Sialyl-di-Lewis.SUP.a .as expressed on glycoproteins but not glycolipids as a functional cancer target and antibodies thereto

Abstract

The present invention relates to an isolated specific binding member capable of binding sialyl-di-Lewis.sup.a, and associated treatments and pharmaceutical compositions for treatment of cancer.

Claims

1. An isolated antibody or antibody fragment specific for sialyl-di-Lewis.sup.a, sialyl-Lewis.sup.a-x, and mono-sialyl-Lewis.sup.a bound to a glycoprotein and which does not bind to mono-sialyl-Lewis.sup.a bound to a glycolipid, wherein the antibody or antibody fragment comprises the following six CDRs: a) QSLLNSGNQKNY (Light chain CDR1) (SEQ ID NO: 5), WAS (Light chain CDR2), and QNDYSSPFT (Light chain CDR3) (SEQ ID NO: 6); and b) GFTFNTYA (Heavy chain CDR1) (SEQ ID NO: 1), IRSKSNNYAT (Heavy chain CDR2) (SEQ ID NO: 2), and VGYGSGGNY (Heavy chain CDR3) (SEQ ID NO: 3).

2. The antibody or antibody fragment according to claim claim 1, wherein the mono-sialyl-Lewis.sup.a is linked to the glycoprotein by a glycan chain comprising at least 4 glycan monomer units.

3. The antibody or antibody fragment according to claim 1, wherein the antibody has: (i) a heavy chain amino acid sequence of: MLLGLKWVFFVVFYQGVHCEVQLVESGGGLVQPKGSLKLSCAASGFTFNTY AMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNN LKKEDTAMYYCVGYGSGGNYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTL GCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRPSETVTCNV AHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDIS KDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVN SAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQW NGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEK SLSHSPGK (SEQ ID NO: 12); and a light chain amino acid sequence of: MESQTQVLMSLLFWVSTCGDIVMTQSPSSLTVTAGEKVTMSCKSSQSLL NSGNQKNYLTWYQQKPGQPPKVLIYWASTRESGVPDRFTGSGSGTDFTLT ISSVQAEDLAVYYCQNDYSSPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGG ASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEY ERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 13); or (ii) a heavy chain amino acid sequence of: MLLGLKWVFFVVFYQGVHCEVQLVESGGGLVQPKGSLKLSCAASGFTF NTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSM LYLQMNNLKKEDTAMYYCVGYGSGGNYWGQGTSVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14); and a light chain amino acid sequence of: MESQTQVLMSLLFWVSTCGDIVMTQSPSSLTVTAGEKVTMSCKSSQSLL NSGNQKNYLTWYQQKPGQPPKVLIYWASTRESGVPDRFTGSGSGTDFTLT ISSVQAEDLAVYYCQNDYSSPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 15).

4. The antibody or antibody fragment according to claim 1, wherein the antibody or antibody fragment is bispecific.

5. The antibody or antibody fragment according to claim 4, wherein the bispecific antibody or antibody fragment is additionally specific for CD3.

6. The antibody or antibody fragment according to claim 1, wherein the antibody or antibody fragment comprises: a light chain variable sequence comprising QSLLNSGNQKNY (Light chain CDR1) (SEQ ID NO: 5), WAS (Light chain CDR2), and QNDYSSPFT (Light chain CDR3) (SEQ ID NO: 6); and a heavy chain variable sequence comprising GFTFNTYA (Heavy chain CDR1) (SEQ ID NO: 1), IRSKSNNYAT (Heavy chain CDR2) (SEQ ID NO: 2), and VGYGSGGNY (Heavy chain CDR3) (SEQ ID NO: 3).

7. The antibody or antibody fragment according to claim 6, wherein the CDRs are carried by a human antibody framework.

8. The antibody or antibody fragment according to claim 6, wherein the antibody or antibody fragment comprises a VH domain comprising residues 1 to 117 (SEQ ID NO: 4) of the amino acid sequence of FIG. 1a or 2a, and/or a VL domain comprising residues 1 to 114 (SEQ ID NO: 7) of the amino acid sequence of FIG. 1b or 2b.

9. The antibody or antibody fragment according to claim 1, wherein the antibody or antibody fragment comprises a human antibody constant region.

10. The antibody or antibody fragment according to claim 1, wherein the antibody fragment is a Fab, (Fab)2, scFv, Fv, Fd or a diabody or wherein the antibody or antibody fragment is provided in the form of a chimeric antigen receptor (CAR).

11. The antibody or antibody fragment according to claim 10, wherein the antibody or antibody fragment is an scFv: (a) comprising in the following order: 1) leader sequence, 2) heavy chain variable region, 3) 3GGGGS (SEQ ID NO: 18) spacer, 4) light chain variable region, and 5) poly-Ala and a 6His tag for purification; (b) comprising in the following order: 1) leader sequence, 2) light chain variable region, 3) 3GGGGS (SEQ ID NO: 18) spacer, and 4) heavy chain variable region, optionally further comprising either 5 or 3 purification tags, in the listed order; and/or (c) in the form of a chimeric antigen receptor (CAR) either in the heavy chain-light chain orientation or the light chain-heavy chain orientation.

12. The antibody or antibody fragment according to claim 1, wherein the antibody or antibody fragment is: (a) a monoclonal antibody; (b) a humanised, chimeric or veneered antibody; and/or (c) a drug conjugate, such as an antibody-drug conjugate (ADC).

13. A pharmaceutical composition comprising the antibody or antibody fragment according to claim 1, and a pharmaceutically acceptable carrier.

14. The pharmaceutical composition according to claim 13, further comprising at least one other active ingredient.

15. A nucleic acid comprising a sequence encoding the antibody or antibody fragment according to claim 1.

16. The nucleic acid according to claim 15, wherein the nucleic acid is a construct in the form of a plasmid, vector, transcription or expression cassette.

17. A recombinant host cell which comprises the nucleic acid according to claim 15.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

Figure Legends

(1) FIG. 1a: Amino acid and nucleotide sequence for the mouse IgG1 heavy chain of the FG129 mAb (SEQ ID NO: 1). Numbers refer to the standardised IMGT system for the numbering of antibody sequences [59]. FIG. 1b: Amino acid and nucleotide sequence for the mouse kappa chain of the FG129 mAb. Numbers refer to the standardised IMGT system for the numbering of antibody sequences [59].

(2) FIG. 2: The chimeric version of the FG129 mAb (original murine variable regions linked to human constant region sequence), produced by a transfected cell line, binds the target cell line (HCT-15). FIG. 2a: Amino acid and nucleotide sequence for the human IgG1 heavy chain of the FG129 mAb. Numbers refer to the standardised IMGT system for the numbering of antibody sequences [59]. FIG. 2b: Amino acid and nucleotide sequence for the human kappa chain of the FG129 mAb. Numbers refer to the standardised IMGT system for the numbering of antibody sequences [59].

(3) FIG. 3a: ELISA screening of FG129 to over 600 glycans arrayed on a glass slide by the CFG. Square represents glucosylamine, circle represents galactose, triangle represents fucose and diamond represents sialic acid.

(4) FIG. 3b: Indirect Western blot analysis of the antigens recognised by mAb FG129 and mAb ch 129 (1 g/ml). Lane M: molecular marker (in red); Lane 1: Colo205 cell lysates (110.sup.5 cells); Lane 2: Colo205 TGL (110.sup.6 cells); Lane 3: HCT-15 cell lysates (110.sup.5 cells); Lane 4: HCT-15 TGL (110.sup.6 cells); Lane 5: BxPc3 cell lysates (110.sup.5 cells); Lane 6: BxPc3 TGL (110.sup.6 cells); Lane 7: LS180 cell lysates (110.sup.5 cells); Lane 8: LS180 TGL (110.sup.6 cells). Negative control consisted of omission of primary antibody. CA19.9 was used as positive control recognising sialyl-Lewis.sup.a on glycolipids as well as glycoproteins.

(5) FIG. 4: ELISA analysis of FG129 and CH129 binding to sialyl-Lewis.sup.a-HSA. CA19.9 was used as positive control recognising sialyl-Lewis.sup.a on glycolipids as well as glycoproteins. Negative controls consisted of an isotype antibody that does not recognise sialyl-Lewis.sup.a, HSA coated wells, uncoated wells where the antigen was omitted, and wells where FG129 was omitted. Error bars represent the meanSD of duplicate wells.

(6) FIG. 5a: Binding of FG129 (1 g/ml) by IHC to colorectal, pancreatic, gastric, ovarian and lung TMAs. Representative images of different staining levels are shown i) negative, ii) weak, iii) moderate and iv) strong (magnification 20).

(7) FIG. 5b: Kaplan-Meier analysis of disease-free survival of pancreatic patients staining with FG129 mAb. Cut-off for high versus low was determined by X-tile.

(8) FIG. 5c: Normal human tissue (AMSBIO) binding of FG129, showing very limited binding in 1) Gallbladder; 2) Ileum; 3) Liver; 4) Oesophagus; 5) Pancreas; 6)Thyroid (magnification 20).

(9) FIG. 6a: Indirect immunofluorescence staining and flow cytometric analysis of FG129 and CH129 (5 g/ml) mAb binding to the cell surface of tumour cell lines.

(10) FIG. 6b: Indirect immunofluorescence staining and flow cytometric analysis of FG129 (5 g/ml) mAb binding to the cell surface of HUVEC normal umbilical cells. An anti-CD55 mAb was used as a positive control and an anti-IgG isotype antibody as a negative control.

(11) FIG. 6c: Indirect immunofluorescence staining and flow cytometric analysis of FG129 and ch129 (5 g/ml) mAbs binding to whole blood. An anti-HLA mAb w6/32 was used as a positive control and an anti-IgG isotype antibody as a negative control.

(12) FIG. 7: Indirect immunofluorescence staining and flow cytometric analysis of titrations of FG129 mAb and CH129 mAb binding to the cell surface of Colo205 (7a), HCT-15 (7b), BxPc3 (7c) and LS180 (7d) cells.

(13) FIG. 8: ADCC killing of Colo205 (8a) and HCT-15 (8b) by FG129 and CH129. Erbitux was used as positive control, while PBMCs and cells alone were used as negative controls. Anova test performed using GraphPad Prism6 shows the significant difference between each concentration and the negative control consisting of cells with PBMCs only.

(14) FIG. 9: CDC killing of Colo205 by FG129 and CH129. Erbitux was used as positive control, while PBMCs and cells alone were used as negative controls. Anova test performed using GraphPad Prism6 shows the significant difference between each concentration and the negative control consisting of cells with PBMCs only.

