HUMANIZED ANTIBODIES AGAINST LEWIS Y

Abstract

The present invention pertains to humanized anti-Lewis Y antibodies which specifically bind to Lewis Y and do not show any cross-reactivity. Especially, the humanized anti-Lewis Y antibodies do not bind to Lewis b or any other blood group carbohydrate antigen. In particular, the present invention is directed to humanized anti-Lewis Y antibodies which are useful in the treatment of cancer.

Claims

1. A humanized antibody which is capable of binding to Lewis Y and which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 10, and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 22.

2. The antibody according to claim 1, wherein the heavy chain variable region comprises the complementarity determining regions CDR-H1 having the amino acid sequence of SEQ ID NO: 12 or 13, CDR-12 having the amino acid sequence of SEQ ID NO: 14 or 15, and CDR-H3 having the amino acid sequence of SEQ ID NO: 16, in particular the complementarity determining regions CDR-H1 having the amino acid sequence of SEQ ID NO: 12, CDR-H2 having the amino acid sequence of SEQ ID NO: 14, and CDR-H3 having the amino acid sequence of SEQ ID NO: 16.

3. The antibody according to claim 1, wherein the heavy chain variable region comprises an amino acid sequence selected from the group of SEQ ID NOs: 1 to 9.

4. The antibody according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11, in particular an amino acid sequence selected from the group of SEQ ID NOs: 1 to 6.

5. The antibody according to claim 1, wherein the light chain variable region comprises the complementarity determining regions CDR-L1 having the amino acid sequence of SEQ ID NO: 24 or 25, CDR-L2 having the amino acid sequence of SEQ ID NO: 26 or 27, and CDR-L3 having the amino acid sequence of SEQ ID NO: 28 or 29, in particular the complementarity determining regions CDR-L1 having the amino acid sequence of SEQ ID NO: 24, CDR-L2 having the amino acid sequence of SEQ ID NO: 26, and CDR-L3 having the amino acid sequence of SEQ ID NO: 28.

6. The antibody according to claim 1, wherein the light chain variable region comprises an amino acid sequence selected from the group of SEQ ID NOs: 17 to 21.

7. The antibody according to claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 23, in particular an amino acid sequence selected from the group of SEQ ID NOs: 17 and 18.

8. The antibody according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 23.

9. The antibody according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 17.

10. The antibody according to claim 1, further comprising an Fc region.

11. The antibody according to claim 10, being an IgG1-type, IgG2-type, IgG3-type or IgG4-type antibody.

12. The antibody according to claim 1, being capable of specifically binding Lewis Y, but not Lewis b.

13. A nucleic acid encoding the antibody according to claim 1.

14. An expression cassette or vector comprising the nucleic acid according to claim 13 and a promoter operatively connected with said nucleic acid.

15. A host cell comprising the nucleic acid according to claim 13.

16. A conjugate comprising the antibody according to claim 1 conjugated to a further agent.

17. The conjugate according to claim 16, wherein the further agent is a cytotoxic agent, tumor-specific antibody or immune checkpoint blocking or activating antibody.

18. The conjugate according to claim 16, being a chimeric antigen receptor.

19. A composition comprising the antibody according to claim 1.

20. The composition according to claim 19 being a pharmaceutical composition which preferably further comprises one or more components selected from the group consisting of solvents, diluents and excipients.

21. (canceled)

22. A method of treating a patient having cancer, an infection, or an immunodeficiency disorder, comprising administering to the patient the antibody of claim 1.

23. The method according to claim 22, wherein the cancer is selected from the group consisting of lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, gastric cancer, leukemia, lymphoma, multiple myeloma, head and neck cancer, pancreatic cancer, liver cancer, prostate cancer and bladder cancer.

24. A method of treating a patient having cancer, an infection, or an immunodeficiency disorder, comprising administering to the patient the conjugate of claim 16.

Description

FIGURES

[0099] FIG. 1 shows a schematic representation of the Lewis carbohydrate antigen family. The Lewis antigens are a related set of glycans that carry fucose in an alpha 1-3 (Lewis X, Y) or an alpha 1-4 (Lewis A, B) linkage to the GlcNAc monosaccharide.

