seems to be the critical event in the induction of psoriatic inflammation and there is experimental evidence that inhibition of this process by ligation of the blood dendritic cell antigen (BDCA)-2 receptor on skin resident PDCs, using the murine predecessor monoclonal antibody AC144, may abrogate the development of psoriatic symptoms [Nestle et al. Plasmacytoid predendritic cells initiate psoriasis through interferon-a production. J Exp Med. 2005 Jul. 4; 202(1): 135-43]. Current monoclonal antibody therapies for patients with psoriasis are based on the neutralization of TNF-alpha, IL-12/IL-23 and IL-17, with an intention to interrupt the activation cycle by neutralizing central inflammatory cytokines. The MB101 approach is believed to influence an earlier phase of the inflammatory cycle and may therefore be an alternative approach to treat plaque psoriasis by preventing the induction of pro-inflammatory cytokines downstream of the IFN-alpha induction.

(93) The mode of action of MB101 was investigated using the murine predecessor molecule monoclonal antibody (mAb) AC144 on human PDCs in vitro. Binding of BDCA-2 antigen by MB101 potently suppresses the induction of IFN-α/(3 production in human PDC, presumably by a mechanism dependent on calcium mobilization and protein-tyrosine phosphorylation by src-family protein-tyrosine kinases [Dzionek et al. BDCA-2, BDCA-3, and BDCA-4: three markers for distinct subsets of dendritic cells in human peripheral blood. J Immunol. 2000 Dec. 1; 165(11): 6037-46.; Dzionek et al. BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen capture and is a potent inhibitor of interferon alpha/beta induction. J Exp Med. 2001 Dec. 17; 194(12): 1823-34.; Dzionek et al. Plasmacytoid dendritic cells: from specific surface markers to specific cellular functions. Hum Immunol. 2002 December; 63(12):1133-48.].

(94) Using mAb AC144, the induction of IFN-α/β production was significantly inhibited by the ligation of BDCA-2, even if the IFN-alpha inducing stimulus was added up to 6 days later and was independent from the stimulus used. This indicates that the ligation of BDCA-2 by mAb AC144 may induce a long-lasting inhibition of IFN-α/β production in PDC, i.e. that there is a difference between the pharmacokinetic and pharmacodynamic half-life of mAb AC144 and therefore MB101 in vivo.

(95) In a recent phase I clinical study (M-2011-255), safety and tolerability of ascending single i.v. doses of MB101 in patients with psoriasis was assessed. Overall, single doses of MB101 in doses of 0.025 to 32.4 μg/kg body weight were safe and wen-tolerated. From dose levels 4.05 μg/kg to 32.4 μg/kg (groups 6 to 9), MB101 concentrations could be quantified in serum. Two subjects (one in dose group 6, one in dose group 8) treated with MB101 showed a strong increase in secretion levels of IFN-alpha at 24 h post dose. With the exception of these subjects, for the dose groups 7-9 a decrease in IFN-alpha production in samples from treated subjects could be observed. IFN-alpha levels at 24 h compared to baseline were lower in the subjects treated with MB101 than in those treated with placebo. In the last dose-group (group 9) the reduction of IFN-alpha production upon CpG-A stimulation was significant and showed up to approx. 80% decrease when compared to the pre-dose control sample from the same subject.

(96) The decrease of MFI values of BDCA-2 staining on PBMCs showed clear dose-response, the effect being most pronounced at higher dose levels. After doses of 16.2 μg/kg and 32.4 μg/kg, Mean fluorescence intensity (MFI) values of BDCA-2 staining decreased to about 10% to 20% of the baseline value. The relationship of MFI values of BDCA-2 staining to the dose showed a sigmoidal course. MFI values of BDCA-2 staining on PBMCs decreased with increasing doses. Hence, there was a clear decrease of BDCA-2 staining on PBMCs with increase of dose. The foregoing data makes it plausible that MB101 can be suitably used for the treatment of an autoimmune disease such as psoriasis.

LIST OF REFERENCES

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