Antibody drug conjugate platform using bispecific antibody

Abstract

The present invention relates to an antibody drug conjugate using a bispecific antibody and its use. The antibody drug conjugate of the present invention can easily form a conjugate of an antibody and a drug without a multistep synthetic process though binding of a conjugate of a bivalent cotinine-peptide and a drug, and an anti-cotinine single chain variable fragment. In addition, the antibody drug conjugate can effectively deliver a drug to a target to which the antibody binds specifically, and can enhance a half-life of a drug in the body, thereby improving a therapeutic effect.

Claims

1. An antibody drug conjugate comprising: a bispecific antibody comprising an anti-cotinine single chain variable fragment (scFv); and a conjugate of bivalent cotinine which is cross-linked with a peptide, and a drug, wherein the anti-cotinine single chain variable fragment (scFv) consists of a heavy chain of SEQ ID NO: 2 and a light chain of SEQ ID NO: 4, wherein the peptide which is cross-linked with bivalent cotinine is 6-18 mer peptide consisting of one or more selected from the consisting of glycine (G), serine (S) and lysine (K).

2. The antibody drug conjugate according to the claim 1, wherein the bivalent cotinine is that two cotinines are cross-linked to the N terminal and C-terminal of a 6-18 mer peptide, respectively.

3. The antibody drug conjugate according to the claim 1, wherein the drug is conjugated with a lysine residue in the 6-18 mer peptide cross-linked with the bivalent cotinine.

4. The antibody drug conjugate according to the claim 1, wherein the bispecific antibody is that a peptide linker (Gly-Gly-Gly-Gly-Ser).sub.3 is inserted between C.sub.H3 domain in heavy-chain constant region of the bispecific antibody and the anti-cotinine single chain variable fragment (scFv).

5. The antibody drug conjugate according to the claim 1, wherein the drug is any one selected from the group consisting of duocarmycin, auristatin, colchicine, anthracycline, calicheamicin, maytansinoid, pyrrolobenzodiazepine, dolastatin, tubulysin, maytansinoid, doxorubicin, cryptophycin, epothilone, safranin, deacetyl colchicine, maytansinol, vedotin, mafodotin, emtansine, mertansine, ravtansine, soravtansine, talirine, tesirine, indolinobenzodiazepines, irinotecan prodrug, exatecan derivative and tubulin inhibitor; or siRNA inhibiting expression of a gene occurring cancer.

6. The antibody drug conjugate according to the claim 1, wherein the bispecific antibody comprises any one selected from the group consisting of cetuximab, trastuzumab, oregovomab, edrecolomab, alemtuzumab, labetuzumab, bevacizumab, ibritumomab, ofatumumab, panitumumab, rituximab, tositumomab, ipilimumab, gemtuzumab, brentuximab, vadastuximab, glembatumumab, depatuxizumab, polatuzumab, denintuzumab, enfortumab, telisotuzumab, tisotumab, pinatuzumab, lifastuzumab, indusatumab, vandortuzumab, sofituzumab, vorsetuzumab, trastuzumab, mirvetuximab, coltuximab, naratuximab, indatuximab, anetumab, lorvotuzumab, cantuzumab, laprituximab, bivatuzumab, vadastuximab, rovalpituzumab, inotuzumab, sacituzumab, labetuzumab, milatuzumab, lupartumab and aprutumab.

7. A pharmaceutical composition for treating cancer comprising the antibody drug conjugate of claim 1.

8. The pharmaceutical composition for treating cancer according to claim 7, wherein the cancer is lung adenocarcinoma having KRAS mutation.

9. A method for treating cancer in a patient comprising a step of administering a therapeutically effective dose of the antibody drug conjugate of claim 1 to the patient.

10. A method for enhancing a half-life of a drug in a patient comprising a step of administering a therapeutically effective dose of the antibody drug conjugate of claim 1 to the patient.

11. A method for preparing an antibody drug conjugate comprising: (s1) a step of preparing a bispecific antibody comprising an anti-cotinine single chain variable fragment (scFv); (s2) a step of preparing a conjugate of a bivalent cotinine which is cross-linked with a peptide and a drug; and (s3) a step of mixing the bispecific antibody produced in the (s1) step and the conjugate produced in the (s2) step.

12. The method for preparing an antibody drug conjugate according to claim 11, wherein the bivalent cotinine is that two cotinines are cross-linked to the N terminal and C-terminal of a 6-18 mer peptide, respectively.