[0100] FIG. 2 shows the results of antigen ELISA assays. Different humanized anti-Lewis Y antibody variants were tested for their binding to Lewis Y, Lewis b and Globo H. A high O.D. signal indicates strong binding of the antibody to the antigen. A: Binding of antibody variants at 25 ng/mL to Lewis Y (LeY), Lewis b (Leb) and Globo H. B: Binding of antibody variants at 25 ng/mL and 12.5 ng/mL to Lewis Y. C: Binding of antibody variants at 100 ng/mL, 50 ng/mL and 25 ng/mL to Lewis b. D: Binding of antibody variants at 100 ng/mL, 50 ng/mL and 25 ng/mL to Globo H. Control (ctrl.+): parent human/mouse chimeric antibody AA9.

[0101] FIG. 3 shows the results of antigen ELISA assays. Binding curves for different humanized anti-Lewis Y antibody variants to Lewis Y (A) and Lewis b (B) were determined. Control 1 (ctrl.+AA9): parent human/mouse chimeric antibody AA9. Control 2 (ctrl.+Chain shuffling): chain shuffling antibody comprising the heavy chain of human/mouse chimeric antibody AA9 and the light chain of human/mouse chimeric antibody CC8.

[0102] FIG. 4 shows the results of antigen ELISA assays. Binding curves for the humanized anti-Lewis Y antibody AA9-3-10.1 to Lewis Y and Lewis b were determined. Control (ctrl.): parent human/mouse chimeric antibody AA9.

[0103] FIG. 5 shows the results of antigen ELISA assays. Binding of the humanized anti-Lewis Y antibody AA9-3-10.1 (humanized AA9) and the parent human/mouse chimeric antibody AA9 (chimeric AA9) to different carbohydrate antigens was tested at an antibody concentration of 50 ng/mL.

[0104] FIG. 6 shows the results of antigen ELISA assays. Different sequence-optimized versions of the humanized anti-Lewis Y antibody AA9-3-10 were tested for their binding to Lewis Y and Lewis b. A high O.D. signal indicates strong binding of the antibody to the antigen. A: Binding of antibody variants at 50 ng/mL to Lewis Y (LeY) and Lewis b (Leb). B: Binding of antibody variants at 50 ng/mL and 25 ng/mL to Lewis Y. C: Binding of antibody variants at 100 ng/mL and 50 ng/mL to Lewis b. Control (pos.-ctrl.-Set2 128+129): parent human/mouse chimeric antibody AA9.

[0105] FIG. 7 shows the results of antigen ELISA assays. Different anti-Lewis Y antibodies were tested for their specificity by analyzing their binding to Lewis Y and other, related carbohydrate antigens. A: humanized anti-Lewis Y antibody AA9-3-10.1; B: anti-Lewis Y antibody h3S193; C: anti-Lewis Y antibody BR96.

[0106] FIG. 8 shows the results of antigen ELISA assays. Binding curves for the anti-Lewis Y antibodies AA9-3-10.1, h3S193 and BR96 to Lewis Y were determined.

[0107] FIG. 9 shows binding of humanized anti-Lewis Y antibody variant AA9-3-10.1, BR96 and h3S193 (as hIgG1) to the tumor cell lines Ls-174T, T-47D, H9D8 and Colo-205. Irrelevant hIgG1 was used as a negative control. Binding is reported as percentage of antibody-positive cells of total live cells.

[0108] FIG. 10 shows binding of humanized anti-Lewis Y antibody variant AA9-3-10.1, BR96 and h3S193 (as mIgG1) to leukocytes isolated from five healthy donors. Irrelevant hIgG1 was used as a reference. Binding to granulocytes, lymphocytes and monocytes is reported as median fluorescence intensity (MFI).

[0109] FIG. 11 shows proliferation inhibition experiments of toxin-coupled AA9-3-10.1 (as hIgG1) and isotype control with different tumor cell lines. Proliferation is reported as percentage proliferation relative to medium control.