(15) FIG. 10: Z-stack confocal microscopy of Alexa Fluor 488 (green) labelled FG129 (panel 10a) and CH129 (panel 10b) internalising in live Colo205, BxPC3 and HCT-15 showing co-localisation with lysosomes. The plasma membrane was labelled with CellMask Orange (red/C), the lysosomes with LysoTracker Deep Red (purple/D) and the nucleus with Hoechst 33258 (blue/A) (magnification 60).

(16) FIG. 11a: Cytotoxicity of Fab-ZAP-FG129 in antigen positive (HCT15, Colo205, BxPC3, ASPC1) and negative (LoVo, LS180) cancer cell lines. The cytotoxicity of internalised FG129 pre-incubated with saporin-linked anti-mouse IgG Fab fragment was evaluated using .sup.3H-thymidine incorporation. Results are presented as percentage of proliferation of cells treated with the primary mAb only. Error bars show the meanSD from four independent experiments.

(17) FIG. 11b: Fab-ZAP-IgG Isotype internalisation assay. Results are presented normalised, as percentage of proliferation of cells treated with the primary mAb only. Error bars show the meanSD from three independent experiments.

(18) FIG. 11c: Cytotoxicity of Fab-ZAP-CH129 Against HCT15, Colo205, BxPC3 cancer Cell lines. The cytotoxicity of internalised CH129 pre-incubated with saporin-linked anti-human IgG Fab fragment was evaluated using .sup.3H-thymidine incorporation. Results are presented normalised, as percentage of proliferation of cells treated with the primary mAb only. Error bars show the meanSD from four independent experiments.

(19) FIG. 11d: Fab-ZAP-IgG Isotype internalisation assay. Results are presented normalised, as percentage of proliferation of cells treated with the primary mAb only. Error bars show the meanSD from three independent experiments.

(20) FIG. 11e: WST8 cytotoxicity assay showing in vitro efficacy of CH129-ADC constructs on Colo205. All three CH129-ADC constructs gave 100% cell killing with the vcE construct giving the highest efficacy (Ec5010.sup.11M) followed by the DM1 and DM4 constructs showing similar efficacy (Ec50s10.sup.10M).

(21) FIG. 11f: WST8 cytotoxicity assay showing in vitro efficacy of CH129-ADC constructs on HCT-15. CH129 constructs show 50-60% cell killing. Rituximab-ADC constructs were used as controls for specific killing. Ritux-vcE and Ritux-DM1 do not show cell killing. Ritux-DM4 shows similar killing activity to the CH129 constructs, indicating non-specific cell killing.

(22) FIG. 11g: WST8 cytotoxicity assay showing bystander killing of the CH129-vcE construct.

(23) FIG. 11h: WST8 cytotoxicity assay showing bystander killing of the CH129-DM4 construct.

(24) FIG. 11i: WST8 cytotoxicity assay showing bystander killing of the CH129-DM1 construct.

(25) FIG. 12a: Sandwich ELISA using FG129 for the detection of secreted sialyl-Lewis.sup.a in sera from pancreatic cancer patients. Negative controls consisted of a normal serum sample from a healthy donor, and 2% BSA-PBS alone. Sialyl-Lewis.sup.a-HSA was used as a positive control.

(26) FIG. 12b: Competition FACS assay showing binding to HCT-15 cell line of pre-incubated FG129 with sera from patients from the pancreatic TMA cohort. Positive controls consisted of normal sera samples from five healthy donors (shown as average between the five), and 2% BSA-PBS pre-incubated with FG129. Negative controls consisted of sialyl-Lewis.sup.a-HSA pre-incubated with FG129 and 2% BSA-PBS alone.

(27) FIG. 13a: Sequence of FG129-scFv, comprised of 1) leader sequence, 2) heavy chain variable region, 3) 3GGGGS spacer, 4) light chain variable region, 5) poly-Ala and 6His tag for purification.

(28) FIG. 13b: ELISA analysis of FG129-scFv and CH129 binding to sialyl-Lewis.sup.a-HSA. Error bars represent the meanSD of duplicate wells.

(29) FIG. 13c: Indirect immunofluorescence staining and flow cytometric analysis of titrations of FG129-scFv binding to the cell surface of Colo205.

DETAILED DESCRIPTION OF THE INVENTION

(30) The invention will now be described further in the following non-limiting examples and accompanying drawings.

(31) Methods

(32) Binding to Tumour Cell Lines:

(33) 110.sup.5 cancer cells were incubated with 50 l of primary antibodies at 4 C. for 1 hr. Cells were washed with 200 l of RPMI 10% new born calf serum (NBCS: Sigma, Poole, UK) and spun at 1,000 rpm for 5 min. Supernatant was discarded and 50 l of FITC conjugated anti-mouse IgG Fc specific mab (Sigma; 1/100 in RPMI 10% NBCS) was used as secondary antibody. Cells were incubated at 4 C. in dark for 1 hr then washed with 200 l RPMI 10% NBCS and spun at 1,000 rpm for 5 min. After discarding supernatant, 0.4% formaldehyde was used to fix the cells. Samples were analysed on a Beckman coulter FC-500 flow cytometer (Beckman Coulter, High Wycombe, UK). To analyse and plot raw data, WinMDI 2.9 software was used. Cellular antibody binding sites for FG129 (used at 30 g/ml) were calculated using the QIFIKIT (Dako UK Ltd) according to the manufacturer's recommendations. Specific antibody binding capacity (SABC) was obtained by subtracting the non-specific binding of an isotype control.

(34) Binding to Blood:

(35) 50 l of healthy donor blood was incubated with 50 l primary antibody at 4 C. for 1 hr. The blood was washed with 150 l of RPMI 10% NBCS and spun at 1,000 rpm for 5 min. Supernatant was discarded and 50 l FITC conjugated anti-mouse IgG Fc specific mAb (1/100 in RPMI 10% NBCS) was used as the secondary antibody. Cells were incubated at 4 C. in the dark for 1 hr then washed with 150 l RPMI 10% NBCS and spun at 1,000 rpm for 5 min. After discarding the supernatant, 50 l/well Cal-Lyse (Invitrogen, Paisley, UK) was used followed by 500 l/well distilled water to lyse red blood cells. The blood was subsequently spun at 1,000 rpm for 5 min. Supernatant was discarded and 0.4% formaldehyde was used to fix the cells. Samples were analysed on a FC-500 flow cytometer (Beckman Coulter). To analyse and plot raw data, WinMDI 2.9 software was used.

(36) Plasma Membrane Glycolipid Extraction:

(37) Colo205 cell pellet (510.sup.7 cells) was resuspended in 500 l of Mannitol/HEPES buffer (50 mM Mannitol, 5 mM HEPES, pH7.2, both Sigma) and passed through 3 needles (23G, 25G, 27G) each with 30 pulses. 5 l of 1M CaCl.sub.2 was added to the cells and passed through 3 needles each with 30 pulses as above. Sheared cells were incubated on ice for 20 min then spun at 3,000 g for 15 min at room temperature. Supernatant was collected and spun at 48,000 g for 30 min at 4 C. and the supernatant was discarded. The pellet was resuspended in 1 ml methanol followed by 1 ml chloroform and incubated with rolling for 30 min at room temperature. The sample was then spun at 1,200 g for 10 min to remove precipitated protein. The supernatant, containing plasma membrane glycolipids, was collected and stored at 20 C.

(38) Glycome Analysis:

(39) To clarify the fine specificities of the FG129 mAbs further, the antibodies were sent to the Consortium for Functional Glycomics where they were screened against 600 natural and synthetic glycans. Briefly, synthetic and mammalian glycans with amino linkers were printed onto N-hydroxysuccinimide (NHS)-activated glass microscope slides, forming amide linkages. Printed slides were incubated with 1 g/ml of antibody for 1 hr before the binding was detected with Alexa488-conjugated goat anti-mouse IgG. Slides were then dried, scanned and the screening data compared to the Consortium for Functional Glycomics database.

(40) Affinity Analysis

(41) Surface Plasmon Resonance (SPR, Biacore X or 3000, GE Healthcare) analysis was used to investigate real-time binding kinetics of the FG129 mAbs. Polyvalent sialyl Le.sup.a-HSA (Isosep AB, Tullinge, Sweden) was coupled onto a CM5 biosensor chip according to the manufacturer's instructions and a reference cell was treated in a similar manner, but omitting the sialyl Le.sup.a conjugate. FG129, CH129 and scFv129 mAbs diluted in HBS-P buffer (10 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 0.005% (v/v) surfactant P20) were run across the chip at a flow rate of 50 l/min and BIAevaluation software 4.1 was used to determine the kinetic binding parameters from which affinities are calculated.

(42) Lewis Antigen and Saliva Sandwich ELISA

(43) ELISA plates were coated overnight at 4 C. with 100 ng/well Lewis-HSA antigens (Isosep), blocked with PBS/BSA and incubated with primary mAbs (direct ELISA). Antibody or Lewis antigen binding was detected using biotinylated secondary mAb (Sigma). Plates were read at 450 nm by Tecan Infinite F50 after incubation with Streptavidin Horseradish Peroxidase (HRPO) conjugate (Invitrogen).

(44) SDS-PAGE and Western Blot Analysis:

(45) Briefly, 110.sup.5 or 10.sup.6 cell equivalents of Colo205 cell lysate, plasma membrane, total lipid extract, plasma membrane lipid extract or HCT-15 cell lysates were analysed for FG129 binding. Tumour cell total and plasma membrane lipid extracts and cell lysates were reduced with dithiothreitol (DTT; Pierce Biotechnology, ThermoFisher, Loughborough, UK) and subjected to SDS-PAGE using NOVEX 4% to 12% Bis-Tris gels (Invitrogen), and transferred to Immobilon-FL PVDF membrane (Merck Millipore, Watford, UK) using 1 transfer buffer (20, Invitrogen) and 20% (v/v) methanol at 30V for 1 hr. Membranes were blocked with 5% (w/v) non-fat dry milk in 0.05% (v/v) Tween-PBS for 1 hr then probed with primary antibodies diluted in Tween-PBS, 2% BSA for 1 hr. Primary antibody binding was detected using biotin-conjugated anti-mouse IgG Fc specific secondary antibody (Sigma; 1/2000 dilution in Tween-PBS, 2% BSA) for 1 hr, and visualized using IRDye 800CW streptavidin (LICOR Biosciences, UK; 1/1000 in Tween-PBS 2% BSA).

(46) Identification of FG129 Heavy and Light Chain Variable Regions.