13. The method for preparing an antibody drug conjugate according to claim 11, wherein the drug is conjugated with a lysine residue in the 6-18 mer peptide cross-linked with the bivalent cotinine.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1a, 1b, 1c and 1d show production and characteristics of bispecific (cetuximab×anti-cotinine) antibody (ERC6). FIG. 1a is a schematic diagram of the antibody drug conjugate (ADC) using the bispecific antibody. The bispecific (cetuximab×anti-cotinine) antibody (ERC6) is designed to have binding specificity to both human EGFR and cotinine payloads. FIG. 1b shows the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) result. The purified ERC6 was inserted to 4-12% (w/v) SDS-PAGE. Bands were visualized by staining the gel with Coomassie Brilliant Blue. The lane 1 or 2 is a sample with or without a reducing agent, respectively. FIG. 1c is size-exclusion chromatography (SEC). The purified ERC6 was analyzed through SEC using high pressure liquid chromatography (SEC-HPLC). FIG. 1d show the pharmacokinetic analysis result of ERC6. 200 μg ERC6 was intravenously injected to Balb/c mice (n=4), and the blood sample was collected through the vein in the orbit of the eye. The circulating serum level of ERC6 was analyzed by enzyme-linked immunosorbent analysis (ELISA). The result was shown as mean±SD obtained from the experiments performed three times.

(2) FIGS. 2a, 2b, 2c and 2d show the binding reactivity of ERC6 to human EGFR and cotinine. FIG. 2a shows the enzyme immunoassay result. Cetuximab, anti-cotinine-IgG and ERC6 were added to wells of a microtiter plate coated with human EGFR or cotinine. The wells were probed with HRP-complexed anti-human IgG (Fab-specific) antibody. FIG. 2b shows the chemical structure of the cross-linked dPEG6 biotin and bivalent cotinine-conjugated peptide (cotinine-biotin). FIG. 2c is the result of adding cetuximab, anti-cotinine-IgG and ERC6 to other wells of the microtiter plate coated with human EGFR and then adding cotinine-biotin and detecting wells with HRP-conjugated streptavidin. The background signal was measured in control wells coated with BSA. The absorbance was measured at 650 nm. FIG. 2d shows the pharmacokinetic analysis of ERC6-complexed cotinine-biotin. 200 μg ERC6 pre-cultured with 1.85 μg cotinine-biotin dissolved in 100 μL sterile PBS was intravenously injected to Balb/c mice (n=4). The blood sample was collected through the vein in the orbit of the eye and the circulating serum level of the ERC6-complexed cotinine-biotin was measured by ELISA. The result was shown as mean±SD obtained from the experiments performed three times.

(3) FIGS. 3a, 3b, 3c, 3d and 3e show the enhanced anti-proliferative effect of the ERC6-complexed cotinine-duocarmycin in the EGFR positive lung adenocarcinoma cell line. FIG. 3a is the flow cytometry result. With or without cotinine-biotin, A549 cells were cultured with cetuximab, anti-cotinine-IgG or ERC6. For analysis, the cells were detected with PE-conjugated streptavidin and FITC-conjugated anti-human Fc. FIG. 3b shows the structure of free duocarmycin. FIG. 3c shows the chemical structure of the bivalent conjugated peptide cross-linked with four duocarmycins (cotinine-duocarmycin). R represents valine-citrulline PAB-linked dimethyl aminoethyl duocarmycin. FIG. 2d is the cell viability analysis result of free duocarmycin. FIG. 3e is the cell viability analysis result of cotinine-duocarmycin. The A549 cell line was treated with palivizumab and DMSO (.circle-solid.); cetuximab and DMSO (.square-solid.); ERC6 and DMSO (.box-tangle-solidup.); palivizumab and duocarmycin (∘); cetuximab and duocarmycin (□); ERC6 and duocarmycin (Δ). Palivizumab was used as the isotype control of bispecific antibody. DMSO was used as the vehicle control of cotinine-duocarmycin. After culturing cells for 72 hrs, the cell ATP content was measured using Cell Titer-Glo reagent, thereby measuring the relative cell viability. The result was shown as mean±SD obtained from the experiments performed three times.

(4) FIGS. 4a, 4b, 4c, 4d and 4e show the enhance anti-proliferative effect of the ERC6-complexed cotinine duocarmycin in the mouse xenograft tumor model. A549 cells were subcutaneously injected to the left and right flanks of each Balb/c-nude mouse. When the tumor volume reached 150 mm.sup.3, mice were divided to three groups randomly (n=4/group) and treated for 5 weeks. Each group was intraperitoneally injected with palivizumab and cotinine-duocarmycin, ERC6 and vehicle or ERC6-complexed cotinine-duocarmycin. Palivizumab was used as the isotype control of bispecific antibody. DMSO was used as the vehicle control of cotinine duocarmycin. FIG. 4a is the result of measuring the tumor volume for 32 days, FIG. 4b is the result of observing the body weight during the treatment period. FIG. 4c is the average tumor volume at the day 32. FIG. 4d is the average mass of dissected tumor when mice were sacrificed. FIG. 4e shows the photograph of tumor of three treatment groups at the end point. The result was shown as mean±SD; compared with the group, *P<0.05, **P<0.01, Student's t test.

