Generation and characterization of mAb 61H9 against junctional adhesion molecule-a with potent antitumor activity

Materials and animal experiments

The JAM-A-Fc cDNA and expression vector pTT5 were supplied by Corebiolab (Wuhan, China), as well as all molecular cloning reagents, including DNA polymerase, DNA ligation, and EcoRI/XhoI. Human ESCC cells (KYSE30, KYSE410) were purchased from Procell Life Science & Technology (Hyderabad, India). Mouse myeloma cells were obtained from Corebiolab.

The nude male mice and BALB/C mice were acquired from Beijing Huafukang Biotechnology Co., Ltd, Beijing, China. The mice were kept in the room with a temperature of 22 ± 2 °C, under light and dark for 12 h. Three to five mice were housed in each cage with free access to food and water. This project used 17 animals for modeling by subcutaneous injection. In the first animal experiment, we used five male BALB/C mice to explore monoclonal preparation. In the following experiment, 12 BALB/c nude male mice were disrupted before grouping. This represented a reasonable method for generating randomized sequences, by randomly dividing the animals into a control group and experimental group, with six mice in each group. The mice used in the experiment were all male, 1–2 months old, and weighed about 20 g. For different experiments, samples were processed in different ways, and all mice were finally sacrificed by intravenous injection of anesthetic drugs. The measurements included mouse weight, serum titer, and tumor size. Euthanasia was carried out when animals lost 25% of their weight, lost appetite completely for 24 h, or could not stand or could only stand with extreme reluctance due to physical weakness for 24 h. Any of the above conditions were the end point of the observation of the project. Animal experiments were conducted in the North Sichuan Medical College (NSMC) animal house without specific pathogens and in accordance with the requirements of the Animal Care and Use Committee of NSMC. The IACUC approval number was ECNSMC202144.

JAM-A-Fc recombinant protein expression and purification

In order to acquire JAM-A-Fc protein, the Fc tag was fused at the C-terminus of the sequence and the sequences were chemically synthesized with optimization for mammalian expression. The cDNA was cloned in expression vector pTT5-Fc and endotoxin-free DNA preparation was done for the pTT5 expression construction obtained. By using a proprietary method, the plasmid was then transiently transfected into HEK293 cells. Culture medium and cells were collected until viability dropped under 50%. Purification was conducted by affinity vs. Protein A resin, equilibration, and washing with PBS buffer (pH 7.5), then elution with 20 mM citric acid buffer (pH 2.7). Analysis was carried out SDS-PAGE of fractions of interest, qualitative and quantitative.

Preparation of mouse monoclonal antibody

Balb/c mice aged 6–8 weeks were purchased for antigen immunization. We mixed 100 μL JAM-A-Fc protein with equal volume Freund’s complete adjuvant for primary immunization on mice via back and intraperitoneal injection. After an interval of 2 weeks, the same treatment was carried out for immunization. One week after the third and fourth immunization, the serum titers of mice were detected by ELISA. If a titer did not reach expectations, the fifth booster immunization was carried out. Seven days after the fourth injection, spleen cells were obtained and fused with myeloma cells. We used 1 mL of 50% polyethylene glycol (PEG) to evenly mix myeloma cells (>10⁷) and immune spleen cell suspension (1 × 10⁸), and then the mix was transferred to a 96-well cell culture plate and cultured in a 37 °C constant temperature incubator with 5% CO2. After fusion, we observed the growth of hybridoma cells, and aspirated the supernatant for ELISA detection when the cells grew to 1/4–1/3 of the hole bottom. P/N ≥ 2.5 was considered as the positive judgment standard, and the serum of the non-immunized mice was the negative control, and the antibody dilution was the blank control. The positive cells were expanded and cloned. A limited dilution method was used for cloning culture. We selected the positive wells with few clones and high OD450 nm values and cloned them again. After one to two cloning operations, when the positive rate of all cloned cell pores reached 100%, the hybridoma cell line secreting specific monoclonal antibody could be determined, expanded, and timely frozen.