EXAMPLES

Example 1: Humanization of the Murine Heavy and Light Chain Variable Regions of Anti-LeY Antibodies

[0110] The nucleic acid sequences coding for the murine heavy and light chain variable regions of two monoclonal anti-LeY antibodies (AA9: SEQ ID NOs: 30 and 31; CC8: SEQ ID NO: 32) were ligated to the genomic sequences of the human constant γ1 region (CH) and the human constant K region (CL), respectively.

[0111] On the basis of these chimeric clones, humanized antibodies were constructed. To this end, point mutations were introduced into the nucleic acid sequences of the murine framework regions of VH and VL in order to generate the corresponding human framework regions. The target human framework regions were selected from a human germ line antibody library. In particular, the most related framework regions were chosen from the library depending on their overall sequence similarity and their CDR loop classification. All data obtained were considered to design a set of different variable sequences of humanized variable light and variable heavy chains of both parent murine antibodies. Some of the variants contain back-mutations to the murine sequence on critical positions. The humanized variants of the light chain variable region were cloned in a κ-chain vector and the humanized variants of the heavy chain variable region were cloned in a γ1-chain vector.

[0112] Antibodies comprising different combinations of the obtained heavy and light chains were produced and screened for their expression and LeY binding profile. The following humanized antibody heavy and light chains variable regions were selected for further analysis.

TABLE-US-00001 TABLE 1 heavy chain SEQ parent light chain SEQ parent variable ID murine variable ID murine region NO antibody region NO antibody VH6 9 AA9 VL1 19 CC8 VH9 7 AA9 VL2 20 CC8 VH10 6 AA9 VL3 17 AA9 VH11 8 AA9 VL6 21 CC8 VH10.1 1 AA9 VL7 18 AA9 VH10.2 2 AA9 VH10.3 3 AA9 VH10.4 4 AA9 VH10.5 5 AA9

Example 2: Binding of the Humanized Antibody Variants to Different Carbohydrate Antigens

[0113] Following expression of the different constructs in NM-H9D8 cells, the titer of the humanized antibody variants was determined and their concentration adjusted. Then, the humanized antibodies were analyzed in an antigen ELISA for binding to Lewis Y, Lewis b and Globo H. Briefly, antigens (coupled to polyacrylamide) were coated to 96-well MaxiSorp plates (Nunc ThermoScientific) overnight. Unspecific binding was blocked and test samples were added at different concentrations. Afterwards, anti-human IgG-Fc POD secondary antibody was added, followed by TMB substrate reaction. Bound antibodies were determined by measurement at 450/630 nm using an EnSpire 2300 multilabel reader (PerkinElmer) or at 450/620 nm using a Tecan SPARK plate reader.

[0114] All variants showed significant binding to Lewis Y similar to that of the parental chimeric antibody. Furthermore, binding to Globo H was negative for all antibodies. However, surprisingly the selected humanized antibody variants showed significantly reduced cross-reactivity towards Lewis b (see FIGS. 2, 3 and 4).

[0115] Humanized antibody variants AA9-3-6 (comprising VL3 and VH6), AA9-3-9 (comprising VL3 and VH9), AA9-3-10 (comprising VL3 and VH10) and CC8-1-11 (comprising VL1 and VH11) were further analyzed for their fine specificity using an antigen ELISA with Lewis Y, Galα1,2-Gal and β-N-acetyl-D-glucosamine-6-sulfate. The analyzed humanized antibodies showed strong, highly specific binding to Lewis Y, but no significant binding to other carbohydrate antigens. Binding to the following carbohydrate antigens was tested at an antibody concentration of 50 ng/mL (see also FIG. 5):