(5) FIG. 5 is the pharmacokinetic analysis result of ERC6-complexed cotinine-biotin. (a) 200 μg of ERC6 pre-cultured with 964 pmole of cotinine-biotin dissolved in 100 μL sterile PBS at the molar ratio of 1:1 was intravenously injected to Balb/c mice (n=4). The peptide has various lengths of 6mer, 12mer or 18mer between two cotinine molecules. The blood sample was collected through the vein in the orbit of the eye, and the circulating serum level of the ERC6-complexed cotinine-biotin was determined by ELISA. (b) The circulating serum level of the total ERC6 was measured by ELISA. The result was shown as mean±SD; compared with the group, *P<0.05, **P<0.01, Student's t test.

(6) FIG. 6 shows the anti-proliferative effect of the ERC6-complexed cotinine-DM1 (emtansine) in the EGFR-positive lung adenocarcinoma cell line. FIG. 6a shows the chemical structure of the bivalent cotinine-conjugated peptide cross-linked with four DM1s (cotinine-DM1). R represents MCC-linked DM1. FIG. 6b is the cell viability analysis result of cotinine-DM1 (emtansine). The A549 cell line was treated with palivizumab and DMSO (.circle-solid.); cetuximab and DMSO (.square-solid.); ERC6 and DMSO (.box-tangle-solidup.); palivizumab and cotinine-DM1 (∘); cetuximab and cotinine-DM1 (□); ERC6 and cotinine-DM1 (Δ). Palivizumab was used as the isotype control of bispecific antibody. DMSO was used as the vehicle control of cotinine-duocarmycin. After culturing cells for 72 hrs, the cell ATP content was measured using Cell Titer-Glo reagent, thereby measuring the relative cell viability. The result was shown as mean±SD obtained from the experiments performed three times.

DETAILED DESCRIPTION

(7) Hereinafter, examples, etc. will be described in detail to facilitate understanding of the present invention. However, the examples according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The examples of the present invention are provided to enable those skilled in the art to more completely understand the present invention.

Example 1: Cell Culture

(8) A549 (human lung adenocarcinoma) cells were obtained from the Korean Cell Line Bank and grown in RPMI-1640 media (Welgene, Seoul, Republic of Korea) supplemented with 10% heat inactivated fetal bovine serum (GIBCO, Grand Island, N.Y., USA), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere with 5% CO.sub.2. HEK293F cells (Invitrogen, Carlsbad, Calif., USA) were grown in FreeStyle™ 293 Expression medium (GIBCO), containing 100 U/mL penicillin and 100 μg/mL streptomycin in Erlenmeyer tissue culture flask with vent cap (Corning Inc., NY, USA) at 37° C. in 70% humidified atmosphere with 7% CO.sub.2 in an orbital shaking incubator (Minitron, INFORS HT, Bottmingen, Switzerland) at 135 rpm.

Example 2: Construction and Purification of Bispecific Cetuximab×Anti-Cotinine Antibody

(9) To construct bispecific cetuximab×anti-cotinine antibody expression vector, genes encoding cetuximab light chain and cetuximab heavy chain-linker(Gly-Gly-Gly-Gly-Ser).sub.3-anti-cotinine single chain variable fragment (scFv) were chemically synthesized (Genscript, Picataway, N.J., USA). Restriction sites, AgeI and XbaI were inserted at the 5′ and 3′ ends of the gene encoding cetuximab light chain, respectively. Additional restriction sites, NheI and BsiWI were inserted at the 5′ end of the gene encoding cetuximab heavy chain and the 3′ end of the gene of anti-cotinine scFv. Light chain and heavy chain-linker-anti-cotinine-scFv was sub-cloned into mammalian expression vector designed for secretion of recombinant protein as the method described in Park S, Lee D H, Park J G, Lee Y T, Chung J. A sensitive enzyme immunoassay for measuring cotinine in passive smokers. Clin Chim Acta 2010; 411(17-18): 1238-42.

(10) As the method described in Boussif O, Lezoualc'h F, Zanta M A, Mergny M D, Scherman D, Demeneix B et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Aced Sci USA 1995; 92(16): 7297-301, the expression vector encoding ERC6 was transfected into HEK293F (Invitrogen) using 25-kDa linear polyethyleneimine (Polyscience, Warrington, Pa., USA). As the method described in Kim H, Park S, Lee H K, Chung J. Application of bispecific antibody against antigen and hapten for immunodetection and immunopurification. Exp Mol Med 2013; 45: e43, the ERC6 was purified from the culture supernatant by affinity chromatography using protein A agarose bead (RepliGen, Waltham, Mass., USA).

Example 3: Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

(11) The analysis was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using NuPage 4-12% Bis-Tris gel (Invitrogen) according to the manufacturer's instructions. The gel was stained with Coomassie Brilliant Blue R-250 (Amresco, Colon, Ohio, USA).

