Recognition molecules for the treatment and detection of tumors

Abstract

The invention relates to recognition molecules directed towards tumors, and it also relates to pharmaceutical compositions comprising said recognition molecules, methods for the production of said recognition molecules, and to the use of said recognition molecules in the diagnosis and therapy of tumor diseases.

Claims

1. A method of providing a molecule for tumor recognition comprising, a. providing a recombinant recognition molecule which comprises a variable heavy chain and a variable light chain and which specifically binds to a glycosylated MUC1 tumor epitope, wherein the variable heavy chain comprises (i) a CDRH1 region that comprises the amino acid sequence of SEQ ID NO: 1 or 2; (ii) a CDRH2 region that comprises the amino acid sequence of SEQ ID NO: 3 or 4; and (iii) a CDRH3 region that comprises the amino acid sequence of SEQ ID NO: 5 or 6; and the variable light chain comprises (i) a CDRL1 region that comprises the amino acid sequence of SEQ ID NO: 7 or 8; (ii) a CDRL2 region that comprises the amino acid sequence of SEQ ID NO: 9 or 10; and (iii) a CDRL3 region that comprises the amino acid sequence of SEQ ID NO: 11 or 12; b. modifying the recombinant recognition molecule by one or more mutations in one or more amino acid sequences set forth in step a., wherein single amino acids are replaced by amino acids having analogous physicochemical properties, c. selecting a recombinant recognition molecule modified in step b. that retains binding specificity towards the glycosylated MUC1 tumor epitope, and d. providing the modified recombinant recognition molecule selected in step c.

2. The method of claim 1, wherein the modification is a replacement by an equivalent canonical structure.

3. The method of claim 1, wherein the modified recombinant recognition molecule comprises a CDRH1 region variant comprising I, Y, W, or V substituted for M at position 4 of SEQ ID NO:1 or 2.

4. The method of claim 1, wherein the modified recombinant recognition molecule comprises a CDRH2 region variant selected from (a) a variant that has K or S substituted for N at position 8 of SEQ ID NO: 4; and (b) a variant that has Y substituted for H at position 9 of SEQ ID NO: 3.

5. The method of claim 1, wherein the modified recombinant recognition molecule comprises a CDRL1 region variant selected from (a) a variant that has P substituted for S at position 2 of SEQ ID NO: 7 or 8; (b) a variant that has F substituted for L at position 15 of SEQ ID NO: 7; and (c) a variant that has L substituted for F at position 15 of SEQ ID NO: 8.

6. The method of claim 1, wherein the modified recombinant recognition molecule comprises a CDRL3 region variant selected from (a) a variant that has P substituted for V at position 6 of SEQ ID NO: 11; and (b) a variant that has P substituted for L at position 6 of SEQ ID NO: 12.

