Abstract
The present invention relates to an anti-CD19 antibody having a variant Fc region having some specific amino acid modifications relative to a wild-type Fc region which confer one or several useful effector functions. The present invention relates in particular to chimeric, humanized or full human anti-CD19 antibodies comprising such a variant Fc region. It relates advantageously to antibodies with an interesting and valuable glycosylation profile, especially a low fucose level and/or a high oligomannose level and low level of sialylated glycoform. The present invention also relates to the use of these antibodies in the treatment, prevention or management of disease or disorder, such as cancer, especially a B-cell malignancy, and auto-immune disease.
Claims
1. An anti-CD19 antibody modified to comprise a variant human .[.IgG.]. .Iadd.IgG1 .Iaddend.Fc region comprising an amino acid substitution at each of the amino acid positions 243, 292, 300, 305, 326, 396, and 333 of the human IgG Fc region, wherein the numbering of the amino acid residues in the Fc region is of the Kabat, wherein the modified antibody is ADCC+ and .[.CDC±.]. .Iadd.CDC+.Iaddend., and wherein the modified antibody comprises the CDRs of the antibodies mR005-1 or mR005-2 whose VH and VL amino acid sequences are depicted on the following table: TABLE-US-00008 Amino acid sequence VH Amino acid sequence VL R005-1 SEQ ID NO: 29 SEQ ID NO: 31 R005-2 SEQ ID NO: 33 SEQ ID NO: 35.
2. The antibody of claim 1, wherein said antibody has been produced in wild-type rodent cells.
3. The antibody of claim 1, which has one or two Fc bearing no (GlcNAc).sub.2(Fuc), +(Man).sub.3(GlcNAc).sub.2 glycan.
4. The antibody of claim 1, which has one or two Fc bearing no (Gal), (GlcNAc).sub.2(Fuc), +(Man).sub.3(GlcNAc).sub.2glycan.
5. The antibody of claim 1, comprising one or two Fc bearing a (Man).sub.5(GlcNAc).sub.2 glycan.
6. The antibody of claim 1, comprising one or two Fc bearing one or two of the following glycans: (Gal), (GlcNAc).sub.2(Fuc), (NeuAc), +(Man).sub.3(GlcNAc).sub.2 (Gal).sub.2(GlcNAc).sub.2(Fuc), (NeuAc), +(Man).sub.3(GlcNAc).sub.2.
.[.7. The antibody of claim 1, wherein the antibody recognizes a non-internalizing epitope on the CD19 antigen..].
8. The antibody of claim 1, which comprises an .Iadd.IgG1 .Iaddend.Fc region in which Phe243 is substituted by Leu, Arg292 is substituted by Pro, Tyr300 is substituted by Leu, Val305 is substituted by Leu, Lys326 is substituted by Ala.Iadd., Glu333 is substituted by Ala, .Iaddend.and Pro396 is substituted by Leu.
9. The antibody of claim 1, wherein said antibody is a chimeric antibody.[., a humanized antibody.]., a full human antibody, a bispecific antibody, .Iadd.or .Iaddend.an antibody drug conjugate.[.or an antibody fragment.]..
10. The antibody of claim 1, that triggers programmed cell death.
.[.11. A pool of antibodies according to claim 1, wherein it comprises less or equal than 15% of such antibodies comprising one or two Fc bearing a (GlcNAc).sub.2(Fuc), +(Man).sub.3(GlcNAc).sub.2 glycan and/or less or equal than 20% of such antibodies comprising one or two Fc bearing a (Gal),(GlcNAc).sub.2(Fuc), +(Man).sub.3(GlcNAc).sub.2 glycan..].
.[.12. The pool of antibodies of claim 11, wherein it comprises at least 15, 20, 30, 40 or 50% of antibodies comprising one or two Fc bearing (Man).sub.5(GlcNAc).sub.2 glycans..].
.[.13. The pool of antibodies of claim 11, wherein it comprises less than 1.5 or 1% of antibodies comprising one or two Fc bearing (Gal),(GlcNAc).sub.2(Fuc),(NeuAc), +(Man).sub.3(GlcNAc).sub.2 and/or less than 2 or 1.5% of antibodies comprising one or two Fc bearing (Gal).sub.2(GlcNAc).sub.2(Fuc), (NeuAc), +(Man).sub.3(GlcNAc).sub.2..].
.[.14. A pharmaceutical composition comprising an antibody of claim 1, and a physiologically acceptable vehicle or excipient..].
.[.15. The antibody of claim 2, wherein the wild-type rodent cells are wild-type CHO cells..].
.[.16. The antibody of claim 1, wherein the IgG Fc region is an IgG1 Fc region..].
.[.17. The composition of claim 14, further comprising an antibody directed against CD20, CD52, CD22, EGF receptor, VEGF receptor, mimics ganglioside GD3, CEA or HER2..].
.[.18. The composition of claim 14, comprising further an antibody directed against CD20..].
.[.19. The antibody of claim 8 wherein Glu333 is substituted by Ala..].
.Iadd.20. A pharmaceutical composition comprising a monoclonal antibody or a fragment thereof, wherein the monoclonal antibody or fragment thereof specifically binds to CD19 and comprises the CDRs of antibody mR005-1 whose VH and VL amino acid sequences are depicted on the following table: TABLE-US-00009 Amino acid sequence VH Amino acid sequence VL R005-1 SEQ ID NO: 29 SEQ ID NO: 31 wherein said monoclonal antibody or fragment thereof triggers programmed cell death; and a physiologically acceptable vehicle or excipient. .Iaddend.
.Iadd.21. The pharmaceutical composition according to claim 20, wherein the antibody or fragment thereof comprises: a) a VH domain comprising: (i) CDR1 of sequence SEQ ID NO: 5; (ii) CDR2 of sequence SEQ ID NO: 6; and (iii) CDR3 of sequence SEQ ID NO: 7; and b) a VL domain comprising: (i) CDR1 of sequence SEQ ID NO: 8; (ii) CDR2 of sequence SEQ ID NO: 9; and (iii) CDR3 of sequence SEQ ID NO: 10. .Iaddend.
.Iadd.22. A pharmaceutical according to claim 20, wherein the antibody or fragment thereof comprises: a) a VH domain comprising: (i) CDR1 of sequence SEQ ID NO: 11; (ii) CDR2 of sequence SEQ ID NO: 12; and (iii) CDR3 of sequence SEQ ID NO: 13; and b) a VL domain comprising: (i) CDR1 of sequence SEQ ID NO: 14; (ii) CDR2 of sequence SEQ ID NO: 15; and (iii) CDR3 of sequence SEQ ID NO: 10. .Iaddend.
.Iadd.23. A pharmaceutical composition according to claim 20, wherein the antibody or fragment thereof comprises: a) a VH domain comprising: (i) CDR1 of sequence SEQ ID NO: 16; (ii) CDR2 of sequence SEQ ID NO: 6; and (iii) CDR3 of sequence SEQ ID NO: 13; and b) a VL domain comprising: (i) CDR1 of sequence SEQ ID NO: 8; (ii) CDR2 of sequence SEQ ID NO: 9; and (iii) CDR3 of sequence SEQ ID NO: 10. .Iaddend.
