Nectin-2 was cloned from Marathon-Ready cDNA of the A549 human lung cancer cell line (BD Biosciences) into mammalian expression vectors pcDNA3.1 (Invitrogen), pEE12.4 (Lonza Biologics), and pEF1/myc-HisA (Invitrogen) to obtain pcDNA3.1-Nectin-2, pEE12.4-Nectin-2, and pEF1/myc-HisA-Nectin-2, respectively. The cDNA encoding human IgG1 and IgG4 Fc region and mouse IgG2a Fc region were cloned from human spleen-derived and mouse spleen-derived Marathon-Ready cDNA (BD Biosciences), respectively. The cDNA encoding Nectin-2 extracellular domain (a.a. 1–361) fused to the human IgG1 Fc region and Nectin-3 extracellular domain (a.a. 1–404) fused to the mouse IgG2a Fc region were inserted into pcDNA3.1 to obtain pcDNA3.1-Nectin-2-ED-hFc and pcDNA3.1-Nectin-3-ED-mFc plasmids, respectively. Similarly, the cDNA encoding C-terminal FLAG-tagged Nectin-2 extracellular domain (a.a. 1–361) and Nectin-3 extracellular domain (a.a. 1–404) were inserted into the pCMV-Tag4a plasmid (Stratagene) to obtain pCMV-Tag4-Nectin-2-ED-FLAG and pCMV-Tag4-Nectin-3-ED-FLAG plasmids, respectively. To obtain an expression plasmid encoding Y-443 with the human IgG4 Fc region, cDNA of the variable regions of the heavy chain or light chain of Y-443 were inserted into the GS expression vector pEE6.4 (Lonza Biologics) with human IgG4 Fc region cDNA or pEE12.4, respectively. The Y-443 IgG4 expression vector was constructed by ligation of the heavy and light chain vector.
OV-90 and MDA-MB-231 cells were purchased from the American Type Culture Collection (ATCC). OV-90 cells were grown in a 1:1 mixture of MCDB 105 medium and Medium 199 containing 15% fetal bovine serum (FBS). MDA-MB-231 cells were grown in Leibovitz’s L-15 medium containing 10% FBS. FM3A (Health Protection Agency Culture Collections (HPACC)) cells were grown in RPMI1640 containing 10% FBS. CHO (Lonza Biologics) and NS0 cells (Lonza Biologics) were cultured in DMEM containing 10% dialyzed FBS. The other cancer cell lines for Nectin-2 expression tests for FCM analysis were purchased from the ATCC, the German Collection of Microorganisms and Cell Cultures (DSMZ), the Japanese Collection of Research Bioresources Cell Bank (JCRB), and HPACC, and were cultured according to the provider’s instructions.
To obtain Nectin-2 stable transfectants, pEF1/myc-HisA-Nectin-2 was transfected into FM3A cells by using a Gene Pulser II (Bio-Rad). The cells were cultured in 96-well plates with selection medium containing Geneticin (Life Technologies). Likewise, pEE12.4/Nectin-2 was transfected into CHO and NS0 cells using a Gene Pulser II. The transfected cells were cultured in 96-well plates with selection medium containing methionine sulfoximine and L-glutamine-free selection medium, respectively. Single colonies grown in the selection media were expanded and the expression level of Nectin-2 on the cell surface was measured by FCM using anti-Nectin-2 poAb. Thus, recombinant cell lines expressing Nectin-2 (Nectin-2/FM3A, Nectin-2/CHO, and Nectin-2/NS0) were obtained.
