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. 2019 Aug 29;11(9):1268.
doi: 10.3390/cancers11091268.

Novel Human Bispecific Aptamer-Antibody Conjugates for Efficient Cancer Cell Killing

Margherita Passariello  1   2 , Simona Camorani  3 , Cinzia Vetrei  1   2 , Laura Cerchia  4 , Claudia De Lorenzo  5   6
Affiliations

Novel Human Bispecific Aptamer-Antibody Conjugates for Efficient Cancer Cell Killing

Margherita Passariello et al. Cancers (Basel). .

Abstract

Monoclonal antibodies have been approved by the Food and Drug Administration for the treatment of various human cancers. More recently, oligonucleotide aptamers have risen increasing attention for cancer therapy thanks to their low size (efficient tumor penetration) and lack of immunogenicity, even though the short half-life and lack of effector functions still hinder their clinical applications. Here, we demonstrate, for the first time, that two novel bispecific conjugates, consisting of an anti-epidermal growth factor receptor (EGFR) aptamer linked either with an anti-epidermal growth factor receptor 2 (ErbB2) compact antibody or with an immunomodulatory (anti-PD-L1) antibody, were easily and rapidly obtained. These novel aptamer-antibody conjugates retain the targeting ability of both the parental moieties and acquire a more potent cancer cell killing activity by combining their inhibitory properties. Furthermore, the conjugation of the anti-EGFR aptamer with the immunomodulatory antibody allowed for the efficient redirection and activation of T cells against cancer cells, thus dramatically enhancing the cytotoxicity of the two conjugated partners. We think that these bispecific antibody-aptamer conjugates could have optimal biological features for therapeutic applications, such as increased specificity for tumor cells expressing both targets and improved pharmacokinetic and pharmacodynamic properties due to the combined advantages of the aptamer and antibody.