Enzyme-linked immunosorbent assay (ELISA)

JAM-A-Fc (1 μg/mL, 100 μL/well) was placed in coated microplates (Corning, Corning, NY, USA), incubated in a 37 °C incubator for 60 min, then removed and washed with PBST three times. Diluted serum was added into each plate and incubated in a 37 °C incubator for 60 min. We added 100 μL secondary antibody goat anti-mouse IgG (Jackson, West Grove, PA, USA), incubated in 37 °C for 1 h, and then washed with PBST (300 μL/well). Finally, TMB was added into the microplates. We put the microplates into the incubator for 5–10 min, then added 2 M HCl into the substrate and later read absorbance at 450 nm.

Immunofluorescence staining

The counted cells were seeded in a six-well culture plate with coverslip so that the coverslip was completely immersed in the medium. The plates were incubated in a 5% CO₂ incubator at 37 °C for 2–3 days. When adherent cells covered 2/3 of the plate bottom, we took out the plates and soaked the slides with PBS three times for 3 min. After fixation with 4% paraformaldehyde for 15 min, normal goat serum was added dropwise to the slides and blocked for 30 min at room temperature; the blocking solution was blotted off using absorbent paper, and adequate primary antibodies were dropped to each slide and put into a wet box and incubated overnight at 4 °C. Then slides were immersed and washed three times for 3 min each with PBST, and the fluorescent secondary antibody was added dropwise after blotting the excess liquid with absorbent paper. After incubating at 20–37 °C for 1 h in a wet box, slides were immersed and washed with PBST three times for 3 min each time. DAPI was then added dropwise and incubated for 5 min in the dark, followed by a blocking solution containing an anti-fluorescent quencher four times for 5 min each to block the sections. Fluorescence microscope was used for observation and acquisition.

To optimally visualize inflammatory cells, our evaluation was based on hematoxylin-eosin staining. The samples were fixed by 95% ethanol for 20 min and washed twice with PBS for 1 min each. Nuclei were stained with hematoxylin stain for 2–3 min and washed with PBS. To stain the cytoplasm, eosin was stained for 1 min and washed with PBS. After blowing or naturally drying the stem cells to climb the pieces, the neutral gums covered the pieces.

Flow cytometric analysis of antigen sites

KYSE30 cells were cultured with RPMI-1640 containing 10% fetal bovine serum, penicillin (100 μ/mL), and streptomycin (100 μ/mL) at 37 °C in saturated humid conditions with 5% CO2. All cells used in the experiment were in the log growth phase. For flow cytometry, normal cultured KYSE30 cells were collected and incubated with corresponding antibodies for 30 min in the dark at 4 °C. After incubation, cells were washed and collected via centrifugation at 1,000 rpm for 5 min. This process was repeated twice. After the last centrifugation, we removed supernatant and added 100 μL PBS to resuspend cells. Finally, the samples were analyzed using NovoExpress software to identify the antigen sites of 61H9G4 mAb.

Cell proliferation assay

KYSE30/KYSE410 cells were placed into 96-well plates and observed daily, and hybridoma supernatant was added to the cell culture plate after gradient dilution (no antibody was added to the control group). We added 10 μL of CCK-8 solution (Dojindo Laboratories, Shanghai, China) into each well and incubated at 37 °C for 3 h. After 24, 48, 72, 96, and 120 h, cell proliferation was assessed by CCK8 assay in each respective group.

Cell apoptosis analysis with flow cytometry

According to FITC and PI fluorescence values, two fluorescence parameters were determined to delimit the cross gate. In the experiment, cells were divided into three subpopulations: living cells that were double negative (Annexin v-FITC-/PI-), early apoptotic cells that were annexin V-FITC single positive (Annexin v-FITC+/PI-), and late apoptotic cells that were double positive for annexin V-FITC and PI (Annexin v-FITC+/PI+). The apoptosis rate of our experimental results was in the upper right quadrant (late apoptosis) and the lower right quadrant (early apoptosis). We added 61H9G4 mAb or IgG to KYSE30/KYSE410 cells, and the next steps were implemented according to the instructions of the Annevix FITC Kit (Vazyme, Nanjing, China). A NovoCyte flow cytometer and NovoExpress software were used for sample analysis and subsequent analysis, separately.