.Iadd.24. The pharmaceutical composition according to claim 20, wherein the antibody further comprises an antibody region selected from the group consisting of human IgG1 Fc, a human kappa region, and a combination thereof. .Iaddend.
.Iadd.25. The pharmaceutical composition according to claim 20, wherein said monoclonal antibody or fragment thereof comprises a VH domain of sequence SEQ ID NO: 29 and a VL domain of sequence SEQ ID NO: 31. .Iaddend.
.Iadd.26. The pharmaceutical composition of claim 20 further comprising an antibody directed against a tumoral antigen that is different from CD19. .Iaddend.
.Iadd.27. The pharmaceutical composition of claim 20 further comprising an antibody directed against CD20. .Iaddend.
.Iadd.28. The pharmaceutical composition of claim 20, wherein said monoclonal antibody further comprises a variant human IgG1 Fc region comprising an amino acid substitution at each of the amino acid positions 243, 292, 300, 305, 326, 396, and 333 of the human IgG Fc region, wherein the numbering of the amino acid residues in the Fc region is of the Kabat, wherein the modified antibody is ADCC+ and CDC+. .Iaddend.
.Iadd.29. The pharmaceutical composition of claim 28, wherein said antibody has been produced in wild-type rodent cells. .Iaddend.
.Iadd.30. The pharmaceutical composition of claim 29, wherein the wild-type rodent cells are wild-type CHO cells. .Iaddend.
.Iadd.31. The pharmaceutical composition of claim 28, wherein said monoclonal antibody comprises an IgG1 Fc region in which Phe243 is substituted by Leu, Arg292 is substituted by Pro, Tyr300 is substituted by Leu, Val305 is substituted by Ile, Lys326 is substituted by Ala, Glu333 is substituted by Ala, and Pro396 is substituted by Leu. .Iaddend.
.Iadd.32. The pharmaceutical composition of claim 28, wherein said monoclonal antibody comprises an IgG1 Fc region in which Phe243 is substituted by Leu, Arg292 is substituted by Pro, Tyr300 is substituted by Leu, Val305 is substituted by Leu, Lys326 is substituted by Ala, Glu333 is substituted by Ala and Pro396 is substituted by Leu. .Iaddend.
.Iadd.33. The pharmaceutical composition of claim 28, further comprising an antibody directed against CD20, CD52, CD22, EGF receptor, VEGF receptor, mimics ganglioside GD3, CEA or HER2. .Iaddend.
.Iadd.34. The pharmaceutical composition of claim 28, further comprising an antibody directed against CD20. .Iaddend.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) FIG. 1: CD19 epitope mapping. Cell lysat was used as CD19 antigen source by testing different combination MAbs used as tracer (biotinylated MAb) or as catcher MAb (purified MAb) respectively.
(2) FIG. 2: Higher level of apoptosis with the murine MAb anti-CD19 R005-1 in Burkitt's lymphoma cell line. The Burkitt's lymphoma cell line Raji was incubated with different MAb concentration for 5 hours. B-cells were double-stained with annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry. Mean+/−SD are shown from two independent experiments.
(3) FIG. 3: The murine MAb R005-1 is one of the best inducer of apoptosis in primary B-CLL cells. Primary B-CLL cells were isolated after ficoll centrifugation. 4×10.sup.6 cells were incubated with interest MAbs and controls for 24 hours at 37° C. in complete media. Then cells were harvested and stained with Annexin-V FITC/Propidium Iodide. 20 000 cells were acquired on LSRII cytometer (BD bioscience, USA) and analysis was done on FlowJo software. Apoptotic cells were defined as annexin V.sup.+/PI.sup.− cells. Data represents the mean+/−SD of five independent experiments.
(4) FIGS. 4A-4D: The nucleotide and amino acids sequences of the murine MAbs R005-1 and R005-2 MAbs, (A): V.sub.H (B): VL. Amino acids are shown as one-letter codes.
(5) TABLE-US-00005 Amino acid Nucleic acid Amino acid Nucleic acid sequence VH sequence VH sequence VL sequence VL mR005-1 SEQ ID SEQ ID SEQ ID SEQ ID NO: 29 NO: 30 NO: 31 NO: 32 mR005-2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 33 NO: 34 NO: 35 NO: 36
(6) FIG. 5: Comparative staining between the native chimeric R005-1 Fc0 and the murine parental mR005-1 MAb on PBMNC cells. Grey lines designated cytofluorometric histograms of negative isotype murine IgG1 or human IgG1 control MAb and black lines showed cytofluorometric histograms obtained peripheral blood lymphocytes or monocytes at 5 μg/ml for 1.10.sup.6 cells/ml. Representative experiments on three independent experiments.
(7) FIG. 6: Cross blocking experiment between the native chimeric R005-1 Fc0 and the murine parental mR005-1 MAb. Cells were pretreated or not with the native chR005-1 Fc0 or with hlgG1 control MAb (5 μg/ml) before the staining with 10 μl/well of FITC conjugated mR005-1 on Raji Burkitt's lymphoma cells. Grey lines designated cytofluorometric histograms of negative isotype mouse IgG1 control MAb and black lines showed cytofluorometric histograms obtained with the FITC conjugated mR005-1. Data represent one representative experiment on three independent experiments.
(8) FIG. 7: The cell labelling with the parental mR005-1 or chR005-1 Fc0 MAb triggered a down modulation of CD19 expression level. B-CLL cells were incubated with biotinylated MAbs at 4° C. for 30 min. At T0, T3h, and T24h, cells were stained with streptavidin-Alexa 488 nm (final dilution 1/1000) 30 min at 4° C. and fixed in formaldehyde 1%. 20 000 cells were then acquired on LSRII cytometer (BD bioscience, USA) and FlowJo analysis were done. Data represent one representative experiment of nine independent experiments.
(9) FIG. 8: The chR005-1 Fc0 MAb display a modest ADCC activity on Burkitt's lymphoma cells. Calcein-AM loaded Raji cells (1×10.sup.5 cells/ml) were incubated with interest MAbs for 20 min at 4° C. Effector cells (PBMNC) were added at the ratio E/T 50:1 for 4 hours at 37° C. under shaking condition. After centrifugation, supernatants were harvested and calcein-AM fluorescence was measured on fluorometer. ADCC lysis level was calculated following the formula: (experimental release−(target+effector spontaneous release)/(maximal release−target spontaneous release)*100. Maximal release value was obtained by treating target cells with Triton X-100. Data represent mean+/−SD of three independent experiments.
(10) FIG. 9: The chR005-1 Fc0 mab display a modest ADCC activity on primary B-CLL cells. Primary B-CLL cells were isolated after ficoll centrifugation. 1×10.sup.5 cells/ml calcein-AM loaded cells were incubated with interest MAbs for 20 min at 4° C. Effector cells (PBMNC) were added at the ratio E/T 50:1 for 4 hours at 37° C. under shaking condition. After centrifugation, supernatants were harvested and calcein-AM fluorescence was measured on fluorometer. ADCC lysis level was calculated following the formula: (experimental release−(target+effector spontaneous release))/(maximal release−target spontaneous release)*100. Maximal release value was obtained by treating target cells with Triton X-100. Data represent mean+/−SD of five independent experiments.