Keywords: antibody; aptamer; cancer; dual targeting; immunotherapy.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Combined treatment of Erb-hcAb and CL4 selectively inhibits tumor cell survival. SK-BR-3 (A), MDA-MB-453 (B), LNCaP (C), NCI-N87 (D), and MCF-7 (E) cells were treated for 72 h with Erb-hcAb or CL4, alone or in combination, at the indicated concentrations. Cell survival after treatments is expressed as percentage of viable treated cells with respect to untreated cells. The scrambled aptamer (CL4Sc) was used in parallel as a negative control. Error bars depict means ± SD. p-values for the indicated mAbs relative to untreated cells, and for CL4 relative to CL4Sc, are: ** p < 0.01; * p < 0.05.
Figure 2
Figure 2
Expression of ErbB2, EGFR, and PD-L1 on tumor cell lines. Cell ELISA assay with a commercial anti-PD-L1 antibody on SK-BR-3, LNCaP, and MCF-7 tumor cells (A) for detection of cell surface PD-L1 expression. Western blotting analyses with the commercial anti-ErbB2 and anti-EGFR mAbs of extracts from SK-BR-3, LNCaP, and MCF-7cells. The intensity of the bands was normalized to actin (B). The ratios of ErbB2/actin and EGFR/actin signal intensities were calculated for each cell extract and found to be about 30 and 5 for SK-BR-3, 2 and 3 for LNCaP and 0.2 and 0.3 for MCF-7, respectively.
Figure 3
Figure 3
Combined treatment of CL4 and anti-PD-L1 mAb efficiently inhibits tumor cell survival. SK-BR-3 (A), LNCaP (B), and MCF-7 (C) cells were treated for 72 h with CL4 or 10_12 mAb, alone or in combination, at the indicated concentrations. Cell survival is expressed as percent of viable treated cells with respect to untreated cells. CL4Sc was used in parallel as a negative control. Error bars depict means ± SD. p-values for the indicated mAbs relative to untreated cells, and for CL4 relative to CL4Sc, are: *** p ≤ 0.001; ** p < 0.01; * p < 0.05.
Figure 4
Figure 4
Cytotoxic effects of the combination of CL4 aptamer and 10_12 mAb on breast cancer cells co-cultured with lymphocytes. (A) SK-BR-3 cells were co-cultured with lymphocytes (effector:target ratio 10:1) left untreated (control) or treated for 24 h with CL4 or 10_12, used alone or in combination, at the indicated concentrations. SK-BR-3 cell survival is expressed as percentage of viable treated cells with respect to untreated cells. (B) SK-BR-3 cell lysis, measured by the LDH release after the incubation with CL4 or 10_12 compounds, used at the indicated concentrations. The levels of LDH are expressed as a percentage of cell lysis with respect to the effects observed in co-cultures of tumor cells and lymphocytes in the absence of the drugs, used as a control. In (A,B), error bars depict means ± SD. p-values for the indicated mAbs relative to untreated cells, and for CL4 relative to CL4Sc, are: *** p ≤ 0.001; ** p < 0.01; * p < 0.05.
Figure 5
Figure 5
Effects of CL4 aptamer and 10_12 mAb on secretion of cytokines from lymphocytes co-cultured with breast cancer cells. SK-BR-3 cells were co-cultured with lymphocytes (effector:target ratio 10:1) and treated for 24 h at 37 °C with CL4 aptamer or 10_12 mAb, used alone (grey column) or in combination (black column), at the indicated concentrations. IL-2 and IFNγ levels (pg/mL) were obtained by ELISA assays performed on cell supernatants. Cells untreated (control) or treated with CL4Sc in the presence of lymphocytes were used as negative controls. Error bars depict means ± SD. p-values for the indicated mAbs relative to untreated cells are: *** p ≤ 0.001; ** p < 0.01.
Figure 6
Figure 6
Schematic representations and binding of Erb-hcAb-CL4 and 10_12-CL4 conjugates. (A) Schematic representations of Erb-hcAb-CL4 (grey) or 10_12-CL4 (black) conjugates, generated by the fusion of CL4 amino-oligonucleotide (anti-EGFR aptamer) with the Fc region of Erb-hcAb (anti-ErbB2 compact antibody) or 10_12 (anti-PD-L1 monoclonal antibody). (B) Binding of Erb-hcAb-CL4 conjugate to SK-BR-3 or MDA-MB-231 cells. As controls, unconjugated Erb-hcAb antibody and CL4 aptamer were tested in parallel by ELISA assays at the same concentration. (C) Binding of 10_12-CL4 to MDA-MB-453 cells and human activated lymphocytes. As a control, unconjugated 10_12 antibody was tested in parallel by ELISA assays at the same concentration. Error bars depict means ± SD. In B and C, p-values for the indicated mAbs relative to untreated cells, are: *** p ≤ 0.001; ** p < 0.01; * p < 0.05. (D) Representative confocal microscopy images of SK-BR-3 and MDA-MB-453 cells incubated with 10_12, 10_12-CL4, as indicated. 10_12 and10_12-CL4 are visualized in green. Nuclei are visualized in blue. Magnification 63×, 1.0× digital zoom. Scale bar = 10 µm.
Figure 7
Figure 7
Effects of Erb-hcAb-CL4 conjugate on tumor cell survival. SK-BR-3 (A) and LNCaP (B) cells were incubated for 72 h in the absence or in the presence of CL4, Erb-hcAb, or Erb-hcAb-CL4 conjugate at the indicated concentrations. Cell survival is expressed as percentage of viable treated cells with respect to untreated cells. The scrambled aptamer was used in parallel as a negative control. Error bars depict means ± SD. p-values for the indicated mAbs relative to untreated cells are: ** p < 0.01; * p < 0.05.
Figure 8
Figure 8
Effects of 10_12-CL4 conjugate on tumor cell survival. SK-BR-3 (A) and LNCaP (B) cells were incubated for 72 h in the absence or in the presence of CL4, 10_12, or 10_12-CL4 conjugate at the indicated concentrations. Cell survival is expressed as percentage of viable treated cells with respect to untreated cells. The scrambled aptamer was used in parallel as a negative control. Error bars depict means ± SD. p-values for the indicated mAbs relative to untreated cells are: *** p ≤ 0.001; ** p < 0.01; * p < 0.05.
Figure 9
Figure 9
Cytotoxic effects of 10_12-CL4 conjugate on breast cancer cells co-cultured with lymphocytes. (A) SK-BR-3 cells were co-cultured with lymphocytes (effector:target ratio 10:1) and treated for 24 h with CL4 aptamer, 10_12 mAb, or 10_12-CL4 conjugate at the indicated concentrations. SK-BR-3 cell survival is expressed as percentage of viable treated cells with respect to untreated cells. (B) SK-BR-3 cell lysis, measured by the LDH release after the incubation with the indicated compounds, used at the concentration of 50 nM. The levels of LDH are expressed as percentage of lysis of treated cells with respect to the effects observed in co-cultures of tumor cells and lymphocytes in the absence of the drugs, used as controls. (C) IL-2 and IFNγ cytokine secretion levels (pg/mL) were measured by ELISA assays performed on cell supernatants. Untreated or treated cells with scrambled CL4 in the presence of lymphocytes were used as negative controls. Error bars depict means ± SD. p-values for the indicated mAbs relative to untreated cells are: *** p ≤ 0.001; ** p < 0.01; * p < 0.05.
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References

    1. Papaioannou N.E., Beniata O.V., Vitsos P., Tsitsilonis O., Samara P. Harnessing the immune system to improve cancer therapy. Ann. Transl. Med. 2016;4:261. doi: 10.21037/atm.2016.04.01. - DOI - PMC - PubMed
    1. Peggs K.S., Quezada S.A., Allison J.P. Cancer immunotherapy: Co-stimulatory agonists and co-inhibitory antagonists. Clin. Exp. Immunol. 2009;157:9–19. doi: 10.1111/j.1365-2249.2009.03912.x. - DOI - PMC - PubMed
    1. Lohrisch C., Piccart M. ERBB2/neu as a predictive factor in breast cancer. Clin. Breast Cancer. 2001;2:129–135. doi: 10.3816/CBC.2001.n.017. - DOI - PubMed
    1. Tagliabue E., Centis F., Campiglio M., Mastroianni A., Martignone S., Pellegrini R., Casalini P., Lanzi C., Ménard S., Colnaghi M.I. Selection of monoclonal antibodies which induce internalization and phosphorylation of p185ERBB2 and growth inhibition of cells with ERBB2/NEU gene amplification. Int. J. Cancer. 1991;47:933–937. doi: 10.1002/ijc.2910470625. - DOI - PubMed
    1. Slamon D.J., Godolphin W., Jones L.A., Holt J.A., Wong S.G., Keith D.E., Levin W.J., Stuart S.G., Udove J., Ullrich A. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science. 1989;244:707–712. doi: 10.1126/science.2470152. - DOI - PubMed