(47) Cell Source and Total RNA Preparation:

(48) Approximately 510.sup.6 cells from hybridomasFG129 were taken from tissue culture, washed once in PBS, and the cell pellet treated with 500 l Trizol (Invitrogen). After the cells had been dispersed in the reagent, they were stored at 80 C. until RNA was prepared following manufacturer's protocol. RNA concentration and purity were determined by Nanodrop. Prior to cDNA synthesis, RNA was DNase I treated to remove genomic DNA contamination (DNase I recombinant, RNase-free, Roche Diagnostics, Burgess Hill, UK) following manufacturer's recommendations.

(49) cDNA Synthesis:

(50) First-strand cDNA was prepared from 3 g of total RNA using a first-strand cDNA synthesis kit and AMV reverse transcriptase following manufacturer's protocol (Roche Diagnostics). After cDNA synthesis, reverse transcriptase activity was destroyed by incubation at 90 C. for 10 mins and cDNA stored at 20 C.

(51) GAPDH PCR to Assess cDNA Quality:

(52) A PCR was used to assess cDNA quality; primers specific for the mouse GAPDH house-keeping gene (5-TTAGCACCCCTGGCCAAGG-3 (SEQ ID NO: 16) and 5-CTTACTCCCTTGGAGGCCATG-3 (SEQ ID NO: 17)) were used with a hot-start Taq polymerase (AmpliTaq Gold 360, Invitrogen) for 35 cycles (95 C., 3 mins followed by 35 cycles of 94 C./30 secs, 55 C./30 secs, 72 C./1 min; final polishing step of 10 mins at 72 C.). Amplified products were assessed by agarose gel electrophoresis.

(53) PCR Primer Design for Cloning FG129 Variable Regions:

(54) Primers were designed to amplify the heavy and light chain variable regions based upon the PCR product sequence data. Primers were designed to allow cloning of the relevant chain into unique restriction enzyme sites in the hIgG1/kappa double expression vector pDCOrig-hIgG1. Each 5 primer was targeted to the starting codon and leader peptide of the defined variable region, with a Kozak consensus immediately 5 of the starting codon. Each 3 primer was designed to be complementary to the joining region of the antibody sequence, to maintain reading frame after cloning of the chain, and to preserve the amino acid sequence usually found at the joining region/constant region junction. All primers were purchased from Eurofins MWG (Ebersberg, Germany).

(55) Heavy Chain Variable Region PCR:

(56) Immunoglobulin heavy chain variable region usage was determined using PCR with a previously published set of primers [60]. Previous results using a mouse mAb isotyping test kit (Serotec, Oxford, UK) had indicated that FG129 were both mouse IgG3 antibodies. Appropriate constant region reverse primers were therefore used to amplify from the constant regions. PCR amplification was carried out with 12 mouse VH region-specific 5 primers and 3 primers specific for previously determined antibody subclass with a hot-start Taq polymerase for 35 cycles (94 C., 5 min followed by 35 cycles of 94 C./1 min, 60 C./1 min, 72 C./2 min; final polishing step of 20 min at 72 C.). Amplified products were assessed by agarose gel electrophoresis. Positive amplifications resulted for the VH4 primer.

(57) Light () Chain Variable Region PCRs:

(58) Immunoglobulin light chain variable region usage was determined using PCR with a previously published set of primers [60]. Previous results using a mouse mAb isotyping test kit had indicated that FG129 used light chains. PCR amplification was carried out with mouse V region-specific 5 and 3 mouse C specific primers with a hot-start Taq polymerase for 35 cycles (94 C., 5 mins followed by 35 cycles of 94 C./1 min, 60 C./1 min, 72 C./2 mins; final polishing step of 20 mins at 72 C.). Amplification products were assessed by agarose gel electrophoresis. Positive amplifications resulted with the VK1 and VK2 primers for FG129.

(59) PCR Product Purification and Sequencing:

(60) PCR products were purified using a Qiaquick PCR purification kit (Qiagen, Crawley, UK). The concentration of the resulting DNA was determined by Nanodrop and the purity assessed by agarose gel electrophoresis. PCR products were sequenced using the originating 5 and 3 PCR primers at the University of Nottingham DNA sequencing facility (http://www.nottingham.ac.uk/life-sciences/facilities/dna-sequencing/index.aspx). Sequences were analysed (V region identification, junction analysis) using the IMGT databasesearch facility (http://www.imgt.rg/IMGT_vquest/vquest?livret-0&Option=mouseIg). Sequencing indicated that FG129 had heavy and light chain variable regions from the following families; heavy chain; IGHV6-6*01, IGHJ1*01, light chain; IGKV12-41*01, IGKJ1*01. Sufficient residual constant region was present in the heavy chain sequences to confirm that FG129 was of the mIgG1 subclass.

(61) Cloning Strategy:

(62) The PCR product for cloning was generated using a proof-reading polymerase (Phusion, New England Biolabs) was cloned into a TA vector (pCR2.1; Invitrogen).

(63) FG129 Heavy/Light Chain PCR for Cloning:

(64) PCR amplification was carried out using a proof-reading polymerase (Phusion; NEB) and the cloning primers described above using the FG129 cDNA template previously described for 35 cycles (98 C., 3 min followed by 35 cycles of 98 C./30 sec, 58 C./30 sec, 72 C./45 sec; final polishing step of 3 min at 72 C.). Successful amplification was confirmed by agarose gel electrophoresis.

(65) TOPO Light Chain Cloning:

(66) Amplified FG129 light chain was treated with Taq polymerase (NEB) for 15 min at 72 C. to add A overhangs compatible with TA cloning. Treated PCR product was incubated with the TOPO TA vector pCR2.1(Invitrogen) and transformed into chemically competent TOP10F cells according to manufacturer's instructions. Transformed bacteria were spread on ampicillin (80 g/ml) supplemented LB agar plates, which were then incubated overnight at 37 C. Colonies were grown in liquid culture (LB supplemented with 80 g/ml ampicillin) and plasmid DNA prepared (spin miniprep kit, Qiagen). Presence of an insert was confirmed by sequential digestion with BsiWI and BamHI and agarose gel electrophoresis. Sequencing was carried out on miniprep DNA from colonies using T7 and M13rev primers. The DNA insert from one such colony had the predicted FG129 light chain sequence; a 300 ml bacterial LB/ampicillin culture was grown overnight and plasmid DNA prepared by maxiprep (plasmid maxi kit, Qiagen). Maxiprep DNA insert was confirmed by sequencing.

(67) TOPO Heavy Chain Cloning:

(68) Amplified FG129 heavy chain was treated with Taq polymerase (NEB) for 15 min at 72 C. to add A overhangs. Treated PCR product was incubated with the TOPO TA vector pCR2.1 and transformed into chemically competent TOP10F cells as above. Transformed bacteria were spread on ampicillin supplemented LB agar plates which were then incubated overnight at 37 C. Colonies were grown in liquid culture (LB/ampicillin) and plasmid DNA prepared (spin miniprep kit). Presence of an insert was confirmed by digestion with HindIII and AfeI and agarose gel electrophoresis. Sequencing was carried out on miniprep DNA from a number of colonies using T7 and M13rev primers. The DNA insert from one such colony had the predicted FG129 heavy chain sequence; a 300 ml bacterial LB/ampicillin culture was grown overnight and plasmid DNA prepared by maxiprep (plasmid maxi kit, Qiagen). Maxiprep DNA insert was confirmed by sequencing.

(69) pDCOrig-hIgG1 Double Expression Vector Light Chain Cloning:

(70) The FG129 light chain was digested from the TOPO vector pCR2.1 by sequential digestion with BsiWI and BamHI and the 400 bp insert DNA agarose gel purified using a QIAquick gel extraction kit (Qiagen) following manufacturer's recommendations. This insert was ligated into previously prepared pDCOrig-hIgG1 vector (see above) and transformed into chemically competent TOP10F cells. Transformations were spread on 35 g/ml Zeocin supplemented LB agar plates which were then incubated overnight at 37 C. Colonies were grown in liquid culture (LB supplemented with 35 g/ml Zeocin) and plasmid DNA prepared (spin miniprep kit, Qiagen). Sequencing was carried out on miniprep DNA from all colonies using a sequencing primer sited in the human kappa constant region. The DNA insert from one of the colonies had the predicted FG129 light chain sequence correctly inserted in pDCOrig-hIgG1; a 300 ml bacterial LB/zeocin culture was grown overnight and plasmid DNA prepared by maxiprep (plasmid maxi kit, Qiagen).

(71) pDCOrig-hIgG1 Double Expression Vector Heavy Chain Cloning:

(72) The FG129 heavy chain insert was digested from the TOPO vector pCR2.1 by digestion with HindIII and AfeI. Vector (pDCOrig-hIgG1-129k) containing the FG129 kappa light chain (prepared above) was also digested with HindIII and AfeI. The vector DNA was then phosphatase treated according to manufacturer's recommendations (Antarctic Phosphatase, NEB). After agarose gel electrophoresis, the 6.5 kb pDCOrig-hIgG1 vector band and 400 bp FG129H insert band were isolated using a QIAquick gel extraction kit (Qiagen) following manufacturer's recommendations. The insert was ligated into the pDCOrig-hIgG1 vector and transformed into chemically competentTOP10F cells. Transformations were spread on 35 g/ml Zeocin supplemented LB agar plates which were then incubated overnight at 37 C. Colonies were grown in liquid culture (LB supplemented with 35 g/ml Zeocin) and plasmid DNA prepared (spin miniprep kit, Qiagen). Presence of an insert was confirmed by digestion with HindIII and AfeI and agarose gel electrophoresis. Sequencing was carried out on miniprep DNA from a number of the colonies using a sequencing primer sited in the human IgG1 constant region. The DNA insert from one of the colonies had the predicted FG129 heavy chain sequence correctly inserted in pDCOrig-hIgG1; a 300 ml bacterial LB/zeocin culture was grown overnight and plasmid DNA prepared by maxiprep (plasmid maxi kit, Qiagen). Sequencing was used to confirm that both heavy and light chain loci.

(73) Expression, Purification and Characterisation of the Chimeric Antibody Constructs.

(74) The methodology for the expression and purification of chimeric antibody described in the present invention can be achieved using methods well known in the art. Briefly, antibodies can be purified from supernatant collected from transiently, or subsequently stable, transfected cells by protein A or protein G affinity chromatography based on standard protocols, for example Sambrook et al. [61].

(75) Cloning, Expression, Purification and Characterisation of the FG129-scFv

(76) The heavy chain and light chain variable region were incorporated in silico into a single scFv sequence in the orientation; leader; heavy chain variable domain; spacer (3GGGGS); light chain variable domain; spacer (6Ala); purification tag (6His) and synthesised. After cloning into a eukaryotic expression vector, Expi293 cells were transfected and allowed to produce protein transiently (6 days). His-tagged scFv was purified from Expi-293 supernatant using immobilised cobalt chromatography (HiTrap Talon 1 ml columns; GE Healthcare). In the binding assays, a biotinilated anti-His tag antibody was used as a secondary antibody (6-His Epitope Tag Antibody, Biotin conjugated, clone HIS.H8; Thermo Fisher).