(11) FIG. 10: The chR005-1 Fc0 did not trigger CDC activity on Burkitt's lymphoma cells. Raji cells (2.10.sup.6 cells/ml) were incubated with interest MAbs for 20 min at 4° C. Then 5 μl of natural human complement was added for 4 hours at 37° C. under shaking condition. After incubation, supernatants were harvested and lactate deshydrogenase (LDH) was measured on fluorometer. CDC lysis level was calculated following the formula: (experimental release−target spontaneous release)/(maximal release−target spontaneous release)*100, where target without natural complement represented spontaneous release. Maximal release value was obtained by treating target cells with Triton X-100. Data represent mean+/−SD of three independent experiments.
(12) FIGS. 11A-11F: The amino acids and nucleic acid sequences of chimeric Fc24 (SEQ ID NO: 3 and 4) or Fc34 (SEQ ID NO: 1 and 2) variant MAb. Amino acids are shown as one-letter codes. According to the literature, the amino acid numbering of Fc region is based to the Kabat® data base, (CH1: aa no 118 to 215; Hinge: aa no 216 to 230; CH2: aa no 231 to 340; CH3: aa no 341 to 447). Variations between the various Fc used in the invention with respect to native Fc (Fc0):
(13) TABLE-US-00006 Name of the mutant Mutations with respect to Fc0 Fc34 F243L/R292P/Y300L/V305L/K326A/P396L Fc24 F243L/R292P/Y300L/V305L/K326A/E333A/P396L Fc39 F243L/R292P/Y300L/V305I/K326A/E333A/P396L Fc18 F243L Fc28 Y300L/V305L Fc19 F243L/R292P/P396L Fc29 F243L/R292PN305L/P396L Fc30 F243L/R292P/Y300L/P396L Fc20 F243L/R292P/Y300L/V305L/P396L Fc23 F243L/R292P/Y300L/V305L/E333A/P396L Fc6 E333A Fc7 K326A/E333A Fc9 K326A
(14) FIG. 12: Enhanced ADCC activity with the chR005-1 Fc24 or chR005-1 Fc34 MAb on Burkitt's lymphoma cells in the presence of PBMNC as effector cells. Calcein-AM loaded Raji cells (1×10.sup.5 cells/ml) were incubated with interest MAbs for 20 min at 4° C. Effector cells (PBMNC) were added at the ratio E/T 50:1 for 4 hours at 37° C. under shaking condition. After centrifugation, supernatants were harvested and calcein-AM fluorescence was measured on fluorometer. ADCC lysis level was calculated following the formula: (experimental release−(target+effector spontaneous release))/(maximal release−target spontaneous release)*100. Maximal release value was obtained by treating target cells with Triton X-100. Data represent mean+/−SD of four independent experiments.
(15) FIG. 13: The chR005-1 Fc24 mediated strong ADCC activity on primary B-CLL cells. Primary B-CLL cells were isolated after ficoll centrifugation. (1×10.sup.5 cells/ml) calcein-AM loaded Raji cells were incubated with interest MAbs for 20 min at 4° C. Effector cells (PBMNC) were added at the ratio E/T 50:1 for 4 hours at 37° C. under shaking condition. After centrifugation, supernatants were harvested and calcein-AM fluorescence was measured on fluorometer. ADCC lysis level was calculated following the formula: (experimental release−(target+effector spontaneous release))/(maximal release−target spontaneous release)*100. Maximal release value was obtained by treating target cells with Triton X-100. Data represent mean+/−SD of five independent experiments.
(16) FIG. 14: The chR005-1 Fc24 or the chR005-1 Fc34 MAb induced CDC activity on Burkitt's lymphoma cell. Raji cells (2.10.sup.6 cells/ml) were incubated with interest MAbs for 20 min at 4° C. Then 5 μl of natural human complement was added for 4 hours at 37° C. under shaking condition. After incubation, supernatants were harvested and lactate deshydrogenase (LDH) was measured on fluorometer. CDC lysis level was calculated following the formula: (experimental release−spontaneous release)/(maximal release−spontaneous release)*100, where target without natural complement represented spontaneous release. Maximal release value was obtained by treating target cells with Triton X-100. Data represent mean+/−SD of three independent experiments.
(17) FIG. 15: The chR005-2 Fc24 also induced CDC activity on Burkitt's lymphoma cell. Raji cells (2.10.sup.6 cells/ml) were incubated with interest MAbs for 20 min at 4° C. Then 5 μl of natural human complement was added for 4 hours at 37° C. under shaking condition. After incubation, supernatants were harvested and lactate deshydrogenase (LDH) was measured on fluorometer. CDC lysis level was calculated following the formula: (experimental release-spontaneous release)/(maximal release−spontaneous release)*100, where target without natural complement represented spontaneous release. Maximal release value was obtained by treating target cells with Triton X-100. Data represent mean+/−SD of one independent experiment.
(18) FIG. 16: The chR005-1 Fc24 MAb mediated strong CDC activity on primary B-CLL cells. B-CLL cells (2-10.sup.6 cells/ml) were incubated with interest MAbs for 20 min at 4° C. Then 5 μl of natural human complement was added for 4 hours at 37° C. under shaking condition. After incubation, supernatants were harvested and lactate deshydrogenase (LDH) was measured on fluorometer. CDC lysis level was calculated following the formula: (experimental release−spontaneous release)/(maximal release−spontaneous release)*100, where target without natural complement represented spontaneous release. Maximal release value was obtained by treating target cells with Triton X-100. Data represent mean+/−SD of one independent experiment.
(19) FIG. 17: Recombinant human C1q binding on chR005-1 wild type or mutant MAb. The binding of human recombinant C1q was assessed by an ELISA binding assay. The murine parental MAb mR005-1 was used as negative control and Rituximab as positive control. Data represent mean+/−SD of two independent experiments.
(20) FIG. 18: Fc24 or Fc34 engineering of anti-CD19 chR005-1 did not influence PCD in Burkitt's lymphoma cell line. The Burkitt's lymphoma cell line Raji was incubated with different MAb concentration for 5 hours. B-cells were double-stained with annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry. Data represent mean+/−SD of two independent experiments.
(21) FIG. 19: The chR005-1 Fc24 or Fc34 engineering of anti-CD19 chR005-1 did not influence PCD in primary B-CLL cells. Cells were incubated with different MAb concentration for 5 hours. B-cells were double-stained with annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry. Data represent mean+/−SD of four independent experiments.
(22) FIG. 20: The mR005-1 MAb combined with chimeric anti-CD20 (Rituximab) synergized their apoptotic effect in Burkitt's lymphoma cells. Daudi cells (4×10.sup.4 cells) were incubated with interest MAbs in combination or not, and controls for 5 hours at 37° C. in complete media. B-cells were double-stained with annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry. Data represent mean+/−SD of three independent experiments.