TABLE-US-00002 TABLE 2 Lac-di-Nac SiaLe.sup.x GlcNAcβ1-3Galβ- 6′-SL Tk β-N-acetyl-D-glucosamine-6-sulfate GalNAcβ1-4Galβ1-4Glcβ- 3′-O-su-Le.sup.a A [type 2]- 3′-OSO3-Le.sup.x B [type ]- 3′-su-LacNAc Galα1-3′Lac 3′-su-Le.sup.c GlcNAcβ1-2′ TF melibiose Galα4GlcNAc Galα1-3′LacNAc Neu5Acβ [Sia].sub.2 α-D-glucose [Sia].sub.3 α-D-galactose Gal2βGal β-D-galactose 6-O-su-LacNAc α-D-mannose core 2 α-D-mannose-6-phosphate H [type 4] α-L-fucose LNT β-N-acetyl-D-glucosamine LNnT α-N-acetyl-D-galactosamine [T.sub.n] Neu5Ac6Gal β-N-acetyl-D-galactosamine GM4 Man.sub.3 A.sub.tri β-D-galactose-3-sulfate B.sub.tri α-N-acetylneuraminic acid A.sub.di 3′SLN B.sub.di Pk, Gb3, GbOse3 H [type 2] Taβ 6′-O-su-LacNAc Galβ3Gal H.sub.di Le.sup.a GlcNAcβ1-3Galβ1-4Glcβ- Le.sup.b di-GalNAcβ Le.sup.d/H type 1 core 3 Le.sup.c core 6 Le.sup.x core 4 Le.sup.y Sia.sub.2 TF La.sup.c 6-SiaTF LacNAc 11-OS, YDS TF 9-OS Fucα3GlcNAc 7-OS Fs-2 3,6-SiaT.sub.n core 5 6′SLN T .sub.aa? maltose 3′-SiaLe.sup.c β-D-glucose T.sub.ββ Fucα4GlcNAc, L.sup.e [GlcNAc].sub.2 Galα2Gal SiaT.sub.n SiaT.sub.n H [type 3]/Globo H GlcNAcβ1-4GalNAcα- 3′-SL 3′-O-su-TF SiaLe.sup.a 3-SiaT.sub.n

Example 3: Generation of Further Humanization Variants Based on Antibody AA9-3-10

[0116] Humanized antibody variant AA9-3-10 (comprising VL3 and VH10) was selected as best candidate for further optimization of the humanized VH sequence. The following humanized antibodies were generated as described in Example 1:

TABLE-US-00003 TABLE 3 heavy chain SEQ ID light chain SEQ ID antibody variable region NO variable region NO AA9-3-10.1 VH10.1 1 VL3 17 AA9-3-10.2 VH10.2 2 VL3 17 AA9-3-10.3 VH10.3 3 VL3 17 AA9-3-10.4 VH10.4 4 VL3 17 AA9-3-10.5 VH10.5 5 VL3 17

[0117] Binding of these humanized antibody variants to Lewis Y and Lewis b was analyzed as described in Example 2. All antibody variants show strong, highly specific binding to Lewis Y. No significant binding to Lewis b could be detected (see FIG. 6).

Example 4: Comparison of Antigen Binding and Specificity of the Humanized Variant AA9-3-10.1 with Known Anti-LeY Antibodies

[0118] Binding specificity of the humanized antibody variant AA9-3-10.1 to different, closely related carbohydrate antigens (βD-galactose, Lewis b, Lewis X, 3′-O-su-Lewis X, lacto-N-tetraose (LNT), Neu5Aca2-5Galβ, and Lewis Y) was compared to that of the known antibodies h3S193 and BR96.

[0119] Briefly, antigens (coupled to polyacrylamide) were coated to 96-well MaxiSorp plates (Nunc ThermoScientific) overnight. Unspecific binding was blocked and test samples were added at different concentrations. Afterwards, anti-human IgG (H+L) POD secondary antibody was added, followed by TMB substrate reaction. Bound antibodies were determined by measurement at 450/630 nm using an EnSpire 2300 multilabel reader (PerkinElmer) or at 450/620 nm using a Tecan SPARK plate reader.

[0120] Humanized antibody variant AA9-3-10.1 shows stronger binding to Lewis Y and a significantly improved specificity. Binding of h3S193 to Lewis Y is much lower at low antibody concentrations and BR96 shows significant binding to βD-galactose and Lewis X. Furthermore, both h3S193 and BR96 bind Neu5Aca2-5Galβ at higher concentrations (see FIGS. 7 and 8).