Example 4: Size-Exclusion Chromatography (SEC)

(12) Purified ERC6 was analyzed by SEC-HPLC, using Agilent 1260 Infinity high pressure liquid chromatography (HPLC) system equipped with Bio SEC-3 column (7.8×300 mm) packaged with 3 μm particles containing *57300 Å pores (Agilent 1260, Agilent technologies Inc., CA, USA). The mobile phase comprised 50 mM sodium phosphate and 150 mM sodium chloride of pH 7.0. 20 μL of ERC6 (1 mg/ml) was injected and eluted isocratically at the flow velocity of 1 mL/min for 30 min. The column effluence was monitored with a ultraviolet ray detector at 280 nm and represented as mAU. The percentage of monomers, aggregates and fragments was quantified on the basis of peak area.

Example 5: Synthesis of Cotinine Conjugates

(13) All peptides used in the present experiment were chemically synthesized using Fmoc-solid-phase peptide synthesis (Peptron, Republic of Korea). Two trans-4′-cotininecarboxylic acids (Sigma-Aldrich, St Louis, Mo., USA) were cross-linked to free amine groups at the N-terminus and C-terminus of GGGGSKGGGGSK and GSKGSKGSKGSKK peptides. After eliminating the allyloxycarbonyl (alloc) group on lysine at the middle of peptides with triphenylphosphine (tetrakis) palladium, dPEG6 (Peptide international Inc., Louisville, Ky., USA) was conjugated to free amine group of GGGGSKGGGGSK peptide. Then, biotin was cross-linked to free amine on dPEG6-conjugated peptide (Peptron). For simplification, the bivalent cotinine-conjugated peptide cross-linked with biotin (cotinine-GGGGSK[(dPEG6)-biotin]GGGGSK-cotinine) was abbreviated to cotinine-biotin.

(14) Four valine-citrulline PAB-linked dimethyl aminoethyl duocarmycins were conjugated to free amino acid groups present in 4 lysines in bivalent cotinine-cross-linked GSKGSKGSKGSKK peptide (Concortis, San Diego, Calif., USA). For simplification, the bivalent cotinine-conjugated peptide cross-linked with 4 duocarmycins (cotinine-[GSK(duocarmycin)].sub.4K-cotinine) was abbreviated to cotinine-duocarmycin.

(15) The cotinine-biotin and cotinine-duocarmycin were purified via reverse-phase using a C18 column. After purification, they were analyzed and validated by mass spectrometry using Agilent 1100 capillary LC and HPLC system equipped with Capcell Pak C18 column (4.6×50 mm, 120 Å) (Shimadzu Corp., Kyoto, Japan).

Example 6: Complexation of ERC6 and Cotinine Payloads

(16) To produce complexes of ERC6 and cotinine-conjugated payloads, the cotinine payloads and ERC6 were mixed though pipetting up and down at the molar ratio of 1:1. Then, the mixture was cultured at the room temperature for 30 min. Then, the complexes were used in vitro or in vivo without additional modifications.

Example 7: Enzyme-Linked Immunosorbent Assay (ELISA)

(17) The wells of a 96-well microtiter plate (Corning) were coated with human EGFR (Sigma) or BSA-conjugated cotinine in coating buffer (0.1M sodium bicarbonate in distilled water, pH 8.6) overnight at 4° C. and blocked with 3% bovine serum albumin (BSA) in PBS for 1 h at 37° C. The antibody at concentrations of 1 μg/mL in 50 μL blocking buffer was added to each well and incubated for 2 hrs at 37° C. After washing with 0.05% Tween 20 in PBS (PBST), HRP-conjugated anti-human IgG (Fab specific) antibodies (Sigma) diluted in the blocking buffer was added and incubated for 1 hr at 37° C. Then, after washing the plate again with 0.05% PBST, 50 μL 3,3′,5,5′-tetramethyl benzidine substrate solution (TMB) (GenDEPOT, Barker, Tex., USA) was added to each well, and the absorbance was measured at 650 nm with a Multiskan Ascent microplate reader (Labsystems, Helsinki, Finland).

(18) To confirm bispecificity of ERC6, the human EGFR-coated wells were cultured with antibodies as above. After washing with 0.05% PBST, the cotinine-biotin was added to each well and cultured for 1 hr at 37° C. Then, HRP-conjugated streptavidin (Thermo Fishers Scientific, Waltham, Mass., USA) diluted in the blocking buffer was added and incubated for 1 hr at 37° C. After washing, TMB was added to each well, and the absorbance was measured at 650 nm.

Example 8: Pharmacokinetic Analysis

(19) All the animals used in the present experiment was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) of National Cancer Center Research Institute, Republic of Korea (Permit number: NCC-15-267). The animals were maintained in the National Cancer Center animal facility in accordance with the AAALAC International Animal Care Policy.