Recombinant protein preparation
Recombinant proteins Nectin-2-ED-FLAG and Nectin-3-ED-FLAG were transiently expressed in FreeStyle 293 F cells (Life Technologies) by transfecting pCMV-Tag4-Nectin-2-ED-FLAG or pCMV-Tag4-Nectin-3-ED-FLAG using 293fectin (Life Technologies), respectively. The Nectin-2-ED-FLAG and Nectin-3-ED-FLAG proteins were then purified from the culture supernatant by anti-FLAG antibody column chromatography (Sigma-Aldrich) followed by a buffer exchange with Dulbecco’s phosphate-buffered saline (D-PBS) using an ultra-filtration system (Amicon). Similarly, recombinant Nectin-2-ED-hFc and Nectin-3-ED-mFc were obtained by the transfection of pcDNA3.1-Nectin-2-ED-hFc or pcDNA3.1-Nectin-3-ED-mFc, respectively. The Nectin-2-ED-hFc and Nectin-3-ED-mFc proteins were purified from the culture supernatant with Protein A Sepharose (GE Healthcare), followed by a buffer exchange with D-PBS using an ultra-filtration system.
Anti-Nectin-2 poAb was raised in New Zealand white rabbits by immunizing with recombinant Nectin-2-ED-FLAG protein emulsified with Freund’s adjuvant every 2 weeks for 3 months. The anti-Nectin-2 poAb was purified from the antiserum by affinity chromatography using a HiTrap NHS-Activated HP column (GE Healthcare) on which Nectin-2-ED-FLAG was immobilized. The purified antibodies were then passed through a Nectin-3-ED-FLAG-affinity column in order to remove antibodies that were cross-reactive to Nectin-3 or FLAG tag. The flow through fraction was used as a Nectin-2-specific poAb.
mRNA expression analysis
Expression levels of Nectin-2 mRNA in normal tissues and cancer tissues were quantified by analyzing Affymetrix U_133 array data from Gene Logic. Nectin-2 mRNA level was calculated by multiplying each expression intensity for a given experiment (a sample hybridized onto a chip) by a global scaling factor. The scaling factor was calculated as follows: (1) from all the non-normalized expression values in the experiment, the largest 2% and smallest 2% of the values were deleted; (2) the mean of the remaining values (trimmed mean) was calculated; (3) the scale factor was calculated as 100/(trimmed mean). The value of 100 used here is the standard target value used by Gene Logic. A genechip probe 232078_at for human Nectin-2 was used for the analysis. Primary cancer tissues were used in the analysis as cancer tissues.
The expression level of Nectin-2 protein in tissues was examined by IHC using paraffin-embedded normal tissue sections and cancer tissue sections from cancer patients (Cybrdi). The tissue sections were deparaffinized, blocked with goat serum (Vector Laboratories) for 20 min at room temperature, and incubated with 1 μg/mL anti-Nectin-2 rabbit poAb for 18 h at 4°C. After 3 washes with D-PBS, the tissue sections were incubated with ENVISION + Rabbit/HRP (Dako) for 30 min and then developed with 3, 3′-diaminobenzidine for 3 min at room temperature. The sections were counterstained with hematoxylin and mounted in Permount (Fisher Scientific). Cases with membranous staining in tumor cells were considered positive for Nectin-2 expression. The antigen-specificity of the positive samples was confirmed by staining without anti-Nectin-2 primary antibody.
Cancer cells were incubated with 3 μg/mL anti-Nectin-2 rabbit poAb for 1 h on ice followed by incubation with 2 μg/mL Alexa488-labeled anti-rabbit IgG (Life Technologies) for 1 h on ice. Cells were washed with D-PBS containing 2% FBS after each antibody incubation step. The fluorescence intensity was measured by using a Cytomics FC 500 instrument (Beckman Coulter). The MFI ratio of the Ab-treated sample to a control sample was used as a measure of the cell surface expression level of Nectin-2 in cancer cells. A control sample was prepared by incubating cells with a rabbit IgG control (Jackson ImmunoResearch Laboratories).
