Specificity and binding activity of mAbs against VEGFR-1. Monoclonal antibodies that bind human VEGFR-1 were generated from mice immunized with recombinant human VEGFR-1 protein. The specificity and binding efficiency of mAbs to human VEGFR-1 were tested by ELISA using recombinant human and mouse VEGFR-1 proteins as antigens. Binding analysis showed that both selected mAbs (4C1 and 3B12) against human VEGFR-1 have strong binding activity to human VEGFR-1 (km = 0.05 and 0.4 µM, respectively), while 3B12 mAb demonstrated binding activity to mouse VEGFR-1 (km = 2.0 µM) (Fig. 1).

ELISA assay of monoclonal antibodies 3B12 and 4C1 to the human (a) and mouse (b) VEGFR-1 target antigen.

Expression of VEGF-A and VEGFR-1 in breast and colon carcinoma cell lines. The expression of VEGF-A and VEGFR-1 was analyzed using RT-PCR in real time on BC cell lines: MCF-7, MDA-MB-231 and CC cell lines: Hutu-80, SW-480, human LoVo, as well as on the mouse BC cell lines EMT-6 and CC cell line CT-26. VEGF-A was expressed at the mRNA level in all the human cell lines tested and was not detected in the mouse cell lines (Fig. 2). VEGFR-1 transcripts were detected in all the cell lines except LoVo cells (Fig. 2), which may be due to the presence of a mutation or deletion in these cells in the selected RT-PCR primer system. Overall, our results indicate that VEGFR-1 and its ligand VEGF-A are widely coexpressed in breast and colon carcinoma cell lines.

Relative levels of VEGFR-1 (a) and VEGF-A (b) mRNA in human and mouse breast cancer (BC) and colon cancer (CC) cell cultures. RT-PCR data are presented as the mean ratio of the target gene mRNA/Actin mRNA ± SD.

All cell lines were positive for cell surface expression of VEGFR-1, which was verified using flow cytometry (Fig. 3). The expression of VEGFR-1 on the surface of Hutu-80 cells shown in Fig. 3c as an example of a representative result. Cytometric visualization of the receptor was carried out using the obtained mAbs to human VEGFR-1 4C1 and 3B12. The results demonstrate more efficient binding of the mAb of the clone 4C1 to the VEGFR-1 epitope on the surface of human cells compared to the mAb of the clone 3B12 (Fig. 3). At the same time, the efficiency of binding of the 3B12 mAb clone to the surface epitope of mouse VEGFR-1 cells is higher compared to the 4C1 mAb clone, which is consistent with ELISA data demonstrating the cross-affinity of the 3B12 mAb to the recombinant mouse VEGFR-1 antigen (Fig. 1b).

Immunofluorescence analysis of surface VEGFR-1 expression by the cell lines Hutu-80, LoVo, SW480, MDA-MB-231, MCF-7, EMT6-HER2, and CT-26. Flow-cytometry data for visualizing VEGFR-1 by mAbs 4C1 (a) and 3B12 (b). Example of flow-cytometry results for Hutu-80 cells (c).

mAbs to human VEGFR-1 inhibit the proliferation of breast and colon cancer cells in vitro. To evaluate the growth of breast or cancer cells when VEGFR-1 was blocked with monoclonal antibodies, the proliferation of all cell lines was analyzed using the xCELLigence DP tool, which allows detection of the cellular index in real time. The use of antibodies 4C1 and 3B12 did not significantly affect the proliferation of cells of the MCF-7 and SW-480 lines and significantly suppressed the growth of cells of the MDA-MB-231, EMT-6, Hutu-80, LoVo, and CT-26 lines (Fig. 4a), which is obviously associated with the significant presence of the VEGFR-1 antigen on the surface of these cells (Figs. 3a and 3b).

Analysis of the proliferative activity of breast- and colon-cancer cell lines when incubated in the presence of mAbs to human VEGFR-1 4C1 and 3B12 (a), the drug Avastin, which binds VEGF-A, and their combination (b). An example of the analysis of proliferation of Hutu-80 cells during coincubation with: 1 is PBS (control), 2 is the mAb 3B12 (1 mg/mL), 3 is the drug Avastin (1 mg/mL), 4 is the combinations Avastin (0.5 mg/mL) with mAb 4C1 (0.5 mg/mL), and 5 is mAb 4C1 (1 mg/mL) (c).

An immunotherapeutic treatment regimen using the drug Avastin, which is a humanized antibody that binds VEGF-A, and a combination of mAb 4C1 to the VEGFR-1 receptor with Avastin was also tested on all cell lines. Avastin did not affect the growth of SW-480 cells, in which the expression level of both VEGFR-1 and VEGF-A was recorded as minimal among all analyzed lines; did not significantly slow down the growth of MCF-7 culture; and significantly suppressed the proliferation of the cell lines Hutu-80, LoVo, MDA-MB-231, and CT-26 (Fig. 4b). When cells were incubated in the presence of a combination of a half dose of both Avastin and mAb 4C1, a synergistic effect was observed compared to the use of Avastin alone for cells of the Hutu-80, LoVo, and CT-26 lines (Figs. 4b and 4c).