[0121] A new method to determine binding constants and affinity is the fluorescence proximity sensing using single stranded DNA (96mer) spotted on a chip on DRX2 instrument (Dynamic Biosensors) and complementary DNA coupled to a ligand. In the present study, streptavidin was used as a ligand to capture biotinylated polyacrylamide-coupled Lewis Y or Lewis b. Binding of humanized antibody variant AA9-3-10.1 or competitors' anti-Lewis Y antibodies to the antigen resulted in a fluorescence change. On- and off-rates can be calculated during association and dissociation. AA9-3-10.1 and competitors' anti-LeY antibodies were diluted to 300, 60, and 12 nM in PE140 buffer and applied to the chip-bound antigen. For the experiment with AA9-3-10.1 on Lewis b, 3000, 600 and 120 nM were used, since very low signals were expected. Binding curves were evaluated by mono-exponential global fit (instrument software). Due to a higher sensitivity, faster interactions can be monitored compared to surface plasmon resonance (SPR). This results in binding kinetics different from SPR but more comparable to the “gold standard” method KinExA, measured in a liquid system.

[0122] The binding assay showed a strong and highly specific binding of AA9-3-10.1 to Lewis Y. The data confirmed the results of the ELISA assays.

TABLE-US-00004 TABLE 4 k.sub.ON k.sub.OFF K.sub.D antibody ligand [M.sup.−1s.sup.−1] [s.sup.−1] [nmol/L] AA9 clgG1 Lewis Y 1.34E+06 7.66E−03 5.72 Lewis b 7.07E+05 3.92E−02 55.45 AA9-3-10.1 hlgG1 Lewis Y 8.67E+05 1.54E−02 17.76 Lewis b n.d. n.d. n.d. AA9-3-10.1 mlgG1 Lewis Y 4.41E+05 9.16E−03 20.77 BR96 mlgG1 Lewis Y 9.46E+05 5.41E−02 57.19 h3S193 mlgG1 Lewis Y n.d. n.d. n.d. n.d.: not determinable

Example 5: Comparison of Tumor Cell Binding of the Humanized Variant AA9-3-10.1 with Known Anti-LeY Antibodies

[0123] The binding properties of humanized anti-Lewis Y antibody variant AA9-3-10.1, BR96 and 35193 (all as hIgG1) to the human cancer cell lines Ls-174T, T-47D, H9D8 and Colo-205 were analyzed by flow cytometry. Irrelevant hIgG1 was used as a negative control. Briefly, tumor cells were harvested and incubated with indicated antibodies in serial dilutions at 4° C. in the dark. Afterwards, cells were washed and incubated with a secondary goat anti-hIgG PE-conjugated antibody at 4° C. in the dark. After an additional washing step, cells were stained with DAPI in order to discriminate between live and dead cells and analyzed via flow cytometry.

[0124] Humanized anti-Lewis Y antibody variant AA9-3-10.1 showed concentration-dependent binding to Lewis Y-positive cell lines Ls-174T and T-47D and no binding to Lewis Y-negative cell lines H9D8 and Colo-205, whereas the competitor anti-Lewis Y antibodies BR96 and 3S193 showed stronger binding than humanized anti-Lewis Y antibody variant AA9-3-10.1 to all four tumor cell lines (see FIG. 9). Stronger binding of BR96 and 3S193 was most likely due to cross-reactivity. For example, Colo-205 is described to be negative for Lewis Y (Westwood et al., 2005), but positive for Lewis b (Noble et al., 2013).

Example 6: Comparison of Blood Cell Binding of the Humanized Variant AA9-3-10.1 with Known Anti-LeY Antibodies

[0125] Binding of humanized anti-Lewis Y antibody variant AA9-3-10.1, BR96 and 3S193 (as mIgG1) to leukocytes was determined using flow cytometry. Therefore, whole blood from five healthy volunteers was used. In a first step, red blood cells were lysed and remaining leukocytes were incubated with indicated antibodies [10 μg/ml] at room temperature. Irrelevant mIgG1 was used as a negative control. Afterwards, cells were washed and incubated with a secondary anti-mIgG AF647-conjugated antibody at room temperature. After a washing step, cells were stained with anti-human CD45 PacificBlue-conjugated antibody at room temperature. After an additional washing step, cells were analyzed via flow cytometry. The immune cell subpopulations, granulocytes, monocytes and lymphocytes, were distinguished by their CD45 expression and granularity.