(77) Immunohistochemistry Assessment for FG129:

(78) To determine the therapeutic value of FG129, it was screened on pancreatic, lung, gastric, ovarian, colorectal cancer tissue microarrays by immunohistochemistry (IHC).

(79) Methodology:

(80) Immunohistochemistry was performed using the standard avidin-biotin peroxidase method. Paraffin embedded tissue sections were placed on a 60 C. hot block to melt the paraffin. Tissue sections were deparaffinised with xylene and rehydrated through graded alcohol. The sections were then immersed in 500 ml of citrate buffer (pH6) and heated for 20 min in a microwave (Whirlpool) to retrieve antigens. Endogenous peroxidase activity was blocked by incubating the tissue sections with endogenous peroxidase solution (Dako Ltd, Ely, UK) for 5 min. Normal swine serum (NSS; Vector Labs, CA, USA; 1/50 PBS) was added to each section for 20 min to block non-specific primary antibody binding. All sections were incubated with Avidin D/Biotin blocking kit (Vector Lab) for 15 min each in order to block non-specific binding of avidin and biotin. The sections were re-blocked with NSS (1/50 PBS) for 5 mins. Then tissue sections were incubated with primary antibody at room temperature for an hour. Anti--2-microglobulin (Dako Ltd; 1/100 in PBS) mAb and PBS alone were used as positive and negative controls respectively. Tissue sections were washed with PBS and incubated with biotinylated goat anti-mouse/rabbit immunoglobulin (Vector Labs; 1/50 in NSS) for 30 min at room temperature. Tissue sections were washed with PBS and incubated with preformed 1/50 (PBS) streptavidin-biotin/horseradish peroxidase complex (Dako Ltd) for 30 min at room temperature. 3, 3-Diaminobenzidine tetra hydrochloride (DAB) was used as a substrate. Each section was incubated twice with 100 l of DAB solution for 5 min. Finally, sections were lightly counterstained with haematoxylin (Sigma-Aldrich, Poole Dorset, UK) before dehydrating in graded alcohols, cleaning by immersing in xylene and mounting the slides with Distyrene, plasticiser, xylene (DPX) mountant (Sigma).

(81) Patient Cohorts:

(82) The study populations include cohorts of a consecutive series of 462 archived colorectal cancer (29) specimens (1994-2000; median follow up 42 months; censored December 2003; patients with lymph node positive disease routinely received adjuvant chemotherapy with 5-flurouracil/folinic acid), 350 ovarian cancer (28) samples (1982-1997; median follow up 192 months: censored November 2005:patients with stage II to IV disease received standard adjuvant chemotherapy which in later years was platinum based), 142 gastric cancer (26) samples (2001-2006; median follow up 66 months; censored January 2009; no chemotherapy) 68 pancreatic and 120 biliary/ampullary cancer (27) samples (1993-2010:median 45 months; censored 2012; 25-46% of patients received adjuvant chemotherapy with 5-fluorouracil/folinic acid and gemcitabine) 220 non small cell lung cancers (01/1996-07/2006: median follow up 36 months censored May 2013; none of the patients received chemotherapy prior to surgery but 11 patients received radiotherapy and 9 patients received at least 1 cycle of adjuvant chemotherapy post surgery) obtained from patients undergoing elective surgical resection of a histologically proven cancer at Nottingham or Derby University Hospitals. No cases were excluded unless the relevant clinico-pathological material/data were unavailable.

(83) Confocal Microscopy:

(84) FG129 and CH129 mAbs were labelled with Alexa-488 fluorophore (A-FG129, A-CH129) according to manufacturer's protocol (Invitrogen). 1.510.sup.5 HCT-15 cells were grown on sterile circular coverslips (22 mm diameter, 0.16-0.19 mm thick) in a 6 well plate for 24 hr in 5% CO.sub.2 at 37 C. 24 hours later, cells on coverslips were treated with 5 g/ml of mAbs for 2 hr at 37 C. in the dark. 2 hours later, excess/unbound mAbs were washed away using PBS. The cells were then fixed using 0.4% paraformaldehyde for 20 min in the dark. 0.4% paraformaldehyde was washed away using PBS. The coverslips were mounted to slides with PBS:glycerol (1:1). The coverslip edge was sealed with clear nail varnish. Localisation of A-FG129 and A-CH129 mAb was visualised under a confocal microscope (Carl Zeiss, Jena, Germany).

(85) ADCC and CDC:

(86) Cells (510.sup.3) were co-incubated with 100 l of PBMCs, 10% autologous serum or media alone or with mAbs at a range of concentrations. Spontaneous and maximum releases were evaluated by incubating the labeled cells with medium alone or with 10% Triton X-100, respectively. After 4 hr of incubation, 50 l of supernatant from each well was transferred to 96 well lumaplates. Plates were allow to dry overnight and counted on a Topcount NXT counter (Perkin Elmer, Cambridge, UK). The mean percentage lysis of target cells was calculated according to the following formula:

(87) $\mathrm{Mean}\%\mathrm{lysis}=100\frac{\mathrm{mean}\mathrm{experimental}\mathrm{counts}-\mathrm{mean}\mathrm{spontaneous}\mathrm{counts}}{\mathrm{mean}\mathrm{maximum}\mathrm{counts}-\mathrm{mean}\mathrm{spontaneous}\mathrm{counts}}$

(88) ADC Assay

(89) ADC was evaluated by measuring the cytotoxicity of immune-complexed mAbs with a mouse Fab-ZAP secondary conjugate (Advanced Targeting Systems) (30). Cells were plated in triplicates overnight into 96-well plates (2,000 cells, 90 l/well). After preincubation (30 minutes at room temperature) of a concentration range of FG129 or CH129 mAbs with 50 ng of the Fab-ZAP conjugate, 10 l of conjugate or free mAb was added to the wells and incubated for 72 hours. Control wells, consisted of cells incubated without conjugate, incubated with secondary Fab-ZAP without primary mAb and incubated with a control mAb in the presence of Fab-ZAP. Cell viability was measured by .sup.3H-thymidine incorporation during the final 24 hours. Results are expressed as a percentage of .sup.3H-thymidine incorporation by cells incubated with conjugate compared with primary mAb only.

(90) To further investigate if CH129 would make a promising ADC candidate in a clinical setting, the mab was chemically conjugated to different payload/linker constructs that were pre-clinically and clinically validated. Thus, three CH129 constructs were produced by ADC Biotechnology: one with the auristatin MMAE linked via a cleavable dipeptide valine-citruline linker and a para-aminobenzylalcohol (PABA) self-immolative spacer, one with the DM4 maytansinoid linked via the intermediately cleavable hindered disulphide linker SPDB and one with the DM1 maytansinoid linked through the non-cleavable SMCC linker. A matched set of control ADC constructs was also produced using the non-targeting mab Rituximab, to be used in relevant assay controls.

(91) The cytotoxic effect of the CH129-ADC constructs was assessed by using the water-soluble tetrazolium salt WST-8 (Sigma) to measure the activity of hydrogenases which is directly proportional with the number of viable cells. Cells were plated in 96-well plates at a density of 2000 cells/90 l/well in 10% FBS-RPMI with Penicillin-Streptomycin (Sigma) and incubated overnight at 37 C., 5% CO2. The ADC constructs were then added to the cells at different concentrations in a final volume of 10 l/well and the plates were incubated at 37 C., 5% CO2 for 72 h with the antibody constructs. The WST-8 was then added (10 l/well) and the plates were further incubated 37 C., 5% CO2 for 3 h. After the 3 h incubation, the plates were read at 450 nm by Tecan Infinite F50. Results are expressed as percentages of control wells, consisting of cells only without any antibody. Cytotoxicity was studied on two colorectal cell lines Colo205 and HCT-15 that express high cell surface densities of the targeted antigen sialyl-lewis-a.

Example 1

(92) Generation and Initial Characterisation of FG129 mAbs

(93) FG129 was produced by immunising Balb/c mice with plasma membrane lipid extracts from LS180 cells (colorectal cell line) incorporated into liposomes, at two-week intervals over a period of 2 months, alpha-galactosylceramide was used as an adjuvant in the first, third and fourth immunisation and anti-CD40 mAb used during the second immunisation.

(94) Analysis of antibody response to immunisations: Antibody titres were initially monitored by lipid enzyme-linked immunosorbent assay (ELISA). Flow cytometry analysis (FACS) was also carried out using LS 180 tumour cells and Western blot using LS180. The mouse considered to have the best response, compared to the pre-bleed serum control, was boosted intravenously (i.v.) with LS180 plasma membrane lipid extract prior to fusion. 8 days after fusion, supernatants were collected and screened against fresh L S 1 8 0 tumour cells by flow cytometry. Hybridomas which demonstrated cell surface binding, using an indirect immunofluorescence assay, were harvested, washed in complete media and spread across 96 well plates at 0.3 cells per well to acquire a clone. The plate was then screened for positive wells and these grown on until a sufficient number of cells was obtained to spread across a 96 well plate at 0.3 cells per well for a second time. If the resulting number of colonies equalled 30 and all hybridomas were positive, the hybridoma was considered a clone. Methods for clonal expansion, bulk culture and antibody purification of antibodies or antibody fragments are available using conventional techniques known to those skilled in the art.

Example 2

(95) Chimerisation of FG129

(96) The term chimeric antibody is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody. Chimeric (or humanised) antibodies of the present invention can be prepared based on the sequence of a murine mAb prepared as described above. The amino acid and nucleotide sequence for the variable and constant regions of the heavy (FIG. 1a) and light chains (FIG. 1b) of the FG129 mAb are shown in FIG. 1. Numbers refer to the standardised IMGT system for the numbering of antibody sequences [49]. The CDR1, CDR 2 and CDR 3 regions are indicated. The FG129 heavy chain belongs to the mouse heavy chain family IGHV10-1*02 (IGHD1-1*01, IGHJ4*01), with three mutations compared to the parental germline gene. The FG129 light chain belongs to the mouse kappa chain family IGKV8-19*01 (IGKJ4*01), with two mutations compared to the parental germline gene.

(97) FG129 heavy and light chain variable regions were cloned into a human IgG1 expression vector. This was transfected into CHO-S or HEK293 cells and human antibody purified on protein G. The chimeric mAbs CH129 bound to the colorectal cell line, Colo205. The amino acid and nucleotide sequence for the heavy and light chains of the human ch129 mAb are shown in FIGS. 2a and 2b respectively.