Inhibition of VEGFR-1 by the specific mAb 3B12 suppresses in vivo growth of mouse colon carcinoma CT-26. To evaluate in vivo whether VEGFR-1 blockade prevents the growth of colon tumors, cells of the CT-26 line were transplanted into DBA/BALB mice and three days after tumor inoculation the following was administered intravenously: PBS (control), mAb 3B12 (1 mg/mouse), mAb 3B12 (3.5 mg/mouse), Avastin (1 mg/mouse), and Avastin (3.5 mg/mouse). The systemic administration of mAb 3B12 at a dose of 1 and 3.5 mg/mouse every 3 days resulted in the statistically significant (p < 0.05) suppression of the growth of CT-26 tumor grafts (Fig. 5a).

Inhibition of VEGFR-1 by monoclonal antibodies slows tumor growth in vivo (a) and increases the life expectancy of animals with transplanted CT-26 tumors (b).

The average life expectancy after the inoculation of tumor cells was: in the control group that did not receive treatment, it was 23 days; in groups receiving the systemic administration of mAb 3B12 in a volume of 1 and 3.5 mg/mouse, it was 37 and 32 days, respectively; and Avastin in a volume of 1 and 3.5 mg/mouse, it was 28 and 39 days, respectively (Fig. 5b). At the same time, significant (р < 0.5), an increase in the life expectancy of animals compared to the control was detected for groups receiving mAb 3B12 (1 mg/mouse) or Avastin (3.5 mg/mouse) (Fig. 5b). Thus, mAb 3B12 to the VEGFR-1 receptor obtained in this study, as well as the mAb included in the drug Avastin and binding VEGF-A, reduce the growth rate of the tumor node in vivo and prolong the survival of mice implanted with CT-26 colon carcinoma cells.

The participation of VEGFR-1 in the induction of angiogenic switching in pathological conditions, the mobilization of stem progenitor cells from the bone marrow, as well as in the growth and migration of tumors confirms the hypothesis of the therapeutic effectiveness of targeting this receptor [4, 8, 16, 18, 29]. In addition to activation in various tumors, VEGFR-1 is expressed in monocytes/macrophages and is involved in their recruitment to tumor sites, where they secrete proangiogenic factors that further stimulate tumor growth and promote resistance to anti-VEGF-A therapy [30]. The selective inhibition of VEGFR-1 by mAbs may enhance the effects of VEGF-A antiangiogenic therapy and counteract the development of resistance to this type of drug [29]. The mechanisms of tumor resistance to bevacizumab include the increased expression of VEGFR-1 (in   tumor cells, endothelial cells, and monocytes/macrophages) and signaling and/or activation of a specific VEGFR-1 ligand, PlGF [1, 6]. Thus, decreased modulation of the PlGF/VEGFR-1 pathway may delay or prevent resistance to anti-VEGF-A agents. The resistance to anti-VEGF-A therapy may also be associated with the formation of blood vessels through mechanisms alternative to angiogenesis, such as intussusception and vasculogenic mimicry [29].

This work describes new mAbs 4C1 and 3B12, which recognize VEGFR-1 and prevent its activation by ligands VEGF-A or PlGF. The resulting mAbs 4C1 and 3B12 are directed against a peptide whose amino-acid sequence is included in the extracellular domain of the receptor, which is confirmed by flow-cytometry data recording the presence of native VEGFR-1 on the surface of a number of tumor lines. The resulting mAbs 4C1 and 3B12 likely inhibit the cellular response that follows the binding of VEGF-A and/or PlGF ligands to the receptor, thereby slowing down the rate of proliferation of a number of tumor cells in vitro. Besides this, mAb 3B12 recognizes both human and mouse VEGFR-1, as demonstrated by ELISA. Thus, it became possible to analyze the effect of treatment with mAb 3B12 on the tumor graft. mAb 3B12 had antitumor activity in vivo. In fact, the effectiveness of five doses of 3B12 at 180 mg/kg was comparable to the effectiveness of five doses of bevacizumab (the drug Avastin) at the same dosage.

Despite its involvement in tumor angiogenesis, VEGFR-1 does not play a significant role in physiological angiogenesis in adults [16, 18]. Therefore, antiangiogenic therapies that selectively target this receptor may exhibit lower systemic toxicity compared with therapies targeting VEGF-A and/or VEGFR-2 [18, 29]. Indeed, the administration of mAb 3B12 at a high concentration of 180 mg/kg in a mouse model was very well tolerated. Besides, in in vitro experiments, the combined effect of mAb 4C1 to the VEGFR-1 receptor and the drug Avastin, which is a humanized antibody that binds VEGF-A, demonstrated a synergistic effect compared to the use of Avastin alone for certain cell lines. On this basis, the simultaneous targeting of VEGFR-1 with mAbs and VEGF-A blockade is likely to result in increased therapeutic efficacy without causing additive toxicity.