7. The method of claim 1, wherein the recombinant recognition molecule comprises: (a) an FRH1 region comprising the following sequence, wherein the amino acid position corresponds to the numbering according to Kabat, TABLE-US-00012 1 E 2 V 3 K 4 L 5 V 6 E 7 S 8 G 9 G 10 G 11 L 12 V 13 Q 14 P 15 G 16 G 17 S 18 M 19 K 20 L 21 S 22 C 23 A or V 24 A, V, S or T 25 S 26 G 27 Y, F, S or D 28 T 29 F, L or I 30 S; (b) an FRH2 region comprising the following sequence, wherein the amino acid position corresponds to the numbering according to Kabat, TABLE-US-00013 36 W 37 V 38 R 39 Q 40 S 41 P 42 E 43 K 44 G 45 L 46 E 47 W 48 V 49 A; (c) an FRH3 region comprising the following sequence, wherein the amino acid position corresponds to the numbering according to Kabat, TABLE-US-00014 66 R 67 F 68 T 69 I 70 S 71 R 72 D 73 D or V 74 S 75 K 76 S 77 S 78 V 79 Y or S 80 L 81 Q 82 M  82a N  82b N  82c L 83 R 84 A or V 85 E 86 D 87 T 88 G 89 I 90 Y 91 Y 92 C 93 T 94 R, G, N, K or S. (d) an FRH4 region comprising the following sequence, wherein the amino acid position corresponds to the numbering according to Kabat, TABLE-US-00015 103 W 104 G 105 Q 106 G 107 T 108 T 109 L 110 T 111 V 112 S 113 S or A; (e) an FRL1 region comprising the following sequence, wherein the amino acid position corresponds to the numbering according to Kabat, TABLE-US-00016 1 D 2 I, V or L 3 V 4 M or L 5 T 6 Q 7 T or A 8 P or A 9 L or F 10 S 11 L or N 12 P 13 V 14 S or T 15 L 16 G 17 D or T 18 Q or S 19 A 20 S 21 I 22 S 23 C; (f) an FRL2 region comprising the following sequence, wherein the amino acid position corresponds to the numbering according to Kabat, TABLE-US-00017 35 W 36 Y 37 L 38 Q 39 K 40 P 41 G 42 Q or L 43 S 44 P 45 K or Q 46 L 47 L 48 I or V 49 Y; (g) an FRL3 region comprising the following sequence, wherein the amino acid position corresponds to the numbering according to Kabat, TABLE-US-00018 57 G 58 V 59 P 60 D 61 R 62 F 63 S 64 G or S 65 S 66 G 67 S 68 G 69 T 70 D 71 F 72 T 73 L 74 K or R 75 I 76 S 77 R 78 V 79 E 80 A 81 E 82 D 83 L or V 84 G 85 V 86 Y 87 Y 88 C; or (h) an FRL4 region comprising the following sequence, wherein the amino acid position to the numbering according to Kabat, TABLE-US-00019  98 F  99 G 100 G or D 101 G 102 T 103 K 104 L 105 E 106 I or L  106a K 107 R 108 A.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

(1) FIG. 1: Vector for cloning and bacterial expression of single-chain antibody fragments.

(2) FIG. 2: Cloning diagram for the preparation of single-chain antibody fragments having different linker length.

(3) FIG. 3: Vector system for cloning and eukaryotic expression of chimeric antibodies in IgG1 or IgM format.

(4) FIG. 4: Binding of the recognition molecules of the invention, mIgG-Panko1 and mIgG-Panko2, to glycosylated and non-glycosylated MUC1 peptide in an ELISA. Binding of the recognition molecules of the invention, mIgG-Panko1 and mIgG-Panko2, to glycosylated and non-glycosylated MUC1 peptide in an ELISA. The non-glycosylated 30mer with the sequence APPAHGVTSAPDTRPAPGSTAPPAHGVTS (SEQ ID NO: 78) and the glycosylated 30mer with the sequence APPAHGVTSAPDT[GalNAcα]R PAPGSTAPPAHGVTSA (SEQ ID NO: 71) were used as antigens and bound in PBS to the plate. The mIgG-Panko1 and mIgG-Panko2 antibodies were employed at a concentration of 0.5 μg/ml in the ELISA.

(5) FIG. 5: Comparison of specific binding of the anti-MUC1 antibodies HMFG-1, C595 and SM3 with mIgG-Panko1 and mIgG-Panko2 to glycosylated and non-glycosylated MUC1 peptide in an ELISA. Comparison of specific binding of the anti-MUC1 antibodies HMFG-1, C595 and SM3 with mIgG-Panko1 and mIgG-Panko2 to glycosylated and non-glycosylated MUC1 peptide in an ELISA. The non-glycosylated 30mer with the sequence APPAHGVTSAPDTRPAPGSTAPPAHGVTS (SEQ ID NO: 78) and the glycosylated 30mer with the sequence APPAHGVTSAPDT[GalNAcα]RPAPGSTAPPAHGVTSA(SEQ ID NO: 71) were used as antigens and dried slightly on the plate in H.sub.2O. The antibodies were employed at a concentration of 10 μg/ml in the ELISA.