Generation of fully human anti-Nectin-2 mAbs
KM mice (10–12-weeks old, male; Kyowa Hakko Kirin) were immunized with Nectin-2-expressing cells (Nectin-2/NS0 or Nectin-2/FM3A), a recombinant protein of Nectin-2 extracellular domain (Nectin-2-ED-hFc or Nectin-2-ED-FLAG), or a combination of Nectin-2/FM3A and the recombinant protein. For cell immunization, Nectin-2/NS0 or Nectin-2/FM3A cells were pre-treated with 20 μg/mL Mitomycin C (Wako) for 30 min at 37°C, and then they were injected intraperitoneally into the mice with Ribi adjuvant at 1 × 107 cells per mouse weekly for 7 weeks. For protein immunization, an emulsion of 50 μg of Nectin-2-ED-hFc or Nectin-2-ED-FLAG and Freund’s complete adjuvant (Difco) was subcutaneously injected into the KM mice. The following immunizations were repeated twice using the same amount of immunogens emulsified with Freund’s incomplete adjuvant at 2-week intervals. Two weeks after the third immunization, 10 μg of Nectin-2-ED-hFc or Nectin-2-ED-FLAG was injected into the tail vein as a final boost. For combination immunization, the first immunization was conducted subcutaneously with Nectin-2-ED-hFc or Nectin-2-ED-FLAG emulsified with Freund’s complete adjuvant followed by the second immunization with the same amount of the proteins emulsified with Freund’s incomplete adjuvant at 2-weeks intervals. In parallel, Mitomycin C treated-Nectin-2/FM3A cells were intraperitoneally injected into the mice every week for 4-weeks after the first protein immunization. Three days after the final protein immunization or 7 days after the final cell immunization, splenocytes were collected from mice that showed a high serum titer against Nectin-2 in a cell ELISA using Nectin-2/CHO and they were fused with mouse myeloma P3X63Ag8U.1 cells (ATCC) using Polyethylene Glycol 1500 (Roche Diagnostics). The fused cells were cultured in a HAT (hypoxanthine-aminopterin-thymidine)-selection media to obtain hybridomas. Hybridomas that secreted anti-Nectin-2 mAb were screened in a Nectin-2/CHO cell ELISA using the culture supernatant. After the selected hybridomas were further sub-cloned, all of the hybridomas that secreted Nectin-2-specific antibody were expanded in medium containing 10% ultra-low IgG FBS (Life Technologies) on a scale of 10–50 mL. Then, each mAb was purified from the culture supernatant using Protein A-Sepharose resin. Antibody isotypes were determined by sandwich ELISA using a plate that was separately coated with 6 different antibodies specific to human IgG1, IgG2, IgG3, IgG4, IgM or the kappa chain.
Epitope binning study
Anti-Nectin-2 mAbs were biotinylated by using a Biotin Labeling Kit-NH2 (Dojindo). Nectin-2/CHO cells (3 × 103) were pre-incubated with 5 μg/mL unlabeled anti-Nectin-2 mAb and 330 ng/mL Streptavidin-Alexa Fluor 647 (Life Technologies) in a 384-well FMAT plate (Applied Biosystems) for 10 min at room temperature. After incubation, the biotinylated anti-Nectin-2 mAb was added to the plate at a final concentration of 100 ng/mL and the plate was further incubated for 1 h at room temperature.
The fluorescence intensity of Alexa Fluor 647 on Nectin-2/CHO cells was detected by using a FMAT8200 Cellular Detection System (Applied Biosystems). The binding inhibition (%) was calculated using the following formula:
where A represents the total fluorescence of the added unlabeled antibody and B represents the total fluorescence in an unlabeled antibody-free well. An epitope binning study was performed with the binding inhibition data by employing Ward’s hierarchical clustering method and using SpotFire DecisionSite for Lead Discovery (TIBCO Software).