(20) Eight-week-old male Balb/c mice were intravenously injected with the complex of ERC6 (200 μg) and cotinine-biotin (1.85 μg) in one-to-one molar ratio dissolved in 100 μL sterile PBS (n=4/group). Blood samples were collected from intra-orbital vein at 0, 1, 24, 48, 72, 96 and 168 hrs post-injection. The samples were left at the room temperature for 2 hrs until the blood coagulated. Subsequently, serum was acquired by centrifuging at 3500 rpm for 15 min at 4° C. The circulating serum levels of total ERC6 and ERC6-complexed cotinine-biotin were determined by enzyme-linked immunosorbent assays (ELISAs).

(21) Total ERC6 in the serum samples was measured as follows: the wells of a 96-well microtiter plate (Corning) were coated with goat anti-human IgG (Fc specific) capturing antibody (EMD Millipore, Darmstadt, Germany) in coating buffer overnight at 4° C. and blocked with 3% BSA in PBS for 1 hr at 37° C. Serum samples diluted in blocking buffer and standard solution were added to each well and incubated for 2 hrs at 37° C. After washing with 0.05% PBST, HRP-conjugated anti-human C.sub.kappa antibody (Thermo Fisher Scientific, Waltham, Mass., USA) diluted in the blocking buffer was added and incubated for 1 hr at 37° C. After washing, TMB (GenDEPOT) was added to each well and the absorbance was measured at 650 nm.

(22) The complex of ERC6 and cotinine-biotin in the serum samples was measured as follows: the wells coated with human EGFR (Sigma) were cultured with serum samples as aforementioned. After washing with 0.05% PBST, HRP-conjugated streptavidin (Thermo Fisher Scientific Pierce) was added and cultured for 1 hr at 37° C. After washing, TMB was added to each well and the absorbance was measured at 650 nm.

Example 9: Flow Cytometry

(23) A549 cells were seeded in a v-bottom 96-well plate (Corning) in the final concentration of 4×10.sup.5 cells per each sample. The cells were treated with 0 or 100 nM of ERC6 and cotinine-biotin diluted in the flow cytometry buffer (0.1% [w/v] sodium azide containing 1% [w/v] BSA in PBS) for 30 min at 37° C. For control experiments, instead of ERC6, palivizumab (Synagis, Boehringer Ingelheim Pharma, Biberach an der Riss, Germany), anti-cotinine-IgG or cetuximab (Erbitux, Merck K GaA, Darmstadt, Germany) was used. After washing with the flow cytometry buffer, the cells were cultured with phycoerythrin (PE)-conjugated streptavidin (BD Biosciences Pharminogen, San Diego, Calif., USA) and FITC-conjugated anti-human IgG (Fc specific) antibody (Thermo Fisher Scientific Pierce) in the dark for 1 hr at 37° C. After additional washing with the same buffer, the cells were resuspended in 200 μL PBS, and analyzed through flow cytometry using FACS Canto II instrument equipped with a 488-nm laser (BD Bioscience, San Jose, Calif., USA). 10,000 cells per measurement were detected and data were analyzed with FlowJo software (TreeStar, Ashland, Oreg., USA).

Example 10: Cell Viability Assays

(24) The effect of ERC6 and cotinine-duocarmycin on tumor cell viability was evaluated using Cell Titer-Glo reagent (Promega Corp., Madison, Wis., USA). A549 cells were seeded in 50 μL RPMI-1640 medium in black-walled 96-well plates (4,000 cells per well) and allowed to adhere overnight at 37° C. in 5% CO.sub.2 and humidified atmosphere. Subsequently, the complexes of ERC6 and cotinine-duocarmycin were serially diluted 10-fold in fresh culture medium (0.02 nM-2000 nM). In the control experiments, instead of ERC6, palivizumab (Boehringer Ingelheim Pharma), or cetuximab (Merck K GaA) was used. The cotinine-duocarmycin and antibody dilutes in 50 μL medium were added to each well, and cultured for 72 hrs. After adding Cell Titer-Glo reagent (Promega Corp.) to each well, luminescence signals were measured using a microplate luminometer (PerkinElmer, Waltham, Mass., USA) according to the manufacturer's instructions. All experiments were conducted in triplicate. The relative cell viability was calculated by dividing by the control luminescence signal [% viability=(Test−Background)/(Control−Background)×100].

Example 11: Xenograft

(25) Six-week-old female Balb/c-nude mice were subcutaneously injected with the A549 (1×10.sup.7 cells) on the left and right flank of each mouse. When the tumor volume reached approximate 150 mm.sup.3, all animals were randomly divided into three groups (n=4/group) and treated for five weeks. Mice were intraperitoneally injected with appropriate controls twice a week for the first two weeks: Group I was administered with palivizumab (2.15 mg/kg) and cotinine-duocarmycin (95 μg/kg); Group II was administered with the ERC6 (3 mg/kg) and dimethyl sulfoxide (DMSO); Group III was administered with the complex of ERC6 (3 mg/kg) and cotinine-duocarmycin (95 μg/kg). Then, three times dose of the drugs were injected thrice a week for the following three weeks. Palivizumab was used as an isotype control of the bispecific antibody. DMSO was used as a vehicle control of cotinine-duocarmycin. The tumor volume was measured using digital calipers twice a week in 32 days after injection. Tumor volume was calculated as length×(width).sup.2×0.5, where the length is the longest axis and the width is the distance perpendicular to the length. Systemic toxicity was evaluated by measuring body weight twice a week. Mice were sacrificed on the day 35 after injection and tumors were dissected and weighted.