(6) FIG. 6: Specific binding of various preferred formats of recognition molecules of the invention in an ELISA, exemplified using non-glycosylated and glycosylated 30mer MUC1 peptide. Specific binding of various preferred formats of recognition molecules of the invention in an ELISA, exemplified using non-glycosylated and glycosylated 30mer MUC1 peptide. The non-glycosylated 30mer with the sequence APPAHGVTSAPDTRPAPGSTAPPAH GVTS (SEQ ID NO: 78) and the glycosylated 30mer with the sequence APPAHGVTSAPDT [GalNAcα]RPAPGSTAPPAHGVTSA (SEQ ID NO: 71) were used as antigens and bound in PBS to the plate. The two scFv formats SEQ ID Nos. 36 and 45 were used with 0.5 μg/ml, the murine IgG with 0.1 μg/ml and the chimeric IgG with 0.01 μg/ml. As different secondary antibodies are used for these various formats, the ELISA data should be assessed merely qualitatively.

(7) FIG. 7: Dependence of binding of the recognition molecules mIgG-Panko1 and mIgG-Panko2 of the invention on the number of tandem repeats in non-glycosylated MUC1 peptides compared to the MUC1-specific antibodies SM3 and C595 in an ELISA. Dependence of binding of the recognition molecules mIgG-Panko1 and mIgG-Panko2 of the invention on the number of tandem repeats in non-glycosylated MUC1 peptides compared to the MUC1-specific antibodies SM3 and C595 in an ELISA. A series of non-glycosylated MUC1 peptides of varying length with the sequence [VTSAPDTRPAPGSTAPPAHG].sub.n wherein n=1 (SEQ ID NO: 72), 3 (SEQ ID NO: 73) and 5 (SEQ ID NO: 74) (TR1, TR3 and TR5), was used as antigens and dried slightly on the plate in H.sub.2O. The antibodies were employed at a concentration of 10 μg/ml.

(8) FIG. 8: Dependence of binding of the recognition molecules mIgG-Panko1 and mIgG-Panko2 of the invention on the number of tandem repeats (multiple glycosylated PDTR regions) compared to the MUC1-specific antibodies SM3 and C595 in an ELISA. A series of glycosylated MUC1 peptides of varying length with the sequence A[HGVTSAPDT(GalNAcα)RPAPGSTAPPA].sub.n, wherein n=1 (SEQ ID NO: 75), 3 (SEQ ID NO: 76) and 5 (SEQ ID NO: 77) (TR1, TR3 and TR5), was used as antigens and dried slightly on the plate in H.sub.2O. The antibodies were employed at a concentration of 10 μg/ml.

(9) FIG. 9: Fluorescence labelling of cells of the tumor cell line T47D (mammary carcinoma) with the MUC1-specific recognition molecule mIgG-Panko2.

(10) FIG. 10: Fluorescence labelling of cells of the tumor cell line K562 (erythroid leukemia) with the MUC1-specific recognition molecule cIgG-Panko1.

(11) FIG. 11A-B: Scatchard diagram for the analysis of cell binding of radiolabelled MUC1-specific recognition molecules. Binding data of the two scFv formats SEQ ID NO. 36 (a) and SEQ ID NO. 45 (b) are exemplified. r: bound molecules per cell, A: employed concentration of radiolabelled recognition molecule [M], x: percentage bound to cells [M]. The difference A−x represents the concentration of free recognition molecules in the batch. The corresponding straight-line equation is given at the top, the slope of the straight-line representing the association constant.

(12) FIG. 12: Specific accumulation of the radiolabelled recognition molecule mIgG-Panko2 in a tumor in a mouse xenotransplant model. Each tumor-bearing mouse (n=5 per point in time) was administered i.v. with 5 μg of .sup.111In-labelled mIgG-Panko2. The mice were sacrificed after the time as indicated, and accumulation in the tumor, relative to injected dose and tumor weight (% ID/g), was determined.