(23) FIG. 21: The chR005-1 Fc24 or the chR005-1 Fc34 MAb combined with chimeric anti-CD20 (Rituxan®) synergized their apoptotic effect in Burkitt's lymphoma cells. Daudi cells (4×10.sup.4 cells) were incubated with interest MAbs in combination or not, and controls for 5 hours at 37° C. in complete media. B-cells were double-stained with annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry. Data represent mean+/−SD of two independent experiments.
(24) FIG. 22: The parental mR005-1 combined with chimeric anti-CD20 (Rituximab®) synergized their apoptotic effect in primary B-CLL cells. Primary B-CLL cells were isolated after ficoll centrifugation. B-CLL (4×10.sup.4 cells) were incubated with interest MAbs in combination or not, and controls for 24 hours at 37° C. in complete media. B-cells were double-stained with annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry. Data represent mean+/−SD of seven independent experiments.
(25) FIG. 23: The chR005-1 Fc24 or the chR005-1 Fc34 combined with chimeric anti-CD20 (Rituxan®) synergized their apoptotic effect in primary B-CLL cells. Primary B-CLL were isolated after ficoll centrifugation. B-CLL (4×10.sup.4 cells) were incubated with interest MAbs in combination or not, and controls for 24 hours at 37° C. in complete media. B-cells were double-stained with annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry. Data represent mean+/−SD of four independent experiments.
(26) FIG. 24: The murine MAb R005-1 triggered apoptosis in primary B-CLL cells Rituxan® refractory. Primary B-CLL cells were isolated after ficoll centrifugation. 4×10.sup.6 cells were incubated with interest MAbs and controls for 24 hours at 37° C. in complete media. Then cells were harvested and stained with Annexin-V FITC/Propidium Iodide. Apoptotic cells were defined as annexin V.sup.+/PI.sup.− cells. Data represent mean+/−SD of two independent experiments.
(27) FIG. 25: The MAb chR005-1 Fc24 or the MAb chR005-1 Fc34 triggered apoptosis in primary B-CLL cells Rituxan® refractory. Primary B-CLL were isolated after ficoll centrifugation. B-CLL (4×10.sup.4 cells) were incubated with interest MAbs in combination or not, and controls for 24 hours at 37° C. in complete media. B-cells were double-stained with annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry. Data represent one representative experiment.
(28) FIG. 26: Similar level of ADCC was observed whatever FcγRIIIA allotypes F/F or V/V. Lymphocytes staining was performed with the MAb unconjugated 3G8 or MEM-154. For each FcγRIIIA—158 polymophism (VV or FF). Data represent mean+/−SD of two independent experiments.
(29) FIG. 27: General characteristics of iDD biotech MAb Heavy or Light chain expression vector. The empty CHO cells were co-transfected with the pcDNA3.3-TOPO expression vector for light chain (Invitrogen) and with the pcDNA3.3 expression vector for heavy chain (Invitrogen) following transient transfection procedure established in our laboratory.
(30) FIG. 28: Glycosylation profile of the MAb chR005-1 Fc0. Antibody oligosaccharides released by PNGase F digestion were analyzed using a MALDI-TOF Voyager DE PRO spectrometer. The m/z value corresponds to the sodium-associated oligosaccharide ion. The sugar composition of each peak shown is detailed in FIG. 32. The schematic oligosaccharide structure of each major peak is illustrated on the right side of the charts: GlcNAc (closed circles), mannose (open squares), galactose (open diamonds) and fucose (open triangles).
(31) FIG. 29: Glycosylation profile of the MAb chR005-1 Fc24. Antibody Fc oligosaccharides released by PNGase F digestion were analyzed using a MALDI-TOF MS spec trometer Reflex III. The m/z value corresponds to the sodium-associated oligosaccharide ion. The sugar composition of each peak shown is detailed in FIG. 32. The schematic oligosaccharide structure of each major peak is illustrated on the right side of the charts: GlcNAc (closed circles), mannose (open squares), galactose (open diamonds) and fucose (open triangles).
(32) FIG. 30: Glycosylation profile of the MAb chR005-1 Fc24. Antibody Fc oligosaccharides released by PNGase F digestion were analyzed using a MALDI-TOF MS spectrometer Reflex III. The m/z value corresponds to the sodium-associated oligosaccharide ion. The sugar composition of each peak shown is detailed in FIG. 32. The schematic oligosaccharide structure of each major peak is illustrated on the right side of the charts: GlcNAc (closed circles), mannose (open squares), galactose (open diamonds) and fucose (open triangles).
(33) FIG. 31: Assignment of carbohydrate structures by comparison with glycan standards following capillary electrophoresis with laser-induced fluorescence detection relative intensities of N-glycan types between the parental chR005-1 Fc0 and the optimized chR005-1 Fc24 or Fc34 MAbs expressed in wild type CHO cells.
(34) FIG. 32: Oligosaccharide analysis of variants MAbs anti-CD19. Comparison of relative intensities of N-glycan types between the parental chR005-1 Fc0 and the optimized chR005-1 Fc24 or Fc34 MAbs expressed in wild type CHO cells.
(35) FIG. 33: Glycan profiles according Fc variants. IgG N-linked glycans of chR005-1 Fc variant antibodies produced from the wild type CHO Easy C cells. Molecular mass are permethylated glycans, detected as [M+Na]+ by Maldi mass spectrometry (A-B). The MAb panel was produced from CHO Easy C cells. Different glycosylation profiles among the Fc variant antibody panel were observed. One representative experiment.
(36) FIGS. 34A-34B: Differential MAb activity according Fc variant. FIG. 34A—Calcein-AM loaded Raji cells (1×10.sup.5 cells/mL) were incubated with interest MAbs for 20 min at 4° C. Effector cells (PBMNC) were added at the ratio E/T 50:1 for 4 hours at 37° C. under shaking condition. After centrifugation, supernatants were harvested and calcein-AM fluorescence was measured on fluorometer. ADCC lysis level was calculated following the formula: [(experimental release−(target+effector spontaneous release))/(maximal release−target spontaneous release)*100]. Maximal release value was obtained by treating target cells with Triton X-100. Data represent mean+/−SD of five independent experiments. FIG. 34B—Raji cells (2-10.sup.6 cells/ml) were incubated with interest MAbs for 20 min at 4° C. Then 5 μl/well of natural human complement was added for 4 hours at 37° C. under shaking condition. After incubation, supernatants were harvested and lactate deshydrogenase (LDH) was measured on fluorometer. CDC lysis level was calculated following the formula: (experimental release−target spontaneous release)/(maximal release−target spontaneous release)*100, where target without natural complement represented spontaneous release. Maximal release value was obtained by treating target cells with Triton X-100. Data represent mean+/−SD of three independent experiments.