[0126] Humanized anti-Lewis Y antibody variant AA9-3-10.1 shows no or only weak binding to leukocyte subsets whereas BR96 binds strongly to granulocytes and 3S193 binds to granulocytes, lymphocytes and monocytes (see FIG. 10). This shows the superior specificity of the humanized anti-Lewis Y antibody variants for tumor cells and their very low cross-reactivity with normal tissue cells.

Example 7: Proliferation Inhibition of Different Tumor Cell Lines Using the Humanized Anti-Lewis Y Antibody Variant AA9-3-10.1 Coupled to a Toxin

[0127] To demonstrate the efficacy of the humanized anti-Lewis Y antibody variants for killing tumor cells, a proliferation inhibition assay with different tumor cell lines was performed. As cytotoxin, MMAE was coupled to Protein G which binds to the antibody and thereby forms an antibody toxin conjugate.

[0128] The cell lines Ls-174T, T-47D, MCF-7 (CSC-enriched), Ovcar-3 and HSC-4 were seeded in culture medium with 5,000 cells/well in a 96-well flat-bottom plate and incubated in presence of humanized anti-Lewis Y antibody variant AA9-3-10.1 (as hIgG1) or irrelevant isotype control in indicated concentrations and ProtG-MMAE for 4 days. Number of viable cells was determined using the commercial CellTiter-Glo Luminescent Cell Viability Assay. Percentage of proliferation was determined relative to a medium only control.

[0129] A toxin-coupled humanized anti-Lewis Y antibody variant AA9-3-10.1 is able to inhibit the proliferation of various Lewis Y-expressing tumor cell lines indicating effective internalization of the antibody (see FIG. 11).

Example 8: Immunohistochemical Staining with Humanized Anti-Lewis Y Antibody Variant AA9-3-10.1 of Different Tumor Tissues from Various Cancer Types

[0130] Binding of humanized anti-Lewis Y antibody variant AA9-3-10.1 to different cancer indications was analyzed by immunohistochemistry. In brief, tissue microarray slides from breast cancer (BRC), non-small cell lung carcinoma (NSCLC), colon carcinoma (CRC), head and neck cancer (HNC), small cell lung carcinoma (SCLC) and ovarian carcinoma (OvCa) were deparaffinized and rehydrated in a descending alcohol series. After antigen retrieval, endogenous peroxidase and unspecific binding was blocked. Binding of humanized anti-Lewis Y antibody variant AA9-3-10.1 as mIgG1 [6.5 μg/ml] was detected with the secondary antibody Envision Flex anti-mouse Ig-HRP and DAB+ staining solution. Finally, slides were counterstained with Mayer's Haematoxylin, mounted and evaluated under the microscope. Binding of humanized anti-Lewis Y antibody variant AA9-3-10.1 is reported using the immunoreactive score (IRS; range 0-12) which is calculated by staining intensity (range 0-3) multiplied by percentage of stained cells (range 0-4).

[0131] Humanized anti-Lewis Y antibody variant AA9-3-10.1 stains tumor tissue of breast cancer (BRC), non-small cell lung carcinoma (NSCLC), colon carcinoma (CRC), head and neck cancer (HNC), small cell lung carcinoma (SCLC) and ovarian carcinoma (OVCa) at a high percentage of cases.