(13) FIG. 13: High specific accumulation of the radiolabelled recognition molecule mIgG-Panko2 in a tumor in a mouse-ZR-75-1 tumor cell model. Each tumor-bearing mouse (n=6 per point in time) was administered i.v. with 5 μg of .sup.111In-labelled mIgG-Panko2. The mice were sacrificed after the time as indicated, and accumulation in the tumor, in serum and organs, relative to injected dose and tumor or organ weight (% ID/g), was determined.

(14) FIG. 14A-D: Inhibition of tumor growth in tumor-bearing mice after treatment with the radiolabelled recognition molecule mIgG-Panko2. On day 8 (14a; small tumors: <0.05 cm.sup.3), on day 36 (14b; medium-sized tumors: about 0.3 cm.sup.3) and on day 57 (14c; large tumors: >0.5 cm.sup.3) after subcutaneous injection of the ZR-75-1 cells, the tumor-bearing mice were given 200 μl into the tail vein. The injection solution contained the .sup.90Y-labelled mIgG-Panko2 (100 μCi per dose; specific activity: 3 mCi/mg antibody) in Ca/Mg-PBS with 4% fetal calf serum to protect from radiolysis. Control groups received the same injection with no radioactively labelled recognition molecule. In the event of small and medium-sized tumors, a second injection (50 μCi) was effected about 3 weeks later.

(15) FIG. 14d shows the treatment of tumor-bearing mice with .sup.90Y-mIgG-Panko2 compared to an irrelevant radiolabelled control antibody .sup.90Y-MOPC21.

(16) FIG. 15A-B: Specific mediation of antibody-dependent cellular cytotoxicity by the recognition molecules of the invention, Panko1 and Panko2. The antibodies were used at a concentration of 0.2 to 25 μg/ml. (a) The cIgG formats of Panko1 and Panko2 show high specific lysis of the MUC1-positive ZR-75-1 cells. (b) The mIgG Panko2 also mediates specific tumor cell lysis and shows a significantly greater effect than the murine anti-MUC1 antibody HMFG-1, with slight lysis being observed only at the highest tested concentration (25 μg/ml) for the latter.

(17) FIG. 16: Analysis of binding of recognition molecules of the invention to human blood cells. Human peripheral blood cells were obtained from the blood of healthy donors by means of density centrifugation. To stimulate human T cells, the isolated blood cells were incubated in RPMI/FCS+1 μg/ml PHA+60 U/ml hIL-2 in an incubator for 3 to 8 days. The isolated blood cells were incubated with mIgG-Panko1, mIgG-Panko2, HMFG1, DF3 or mIgG1 (control) and stained with Cy3-conjugated goat anti-mouse Ig (Dianova) and/or with a CD marker-specific (CD3, CD14, CD19), FITC-labelled antibody. Following washing, the cells were analyzed using flow cytometry.

(18) The recognition molecules Panko1 and Panko2 show no or only low binding to human blood cells. In contrast, the MUC1-specific antibodies HMFG-1 and DF3 bind strongly to PHA-stimulated T cells, and HMFG-1 also binds to B cells and monocytes.