(37) FIGS. 35A-35B: Differential MAb activity according Fc variant. FIG. 35A—Calcein-AM loaded Raji cells (1×10.sup.5 cells/mL) were incubated with interest MAbs for 20 min at 4° C. Effector cells (PBMNC) were added at the ratio E/T 50:1 for 4 hours at 37° C. under shaking condition. After centrifugation, supernatants were harvested and calcein-AM fluorescence was measured on fluorometer. ADCC lysis level was calculated following the formula: [(experimental release−(target+effector spontaneous release))/(maximal release−target+spontaneous release)*100]. Maximal release value was obtained by treating target cells with Triton X-100. Data represent mean+/−SD of three independent experiments. FIG. 35B—Calcein-AM loaded Raji cells (1×10.sup.5 cells/mL) were incubated with interest MAbs for 20 min at 4° C. 50 μl per well of effector cells from whole blood were added for 4 hours at 37° C. under shaking condition. After centrifugation, supernatants were harvested and calcein-AM fluorescence was measured on fluorometer. ADCC ILysis level was calculated following the formula: [(experimental release−(target+effector spontaneous release))/(maximal release−target spontaneous release)*100]. Maximal release value was obtained by treating target cells with Triton X-100. One representative experiment.
(38) FIG. 36: Differential MAb activity according Fc variant. Calcein-AM loaded Raji cells (1×10.sup.5 cells/mL) were incubated with interest MAbs for 20 min at 4° C. 50 μl per well of effector cells from whole blood were added for 4 hours at 37° C. under shaking condition. After centrifugation, supernatants were harvested and calcein-AM fluorescence was measured on fluorometer. ADCC lysis level was calculated following the formula: [(experimental release−(target+effector spontaneous release))/(maximal release−target spontaneous release)*100]. Maximal release value was obtained by treating target cells with Triton X-100. One representative experiment.
(39) FIGS. 37A-37B: Inhibition of tumour growth in vivo by the chR005-1 MAb Fc24 or chR005-1 Mab Fc34. Mice injected with Raji cells subcutaneously were treated by intravenous MAb infusion once a week starting on day 21 at 100 mg/kg with or without leukocytes. The tumors were measured twice a week.
(40) FIG. 38: Complement human serum binding on chR005-1 wild type or mutant MAb. The binding of human complement serum to MAbs was assessed by an ELISA binding assay. The 96-well plates (Nunc) were coated overnight at 4° C. with varying MAb concentrations. After washing, the plates were blocked with PBS—5% BSA for 1 h, and incubated for 1 h with 2.5 μl of natural human complement (Sigma). Then, 100 μl of a 1/500 dilution of sheep anti-human C1q peroxidase-conjugated Ab (Abd Serotec) added and incubated for 1 h. The plates were developed with 100 μl per well of TMB substrate (Uptima Interchim). After H.sub.2SO.sub.4 addition, the OD was measured at 450 nm/630 nm using a MRX II microplate reader. Data represent mean+/−SD of two independent experiments.
(41) FIG. 39: The growth of Rituxan refractory RL established tumor was inhibited with the Fc24 optimized chR005-1 MAb. Mice injected with refractory Rituxan RL cells subcutaneously were treated by intravenous MAb infusion once a week starting on day 20 at 100 mg/kg.
(42) FIG. 40: The combination of the Fc24 optimized chR005-1 MAb and the drug Doxorubicine increased the level of growth inhibition Raji established tumor. Mice bearing established SC Burkitt's lymphoma tumors were treated weekly. The control group received vehicle (NaCl 0.9%) while the treated groups received one of the following: chR005-1 Fc24 MAb 100 mg/kg, doxorubicine 2 mg/kg, chR005-1 Fc24 MAb+doxorubicine.
MATERIALS AND METHODS
(43) Cells: The CHO dhfr.sup.−/− cell line was obtained for research purpose from ATCC (American Type Culture Collection, USA). Burkitt's lymphoma Raji or Daudi cell lines as the T lymphoma Cem cells were obtained from ECACC (European Collection of Cell Culture, UK). Human PBMNCs were purified from leukapheresis of anonymous healthy volunteer donors (Blood Center, Lyon France) using Ficoll-Histopaque density gradient (Sigma, Saint Quentin Fallavier, France). All B-CLL patients enrolled in this study had been defined immunophenotypically as outlined by criteria from the National Cancer Institute Working Group in 1996 (Hallek et al., 2008). Blood was obtained from patients after written informed consent in accordance with the Declaration of Helsinki.
(44) Reagents and antibodies: The IgG1 chimeric negative control was produced at iDD biotech (Dardilly, France). The murine parental MAb mR005-1 or mR005-2 were isolated from iDD biotech hybridoma library. The wild type chR005-1 Fc0 (also called native IgG1) and all the modified antibodies were generated and produced by iDD biotech (Dardilly, France). FITC labelled annexin-V and propidium iodide (PI) were purchased from BD Biosciences (Pont de Claix, France) and Sigma (Saint Quentin Fallavier, France), respectively. Goat Fab′ 2 anti-human RPE IgG antibody and Goat anti-mouse FITC Ig antibody were respectively purchased by Sigma (Saint Quentin Fallavier, France) and MP Biomedical (Illkirch, France). Rituximab were produced by Genentech and purchased commercially. Others MAbs anti-CD19 4G7 (IgG1), Bu12 (IgG1), HD37 (IgG1), B4 (IgG1) were purchased by Santa Cruz Biotechnology (California, USA), Abd Serotec (Düsselldorf, Germany), Santa Cruz Biotechnology (California, USA), Biogenex (San Ramon, USA) respectively. The Camptothecin used as positive control for apoptosis assays was purchased by Sigma (Saint Quentin Fallavier, France).
(45) Murine antibody generation: The Balb/c mice were immunised with CD19 expressing cells such as human chronic lymphoid leukaemia cells. Murine MAbs were generated using standard hybridoma techniques (Zola et al., 1987) and screened initially for their ability to bind by flow cytometry only CD19 positive cell line. Purified MAbs were produced following ascitis purification then purified by using protein-A Sepharose (Pharmacia, Uppsala, Sweden).
(46) Conversion of murine MAb to native chimeric MAb: cDNA corresponding to the variable region of the hybridoma was obtained using two approaches, the first approach consist to the utilisation in PCR of the degenerate N-term amino acid related primer set generate since the N-Terminal sequencing and the second approach consist to the utilisation in PCR of degenerate primer set generate by IMGT® primer database and specific primers previously described (Essono et al., 2003; Wang et al., 2000). The sequence of N-terminal variable region was determined by Edman degradation. Total RNA extraction was carried out using the Tri Reagent kit according to the protocol described by the supplier Sigma. The amplified VL and VH fragments were cloned into the TOPO-TA cloning vector (Invitrogen) for sequence analyses by the dideoxytermination method (Sanger et al., 1977). Then antibody variants constructs were amplified by PCR and cloned into the vector pcDNA3.3.
(47) Construction of antibody variants: Substitutions in the Fc domain were introduced using “megaprimer” method of site-directed mutagenesis (Sarkar et al., 1990). Positions are numbered according to the Kabat® index (Identical V region amino acid sequences and segments of sequences in antibodies of different specificities). Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites were analyzed (Kabat et al., 1991). Heavy and light chain constructs were co-transfected into CHO DG44 (ATCC) suitable for MAb screening. Antibodies were purified using protein A affinity chromatography (GE Healthcare).