Example 12: Immunofluorescence

(26) Balb/c-nude mice were subcutaneously injected with the A549 (1×10.sup.7 cells) on the left and right flank. When the tumor volume reached 500 mm.sup.3, mice having tumors were administered by the following single intraperitoneal (i.p.) injection: Group I was administered with 144 μg palivizumab and 1.85 μg cotinine-biotin; Group II was administered with 200 μg ERC6 and vehicle (distilled water); Group III was administered with the complex of ERC6 (200 μg) and cotinine-biotin (1.85 μg). In 24 hrs after injection, mice were anesthetized with isoflurane and euthanized through transcardial perfusion with 10 ml of 4% [w/v] paraformaldehyde in PBS. The dissected tumors were equilibrated in cryopreservation solution containing 30% [w/v] sucrose in PBS for 24 hrs at 4° C., and frozen in the optimum cutting temperature embedding medium (Sakura Finetek, Torrance, Calif., USA) in liquid nitrogen, and stored at −80° C. until cutting. For immunofluorescent staining, cryosections were prepared in 4 μm thickness, and fixed with 4% paraformaldehyde in PBS for 10 min at the room temperature. After washing with PBS, the sections were blocked with 10% [v/v] normal goat serum (CST, Danvers, Mass., USA) in IHC-Tek antibody diluted solution (pH 7.4) (IHC WORLD, Woodstock, Md., USA) for 1 hr at the room temperature. The tissue sections were cultured with Alexa Fluor 488-conjugated streptavidin (Molecular Probes Inc., Eugene, Oreg., USA for 8 hrs, and stained with Alexa Fluor 594-conjugated anti-human IgG antibody (Molecular Probes Inc.) in the dark and humidified chamber of 4° C. for 16 hrs. After washing with PBS, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Pierce, Rockford, Ill., USA) according to the manufacturer's instructions. The sections were fixed on slides with fluorescence fixed medium (DAKO, Glostrup, Denmark) and 40-fold enlarge images were obtained with FV10 ASW software using FV1000 laser scanning microscope (Olympus, Tokyo, Japan). The emission and excitation filters were arranged so as to be imaged in three colors.

(27) Statistical Analysis

(28) Statistics used in the experiments were carried out using GraphPad Prism version 5.0 software (GraphPad Software Inc., San Diego, Calif., USA). Results were expressed as mean values of ±standard deviations (SD) for the indicated number of independent measurements. Statistical significance was determined using two tailed unpaired Student's t-test and P-values of less than 0.05 were considered statistically significant. P values are indicated in the figures and in their legends.

Experimental Example 1: Design of Antibody Drug Conjugate Platform Using Tetravalent Bispecific Antibody Format

(29) Bispecific antibodies were designed in the IgG-based tetravalent format by fusing two single chain variable fragments (scFvs) to C.sub.H3 domain of heavy chain (FIG. 1a). For flexibility, the glycine and serine rich peptide linker [(Gly-Gly-Gly-Gly-Ser).sub.3] was inserted between the C.sub.H3 domain and scFv. Hereby, the tetravalent antibodies containing 2 of Fab arms of bispecific antibody and 2 of scFvs of C-terminus of heavy chain were produced, thereby simultaneously targeting epidermal growth factor receptor (EGFR) and cotinine, respectively. To develop bispecific cetuximab×anti-cotinine scFv antibodies (ERC6), the gene structure encoding cetuximab IgG, linker and anti-cotinine scFv were cloned into the eukaryotic expression vector, ERC6 was purified from HEK393F culture supernatant transfected temporarily using protein A affinity column chromatography.

(30) Conventional antibody drug conjugates require multistep procedures for conjugates to conjugate the drug to the antibody, and commonly the optimization procedure is necessary. On the other hand, the target delivery of drug using the cotinine-conjugated bispecific antibody requires conjugation of cotinine and drug. Thus, if the carboxycotinine(trans-4-cotininecarboxylic acid) chemical cross-link procedure is established, there will be an advantage for platform than conventional ADCs. Two carboxycotinines were cross-linked in N-terminus and C-terminus of the peptide in 13 amino acids length. The binding capacity of the bispecific antibody to the bivalent cotinine cross-linked peptide was more stable by the valency effect (FIG. 5). Various payloads can be conjugated to ε-amino acid chain of lysine in the peptide depending on purposes. In the present invention, biotin or duocarmycin was conjugated to the bivalent cotinine conjugated-peptide (FIG. 2b, 3c).