TABLE-US-00005 TABLE 5 IRS BRC NSCLC CRC HNC SCLC OvCa negative 0 1 10 10 22 24 59 weakly positive 1-3 3 12 9 23 18 27 moderately positive 4-8 4 11 11 21 3 14 strongly positive  9-12 2 2 5 3 0 0 positive cases/total cases 9/10 25/35 25/35 47/69 21/45 41/100 % positive cases 90% 71.4% 71.4% 68.1% 46.7% 41%

TABLE-US-00006 SEQUENCE LISTING SEQ ID NO sequence 1 QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYNID WVRQTPGKGLEWMG YIYPYQGYSDYNQKFKS KATMTVDKSTSTAYMELRSLRSDDTAVYYCAR QLGPGTF WGQGTLVTVS 2 QVQLVQSGPEVVKPGASVKVSCKASGYTFT DYNID WVRQTPGKGLEWMG YIYPYQGYSDYNQKFKS KATMTVDTSTSTAYMELRSLRSDDTAVYYCAR QLGPGTF WGQGTLVTVS 3 QVQLVQSGPEVVKPGASVKVSCKASGYTFT DYNID WVRQAPGKGLEWMG YIYPYQGYSDYNQKFKS KATMTVDTSTSTAYMELRSLRSDDTAVYYCAR QLGPGTF WGQGTLVTVS 4 QVQLVQSGPEVVKPGASVKVSCKASGYTFT DYNID WVRQTPGKGLEWMG YIYPYQGYSDYNQKFKS RVTMTVDKSTSTAYMELRSLRSDDTAVYYCAR QLGPGTF WGQGTLVTVS 5 QVQLVQSGPEVVKPGASVKVSCKASGYTFT DYNID WVRQAPGQGLEWMG YIYPYQGYSDYNQKFKS KATMTTDTSTSTAYMELRSLRSDDTAVYYCAR QLGPGTF WGQGTLVTVS 6 QVQLVQSGPEVVKPGASVKVSCKASGYTFT DYNID WVRQTPGKGLEWMG YIYPYQGYSDYNQKFKS KATMTVDKSTSTAYMELRSLRSDDTAVYYCAR QLGPGTF WGQGTLVTVS 7 QVQLVQSGPEVVKPGASVKVSCKASGYTFT DYNMD WVRQTPGKGLEWMG YIYPYNGYSDYNQKFKS KATMTVDKSTSTAYMELRSLRSDDTAVYYCAR QLGPGTF WGQGTLVTVS 8 KVQLVQSGPEVVKPGASVKVSCKASGYTFT DYNMD WVKQTPGKSLEWIG YIYPYNGYSDYNQKFKS KATLTVDKSTSTAYMELRSLRSDDTAVYYCAR QLGPGTF WGQGTLVTVS 9 KVQLVQSGAEVKKPGASVKVSCKASGYTFT DYNMD WVRQAPGQGLEWMG YIYPYNGYSDYNQKFKS KATLTTDTSTSTAYMELRSLRSDDTAVYYCAR QLGPGTF WGQGTLVTVS 10 XVQLVQSGXEVXKPGASVKVSCKASGYTFT DYNXD WVXQXPGXXLEWMG YIYPYNGYSDYNQKFKS NNTNTNDNSTSTAYMELRSLRSDDTAVYYCAR QLGPGTF WGQGTLVTVS with X1: Q/K, X9: A/P, X12: K/V, X34: I/M, X38: R/K, X40: T/A, N43: K/Q, X44: G/S, X55: Q/N, X67: K/R, X68: A/V, X70: M/L, X72: V/T, X74: K/T 11 QVQLVQSGXEVXKPGASVKVSCKASGYTFT DYNID WVRQXPGXGLEWMG YIYPYQGYSDYNQKFKS NNTMTNDNSTSTAYMELRSLRSDDTAVYYCAR QLGPGTF WGQGTLVTVS with X9: A/P, X12: K/V, X40: T/A, X43: K/Q, N67: K/R, N68: A/V, N72: V/T, X74: K/T 12 DYNID 13 DYNMD 14 YIYPYQGYSDYNQKFKS 15 YIYPYNGYSDYNQKFKS 16 QLGPGTF 17 DIVMTQTPLSLSVTPGQPASISC KSSQSLLHGNGKTYLN WLLQKPGQSPKLLIY