(48) CD19 MAb ELISA competition: For competition binding ELISA experiments, cell lysate was used as CD19 antigen source by testing different combination MAbs used as tracer (biotinylated MAb) or as catcher MAb (purified MAb) respectively.
(49) FACS analysis of MAb binding to human CD19: The binding to human CD19 of all MAb generated in the present study were measured with a FACScan (BD Biosciences, Pont de Claix, France) by using the goat anti-mouse FITC Ig from Sigma (Saint Quentin Fallavier, France) for the detection of murine MAbs and with the goat anti-human PE Ig from Sigma (Saint Quentin Fallavier, France) for the detection of chimeric MAbs. For competition binding experiments, cells were pre-incubated with an excess of either the parental MAb or a mouse IgG1 isotype control antibody.
(50) Laser scanning confocal microscopy by using biotinylated antibodies: Cell fluorescence has been visualized using an Inverted Zeiss Axiovert 100M LSM 510 Meta confocal microscope following cell labelling with biotinylated MAbs.
(51) Glycosylation analysis: Release of N-glycans & permethylation were carried out following standard procedures (Ciucanu et al., 1984). Analysis of protein glycosylation was determined by mass spectrometry (Morelle et al., 2007).
(52) Antibody affinity: Determination of antibody KD values was performed as previously described (Benedict et al., 1997) by using binding assay analyzed by flow cytometry to detect cell-bound antibody.
(53) Assessment of apoptosis by flow cytometry: The apoptosis of cells after incubation with antibodies was measured using annexin V/PI staining followed by FACS analysis. The apoptotic cells were determined in the gate showing a positive staining for annexin V and negative staining for propidium iodide.
(54) Antibody dependent cell cytoxicity Assay (ADCC): Primary B-CLL cells or B-cell lines (Raji) (Target cells) were loaded with 12.5 μM Calcein-AM dye (Sigma, France). 5 000 target cells per well were the pre-incubated with different concentration of interest MAbs and controls for 20 min at +4° C. Effector cells were then added to the target cells at the ratio E/T equal to 50:1. Specific ADCC lysis was calculated using the formula above: (experimental release−(spontaneous release Target+Effector))/(maximal release−spontaneous release target)*100, where target and effector cells without antibody represented spontaneous release. Maximal release value was obtained by treating target cells with Triton X-100.
(55) Complement dependent cytoxicity Assay (CDC): Target cells (50 000 cells per well) either primary B-CLL cells or B-cell lines (Ramos, Raji, and Daudi cell lines) were incubated with various MAbs concentration. Then, human normal serum were added the culture and then cells were incubated 4 hours at +37° C. under shaking condition. At the end of incubation, lactate deshydrogenase present in supernatant was measured with LDH assay kit (Promega, France). Fluorescence was recorded at the 590 nm excitation wavelength. Specific CDC lysis was calculated using the formula above: (experimental release−target spontaneous release)/(maximal release−target spontaneous release)*100, where target and effector cells without antibody represented spontaneous release. Maximal release value was obtained by treating target cells with Triton X-100.
(56) Complement binding assay: The binding of human C1q to MAbs was assessed by an ELISA binding assay. The 96-well plates (Nunc) were coated overnight at 4° C. with varying MAb concentrations. After washing, the plates were blocked with PBS-5% BSA for 1 h, and incubated for 1 h with 0.2 μg/ml of recombinant human C1q (Abd Serotec) or 2.5 μl of natural human complement (Sigma). Then, 100 μl of a 1/500 dilution of sheep anti-human C1q peroxidase-conjugated Ab (Abd Serotec) added and incubated for 1 h. The plates were developed with 100 μl per well of TMB substrate (Uptima Interchim). After H.sub.2S0.sub.4 addition, the OD was measured at 450 nm/630 nm using a MRX II microplate reader.
(57) Detection VV or FF polymorphism by flow cytometry: The FcγRIIIA—158 V/F polymorphism was based on the MEM-154/3G8 fluorescence ratio as described (Böttcher et al., 2005). Human blood samples (50 μl) were incubated 15 min in the dark at RT with 10 μg/ml of unconjugated 3G8 (Becton Dickinson, USA), MEM-154 (Abcam, USA) or with the isotype control MAb. 2 ml of a red blood lysing buffer (BD biosciences, USA) diluted at 1/10 is added during 5 min. After washes, goat anti-mouse FITC Ig from Sigma (Saint Quentin Fallavier, France) is added (100 μl diluted to 1/800). Cells were incubated a further 30 min at RT then washed twice and assayed using a FACScan (BD Biosciences, Pont de Claix, France).
(58) Results
(59) 1. CD19 Epitope Mapping.
(60) By ELISA competition, we determined the CD19 epitope mapping by using a MAb panel anti-CD19 including the MAb mR005-1, mR005-2, 4G7, B4, Bu12, HD37. As shown in FIG. 1, a strong competition was observed by using as tracer/catcher MAbs the combination 4G7/mR005-1 and HD37/mR005-1 revealing that these antibodies recognized the same epitope or an adjacent epitope. By contrast, no significant competition was observed with the mR005-2 or BU12 MAbs.
(61) 2. The Parental Murine MAb mR005-1 Triggers Apoptosis in Burkitt's Lymphoma Cell Line or in Primary B-CLL Cells.
(62) The murine MAb mR005-1 was highly effective at inducing PCD than the murine MAb mR005-2 in Burkitt's lymphoma Raji cell line (FIG. 2). It is tempting to suggest that the superior apoptotic effect of the mR005-1 MAb could be related to the fine specificity of these molecules. Our results also demonstrated that the mR005-1 MAb was more efficient to trigger PCD than rituximab.
(63) 3. The Murine MAb Biological Activity to Trigger Apoptosis is Linked to Epitope on CD19.
(64) A panel of murine MAb against human CD19 was tested to evaluate the apoptotic potential of various clones. We used the annexin-V/PI approach to determine the level of apoptosis induced by these antibodies at 24 hours post incubation. We have found that mR005-1 is one of the best MAbs as inducer of apoptotic process in B-CLL cells isolated from patients (FIG. 3). Similar data was observed by using the Burkitt's lymphoma cell line (data not shown). Our observations presented here suggest that treatment of B-CLL tumor cells with derivated chimeric MAb related to the mR005-1 MAb, which are capable of inducing an apoptotic signal, may contribute to their direct killing and elimination.
(65) 4. Chimerisation of the Murine MAb R005-1 and of the Murine MAb R005-2.
(66) In comparison with the IMGT® database, the sequence of VL mR005-1 and the sequence of VH mR005-1 were validated recognizing at least 96% and at least 92% respectively (FIGS. 4A-4D). The sequences of VL mR005-2 and VH mR005-2 were also validated (99% and 98%, respectively). The authenticity of the VH and VL sequences obtained by cDNA cloning were also confirmed by N-terminal amino acid sequencing of the target mouse monoclonal antibody. Heavy and light chains were separated before amino acid sequencing by polyacrylamide gel electrophoresis under reducing conditions and the 20 first amino were sequencing by Edman degradation. Then sequences were then cloned in the Light chain expression vector (VL mR005-1 or VL mR005-2) and in the Heavy MAb expression vectors (VH mR005-2 or VH mR005-2) encoding also for a native Fc region named Fc0. Transient transfection on CHO dhfr.sup.−/− cells by lipofection was performed.