Example 3

(98) Defining the Epitopes Recognised by FG129 and CH129 mAbs

(99) MAb FG129 is a mouse IgG1k isotype that was generated by immunising Balb/c mice with glycolipid extracts from colorectal cell line LS180. Glycan profiling analysis done by CFG on 600 natural and synthetic glycans shows a high specificity of FG129 binding sialyl-di-Lewis.sup.a (100%) and sialyl-Lewis.sup.a-x (89%). It can also bind to mono-sialyl-Lewis.sup.a (89%), but only if presented on a long carrier (sp8) and not on a short carrier (sp0), suggesting that it requires at least 4 carbohydrates or sufficient space to allow the three carbohydrate residues to insert into the antibody sequence presented in the correct conformation to bind (FIG. 3a).

(100) To analyse if these glycans' were expressed on glycoproteins or glycolipids from tumour cell lines FG129 binding was assessed by Western blotting (FIG. 3b). Tumour lysates or tumour glycolipid extracts from colorectal (Colo205 HCT-15 and LS180) and pancreatic cells lines (BxPc3), were blotted with FG129, CH129 mAb, secondary antibody alone or CA19.9 (anti-sialyl lewis a Mab). FG129 and CH129 bound to a wide range of glycoproteins in Colo205 and HCT-15 lysate and to a smaller number of glycoproteins in BxPc3 and LS180 lystates. FG129 failed to bind to any of the tumour glycolipid extracts. In contrast, CA19.9 showed binding to a wider range of glycoproteins in BxPc3, Colo205 and LS180 and to glycolipids from BxPc3 and HCT-15 cells. These results suggest that FG129 prefers to bind to six carbohydrate residues and prefers sialyl-di-Lewis.sup.a which is predominantly expressed on proteins. In contrast, CA19.9 which prefers the 3 carbohydrate residue glycan, sialyl-Lewis.sup.a, binds to both lipids and proteins.

(101) As mAbs require strong affinity to localise within tumours the affinity of FG129 mAb was assessed by Biacore and ELISA. Affinity measurements using SPR (Biacore X or 3000) on a sialyl-Lewis.sup.a (as sialyl-di-Lewis.sup.a is unavailable) coupled chip revealed two possible functional affinitiesa dominant one (K.sub.d10.sup.7M) accounting for 80% of the population and another very high affinity (K.sub.d10.sup.13M) with fast association (10.sup.4 l/Ms) and very slow dissociation rate (K.sub.d-10.sup.8 l/s) (Table 1a). In particular, the affinity measurements revealed subnanomolar functional affinity for FG129 and nanomolar affinity for CH129, both showing relatively fast on-rates and slow off-rates for sialyl-Lewis-a binding (Table 1b). The monovalent binding affinity of the scFv129 was lower (10.sup.7M), with a slower on-rate but similar off-rate, suggesting bivalent binding on the chip by FG129 and CH129.

(102) TABLE-US-00001 TABLE 1a Determination of kinetic sialyl-Lewis.sup.a binding parameters by SPR Equilibrium dissociation constant Association rate Dissociation rate K.sub.d (M) k.sub.a (1/Ms) k.sub.d (1/s) Major K.sub.d1 (80%)~1.3 10.sup.7 k.sub.a1~1.97 10.sup.4 k.sub.d1~2.57 10.sup.3 Minor K.sub.d2 (20%)~1.4 10.sup.13 k.sub.a2~8.85 10.sup.4 k.sub.d2~1.35 10.sup.8

(103) TABLE-US-00002 TABLE 1b Determination of kinetic sialyl-Lewis-a binding parameters by SPR SPR Real-time sialyl Le.sup.a-HSA binding Association Dissociation Dissociation Rate Rate Constant mAb k.sub.on (1/Ms) k.sub.off (1/s) Kd (M) FG129 6.2 10.sup.5 1.1 10.sup.4 0.2 10.sup.9 CH129 1.3 10.sup.5 2.6 10.sup.4 2.1 10.sup.9 FG129-scFv 3.0 10.sup.3 5.0 10.sup.4 1.7 10.sup.7

(104) Additionally, antigen binding was assessed by ELISA using sialyl-Lewis.sup.a-HSA which revealed a FG129 and CH129 dose dependent response, confirmed specific sialyl-Lewis.sup.a binding with a subnanomolar Ec.sub.50 (10.sup.10M) and also showed no binding to HSA and plastic (FIG. 4).

Example 4

(105) Immunohistochemistry Assessment of FG129 and CH129 mAbs.

(106) To determine the therapeutic value of FG129, it was screened on colorectal, gastric, pancreatic, lung, and ovarian tumour tissue microarrays (TMAs) by immunohistochemistry (IHC).

(107) The tumour tissue binding of FG129 was assessed by IHC on tumour TMAs. The mAb bound to 74% (135/182) of pancreatic tumours, 50% (46/92) of gastric tumours, 36% (100/281) of colorectal tumours, 27% (89/327) of ovarian and 21% (42/201) of NSCLC tumours (Table 1).

(108) TABLE-US-00003 TABLE 2 Binding of FG129 (1 g/ml) by IHC to gastric, colorectal, pancreatic, ovarian and lung TMAs by staining intensity Pancreatic + Lung bilary/ (adeno- Gastric Colorectal ampullary Ovarian carcinoma) Staining Number % Number % Number % Number % Number % Negative 46 50 181 64 45 25 238 73 159 79 Weak 25 27 72 26 37 21 63 19 21 10 Moderate 10 11 25 9 61 34 21 6 9 4 Strong 11 12 3 1 37 21 5 2 12 6

(109) Representatives of different staining levels of tumour tissues with FG129 are shown in FIG. 5a. In pancreatic cancer cohort, Kaplan-Meier analysis of disease-free survival of pancreatic patients revealed a significantly lower mean survival time in the high FG129 binding group (mean survival: 30 months (n=94)) compared to the lower FG129 binding group (mean survival: 90 months (n=82)), p=0.004, Log-Rank test. On multivariate analysis using Cox regression, high FG129 antigen expression in pancreatic cancer was a marker for poor prognosis which was independent of perineural invasion (p=0.00.sup.3) (FIG. 5b).

(110) In normal tissue, FG129 had a very restricted binding pattern and did not bind most normal tissues like heart, brain, stomach, and kidney (table 1). Very limited binding was seen in gallbladder (weak), ileum (1%), liver (1%), oesophagus (5%), pancreas (10%), and thyroid (weak: (FIG. 5c). This is in direct contrast to CA19.9 mAb which recognizes sialyl Lewis.sup.a on both lipids and proteins. It binds strongly (3+) to oesophagus, gallbladder and liver, moderately (2+) to breast and weakly (1+) to rectum. FG129 displays strong tumour tissue binding with low normal tissue reactivity, and is associated with poor prognosis in pancreatic cancer patients.

(111) TABLE-US-00004 TABLE 3 Summary of FG129 and CA19.9 binding to a panel of normal tissues using paraffin-fixed sections. Intensity of staining is shown as 0, 1, 2 or 3, relating to negative, weak, moderate or strong binding. Tissue Type FG129 CA19.9 Oesophagus 0 3 Oesophagus 1 3 Rectum 0 1 Rectum 0 1 Gallbladder 1 3 Gallbladder 1 1 Skin 0 0 Skin 0 0 Adipose 0 0 Adipose 0 0 Heart 0 0 Heart 0 0 Skeletal 0 0 Skeletal 0 0 Bladder 0 0 Bladder 0 0 Ileum 1 0 Ileum 1 0 Spleen 0 0 Spleen 0 0 Brain 0 0 Brain 0 0 Jejunum 0 0 Jejunum 0 0 Stomach 0 0 Stomach 0 0 Breast 0 2 Breast 0 2 Kidney 0 0 Kidney 0 0 Testis 0 0 Testis 0 0 Cerebellum 0 0 Cerebellum 0 0 Liver 3% at 1 3 Liver 3% at 1 3 Thymus 0 1 Thymus 0 2 Cervix 0 0 Cervix 0 0 Lung 0 0 Lung 0 0 Smooth Muscle 0 0 Smooth Muscle 0 0 Colon 0 2 Colon 0 1 Ovary 0 0 Ovary 0 0 Tonsil 0 1 Tonsil 0 0 Diaphragm 0 0 Diaphragm 0 0 Pancreas 1 3 Pancreas 1 2 Uterus 0 0 Uterus 0 0 Duodenum 0 0 Duodenum 0 0 Thyroid 1 0 Thyroid 1 0

(112) In normal tissue, CH129 had a very restricted binding pattern and did not bind most normal tissues like heart, brain, stomach, and kidney (table 1). Very limited binding was seen in gallbladder (weak), ileum (1%), liver (1%), oesophagus (5%), pancreas (10%), and thyroid (weak: (FIG. 5a).

Example 5

(113) FG129 and CH129 mAbs Binding Studies

(114) To determine if any cell line is a good model for tumours expressing sialyl-di-Lewis.sup.a a range of cell lines and normal cells were screened for cell surface binding of FG129. FG129 and CH129 showed strong binding (geometric mean (Gm)1000) to tumour cell lines HCT-15, Colo205, moderate binding (Gm 100) to BxPc3, ASPC1, LS180, DLD1, and DMS79 and no binding to AGS, SW480, EKVX, MCF-7, LoVo, DU4475, OVCAR3, OVCAR4 and OVCA433. This suggests that HCT-15, Colo205, ASPC1, BxPc3, LS180, DLD1, and DMS79 would be good models for assessing the sensitivity of tumour cells with different cell densities of sialyl-Lewis.sup.a to FG129 therapy (FIG. 6a). FG129 failed to bind to normal HUVEC cells (FIG. 6b). For comparison, an anti-CD55 mAb was used as a positive control and an anti-IgG isotype antibody as a negative control. Importantly, FG129 and CH129 did not bind to PBMCs from a range of healthy donors (FIG. 6c). These results identified several cell lines as models of human tumours for in vitro studies and showed that FG129 did not bind to normal blood or endothelial cells suggesting that they would not prevent FG129 localising within tumours.

(115) The antigen density (SABC) was calculated to be 985,813 and 1,570,563 for HCT-15 and COLO205, respectively. Moderately binding cells included BxPc3 and LS180 (SABC: 300,036 and 469,272 respectively).

(116) To estimate the affinity of binding to tumour cell lines, varying concentration of FG129 and CH129 mAbs were added to Colo205, HCT-15, BxPC3 and LS180 and binding was detected by indirect immunofluorescence analysis and flow cytometric analysis (FIG. 7). Both FG129 and CH129 bound to the high expressing cell lines with Kd of 6-20 nM and to low expressing cell lines with a Kd of 30-50 nM. This is higher than binding to sialyl lewis.sup.a-HSA and probably reflects the complexity of glycan expression on the cell surface.