Experimental Example 2: Property of Bispecific Cetuximab×Anti-Cotinine scFv Antibody (ERC6)

(31) To analyze the purity and molecular weight of purified ERC6, ERC6 was visualized in a sodium dodecyl sulfate (SDS) polyacrylamide gel stained with Coomassie Brilliant Blue. The major band having the molecular weight of 206 kDa was observed in non-reducing condition, and two major bands of 78 kDa and 25 kDa were observed in non-reducing condition (FIG. 1b). The recombinant protein having the molecular weight of 206 kDa corresponded to the completely assembled ERC6, expected by ProtParam tool (ExPASy). The non-modified light chain of 25 kDa and the heavy chain fused with one scFv on the C.sub.H3 domain of 75 kDa were visualized by reduction of disulfide bonds. Therefore, data demonstrated that bispecific antibodies were pure and prepared without damage.

(32) The physicochemical properties of purified recombinant protein were analyzed with size-exclusion chromatography using high pressure liquid chromatography (SEC-HPLC). The ERC6 was shown as the single major peak having the clear molecular weight corresponding to the rightly assembled form. These data showed that the ERC6 did not produce a fragment, and it was not aggregated, and it produced multi-mers (FIG. 1c).

(33) In addition, to measure the stability of ERC6 in vivo, the pharmacokinetic analysis was performed. The serum half-life of ERC6 was determined in Balb/c mice (n=4). The circulating serum level of ERC6 was determined by enzyme-linked immunosorbent assay (ELISA) using blood samples collected from the vein in the orbit of an eye. The ERC6 intravenously injected was stable in the mouse serum until day 5, and this was similar to the result of cetuximab IgG.

Experimental Example 3: Binding Reactivity of ERC6 to Human EGFR and Cotinine

(34) To experiment the reactivity of ERC6 to EGFR and cotinine, the enzyme immunoassay was applied (FIG. 2a). As the binding activity of ERC6 to EGFR was confirmed, it was demonstrated that the reactivity to EGFR was independent to an additional scFv. In addition, it was confirmed that the affinity of the anti-cotinine scFv module to cotinine was maintained (FIG. 2a).

(35) To determine whether ERC6 binds human EGFR and cotinine simultaneously, the additional enzyme immunoassay was performed using a streptavidin-biotin detection system. After culturing the human EFGR-coated microtiter plate with ERC6, the bivalent cotinine-conjugated peptide cross-linked with biotin (cotinine-biotin) was added to each well (FIG. 2b), and then the HRP-conjugated streptavidin was added. The ERC6 was simultaneously conjugated to EGFR and cotinine (FIG. 2c).

(36) To evaluate the stability of cotinine payloads when forming the complex with ERC6, the pharmacokinetic analysis was performed. The ERC6 pre-cultured with cotinine-biotin was intravenously injected to mice (n=4) and the circulating serum level was measured by ELISA. The cotinine payload was rapidly removed in the mouse blood stream, due to its low molecular weight (t.sub.1/2=0.557 h). On the other hand, the circulating half-life of the cotinine-biotin was extended by binding to ERC6 (t.sub.1/2=18 hrs) (FIG. 2d). The duality of ERC6 to cotinine enhanced the stability of ERC6-complexed cotinine-biotin by increased binding capacity (FIG. 6). In addition, the clearance pattern of cotinine-biotin was matched with the decomposition pattern of ERC6, and this means that the half-life of the conjugate mostly depends on pharmacokinetics of ERC6 (FIG. 2d).

Experimental Example 4: ERC6-Complexed Cotinine-Duocarmycin Inducing Strong Cytotoxic Effect on Lung Adenocarcinoma Cells with KRAS Mutation

(37) A549 is a lung adenocarcinoma cell line that expresses wild-type EGFR with a KRAS mutation, showing primary resistance to EGFR targeted treatment such as cetuximab. Thus, to test the efficacy of ERC6-conjugated cotinine cytotoxic agent on cancer cells having wild-type EGFR and KRAS mutation, the cell line was used.

(38) Before the cytotoxicity analysis, the expression of EGFR on the A549 cell surface was confirmed by flow cytometry analysis (FIG. 3a). The ERC6 showed the binding activity similar to the EGFR expressed in the plasma membrane in the similar level to cetuximab. As predicted, the cotinine-biotin showed the binding activity to the A549 cell, when the ERC6 was present. The mixture of the cotinine-biotin and anti-cotinine IgG or cetuximab did not produce any significant signal derived from streptavidin-PE. Additionally, the cotinine-biotin did not affect the binding capacity of ERC6 to EGFR. These data demonstrated that the ERC6 mediated the delivery of cotinine-biotin to EGFR positive cells in the target specific manner by simultaneous binding activity.

(39) The cytotoxic activity to the A549 cell was investigated by cell viability analysis FIG. 3e). The relative cell viability was determined by measuring the cell ATP content directly related to the number of survivable cells. Duocarmycin without payloads (free duocarmycin) showed the stronger anti-tumor effect than the cotinine-duocarmycin (FIG. 3d, 3e). If the toxicity of cotinine-duocarmycin is lower than free duocarmycin, the penetration via the cytoplasmic membrane may be less effective.