(67) 5. Authenticity and Selection of VH and VL chR005-1 Chains Confirmed by Flow Cytometry Analysis.
(68) The native chimeric MAb chR005-1 Fc0 constructed from the VH1 and VL1 sequences was compared directly with the parental murine MAb mR005-1 for staining and specificity. As shown in FIG. 5, comparable staining was observed on peripheral blood lymphocytes. Cell binding competition between the murine parental mR005-1 and the native chR005-1 Fc0 was also showed in FIG. 6. The chR005-1 Fc0 completely blocked the murine parental mR005-1 MAb binding.
(69) 6. The Induction of CD19 Internalization Following MAb Binding is not a General Effect and could be Related to the Different MAb Anti-CD19 Used.
(70) By an indirect staining in flow cytometry, the presence or not of the naked antibody at B-CLL cell surface following incubation at 4° C. or at 37° C. was determined (FIG. 7). A significant shift of geometric mean fluorescence intensity was observed with rituximab at 37° C. for an extended time period beyond 3 or 24 hours. Similar effect was noticed with the parental murine MAb mR005-1, although a lower modulation of geometric mean fluorescence intensity was observed with the wild type chimeric MAb chR005-1 Fc0.
(71) 7. Characterization of the Native chR005-1 Fc0 Cytotoxicity Activity.
(72) In many applications, chimeric antibodies have demonstrated improved effector function in complement-mediated tumor cells lysis and in antibody-dependent cellular cytotoxicity assays as compared to the parental murine monoclonal antibody (Liu et al., 1987; Nishimura et al., 1987; Hamada et al., 1990). The native chimeric MAb chR005-1 Fc0 induced modest ADCC against Burkitt's lymphoma cell line (FIG. 8) or against ex vivo B-CLL cells from patients (FIG. 9). In a standard cytotoxicity assay complement dependent, only rituximab used as positive control killed the Raji cells, whereas the chR005-1 Fc0 failed to trigger cell cytotoxicity (FIG. 10).
(73) 8. Generation of Variants for Human IgG1 C.sub.H2 Domain.
(74) As used herein, the term “heavy chain” is used to define the heavy chain of an IgG antibody. In an intact, native IgG, the heavy chain comprises the immunoglobulin domains VH, CH1, Hinge, CH2 and CH3. Throughout the present specification, the numbering of the residues in an IgG heavy chain is that of the EU index (Kabat et al, 1991), expressly incorporated herein by references. The “EU index as in Kabat” refers to the numbering of the human IgG1 EU antibody.
(75) We constructed several variants including single, double, three, four or five substitution variants to enhance ability to mediate effector function, (FIGS. 11A-11F). Fc34 LPLLAL F243L/R292P/Y300L/V305L/K326A/P396L Fc24 LPLLAAL F243L/R292P/Y300L/V305L/K326A/E333A/P396L
(76) 9. The Fc24 or Fc34 Variant chR005-1 Efficiently Triggered ADCC.
(77) The MAb activity to mediate ADCC from the MAb variant panel was measured using Raji target cells firstly in whole blood based assays (FIG. 12). The potency and efficacy of the chR005-1 Fc24 or Fc34 MAb was notably higher when compared to parental chR005-1 Fc0.
(78) The activity of chR005-1 Fc24 was also assessed by using ex vivo B-CLL cells from patients (FIG. 13). Similar to observations with ADCC on Raji cell line, the potency and efficacy of the chR005-1 Fc20 MAb was notably higher when compared to parental chR005-1 Fc0.
(79) 10. Investigation on the MAb Variant Panel to CDC Using B-Cell Lymphoma.
(80) As shown in FIG. 14, the six mutants Fc34 (F243L/R292P/Y300L/V305L/K326A/P396L) or the seven mutants Fc24 (F243L/R292P/Y300L/V305L/K326A/E333A/P396L) generated from the wild type chR005-1 Fc0 induced CDC using the Raji cell line.
(81) As shown in FIG. 15, the five mutants Fc24 (F243L/R292P/Y300L/V305L/K326A/E333A/P396L) generated from the wild type chR005-2 Fc0 also induced CDC using the Raji cell line.
(82) The six mutants Fc34 (F243L/R292P/Y300L/V305L/K326A/P396L) or the five mutants Fc24 (F243L/R292P/Y300L/V305L/K326A/E333A/P396L) generated from the wild type chR005-1 Fc0 also induced CDC when targeting ex vivo B-CLL (FIG. 16).
(83) 11. The Variants chR005-1 Fc24 or Fc34 Bound the Complement with a Higher Efficient for the chR005-1 Fc34.
(84) The human or the natural complement present in serum (FIG. 17) bound the variants chR005-1 Fc24 or chR005-1 Fc34 revealing a higher efficacy with the chR005-1 Fc34 variant.
(85) 12. The MAb Variants Triggered Similar Level of Cell Apoptosis.
(86) The biological activity on programmed cell death of chR005-1 Fc24 or chR005-1 Fc34 MAb was tested on Raji CD19 positive cell line (FIG. 18) or on ex vivo B-CLL (FIG. 19). The Fc MAb engineering did not influence CD19 triggered apoptosis.
(87) 13. Monoclonal Antibodies May be Used in Combination.
(88) The MAb activity to mediate PCD alone or in combination was compared using Daudi target cells. A higher level of apoptotic cells was observed in the presence of murine parental mR005-1 (FIG. 20) or with the variant Fc24 or Fc34 chR005-1 MAb (FIG. 21), combined with rituximab demonstrated the feasibility and the benefit of the MAb combination.
(89) The MAb activity to mediate PCD alone or in combination was compared using on ex vivo B-CLL. A higher level of apoptotic cells was observed in the presence of the murine parental mR005-1 (FIG. 22) or with the variant Fc24 or Fc34 chR005-1 MAb (FIG. 23), combined with rituximab demonstrated the feasibility and the benefit of the MAb combination.
(90) 14. The MAb Directed Against CD19 can be Used in Patient with Rituximab Recurrent and Refractory Disease.
(91) Ex vivo B-CLL samples from patients with recurrent and refractory disease following rituximab treatment were treated in vitro with the murine parental mR005-1 (FIG. 24) or with MAb chR005-1 Fc24 or chR005-1 Fc34 (FIG. 25). The higher level of apoptosis observed with MAbs against CD19 compared with rituximab demonstrated the feasibility and the efficacy to use MAbs directed another antigen than CD20.
(92) 15. No Influence of Fc Polymorphism.
(93) Similar level of ADCC was observed whatever FcγRIIIA allotypes F/F or V/V. Lymphocytes staining was performed with the MAb unconjugated 3G8 or MEM-154 (FIG. 26).
(94) 16. MAb Expression.