Cell proliferation assay
OV-90 cells were seeded at 3 × 103
cells/well in a 96-well plate in the presence of 1% FBS and 30 μg/mL anti-Nectin-2 poAb or mAbs and then cultured for 6 days at 37°C. Cell proliferation was measured by using Cell Counting Kit-8 (Dojindo). The efficacy of the anti-Nectin-2 mAbs was evaluated with various concentrations of the antibodies under the same conditions. Cell growth inhibition (%) was calculated using the following formula:
Nectin-2-Nectin-2 and Nectin-2-Nectin-3 interaction inhibition assays
Nectin-2-Nectin-3 interaction inhibition was quantitatively assessed using a Biacore2000 (GE Healthcare). In brief, Nectin-3-ED-mFc protein was immobilized on sensor chip CM5 (GE Healthcare) by using an Amine Coupling Kit (GE Healthcare). A mixture of Nectin-2-ED-hFc at 40 μg/mL and anti-Nectin-2 mAb at 30 μg/mL was passed through the Nectin-3-ED-mFc immobilized chip. Nectin-2-Nectin-3 interaction inhibition was calculated as a percentage of the decrease in the maximum response unit compared to a human IgG control (Jackson ImmunoResearch Laboratories).
Nectin-2-Nectin-2 interaction inhibition was measured by using an ELISA-based time-resolved fluorescence spectroscopy assay. Nectin-2-ED-hFc protein was conjugated with europium N1 ITC chelate by using a DELFIA Eu-Labeling Kit (PerkinElmer). Unlabeled Nectin-2-ED-hFc protein was immobilized on to a Delfia Clear Strip Plate (PerkinElmer). After blocking with PBS containing 2% bovine serum albumin, the Eu-labeled Nectin-2-ED-hFc and anti-Nectin-2 mAb were simultaneously added at final concentrations of 3.2 μg/mL and 30 μg/mL, respectively, followed by incubation for 1.5 h at room temperature. After washing with D-PBS containing 0.05% Tween20, Enhancement Solution (PerkinElmer) was added to each well. The fluorescence intensity was measured at 615 nm with an excitation wavelength of 340 nm and a delayed time of 400 μs by using an ARVO1420 Multilabel Counter (PerkinElmer). Nectin-2-Nectin-2 interaction inhibition was calculated as a percentage of the decrease in the fluorescence signal compared to a human IgG control.
Human peripheral blood mononuclear cells (PBMC) from AllCells were cultured in RPMI1640 medium containing 10% FBS, 0.1 nM human IL-2 (DIACLONE Research), and 55 μM 2-mercaptoethanol for 24 h. PBMC were incubated with 51
Cr-labeled OV-90 cells or 51
Cr-labeled MDA-MB-231 cells at a ratio of 50:1 in the presence of anti-Nectin-2 mAb for 4 h at 37°C. After incubation, the radioactivity of 51
Cr that had been released into the culture medium from the target cells was measured by using an AccuFLEX γ 7000 (Aloka). Specific lysis by anti-Nectin-2 mAb was calculated using the following formula:
where A represents the radioactivity of the test supernatant, B represents the radioactivity of the target cells alone, and C represents the radioactivity of the maximum 51Cr release from the target cells that were lysed with 1% Triton X-100.
MDA-MB-231 cells (5 × 103
) were incubated with 10-fold diluted human serum complement (Quidel) and various concentrations of Y-443 in a black wall/transparent bottom 96-well plate (BD Biosciences) for 60 min at 37°C. The cells were stained with 10 μM propidium iodide (PI), and the PI-positive cells (CDC-damaged cells) were detected using an Acumen ex3 instrument (TTP Labtech). As a positive control, rituximab (Roche) was used in combination with Daudi cells (ATCC). CDC activity was calculated using the following formula:
where A represents the number of PI-positive cells in the presence of mAb and human serum, B represents that in the absence of mAb, and C represents the number of PI-positive cells that were incubated with 0.1% Triton X-100.