REFERENCES

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DESCRIPTION OF SEQUENCES

(20) TABLE-US-00011 CDR sequences SEQ ID NO. 1  DAWMD SEQ ID NO. 2  NYWMN SEQ ID NO. 3  EIRSKANNHATYYAESVKG SEQ ID NO. 4  EIRLKSNNYTTHYAESVKG SEQ ID NO. 5  GGYGFDY SEQ ID NO. 6  HYYFDY SEQ ID NO. 7  RSSQSIVHSNGNTYLE SEQ ID NO. 8  RSSKSLLHSNGITYFF SEQ ID NO. 9  KVSNRFS SEQ ID NO. 10 QMSNLAS SEQ ID NO. 11 FQGSHVPLT SEQ ID NO. 12 AQNLELPPT CDR sequences (canonical structure variants) SEQ ID NO. 13 NYWVN SEQ ID NO. 14 NYWIN SEQ ID NO. 15 NYWYN SEQ ID NO. 16 NYWWN SEQ ID NO. 17 DAWID SEQ ID NO. 18 DAWVD SEQ ID NO. 19 DAWYD SEQ ID NO. 20 DAWWD SEQ ID NO. 21 EIRSKANNYATYYAESVKG SEQ ID NO. 22 EIRLKSNKYTTHYAESVKG SEQ ID NO. 23 EIRLKSNSYTTHYAESVKG SEQ ID NO. 24 RPSQSIVHSNGNTYLE SEQ ID NO. 25 RSSQSIVHSNGNTYFE SEQ ID NO. 26 RPSQSIVHSNGNTYFE SEQ ID NO. 27 RPSKSLLHSNGITYFF SEQ ID NO. 28 RSSKSLLHSNGITYLF SEQ ID NO. 29 RPSKSLLHSNGITYLF SEQ ID NO. 30 FQGSHPPLT SEQ ID NO. 31 AQNLEPPPT variable heavy chains SEQ ID NO. 32 EVKLVESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE IRSKANNHATYYAESVKGRFTISRDVSKSSVYLQMNNLRAEDTGIYYCTR GGYGFDYWGQGTTLTVSS SEQ ID NO. 33 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE IRLKSNNYTTHYAESVKGRFTISRDDSKSSVSLQMNNLRVEDTGIYYCTR HYYFDYWGQGTTLTVSS variable light chains SEQ ID NO. 34 DIVLTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPK LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP LTFGDGTKLELKRA SEQ ID NO. 35 DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYFFWYLQKPGLSPQ LLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELP PTFGGGTKLEIKRA V.sub.H/V.sub.L pairs Panko1 SEQ ID NO. 32 SEQ ID NO. 34 Panko2 SEQ ID NO. 33 SEQ ID NO. 35 various single chain Fv formats SEQ ID NO. 36 EVKLVESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE IRSKANNHATYYAESVKGRFTISRDVSKSSVYLQMNNLRAEDTGIYYCTR GGYGFDYWGQGTTLTVSSASSGGGGSGGGGSGGSARDIVLTQTPLSLPVS LGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVP DRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGDGTKLELKRA AAHHHHHHGAAEQKLISEEDLNGAA SEQ ID NO. 37 EVKLVESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE IRSKANNHATYYAESVKGRFTISRDVSKSSVYLQMNNLRAEDTGIYYCTR GGYGFDYWGQGTTLTVSSASSGSGSSADIVLTQTPLSLPVSLGDQASISC RSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSG TDFTLKISRVEAEDLGVYYCFQGSHVPLTFGDGTKLELKRAAAHHHHHHG AAEQKLISEEDLNGAA SEQ ID NO. 38 EVKLVESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE IRSKANNHATYYAESVKGRFTISRDVSKSSVYLQMNNLRAEDTGIYYCTR GGYGFDYWGQGTTLTVSSASSGGSSADIVLTQTPLSLPVSLGDQASISCR SSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGT DFTLKISRVEAEDLGVYYCFQGSHVPLTFGDGTKLELKRAAAHHHHHHGA AEQKLISEEDLNGAA SEQ ID NO. 39 EVKLVESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE IRSKANNHATYYAESVKGRFTISRDVSKSSVYLQMNNLRAEDTGIYYCTR GGYGFDYWGQGTTLTVSSASSGSSADIVLTQTPLSLPVSLGDQASISCRS SQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTD FTLKISRVEAEDLGVYYCFQGSHVPLTFGDGTKLELKRAAAHHHHHHGAA EQKLISEEDLNGAA SEQ ID NO. 40 EVKLVESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE IRSKANNHATYYAESVKGRFTISRDVSKSSVYLQMNNLRAEDTGIYYCTR GGYGFDYWGQGTTLTVSSASSSSADIVLTQTPLSLPVSLGDQASISCRSS QSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDF TLKISRVEAEDLGVYYCFQGSHVPLTFGDGTKLELKRAAAHHHHHHGAAE QKLISEEDLNGAA SEQ ID NO. 41 EVKLVESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE IRSKANNHATYYAESVKGRFTISRDVSKSSVYLQMNNLRAEDTGIYYCTR GGYGFDYWGQGTTLTVSSASSSADIVLTQTPLSLPVSLGDQASISCRSSQ SIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFT LKISRVEAEDLGVYYCFQGSHVPLTFGDGTKLELKRAAAHHHHHHGAAEQ KLISEEDLNGAA SEQ ID NO. 42 EVKLVESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE IRSKANNHATYYAESVKGRFTISRDVSKSSVYLQMNNLRAEDTGIYYCTR GGYGFDYWGQGTTLTVSSASSADIVLTQTPLSLPVSLGDQASISCRSSQS IVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTL KISRVEAEDLGVYYCFQGSHVPLTFGDGTKLELKRAAAHHHHHHGAAEQK LISEEDLNGAA SEQ ID NO. 43 EVKLVESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE IRSKANNHATYYAESVKGRFTISRDVSKSSVYLQMNNLRAEDTGIYYCTR GGYGFDYWGQGTTLTVSSASADIVLTQTPLSLPVSLGDQASISCRSSQSI VHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLK ISRVEAEDLGVYYCFQGSHVPLTFGDGTKLELKRAAAHHHHHHGAAEQKL ISEEDLNGAA SEQ ID NO. 44 EVKLVESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE IRSKANNHATYYAESVKGRFTISRDVSKSSVYLQMNNLRAEDTGIYYCTR GGYGFDYWGQGTTLTVSSAADIVLTQTPLSLPVSLGDQASISCRSSQSIV HSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQGSHVPLTFGDGTKLELKRAAAHHHHHHGAAEQKLI SEEDLNGAA SEQ ID NO. 45 EVKLVESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE IRSKANNHATYYAESVKGRFTISRDVSKSSVYLQMNNLRAEDTGIYYCTR GGYGFDYWGQGTTLTVSSADIVLTQTPLSLPVSLGDQASISCRSSQSIVH SNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIS RVEAEDLGVYYCFQGSHVPLTFGDGTKLELKRAAAHHHHHHGAAEQKLIS EEVHQ SEQ ID NO. 46 EVKLVESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE IRSKANNHATYYAESVKGRFTISRDVSKSSVYLQMNNLRAEDTGIYYCTR GGYGFDYWGQGTTLTVSSDIVLTQTPLSLPVSLGDQASISCRSSQSIVHS NGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR VEAEDLGVYYCFQGSHVPLTFGDGTKLELKRAAAHHHHHHGAAEQKLISE EDLNGAA SEQ ID NO. 47 EVKLVESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE IRSKANNHATYYAESVKGRFTISRDVSKSSVYLQMNNLRAEDTGIYYCTR GGYGFDYWGQGTTLTVSDIVLTQTPLSLPVSLGDQASISCRSSQSIVHSN GNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRV EAEDLGVYYCFQGSHVPLTFGDGTKLELKRAAAHHHHHHGAAEQKLISEE DLNGAA SEQ ID NO. 48 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE IRLKSNNYTTHYAESVKGRFTISRDDSKSSVSLQMNNLRVEDTGIYYCTR HYYFDYWGQGTTLTVSSASSGGGGSGGGGSGGSARDIVMTQAAFSNPVTL GTSASISCRSSKSLLHSNGITYFFWYLQKPGLSPQLLIYQMSNLASGVPD RFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPPTFGGGTKLEIKRAA AHHHHHHGAAEQKLISEEDLNGAA SEQ ID NO. 49 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE IRLKSNNYTTHYAESVKGRFTISRDDSKSSVSLQMNNLRVEDTGIYYCTR HYYFDYWGQGTTLTVSSASSGSGSSADIVMTQAAFSNPVTLGTSASISCR SSKSLLHSNGITYFFWYLQKPGLSPQLLIYQMSNLASGVPDRFSSSGSGT DFTLRISRVEAEDVGVYYCAQNLELPPTFGGGTKLEIKRAAAHHHHHHGA AEQKLISEEDLNGAA SEQ ID NO. 50 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE IRLKSNNYTTHYAESVKGRFTISRDDSKSSVSLQMNNLRVEDTGIYYCTR HYYFDYWGQGTTLTVSSASSGGSSADIVMTQAAFSNPVTLGTSASISCRS SKSLLHSNGITYFFWYLQKPGLSPQLLIYQMSNLASGVPDRFSSSGSGTD FTLRISRVEAEDVGVYYCAQNLELPPTFGGGTKLEIKRAAAHHHHHHGAA EQKLISEEDLNGAA SEQ ID NO. 51 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE IRLKSNNYTTHYAESVKGRFTISRDDSKSSVSLQMNNLRVEDTGIYYCTR HYYFDYWGQGTTLTVSSASSGSSADIVMTQAAFSNPVTLGTSASISCRSS KSLLHSNGITYFFWYLQKPGLSPQLLIYQMSNLASGVPDRFSSSGSGTDF TLRISRVEAEDVGVYYCAQNLELPPTFGGGTKLEIKRAAAHHHHHHGAAE QKLISEEDLNGAA SEQ ID NO. 52 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE IRLKSNNYTTHYAESVKGRFTISRDDSKSSVSLQMNNLRVEDTGIYYCTR