Cell cycle analysis with flow cytometry

Different antibodies were added to KYSE30/410 cells for 72 h. Cells were collected and washed once with PBS. Cell suspension was added into pre-cooled ethanol at −20 °C, and then was fixed at 4 °C overnight, 1,000 rpm * 5 min. We removed the ethanol, added 200 μL DNA dye to each tube, kept them away from light at room temperature for 15 min, and used the NovoCyte flow cytometer for detection.

Scratch-migration assay

Two × 105 cells were seeded into six-well plates and grown to confluency. After cells were plated into the plate bottom, a cell scratch was made with a 10 μL pipette tip perpendicular to the well plate, ensuring that individual scratch widths were consistent. The cell culture medium was aspirated, well plates were rinsed with PBS three times, cell debris generated by the scratch was washed away, and plates were observed and imaged after 0, 6, 24 and 48 h under microscope (IX71; Olympus, Shinjuku City, Tokyo, Japan).

In vivo experiments

BALB/c nude mice (male, 5 weeks) were randomly divided into anti-JAM-A and control groups (n = 6 each group) subcutaneously for a total of 3 × 10⁶ KYSE30 cells to initiate tumors. Thirteen days after cell inoculation, control and treated mice were intratumorally and peripherally injected with PBS and 61H9G4, respectively, every 2 days for a total of six injections. Tumor volume size was measured every 3 days. The mice were sacrificed after 39 days, and the tumor tissues were collected.

Statistical analyses

GraphPad prism 9.0 was used for statistical analysis and plotting, the measurement data were expressed by mean ± S.D. T-test or one-way ANOVA were used to compare two or more groups. The difference was statistically significant with P<0.05 (*P < 0.05, **P < 0.01, ***P < 0.001). NS stands for no statistical significance. The assumptions for the t-test included normal distribution, homogeneity of variance, and independence. The Shapiro Wilk test was used to check for normality, and the Levene test was used to check for homogeneity of variance.

Expression and purification of JAM-A-Fc recombinant protein

The antigen sequences were chemically synthesized (Corebiolab, Wuhan, China) and the structure of JAM-A gene in a linear model was presented (Fig. 1A). The constructed JAM-A expression plasmids pTT5-JAM-A-Fc were transfected into the mammalian expression cell HEK293 with transfection reagent, and on the 10th day after transfection, we collected the cell supernatant for purification. The cell supernatant was purified by affinity vs. protein A resin, the purity of JAM-A-Fc protein was about 90% and its molecular weight was about 56.4 kDa with a yield of 3.93 mg (Fig. 1B).

Preparation and identification of anti-JAM-A monoclonal antibodies

Eight fusion cells were screened by limited dilution assay. Clones with OD450 > 0.5 were selected and retested in new wells. Fc labeled antigen was added as negative screen control to eliminate Fc labeled antibody. Finally, four monoclonal strains with high JAM-A-Fc OD450 value and low Fc OD450 value were obtained and named 61H9, 70E5, 71A8, and 74H3, respectively. Immunofluorescence staining assay showed 61H9 was the most suitable cell line for mAb production as its fluorescence signal was the strongest (Figs. 2A, 2B).

The obtained anti-human JAM-A mouse mAb hybridoma variable region sequence (61H9) was subjected to antibody full-length recombinant expression following splicing to the constant region sequence. The above two pieces of sequences were respectively constructed into the expression vector pTT5 and transfected into CHO cells for antibody expression, and supernatant and cells were harvested for protein A antibody affinity purification. After using the reductive loading buffer to open the disulfide bond, there were approximately 55 (heavy chain) and 25 kD (light chain) bands, indicating that the antibody is an IgG type antibody (Fig. S1A), and purified antibodies were also present under non-reductive conditions (Fig. S1B). Finally, 23 mg of anti-JAM-A (61H9G4) was obtained for subsequent antibody function experiments.

Evaluation of antibody binding site of 61H9G4

To compare the difference between 61H9G4 and the commercial antibody AB275688 that binds to JAM-A, we carried out flow cytometric analysis. By adding fluorescein, the %parent of 61H9G4 and AB275688 binding to JAM-A was ascertained. The experimental results indicated that the % parent of the control group, AB275688, and 61H9G4 were 0.09%, 99.98%, and 99.33%, respectively (Figs. 3A–3C). Competitive inhibition binding assays was performed by first incubating homemade antibodies and then incubating commercial antibodies with a %parent of 30.89% (Fig. 3D), which showed that the recognition sites of JAM-A antigen by self-made antibody and commercialized antibody had some differences. Additionally, the binding activity of 61H9G4 to the JAM-A site was similar to that of AB275688, both of them have significantly higher %parent than control group (Fig. 3E).