(117) The antigen density (SABC) was calculated to be 985,813 and 1,570,563 for HCT-15 and COLO205, respectively. Moderately binding cells included BxPc3 and LS180 (SABC: 300,036 and 469,272 respectively).

Example 6

(118) In Vitro Anti-Tumour Activity of FG129 and CH129

(119) The ability of FG129 and CH129 to induce Colo205 and HCT-15 tumor cell death in the presence of human PBMCs through ADCC was investigated. Both the mouse FG129 and chimeric CH129 mAb induced potent cell lysis of both cell lines in a concentration-dependent manner. CH129 had 2-4 increase in killing when compared to the mouse mAb with an EC.sub.50 value of 10.sup.10M (FIG. 8). The ability of FG129 and CH129 to induce Colo205, tumor cell death in the presence of complement through CDC was investigated. Chimeric but not mouse mabs showed good CDC (FIG. 9).

Example 7

(120) Internalisation and ADC (Antibody Dependent Drug Cytotoxicity)

(121) To further determine the therapeutic ability of the FG129 and CH129 the mAbs were screened for their ability to act as a drug carrier by internalising and delivering drug to lysosomes. Cellular internalisation was assessed by confocal microscopy, which showed internalisation of both 129 mAbs over a period of 90 minutes and co-localisation within the lysosomes. The nucleus was stained in blue, plasma membrane in red, lysosomal compartments in purple and the 129 antibodies in green. Internalisation is seen on high cell surface antigen density colorectal cell lines Colo205 and HCT-15 and on pancreatic cell line BxPC3 (FIGS. 10a and b).

(122) Internalisation was confirmed by ADC assays using Fab-ZAP, an anti-mouse IgG or anti-human IgG linked to the ribosome inactivating protein saporin, which killed the cells that internalised the Fab-ZAP-FG129/CH129 immune complex, but left the cells that did not internalise unaffected. Internalisation of Fab-ZAP-FG129 or CH129 led to a dose-dependent decrease in cell viability (Ic5010.sup.12M) on high binding cells Colo205 and HCT-15 but not BxPc3 or ASPC1 (FIGS. 11a and 11c). No killing of low expressing cell lines LS180 or antigen negative cell line LoVo was observed (FIG. 11a). Fab-ZAP alone or Fab-ZAP pre-incubated with an isotype-matched IgG1 antibody against an antigen not expressed by cells, did not kill the cells (FIGS. 11b and 11d).

(123) Additionally, to investigate if CH129 would make a promising ADC candidate in a clinical setting, the mab was chemically conjugated to different payload/linker constructs that were pre-clinically and clinically validated. Thus, three CH129 constructs were produced by ADC Biotechnology: one with the auristatin MMAE linked via a cleavable dipeptide valine-citruline linker and a para-aminobenzylalcohol (PABA) self-immolative spacer (CH129-vcE), one with the DM4 maytansinoid linked via the intermediately cleavable hindered disulphide linker SPDB (CH129-DM4) and one with the DM1 maytansinoid linked through the non-cleavable SMCC linker (CH129-DM1). A matched set of control ADC constructs was also produced using the non-targeting mab Rituximab, to be used in relevant assay controls. Cytotoxicity was studied on two colorectal cell lines Colo205 and HCT-15 that express high cell surface densities of the targeted antigen sialyl-lewis-a.

(124) CH129-ADC constructs give high in vitro target dependant efficacy. Results show a dose dependant decrease in cell death directly related with the decrease in antibody concentration on both cell lines. Cell killing was also target dependent, with higher killing being seen on the higher antigen expressing cell line Colo205, compared to HCT-15. On Colo205 (FIG. 11e) all three CH129-ADC constructs gave 100% cell killing with the vcE construct giving the highest efficacy (Ec5010.sup.11M) followed by the DM1 and DM4 constructs showing similar efficacy (Ec50s10.sup.10M).

(125) On HCT-15 (FIG. 11f) only 50-60% of the cells were killed at the highest concentrations, with CH129-DM4 giving the best Ec50 of 210.sup.9M, while DM1 gave an Ec50 of 610.sup.9M and vcE giving an Ec50 of 10.sup.8M. Matched Rituximab-ADC constructs which did not bind the cell line were used as controls to assess the specificity of the killing. The absence of activity of the vcE and DM1 Rituximab constructs, indicates that the activity seen with the targeted constructs is specific, and not due to systemic release of free drug. However, Rituximab-DM4 shows similar activity to the CH129 constructs, suggesting non-specific killing.

(126) In order to determine if the ADCs with cleavable linkers would kill antigen negative cells from the surroundings of antigen positive cells, the ADC constructs were tested on a mixture of antigen positive and antigen negative cells, and as well on cell lines with heterogeneous tumour antigen expression.

(127) ADCs with Cleavable Linkers Give Bystander Killing Compared with Uncleavable Linkers.

(128) The bystander killing effect of the ADC constructs was evaluated on different cell ratio mixtures of high tumour antigen expressing cells Colo205 with cells that do not express the antigenAGS. Cells were mixed at ratios of 2:1, 5:1 and 10:1 AGS to Colo205. Colo205 only, and AGS only were used as positive and negative controls respectively. Since AGS is an antigen negative cell line, the killing see on this cell line is non-specific, therefore concentrations at which killing is observed on AGS were not considered when assessing bystander killing. Specific killing is shown in FIGS. 11g, 11h and 11i highlighted by the rectangle. DM1 was the most stable in this aspect, as it showed killing at concentrations higher than 10 nM, while DM4 at 3 nM and vcE were less stable showing non-specific killing from 1 nM.

(129) As DM1 is linked with a non-cleavable linker, it consisted the negative control for bystander killing. The difference between the killing given by DM1 and DM4/vcE at the circled concentrations could be due to bystander killing. Thus, DM4 gave a specific killing of 90%, vcE of 50-80% while DM1 of 20% of the cells.

Example 8

(130) Expression of Sialyl-Lewis A on Secreted Antigens within Sera from Cancer Patients

(131) The presence of secreted FG129 antigen in pancreatic patients sera was investigated by sandwich ELISA, which showed that FG129 bound to 33% (7/21) of sera (FIG. 12a). When tumours from these patients were analysed by IHC for binding of sialyl-di-Lewis.sup.a on the tumour cells or within the stroma, all but one tumour was positive but only 6 tumours displayed stromal staining. The presence of secreted antigen was significantly associated with stromal tissue staining from tumours resected from these patients (p=0.023, correlation coefficient=0.621) suggesting that staining of resected tumours could predict patients in whom antigen may be present in the serum (Table 4).

(132) TABLE-US-00005 TABLE 4 Tumour and stromal H score by IHC and pancreatic serum binding by sandwich ELISA of FG129 Sandwich ELISA Panc Tumor H Stromal H serum binding score score to FG129 OD P4 200 100 0.2 P5 180 0 0.05 P9 285 150 0.11 P10 120 0 0.07 P11 60 0 0.05 P12 150 100 0.06 P18 250 40 0.13 P20 0 0 0.05 P23 260 0 0.06 P32 110 0 0.05 P36 120 0 0.06 P40 280 25 0.06 P41 130 70 0.13

(133) In order to mimic the in vivo setting, it was investigated if at 37 C. FG129 would bind preferentially to secreted antigen or to tumour cell surface. Binding of FG129 to secreted antigen or tumour cells was analysed in a competition FACS assay on HCT-15 cells at 37 C. All serum reduced binding to HCT-15 cells but there was no association with secreted sialyl-Lewis.sup.a antigen suggesting the viscosity of the serum reduced the kinetics of mAb binding. Serum from a normal donor which did not have secreted sialyl-Lewis.sup.a antigen also showed a reduction in binding to HCT-15 cells (Gm x to 1200). Antigen positive patient sera also reduced binding (Gm 600-1000) as did antigen negative patent sera (Gm 650-1500). Even though FG129 was pre-incubated with the pancreatic sera, the mAb showed a strong preference for binding to the cells and not to the secreted antigen from the sera (FIG. 12b). This suggests that secreted antigen should not prevent FG129 from localising within tumours.

Example 9

(134) Cloning, Expression, Purification and Characterization of the FG129-scFv

(135) With its limited normal tissue binding and the very high tumour tissue binding, the FG129 antibody makes an attractive candidate to be used in the context of a chimeric antigen receptor (CAR) as a scFv in order to induce anti-tumour T cell responses.

(136) To determine if the scFv would maintain the binding characteristics of the FG129 full antibody, the heavy chain and light chain variable region were incorporated in silico into a single scFv sequence in the orientation: leader, heavy chain variable domain, spacer (3GGGGS), light chain variable domain, spacer (6Ala); purification tag (6His) and synthesized (FIG. 13a). After cloning into a eukaryotic expression vector, Expi293 cells were transfected and allowed to produce protein transiently (6 days). His-tagged scFv was purified from Expi-293 supernatant using immobilised cobalt chromatography. The scFv was then characterised in terms of its binding properties to the sialyl-Lewis-a antigen or to cells expressing the antigen.

(137) The antigen binding affinity of the FG129-scFv was measured by SPR and by ELISA on sialyl-Lewis-a. In antigen binding assay by ELISA, the FG129-scFv showed specific sialyl-Lewis-a binding that titrated down with decrease in scFv concentration (Ec50=10.sup.6M) (FIG. 13b). Antigen binding affinity was also measured by SPR which gave a Kd of 10.sup.7M (Table 1). In cell binding assays, on Colo205, the FG129-scFv showed a high binding (Gm 400) and gave a dose dependent response with a submicromolar Kd (10.sup.7M) (FIG. 13c). Therefore the FG129-scFv maintains a high specific binding comparable to the binding of the full antibody and also displays a high binding affinity (Kd10.sup.7M) despite having only one binding instead of the two binding arms of the full FG129 mab.

(138) The Sequence Listing is submitted as an ASCII text file in the form of the file named Sequence_Listing.txt, which was created on Feb. 23, 2018, and is 21,700 bytes, which is incorporated by reference herein.