(40) The ERC6 or cetuximab also could not significantly inhibit the proliferation of the A549 cell, due to the primary resistance to the EGFR targeted treatment of the A549 cell (FIG. 3e). However, when the ERC6 and cotinine-duocarmycin were combined, the cytotoxic effect was significantly enhanced than the cotinine-duocarmycin mixture, compared to cetuximab or antibody of negative control.

(41) The half of the maximum inhibitory concentration (IC.sub.50) was 0.3 nM. The experimental result demonstrated that the ERC6 effectively facilitated internalization of cotinine-duocarmycin with the EGFR positive cell, through receptor-mediated endocytosis. As a result, KRAS-mediated primary resistance of the ERC6-complexed cotinine-duocarmycin was overcome.

(42) In addition, through additional experiments, the cytotoxic effect of the antibody drug conjugate, in which the ERC6 and cotinine-emtansine (Cot-DM1) were combined, was experimented. This antibody drug conjugate also showed the significantly high cytotoxic effect to the A549 cell line, as same as the cotinine-duocarmycin-combined conjugate (FIG. 6b).

Experimental Example 5: ERC6-Complexed Cotinine-Duocarmycin Inhibiting Tumor Growth in Animal Model Having Lung Adenocarcinoma

(43) To evaluate the in vivo efficacy of ERC6-complexed cotinine-duocarmycin, cetuximab-refractory A549 cells were grafted to mice (n=4/group). When the tumor volume reached 150 mm.sup.3, mice were intraperitoneally injected with palivizumab and cotinine-duocarmycin, ERC6 and vehicle or ERC6-complexed duocarmycin for five weeks. Drugs were administered to the mice twice a week for two weeks, and then thrice a week for three weeks.

(44) The mice receiving ERC6-complexed cotinine-duocarmycin showed inhibition of tumor growth, in comparison with animals treated with cotinine-duocarmycin or ERC6 alone (FIG. 4a). Although cotinine-duocarmycin showed potent anti-proliferative effect on A549 in vitro, the isotype control of bispecific antibody did not inhibit the tumor growth in vivo. This observation result showed that only the cotinine-duocarmycin could not be selectively delivered to the tumor site and inhibit the tumor growth. The rapid removal from the mouse bloodstream due to non-specificity and low molecular weight can explain this efficacy insufficiency. On the other hand, the ERC6 allowed the cotinine-duocarmycin to extend the circulating half-life and be delivered to EGFR expressing tumor tissues. Thus, the ERC6 and cotinine-duocarmycin synergistically increase the anti-proliferative efficacy in EGFR-positive cetuximab-refractory tumor in vivo.

(45) In addition, there was no significant body weight decrease in mice during the treatment period of 5 weeks (FIG. 4b). This observation result indirectly means that the ERC6 and cotinine-duocarmycin did not have systemic toxicity. Consequentially, these data indicate that the ERC6 can function as a drug delivery system and selectively deliver a cotinine-binding cytotoxic drug to EGFR expressing tumor tissues in the target specific manner without systemic toxicity.

Experimental Example 6: Tissue Distribution of ERC6-Complexed Cotinine Payloads in Mouse Xenograft Tumor Model

(46) The pharmacokinetics demonstrated that the circulating half-life of the ERC6-complexed payloads was extended by binding to ERC6. To investigate the specific delivery of cotinine payloads into antigen expressing tumor tissues, immunofluorescence analysis was performed in the A549 xenograft mouse model. The A549 cell was subcutaneously injected to the left flank of each Balb/c-nude mouse. When the tumor reached 500 mm.sup.3, palivizumab and cotinine-biotin, cetuximab and vehicle or ERC6-complexed cotinine-biotin was intraperitoneally injected to mice having tumor. In 24 hrs after injection, animals were sacrificed, and dissected tumor tissues were imaged ex vivo. Cotinine payloads and antibodies on tumor tissues were detected through fluorescence-labeled secondary antibody. The antibody was detected by Alexa 594-labeled anti-human Fc (red), and the cotinine-biotin was detected by Alexa 488-labeled streptavidin (green). For reference, nuclei were stained by DAPI (blue), and the image magnification was x40. The tissue distribution of ERC6-complexed cotinine-biotin was observed through a confocal microscope.

(47) Compared to palivizumab used as the isotype control of bispecific antibody, the accumulation of ERC6 was observed in the human EGFR positive tumor tissue. In addition, the position of the cotinine-biotin in tumor was observed only in case of injecting ERC6. The cotinine-biotin without ERC6 can be removed rapidly in blood stream due to its low molecular weight. This observation result demonstrates that the ERC6 selectively delivers cotinine payloads to the desired tumor site in the target specific manner in vivo.