61H9G4 inhibited proliferation and migration, and promoted apoptosis of ESCC cells

In the CCK-8 assays, cell viability decreased 24, 48, 72, 96, and 120 h after 61H9G4 treatment compared with the control groups (0 μg/mL). Specifically, when the concentration of 61H9G4 was 40 μg/mL, the cell viability decreased fastest. This indicated that 61H9G4 exerted a proliferation-suppressive effect on both KYSE30 and KYSE410 cells (Fig. 4A). To explore whether 61H9G4 affected cell apoptosis, we used flow cytometry to compare the capacity of 61H9G4 vs. IgG to induce apoptosis. From the results, we found that 61H9G4 significantly promoted cell apoptosis compared with IgG (P < 0.001) (Fig. 4B). Furthermore, we explored the influence of 61H9G4 on cell cycles using flow cytometry. Notably, 61H9G4 arrested cells in the G0/G1 phase, which may promote cell apoptosis (P < 0.01 or P < 0.001) (Fig. 4C). Meanwhile, we analyzed scratch wound healing, and calculated the relative migration distance at 0, 6, 24, and 48 h. As shown in Fig. 5A, cell migration ability significantly decreased in the 61H9G4 group compared to the IgG group (P < 0.05). The cell migration ability in the 61H9G4 group was significantly weaker than the control group at 24 h (P < 0.01). This showed that 61H9G4 could suppress migration of esophageal squamous cell carcinoma. In the transwell assay, after 24 h of cell culture, the cell counts of the 61H9G4 group was significantly decreased compared with the IgG group (P < 0.01) (Fig. 5B).

Effects of 61H9G4 on cell proliferation and invasion related pathways

We used WBto reveal the possible proliferation- and invasion- related signaling pathways affected by anti-JAM-A (Figs. 6A, 6B). In proliferation-related analysis, we found that 61H9G4 can significantly inhibit the expression levels of CyclinD1 and BCL2 in cells (Figs. 6C, 6D). CyclinD1 is a key protein in regulating the G1 phase of the cell cycle, and overexpression of CyclinD1 is closely related to the occurrence and development of malignant tumors. BCL2 can inhibit apoptosis occurrence. In addition to that, 61H9G4 could significantly increase the expression levels of p53 and caspase-3 in cells, and p53 acts as a typical tumor suppressor gene and can inhibit cell proliferation and migration. Additionally, cell apoptosis is accompanied by caspase-3 activation.

The NF-κB pathway is a classical signal pathway that promotes tumor genesis and metastasis. By detecting the phosphorylation level of IκBα and P65, two typical indicators of this pathway, we found that 61H9G4 can significantly inhibit phosphorylation of IκBα and P65 proteins, which may inhibit NF-κB pathway activation and thereby regulate cell related phenotypic changes such as proliferation and migration. The above results indicated that 61H9G4 was closely related to the regulatory pathways that inhibit the proliferation and invasion of ESCC.

61H9G4 effectively inhibited KYSE30 cell growth and decreased BCL-2 and IκBα expression

We further investigated the antitumor efficacy of 61H9G4 in vivo. After 39 days implantation, the tumors were dissected from the BALB/c nude mice to measure the volume (Fig. 7A). As shown in Fig. 7B, from the 30th day of cell inoculation, the tumor growth of the treatment group began to slow down, and the tumor volume was significantly inhibited from the 33rd day. On the 39th day, the inhibition rate of tumor growth in the 61H9G4 treatment group was about 50% compared with the control group. This suggested that 61H9G4 treatment effectively inhibited tumor growth in mice.

Additionally, compared with the control group, HE staining showed that cancer cells in the 61H9G4 group had small nuclei, light staining, and reduced mitotic stages (Fig. 7C). Consistent with the in vitro experimental results, immunohistochemistry analysis showed that anti-JAM-A group had significantly lower expression of BCL-2 and IκBα than control group in nude mice tumor tissue (Figs. 7D, 7E), was related to the tumor volume of nude mice.