(139) Sequences

(140) TABLE-US-00006 (SEQIDNO:8) MouseFG129/29IgG1heavychain. atgctgttgggg ctgaagtgggttttctttgttgttttttatcaaggtgtgcattgt gaggtgcagcttgttgagtctggtgga...ggattggtgcagcct aaagggtcattgaaactctcatgtgcagcctctggattcaccttc ............aatacctacgccatgaactgggtccgccaggct ccaggaaagggtttggaatgggttgctcgcataagaagtaaaagt aataattatgcaacatattatgccgattcagtgaaa...gacagg ttcaccatatccagagatgattcacaaagcatgctctatctgcaa atgaacaacttgaaaaaggaggacacagccatgtattactgtgta gggtacggtagtgggggaaactactggggtcaagga......... acctcagtcaccgtctcctcagccaaaacgacacccccatctgtc tatccactggcccctggatctgctgcccaaactaactccatggtg accctgggatgcctggtcaagggctatttccctgagccagtgaca gtgacctggaactctggatccctgtccagcggtgtgcacaccttc ccagctgtcctggagtctgacctctacactctgagcagctcagtg actgtcccctccagccctcggcccagcgagaccgtcacctgcaac gttgcccacccggccagcagcaccaaggtggacaagaaaattgtg cccagggattgtggttgtaagccttgcatatgtacagtcccagaa gtatcatctgtcttcatcttccccccaaagcccaaggatgtgctc accattactctgactcctaaggtcacgtgtgttgtggtagacatc agcaaggatgatcccgaggtccagttcagctggtttgtagatgat gtggaggtgcacacagctcagacgcaaccccgggaggagcagttc aacagcactttccgctcagtcagtgaacttcccatcatgcaccag gactggctcaatggcaaggagttcaaatgcagggtcaacagtgca gctttccctgcccccatcgagaaaaccatctccaaaaccaaaggc agaccgaaggctccacaggtgtacaccattccacctcccaaggag cagatggccaaggataaagtcagtctgacctgcatgataacagac ttcttccctgaagacattactgtggagtggcagtggaatgggcag ccagcggagaactacaagaacactcagcccatcatgaacacgaat ggctcttacttcgtctacagcaagctcaatgtgcagaagagcaac tgggaggcaggaaatactttcacctgctctgtgttacatgagggc ctgcacaaccaccatactgagaagagcctctcccactctcctggt aaa (SEQIDNO:9) MouseFG129/29kappachain. atggaatcacag actcaggtcctcatgtccctgctgttctgggtatctacctgtggg gacattgtgatgacacagtctccatcctccctgactgtgacagca ggagagaaggtcactatgagctgcaagtccagtcagagtctgtta aacagtggaaatcaaaagaactacttgacctggtaccagcagaaa ccagggcagcctcctaaagtgttgatctactgggca......... ............tccactagggaatctggggtccct...gatcgc ttcacaggcagtgga......tctggaacagatttcactctcacc atcagcagtgtgcaggctgaagacctggcagtttattactgtcag aatgattatagttctccattcacgttcggctcggggacaaagttg gaaataaaacgggctgatgctgcaccaactgtatccatcttccca ccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgc ttcttgaacaacttctaccccaaagacatcaatgtcaagtggaag attgatggcagtgaacgacaaaatggcgtcctgaacagttggact gatcaggacagcaaagacagcacctacagcatgagcagcaccctc acgttgaccaaggacgagtatgaacgacataacagctatacctgt gaggccactcacaagacatcaacttcacccattgtcaagagcttc aacaggaatgagtgt (SEQIDNO:10) MouseFG129/29heavychainchimerisedtohIgG1constantregion atgctgttgggg ctgaagtgggttttctttgttgttttttatcaaggtgtgcattgt gaggtgcagcttgttgagtctggtgga...ggattggtgcagcct aaagggtcattgaaactctcatgtgcagcctctggattcaccttc ............aatacctacgccatgaactgggtccgccaggct ccaggaaagggtttggaatgggttgctcgcataagaagtaaaagt aataattatgcaacatattatgccgattcagtgaaa...gacagg ttcaccatatccagagatgattcacaaagcatgctctatctgcaa atgaacaacttgaaaaaggaggacacagccatgtattactgtgta gggtacggtagtgggggaaactactggggtcaagga......... acctcagtcaccgtctccagcgcttccaccaagggcccatcggtc ttccccctggcaccctcctccaagagcacctctgggggcacagcg gccctgggctgcctggtcaaggactacttccccgaaccggtgacg gtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttc ccggctgtcctacagtcctcaggactctactccctcagcagcgtg gtgaccgtgccctccagcagcttgggcacccagacctacatctgc aacgtgaatcacaagcccagcaacaccaaggtggacaagaaagtt gagcccaaatcttgtgacaaaactcacacatgcccaccgtgccca gcacctgaactcctggggggaccgtcagtcttcctcttcccccca aaacccaaggacaccctcatgatctcccggacccctgaggtcaca tgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaag ccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtc ctcaccgtcctgcaccaggactggctgaatggcaaggagtacaag tgcaaggtctccaacaaagccctcccagcccccatcgagaaaacc atctccaaagccaaagggcagccccgagaaccacaggtgtacacc ctgcccccatcccgggatgagctgaccaagaaccaggtcagcctg acctgcctggtcaaaggcttctatcccagcgacatcgccgtggag tgggagagcaatgggcagccggagaacaactacaagaccacgcct cccgtgctggactccgacggctccttcttcctctacagcaagctc accgtggacaagagcaggtggcagcaggggaacgtcttctcatgc tccgtgatgcatgaggctctgcacaaccactacacgcagaagagc ctctccctgtctccgggtaaa (SEQIDNO:11) MouseFG129/29kappachainchimerisedtohIgkconstantregion atggaatcacag actcaggtcctcatgtccctgctgttctgggtatctacctgtggg gacattgtgatgacacagtctccatcctccctgactgtgacagca ggagagaaggtcactatgagctgcaagtccagtcagagtctgtta aacagtggaaatcaaaagaactacttgacctggtaccagcagaaa ccagggcagcctcctaaagtgttgatctactgggca......... ............tccactagggaatctggggtccct...gatcgc ttcacaggcagtgga......tctggaacagatttcactctcacc atcagcagtgtgcaggctgaagacctggcagtttattactgtcag aatgattatagttctccattcacgttcggctcggggacaaagttg gaaataaaacgtacggtagcggccccatctgtcttcatcttcccg ccatctgatgagcagttgaaatctggaactgcctctgttgtgtgc ctgctgaataacttctatcccagagaggccaaagtacagtggaag gtggataacgccctccaatcgggtaactcccaggagagtgtcaca gagcaggacagcaaggacagcacctacagcctcagcagcaccctg acgctgagcaaagcagactacgagaaacacaaagtctacgcctgc gaagtcacccatcagggcctgagctcgcccgtcacaaagagcttc aacaggggagagtgt

(141) FIG. 1a: Complete amino acid sequence of mouse F129/29 IgG1 heavy chain. (SEQ ID NO: 12)

(142) TABLE-US-00007 -19MLLGLKWVFFVVFYQGVHC 1EVQLVESGGGLVQPKGSLKLSCAASGFTF 31NTYAMNWVRQAPGKGLEWVARIRSKS 61NNYATYYADSVKDRFTISRDDSQSMLYLQ 91MNNLKKEDTAMYYCVGYGSGGNYWGQG 121TSVTVSSAKTTPPSVYPLAPGSAAQTNSMV 151TLGCLVKGYFPEPVTVTWNSGSLSSGVHTF 181PAVLESDLYTLSSSVTVPSSPRPSETVTCN 211VAHPASSTKVDKKIVPRDCGCKPCICTVPE 241VSSVFIFPPKPKDVLTITLTPKVTCVVVDI 271SKDDPEVQFSWFVDDVEVHTAQTQPREEQF 301NSTFRSVSELPIMHQDWLNGKEFKCRVNSA 331AFPAPIEKTISKTKGRPKAPQVYTIPPPKE 361QMAKDKVSLTCMITDFFPEDITVEWQWNGQ 391PAENYKNTQPIMNTNGSYFVYSKLNVQKSN 421WEAGNTFTCSVLHEGLHNHHTEKSLSHSPG 451K

(143) FIG. 1b: Complete amino acid sequence of mouse F129/29 kappa chain. (SEQ ID NO: 13)

(144) TABLE-US-00008 -19MESQTQVLMSLLFWVSTCG 1DIVMTQSPSSLTVTAGEKVTMSCKSSQSLL 31NSGNQKNYLTWYQQKPGQPPKVLIYWA 61STRESGVPDRFTGSGSGTDFTLT 91ISSVQAEDLAVYYCQNDYSSPFTFGSGTKL 121EIKRADAAPTVSIFPPSSEQLTSGGASVVC 151FLNNFYPKDINVKWKIDGSERQNGVLNSWT 181DQDSKDSTYSMSSTLTLTKDEYERHNSYTC 211EATHKTSTSPIVKSFNRNEC

(145) FIG. 2a: Complete amino acid sequence of mouse FG129/29 heavy chain variable region chimerised to human IgG1 heavy chain constant region. (SEQ ID NO: 14)

(146) TABLE-US-00009 -19MLLGLKWVFFVVFYQGVHC 1EVQLVESGGGLVQPKGSLKLSCAASGFTF 31NTYAMNWVRQAPGKGLEWVARIRSKS 61NNYATYYADSVKDRFTISRDDSQSMLYLQ 91MNNLKKEDTAMYYCVGYGSGGNYWGQG 121TSVTVSSASTKGPSVFPLAPSSKSTSGGTA 151ALGCLVKDYFPEPVTVSWNSGALTSGVHTF 181PAVLQSSGLYSLSSVVTVPSSSLGTQTYIC 211NVNHKPSNTKVDKKVEPKSCDKTHTCPPCP 241APELLGGPSVFLFPPKPKDTLMISRTPEVT 271CVVVDVSHEDPEVKFNWYVDGVEVHNAKTK 301PREEQYNSTYRVVSVLTVLHQDWLNGKEYK 331CKVSNKALPAPIEKTISKAKGQPREPQVYT 361LPPSRDELTKNQVSLTCLVKGFYPSDIAVE 391WESNGQPENNYKTTPPVLDSDGSFFLYSKL 421TVDKSRWQQGNVFSCSVMHEALHNHYTQKS 451LSLSPGK

(147) FIG. 2b: Complete amino acid sequence of mouse FG129/29 kappa chain variable region chimerised to human kappa chain constant region. (SEQ ID NO: 15)

(148) TABLE-US-00010 -19MESQTQVLMSLLFWVSTCG 1DIVMTQSPSSLTVTAGEKVTMSCKSSQSLL 31NSGNQKNYLTWYQQKPGQPPKVLIYWA 61STRESGVPDRFTGSGSGTDFTLT 91ISSVQAEDLAVYYCQNDYSSPFTFGSGTKL 121EIKRTVAAPSVFIFPPSDEQLKSGTASVVC 151LLNNFYPREAKVQWKVDNALQSGNSQESVT 181EQDSKDSTYSLSSTLTLSKADYEKHKVYAC 211EVTHQGLSSPVTKSFNRGEC