(95) The empty CHO dhfr−/− cells (purchased by the ATCC collection) were co-transfected with the pcDNA3.3 expression vector for light chain and with the pcDNA3.3 expression vector for heavy chain following transient transfection procedure established in our laboratory. General characteristics of this MAb expression vector are shown in FIG. 27. The empty CHO cells were co-transfected with the pcDNA3.3-expression vector for light chain (Invitrogen) and with the pcDNA3.3 expression vector for heavy chain (Invitrogen) following transient transfection procedure established in our laboratory. General characteristics of this research MAb expression vector are shown in FIG. 27. By using the pcDNA3.3 vector, expression of these chimeric antibody chains in mammalian cells was controlled by the full-length human CMV immediate early promoter/enhancer. Secretion of H and L chains were enabled by the respective human IgH leader sequence. And in the 3′ region, a Herpes Simplex Virus thymidine kinase polyA tail allows for efficient induction and stabilization of mRNA. The coding regions for light and heavy chains of MAb anti-CD19 are introduced into the expression vector pcDNA3.3-TOPO in the TOPO cloning site. The transformants are analyzed for correct orientation and reading frame, the expression vector may be transfected into CHO cell line
(96) 17. Generation of Low Fucosylated Anti-CD19 MAb chR005-1.
(97) The sugar core found in the Fc region of IgG is a bi-antennary complex [Asn297-GN-GN-M-(M-GN)2] where GN is N-acetylglucosamine, and M is mannose. Oligosaccharides can contain zero (G0), one (G1) or two (G2) galactose (G). Variations of IgG glycosylation patterns can include core fucosylation (F). As shown in FIGS. 28 and 31. the three major peaks in the chR005-1 Fc0 sample correspond to masses of fucosylated oligosaccharides with (GlcNAc).sub.2(Fuc)1+(Man).sub.3 (GlcNAc).sub.2(m/z 1836), (Gal)1 (GlcNAc)2 (Fuc)1+(Man).sub.3(GlcNAc).sub.2(m/z 2040) and (Gal).sub.2(GlcNAc).sub.2(Fuc)1+(Man).sub.3(GlcNAc).sub.2(m/z 2245). By contrast, as shown in FIG. 29-30, the two peaks G0F and G1F were present at much lower levels in the chR005-1 Fc24 or chR005-1 Fc34 antibody (7.2% or 5.2% and 16.8% or 16.1% respectively) compared to the native chR005-1 Fc0 (28% or 38.3% respectively). No significant impact was observed on the peak G2F. A higher level of oligomannoses between (Man).sub.5(GlcNAc).sub.2(m/z 1579), (Man).sub.6(GlcNAc).sub.2 (m/z 1784) (Man).sub.7(GlcNAc).sub.2(m/z 1988), (Man).sub.8(GlcNAc).sub.2(m/z 2192) and (Man).sub.9(GlcNAc).sub.2(m/z 2396) was observed (29.9% or 27.9% versus 0%) as also a higher level of sialylated glycoforms (1.6%% or 2.4% versus 0.8%). Including (Gal)1 (GlcNAc).sub.2(Fuc).sub.1(NeuAc).sub.1+(Man).sub.3(GlcNAc).sub.2. (m/z 2401), (Gal).sub.2(GlcNAc).sub.2(Fuc).sub.1(NeuAc).sub.1+(Man).sub.3(GlcNAc).sub.2. (m/z 2605) and (Gal).sub.3 (GlcNAc).sub.2(Fuc).sub.1(NeuAc).sub.1+(Man).sub.3(GlcNAc).sub.2. (m/z 2966) in the chR005-1 Fc24 or chR005-1 Fc34 antibody compared to the native chR005-1 Fc0.
(98) As shown in FIG. 32, data demonstrated accumulation of non-fucosylated in the optimized antibody from MAb chR005-1 Fc24 or chR005-1 Fc34 instead of fucosylated structures in the native chimeric chR005-1 Fc0 antibody.
(99) 18. Determination of the Binding MAb Affinity.
(100) The binding properties of MAbs mR005-1, chR005-1 Fc0, chR005-1 Fc24 and chR005-1 Fc34 were examined by flow cytometry analysis and binding equilibrium studies on CD19 positive Raji cell line. Therefore neither the chimerization nor MAb optimization resulted in significant changes in MAb affinity:
(101) TABLE-US-00007 MAb KD nM mR005-1 2.82 chR005-1 Fc0 1.83 chR005-1 Fc24 1.26 chR005-1 Fc34 2.63
(102) 19. Different Glycan Profile According Fc Variant
(103) FIG. 33 shows Fc variant impact on glycans such as fucosylated oligosaccharides (m/z 1836, 2040, 2245), oligomannoses (m/z 1579, 1784, 1988, 2192, 2396) and/or sialylated glycoforms (2401), 2605, 2966) compared to the native chR005-1 Fc0.
(104) 20. Differential MAb Activity According Fc Variant.
(105) FIGS. 34A-34B show different Fc variant anti-CD19 antibodies and their ability to trigger CDC and/or ADCC with isolated PBMNC. The present invention provided that only the chR005-1 Fc24 or chR005-1 Fc34 variant triggered both ADCC and CDC MAb activities.
(106) 21. FIGS. 35A-35B show Different Fc Variant Anti-CD19 Antibodies (Fc14, chR005-1, Fc34) and their Ability to Trigger CDC and/or ADCC in the Presence of Whole Blood. Only the chR005-1 Fc34 Triggered ADCC in the Presence of Natural Circulating Immunoglobulins.
(107) 22. FIG. 36 Shows Different Fc Variant Anti-CD19 Antibodies (chR005-1 Fc24; Fc39) and their Ability to Trigger ADCC in the Presence of Whole Blood at Different Levels According the Optimized Fc.
(108) 23. The Invention Includes, that the Improved ADCC with the chR005-1 Fc34 was Not Limited in CD16A Off Rates.
(109) FIGS. 37A-37B show in vivo comparative effect of the chR005-1 MAb Fc24 or Fc 34 optimized variant on tumour clearance in established mouse Raji lymphoma xenograft model with or without human leukocyte inoculation. The invention includes, that the improved ADCC with the chR005-1 Fc34 was not limited in CD16A off rates compared to the chR005-1 Fc24 containing further the E333A modification.
(110) 24. Impact of Sialylation on CDC Ability.
(111) FIG. 38 shows that the modification at position 243 leading additional sialylation was not enough sufficient to restore recognition target cell lysis by complement. The present invention reveals that only the chR005-1 Fc24 or chR005-1 Fc34 MAbs which exhibits around the same chR005-1 Fc20 sialylated glycoform pattern but with a different protein sequence and a higher level of bound complement induced a target cell lysis by complement.
(112) 25. Impact of the MAb Anti-CD19 on Rituxan Refractory Follicular Lymphoma.
(113) FIG. 39 shows that the growth of Rituxan refractory RL established tumour was inhibited with the Fc24 optimized chR005-1 MAb.
(114) 26. Influence of MAb and Drug Combination
(115) FIG. 40 shows that the combination of the Fc24 optimized chR005-1 MAb and the drug Doxorubicine increased the level of growth inhibition Raji established tumour.
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