Preparation of anti-Nectin-2 mAbs for in vivo study
The hybridomas that produced Y-187 and Y-443 were cultured in Daigo’s T medium (Nihon Pharmaceutical) containing 10% Ultra Low IgG FBS (Life Technologies). The culture supernatant was filtered, concentrated, and then purified with a Protein A-column. The antibody fraction was separated on a Superdex 200 26/60 column (GE Healthcare) to obtain the monomer fraction and endotoxins were removed by using an ActiClean Etox column (Sterogene). The recombinant IgG4 form of Y-443 was prepared as follows. CHOK1SV cells (Lonza Biologics) were maintained in CD-CHO medium (Life Technologies) containing 6 mM L-glutamine. Linearized Y-443 IgG4 expression vector was transfected into CHOK1SV cells with a Gene Pulser II. The cells were seeded into 96-well plates at 37°C in a humidified 8% CO2. After 1 day of culturing, methionine sulfoximine (MSX) was added at a final concentration of 25 μM. After selection of the culture, a recombinant CHOK1SV clone that produced high levels of Y-443 IgG4 was selected and the cells were expanded into T75 flasks in serum-free CD-CHO medium containing 25 μM MSX and then adapted into a suspension culture. Y-443 IgG4 was purified from the supernatant of a 1 L fed-batch culture by chromatography with Protein A Sepharose FF (GE Healthcare), followed by Superdex200 26/60.
In vivo study
For the OV-90 subcutaneous mouse xenograft model, 8 × 106
OV-90 cells were subcutaneously inoculated into a flank of BALB/cAJcl-nu/nu mice (6 weeks old, females) from CLEA Japan. The mice were randomly grouped (n = 13–17). Vehicle (D-PBS), Y-187, or Y-443 (15 mg/kg) was intravenously injected twice weekly for 5 weeks from the day of the cancer cell inoculation. Tumor diameters were measured with a caliper twice weekly and approximate tumor volumes were calculated as 0.5 × length × (width)2
during the treatment period to monitor tumor growth. The TGI was calculated by the following formula:
where A is the average tumor volume for the Ab-treated group and B is the average tumor volume for the vehicle group.
The difference in the average tumor volume between the Ab-treated groups and the vehicle group was analyzed using the Steel test.
For the MDA-MB-231 subcutaneous xenograft model, 3 × 106 MDA-MB-231 cells were subcutaneously inoculated with Matrigel (Becton Dickinson) into BALB/cAJcl-nu/nu mice (6 weeks old, female) as described above. The mice were grouped 36 days after the inoculation when the average sizes of the tumors reached approximately 200 mm3 and then vehicle (D-PBS), Y-443 (0.3 and 1 mg/kg), or its IgG4 form antibody (0.3, 1, and 3 mg/kg) was intravenously injected once per week for 3 weeks. The tumor volumes were measured and the TGI for each treatment group was determined at day 57 as described above. The difference in the average tumor volume between the Ab-treated groups and the vehicle group was analyzed using Williams test.
The MDA-MB-231 lung metastasis model was carried out as follows. Briefly, 100 μL of 1 × 106 cultured MDA-MB-231 cancer cell suspension in Hank’s buffered salt solution was intravenously injected into the tail vein of SCID mice (5–6 weeks old). The mice were randomly grouped (n = 5) 33 days after the inoculation and were treated with vehicle (D-PBS) or Y-443 (0.001, 0.01, 0.1, 1, or 10 mg/kg) weekly on days 33, 40, 47, and 54. On day 61, the mice were sacrificed to excise the lungs, and 0.2% Evans blue dye was injected intratracheally to visualize their tumor colonies. The lungs were subsequently fixed with a mixture of picric acid, 10% neutral buffered formalin, and acetic acid (15:5:1), and the number of colonies on the diaphragmatic surface of the lungs was counted macroscopically in a blind manner. The difference between the colony numbers of the Ab-treated group and the vehicle group was analyzed using the one-tailed Shirley-Williams test.
All of the animal studies including immunization were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Takeda Pharmaceutical Company Ltd. (Permit Numbers: 2802, 2701, and E1-0311).