REFERENCES CITED IN THE DESCRIPTION

(149) 1. Hakomori S. Glycosylation defining cancer malignancy: new wine in an old bottle. Proc Natl Acad Sci USA. 2002; 99:10231-3. 2. Fuster M M, Esko J D. The sweet and sour of cancer: glycans as novel therapeutic targets. Nat Rev Cancer. 2005; 5:526-42. 3. Varki A, Kannagi R, Toole B P. Glycosylation Changes in Cancer. In: Varki A, Cummings R D, Esko J D, et al., editors. Essentials of Glycobiology. 2010/03/20 ed: Cold Spring Harbor (N.Y.); 2009. 4. Pour P M, Tempero M M, Takasaki H, Uchida E, Takiyama Y, Burnett D A, et al. Expression of blood group-related antigens ABH, Lewis A, Lewis B, Lewis X, Lewis Y, and CA 19-9 in pancreatic cancer cells in comparison with the patient's blood group type. Cancer Res. 1988; 48:5422-6. 5. Rabu C, McIntosh R, Jurasova Z, Durrant L. Glycans as targets for therapeutic antitumor antibodies. Future Oncol. 2012; 8:943-60. 6. Tang H, Singh S, Partyka K, Kletter D, Hsueh P, Yadav J, et al. Glycan motif profiling reveals plasma sialyl-lewis x elevations in pancreatic cancers that are negative for sialyl-lewis a. Molecular & cellular proteomics: MCP. 2015; 14:1323-33. 7. Inagaki H, Sakamoto J, Nakazato H, Bishop A E, Yura J. Expression of Lewis(a), Lewis(b), and sialated Lewis(a) antigens in early and advanced human gastric cancers. J Surg Oncol. 1990; 44:208-13. 8. Sakamoto J, Furukawa K, Cordon-Cardo C, Yin B W, Rettig W J, Oettgen H F, et al. Expression of Lewis.sup.a, Lewisb, X, and Y blood group antigens in human colonic tumors and normal tissue and in human tumor-derived cell lines. Cancer Res. 1986; 46:1553-61. 9. Hakomori S, Andrews H D. Sphingoglycolipids with Leb activity, and the co-presence of Lea-, Leb-glycolipids in human tumor tissue. Biochim Biophys Acta. 1970; 202:225-8. 10. Koprowski H, Steplewski Z, Mitchell K, Herlyn M, Herlyn D, Fuhrer P. Colorectal carcinoma antigens detected by hybridoma antibodies. Somatic cell genetics. 1979; 5:957-71. 11. Stanley P, Cummings R D. Structures Common to Different Glycans. In: Varki A, Cummings R D, Esko J D, et al., editors. Essentials of Glycobiology. 2010/03/20 ed: Cold Spring Harbor (N.Y.); 2009. 12. Kannagi R. Carbohydrate antigen sialyl Lewis aits pathophysiological significance and induction mechanism in cancer progression. Chang Gung medical journal. 2007; 30:189-209. 13. Kishimoto T, Ishikura H, Kimura C, Takahashi T, Kato H, Yoshiki T. Phenotypes correlating to metastatic properties of pancreas adenocarcinoma in vivo: the importance of surface sialyl Lewis(a) antigen. International journal of cancer Journal international du cancer. 1996; 69:290-4. 14. Sato M, Narita T, Kimura N, Zenita K, Hashimoto T, Manabe T, et al. The association of sialyl Lewis(a) antigen with the metastatic potential of human colon cancer cells. Anticancer research. 1997; 17:3505-11. 15. Matsui T, Kojima H, Suzuki H, Hamajima H, Nakazato H, Ito K, et al. Sialyl Lewisa expression as a predictor of the prognosis of colon carcinoma patients in a prospective randomized clinical trial. Japanese journal of clinical oncology. 2004; 34:588-93. 16. Nakayama T, Watanabe M, Katsumata T, Teramoto T, Kitajima M. Expression of sialyl Lewis(a) as a new prognostic factor for patients with advanced colorectal carcinoma. Cancer. 1995; 75:2051-6. 17. O'Brien D P, Sandanayake N S, Jenkinson C, Gentry-Maharaj A, Apostolidou S, Fourkala E O, et al. Serum CA19-9 is significantly upregulated up to 2 years before diagnosis with pancreatic cancer: implications for early disease detection. Clinical cancer research: an official journal of the American Association for Cancer Research. 2015; 21:622-31. 18. Sawada R, Sun S M, Wu X, Hong F, Ragupathi G, Livingston P O, et al. Human monoclonal antibodies to sialyl-Lewis (CA19.9) with potent CDC, ADCC, and antitumor activity. Clinical cancer research: an official journal of the American Association for Cancer Research. 2011; 17:1024-32. 19. Huehls A M, Coupet T A, Sentman C L. Bispecific T-cell engagers for cancer immunotherapy. Immunology and cell biology. 2015; 93:290-6. 20. Kontermann R E. Dual targeting strategies with bispecific antibodies. mAbs. 2012; 4:182-97. 21. Ward E S, Gussow D, Griffiths A D, Jones P T, Winter G. Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature. 1989; 341:544-6. 22. Bird R E, Hardman K D, Jacobson J W, Johnson S, Kaufman B M, Lee S M, et al. Single-chain antigen-binding proteins. Science. 1988; 242:423-6. 23. Huston J S, Levinson D, Mudgett-Hunter M, Tai M S, Novotny J, Margolies M N, et al. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci USA. 1988; 85:5879-83. 24. Holliger P, Prospero T, Winter G. Diabodies: small bivalent and bispecific antibody fragments. Proc Natl Acad Sci USA. 1993; 90:6444-8. 25. Holliger P, Winter G. Engineering bispecific antibodies. Current opinion in biotechnology. 1993; 4:446-9. 26. Traunecker A, Lanzavecchia A, Karjalainen K. Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells. The EMBO journal. 1991; 10:3655-9. 27. Karlin S, Altschul S F. Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. Proc Natl Acad Sci USA. 1990; 87:2264-8. 28. Karlin S, Altschul S F. Applications and statistics for multiple high-scoring segments in molecular sequences. Proc Natl Acad Sci USA. 1993; 90:5873-7. 29. Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. Basic local alignment search tool. Journal of molecular biology. 1990; 215:403-10. 30. Altschul S F, Madden T L, Schaffer A A, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic acids research. 1997; 25:3389-402. 31. Myers E W, Miller W. Approximate matching of regular expressions. Bulletin of mathematical biology. 1989; 51:5-37. 32. Torelli A, Robotti C A. ADVANCE and ADAM: two algorithms for the analysis of global similarity between homologous informational sequences. Computer applications in the biosciences: CABIOS. 1994; 10:3-5. 33. Pearson W R, Lipman D J. Improved tools for biological sequence comparison. Proc Natl Acad Sci USA. 1988; 85:2444-8. 34. Marks J D, Griffiths A D, Malmqvist M, Clackson T P, Bye J M, Winter G. By-passing immunization: building high affinity human antibodies by chain shuffling. Bio/technology. 1992; 10:779-83. 35. Stemmer W P. Rapid evolution of a protein in vitro by DNA shuffling. Nature. 1994; 370:389-91. 36. Gram H, Marconi L A, Barbas C F, 3rd, Collet T A, Lerner R A, Kang A S. In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library. Proc Natl Acad Sci USA. 1992; 89:3576-80. 37. Barbas C F, 3rd, Hu D, Dunlop N, Sawyer L, Cababa D, Hendry R M, et al. In vitro evolution of a neutralizing human antibody to human immunodeficiency virus type 1 to enhance affinity and broaden strain cross-reactivity. Proc Natl Acad Sci USA. 1994; 91:3809-13. 38. Schier R, McCall A, Adams G P, Marshall K W, Merritt H, Yim M, et al. Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site. Journal of molecular biology. 1996; 263:551-67. 39. Sidman K R, Steber W D, Schwope A D, Schnaper G R. Controlled release of macromolecules and pharmaceuticals from synthetic polypeptides based on glutamic acid. Biopolymers. 1983; 22:547-56. 40. Langer R, Brem H, Tapper D. Biocompatibility of polymeric delivery systems for macromolecules. Journal of biomedical materials research. 1981; 15:267-77. 41. R. P. R. Remington's Pharmaceutical Sciences. 16th ed: Mack Pub. Co; 1980. 42. Stewart J M. Solid Phase Peptide Synthesis. 2nd ed: Pierce Chemical Co; 1984. 43. Miklos Bodanszky A B, Barry M. Trost The Practice of Peptide Synthesis 1984. 44. Pluckthun A. Antibody engineering: advances from the use of Escherichia coli expression systems. Bio/technology. 1991; 9:545-51. 45. Reff M E. High-level production of recombinant immunoglobulins in mammalian cells. Current opinion in biotechnology. 1993; 4:573-6. 46. Trill J J, Shatzman A R, Ganguly S. Production of monoclonal antibodies in COS and CHO cells. Current opinion in biotechnology. 1995; 6:553-60. 47. Sambrook J. Molecular Cloning: A Laboratory Manual. 2nd ed; 1989. 48. Ausubel F M. Short Protocols in Molecular Biology; 1992. 49. Lefranc M P, Giudicelli V, Ginestoux C, Jabado-Michaloud J, Folch G, Bellahcene F, et al. IMGT, the international ImMunoGeneTics information system. Nucleic acids research. 2009; 37:D1006-12. 50. Jones S T, Bendig M M. Rapid PCR-cloning of full-length mouse immunoglobulin variable regions. Bio/technology. 1991; 9:579. 51. Simpson J A, Al-Attar A, Watson N F, Scholefield J H, Ilyas M, Durrant L G. Intratumoral T cell infiltration, MHC class I and STAT1 as biomarkers of good prognosis in colorectal cancer. Gut. 2010; 59:926-33. 52. Duncan T J, Rolland P, Deen S, Scott I V, Liu D T, Spendlove I, et al. Loss of IFN gamma receptor is an independent prognostic factor in ovarian cancer. Clinical cancer research: an official journal of the American Association for Cancer Research. 2007; 13:4139-45. 53. Abdel-Fatah T, Arora A, Gorguc I, Abbotts R, Beebeejaun S, Storr S, et al. Are DNA repair factors promising biomarkers for personalized therapy in gastric cancer?Antioxidants & redox signaling. 2013; 18:2392-8. 54. Storr S J, Zaitoun A M, Arora A, Durrant L G, Lobo D N, Madhusudan S, et al. Calpain system protein expression in carcinomas of the pancreas, bile duct and ampulla. BMC cancer. 2012; 12:511. 55. Kohls M D, Lappi D A. Mab-ZAP: a tool for evaluating antibody efficacy for use in an immunotoxin. BioTechniques. 2000; 28:162-5.