HYYFDYWGQGTTLTVSSASSSSADIVMTQAAFSNPVTLGTSASISCRSSK SLLHSNGITYFFWYLQKPGLSPQLLIYQMSNLASGVPDRFSSSGSGTDFT LRISRVEAEDVGVYYCAQNLELPPTFGGGTKLEIKRAAAHHHHHHGAAEQ KLISEEDLNGAA SEQ ID NO. 53 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE IRLKSNNYTTHYAESVKGRFTISRDDSKSSVSLQMNNLRVEDTGIYYCTR HYYFDYWGQGTTLTVSSASSSADIVMTQAAFSNPVTLGTSASISCRSSKS LLHSNGITYFFWYLQKPGLSPQLLIYQMSNLASGVPDRFSSSGSGTDFTL RISRVEAEDVGVYYCAQNLELPPTFGGGTKLEIKRAAAHHHHHHGAAEQK LISEEDLNGAA SEQ ID NO. 54 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE IRLKSNNYTTHYAESVKGRFTISRDDSKSSVSLQMNNLRVEDTGIYYCTR HYYFDYWGQGTTLTVSSASSADIVMTQAAFSNPVTLGTSASISCRSSKSL LHSNGITYFFWYLQKPGLSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLR ISRVEAEDVGVYYCAQNLELPPTFGGGTKLEIKRAAAHHHHHHGAAEQKL ISEEDLNGAA SEQ ID NO. 55 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE IRLKSNNYTTHYAESVKGRFTISRDDSKSSVSLQMNNLRVEDTGIYYCTR HYYFDYWGQGTTLTVSSASADIVMTQAAFSNPVTLGTSASISCRSSKSLL HSNGITYFFWYLQKPGLSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRI SRVEAEDVGVYYCAQNLELPPTFGGGTKLEIKRAAAHHHHHHGAAEQKLI SEEDLNGAA SEQ ID NO. 56 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE IRLKSNNYTTHYAESVKGRFTISRDDSKSSVSLQMNNLRVEDTGIYYCTR HYYFDYWGQGTTLTVSSAADIVMTQAAFSNPVTLGTSASISCRSSKSLLH SNGITYFFWYLQKPGLSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRIS RVEAEDVGVYYCAQNLELPPTFGGGTKLEIKRAAAHHHHHHGAAEQKLIS EEDLNGAA SEQ ID NO. 57 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE IRLKSNNYTTHYAESVKGRFTISRDDSKSSVSLQMNNLRVEDTGIYYCTR HYYFDYWGQGTTLTVSSADIVMTQAAFSNPVTLGTSASISCRSSKSLLHS NGITYFFWYLQKPGLSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISR VEAEDVGVYYCAQNLELPPTFGGGTKLEIKRAAAHHHHHHGAAEQKLISE EDLNGAA SEQ ID NO. 58 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE IRLKSNNYTTHYAESVKGRFTISRDDSKSSVSLQMNNLRVEDTGIYYCTR HYYFDYWGQGTTLTVSSDIVMTQAAFSNPVTLGTSASISCRSSKSLLHSN GITYFFWYLQKPGLSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRV EAEDVGVYYCAQNLELPPTFGGGTKLEIKRAAAHHHHHHGAAEQKLISEE DLNGAA SEQ ID NO. 59 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE IRLKSNNYTTHYAESVKGRFTISRDDSKSSVSLQMNNLRVEDTGIYYCTR HYYFDYWGQGTTLTVSDIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNG ITYFFWYLQKPGLSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVE AEDVGVYYCAQNLELPPTFGGGTKLEIKRAAAHHHHHHGAAEQKLISEED LNGAA Murine Antibodies light chain of a murine IgG1 (kappa chain) SEQ ID NO. 60 DIVLTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPK LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP LTFGDGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDIN VKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCE ATHKTSTSPIVKSFNRNEC SEQ ID NO. 61 DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYFFWYLQKPGLSPQ LLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELP PTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDIN VKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCE ATHKTSTSPIVKSFNRNEC heavy chain of murine IgG1 SEQ ID NO. 62 EVKLVESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE IRSKANNHATYYAESVKGRFTISRDVSKSSVYLQMNNLRAEDTGIYYCTR GGYGFDYWGQGTTLTVSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF PEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRPSETVTCN VAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLT PKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSEL PIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKE QMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFV YSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO. 63 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE IRLKSNNYTTHYAESVKGRFTISRDDSKSSVSLQMNNLRVEDTGIYYCTR HYYFDYWGQGTTLTVSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFP EPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRPSETVTCNV AHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTP KVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELP IMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQ MAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVY SKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK murine antibodies mIgG-Panko1  SEQ ID NO. 60 SEQ ID NO. 62 mIgG-Panko2  SEQ ID NO. 61 SEQ ID NO. 63 Chimeric antibodies (mouse/human) heavy chain of chimeric IgG1 (gamma chain) SEQ ID NO. 64 EVKLVESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE IRSKANNHATYYAESVKGRFTISRDVSKSSVYLQMNNLRAEDTGIYYCTR GGYGFDYWGQGTTLTVSGSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 65 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE IRLKSNNYTTHYAESVKGRFTISRDDSKSSVSLQMNNLRVEDTGIYYCTR HYYFDYWGQGTTLTVSGSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK heavy chain of a chimeric IgM (μ chain) SEQ ID NO. 66 EVKLVESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE IRSKANNHATYYAESVKGRFTISRDVSKSSVYLQMNNLRAEDTGIYYCTR GGYGFDYWGQGTTLTVSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDF LPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTD EHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLI CQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLT IKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFAS IFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFS AVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYL LPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPM PEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKST GKPTLYNVSLVMSDTAGTCY SEQ ID NO. 67 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE IRLKSNNYTTHYAESVKGRFTISRDDSKSSVSLQMNNLRVEDTGIYYCTR HYYFDYWGQGTTLTVSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFL PDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDE HVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLIC QATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTI KESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASI FLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSA VGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLL PPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMP EPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTG KPTLYNVSLVMSDTAGTCY light chain of a chimeric IgG1 or IgM (kappa chain) SEQ ID NO. 68 DIVLTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPK LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP LTFGDGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC SEQ ID NO. 69 DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYFFWYLQKPGLSPQ LLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELP PTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC chimeric antibodies cIgG-Panko1 SEQ ID NO. 64 SEQ ID NO. 68 cIgG-Panko2 SEQ ID NO. 65 SEQ ID NO. 69 cIgM-Panko1 SEQ ID NO. 66 SEQ ID NO. 68 cIgM-Panko1 SEQ ID NO. 67 SEQ ID NO. 69