LVSNLESGVPDR FSGSGSGTDFTLKISRVEAEDVGVYYC LQATHFPLT FGQGTKLEIK 18 DIVMTQTPLSLSVTPGQPTSISC KSSQSLLHGNGKTYLN WLLQKPGQSPKLLIY LVSNLESGVPDR FSGSGSGTDFTLKISRVEAEDLGVYYC LQATHFPLT FGAGAKLEIK 19 DIVMTQTPLSLSVTPGQPASISC TSSQSLVHSNGNSYLD WYLQKPGQSPQLLIY EVSKRNSGVPDR FSGSGSGTDFTLKISRVEAEDVGVYYC FQRTHLPLT FGQGTKLEIK 20 DIVLTQTPLSLSVTPGQPASISC TSSQSLVHSNGNSYLD WYLQKPGQSPQLLIY EVSKRNSGVPDR FSGSGSGKDFTLKISRVEAEDVGVYYC FQRTHLPLT FGQGTKLEIK 21 DIVLTQTPLSLSVTPGQPASISC TSSQSLVHSNGNSYLD WYLQKPGQSLQLLIY EVSKRNSGVPDR FSGSGSGTDFTLKISRVEAEDVGVYYC FQRTHLPLT FGAGTKLEIK 22 DIVXTQTPLSLSVTPGQPXSISC NSSQSLNHNNGNNYLN WNLQKPGQSNNLLIY NVSNNNSGVPDR FSGSGSGXDFTLKISRVEAEDXGVYYC XQXTHXPLT FGXGXKLEIK with X4: M/L, X19: A/T, N24: K/T, X30: L/V, X32: G/S, X35: K/N, N36: T/S, X39: N/D, X41: L/Y, N49: P/L, X50: K/Q, N55: L/E, X58: N/K, N59: L/R, X60: E/N, X74: T/K, N88: V/L, X94: L/F, N96: A/R, X99: F/L, X105: Q/A, X107: T/A 23 DIVMTQTPLSLSVTPGQPNSISC KSSQSLLHGNGKTYLN WLLQKPGQSPKLLIY LVSNLES GVPDRFSGSGSGTDFTLKISRVEAEDXGVYYC LQATHFPLT FGXGXKLEIK with X19: A/T, X88: V/L, X105: Q/A, X107: T/A 24 KSSQSLLHGNGKTYLN 25 TSSQSLVHSNGNSYLD 26 LVSNLES 27 EVSKRNS 28 LQATHFPLT 29 FQRTHLPLT 30 KVKLQQSGPDLVKPGASVKISCKASGYTFT DYNMD WVKQTHAKSLEWIG YIYPYNGYSDYNQKFKS KATLTVDKSSSTAYMELHSLTSEDSAIYYCAR QLGPGTF WGQGTLVTVS 31 DIVMTQTPLTLSVTIGQPTSISC KSSQSLLHGNGKTYLN WLLQRPGQSPKLLIY LVSNLESGVPDR FSGSGSGTDFTLKISRVEAEDLGVYYC LQATHFPLT FGAGAKLELK 32 DIVLTQSPLFLHVSLGDQASISC TSSQSLVHSNGNSYLD WHLQKSDQSLQLLIY EVSKRNSGVPDR FSGSGSGKDFTLKISRVEPEDLGIYYC FQRTHLPLT FGAGTKLEIK

Identification of the Deposited Biological Material

[0132] The cell lines DSM ACC 2806, DSM ACC 2807 and DSM ACC 2856 were deposited at the DSMZ—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7B, 38124 Braunschweig (DE) by Glycotope GmbH, Robert-Rössle-Str. 10, 13125 Berlin (DE) on the dates indicated in the following table.

TABLE-US-00007 Name of the Cell Accession Date of Line Number Depositor Deposition NM-H9D8 DSM ACC 2806 Glycotope GmbH Sep. 15, 2006 NM-H9D8-E6 DSM ACC 2807 Glycotope GmbH Oct. 5, 2006 NM-H9D8-E6Q12 DSM ACC 2856 Glycotope GmbH Aug. 8, 2007