Differential Antitumor Effects of IgG and IgM Monoclonal Antibodies and Their Synthetic Complementarity-Determining Regions Directed to New Targets of B16F10-Nex2 Melanoma Cells

Abstract

Malignant melanoma has increased incidence worldwide and causes most skin cancer-related deaths. A few cell surface antigens that can be targets of antitumor immunotherapy have been characterized in melanoma. This is an expanding field because of the ineffectiveness of conventional cancer therapy for the metastatic form of melanoma. In the present work, antimelanoma monoclonal antibodies (mAbs) were raised against B16F10 cells (subclone Nex4, grown in murine serum), with novel specificities and antitumor effects in vitro and in vivo. MAb A4 (IgG2ak) recognizes a surface antigen on B16F10-Nex2 cells identified as protocadherin beta(13). It is cytotoxic in vitro and in vivo to B16F10-Nex2 cells as well as in vitro to human melanoma cell lines. MAb A4M (IgM) strongly reacted with nuclei of permeabilized murine tumor cells, recognizing histone 1. Although it is not cytotoxic in vitro, similarly with mAb A4, mAb A4M significantly reduced the number of lung nodules in mice challenged intravenously with B16F10-Nex2 cells. The V(H) CDR3 peptide from mAb A4 and V(L) CDR1 and CDR2 from mAb A4M showed significant cytotoxic activities in vitro, leading tumor cells to apoptosis. A cyclic peptide representing A4 CDR H3 competed with mAb A4 for binding to melanoma cells. MAb A4M CDRs L1 and L2 in addition to the antitumor effect also inhibited angiogenesis of human umbilical vein endothelial cells in vitro. As shown in the present work, mAbs A4 and A4M and selected CDR peptides are strong candidates to be developed as drugs for antitumor therapy for invasive melanoma.

Figure 1

Reactivity of mAb A4 and mAb A4M with B16F10-Nex2 tumor cells. (A) mAb binding to melanoma cells evaluated in CLELISA. (B) Immunofluorescence of B16F10-Nex2 cells with mAb A4 and mAb A4M. Negative control, melanoma cells incubated with anti-Ig FITC alone.

Figure 2

MAb A4 is cytotoxic in melanoma cell lines. (A) Inhibitory mAb A4 effect against human melanoma cell lines in the absence of complement. Cell viability was measured by MTT. Data are shown as mean percentage variation of cell death normalized to control. Control, cells incubated with an irrelevant antibody at 20 µg/well; *P < .001. (B) MAb A4 induced DNA degradation in B16F10-Nex2 and human melanoma cell lines. STD, 1 Kb Plus DNA Ladder; Ctrl, cells incubated with irrelevant antibody at the same concentration. (C) B16F10-Nex2 cells were treated with mAb A4, cisplatin, or an irrelevant antibody as described in Materials and Methods for 6 and 12 hours. Increased numbers of Annexin and propidium iodide (PI) double-positive cells were observed after mAb A4 and cisplatin incubation.

Figure 3

MAb A4 recognizes protocadherin β₁₃ on melanoma cells. (A) TL of B16F10-Nex2 was examined for reactivity with mAb A4. TL, a band at 87 kDa was seen on SDS-PAGE; WB, a protein of identical molecular mass was recognized by mAb A4 in Western blot analysis; IP, the 87-kDa component was immunoprecipitated by mAb. (B) A mAb A4-immunoprecipitated sample was analyzed by LC-MS/MS, and the peptide shown underlined in bold italic and below was fully identified, being a characteristic of protocadherin β₁₃ (complete sequence shown, 796 aa, calculated mol. wt. = 87,458).

Figure 4

MAb A4M reacts with histone 1 in B16F10-Nex2 cells. (A) Western blot analysis of B16F10-Nex2 nuclear extract (NE) and H1 commercially purified calf thymus histone (Sigma) with mAb A4M and anti-pan-histone antibody. Negative control, with irrelevant mAb. (B) Confocal microscopy showing the mAb A4M reactivity on the nuclei of B16F10-Nex2 cells compared with antihistone antibody. Cells untreated (Histone +) and treated with sulfuric acid to remove histones (Histone -) are compared. Bottom panel: Colocalization of mAb A4M and antihistone reactions (MERGE). Red indicates phalloidin-rhodamine; blue, DAPI staining.

Figure 5

Antitumor effects of mAb A4 and mAb A4M. (A) MAb A4 injected i.p. led to inhibition of B16F10-Nex2 subcutaneous tumor growth (black circles, n = 5). Controls, animals injected i.p. with irrelevant antibody (white circles, n = 5). (B) Survival record of both groups treated with mAb A4 and the control group (P < .001). (C) Effect of the mAb A4 and mAb A4M-treatment on experimental lung metastases showing a significant decrease in their number with both mAbs (P = .05 and P < .05, respectively). (D) Representative lungs from control and mAb-treated animals after 25 days of intravenous tumor challenge. Control, animals treated with irrelevant antibody at the same concentration.

Figure 6

MAb A4 V_H CDR3 (H3) peptide is the only inhibitory CDR of mAb A4 (A). A4 H3 peptide induces DNA degradation in B16F10- Nex2 cells (B). STD, 1 Kb Plus DNA Ladder (Invitrogen). (C) Cyclic-extended and linear mAb A4 H3 peptides were tested for competition with mAb A4 in CL-ELISA. Peptides were incubated with tumor cells previously to addition of mAb A4 as described in Materials and Methods. (D) In vitro titration of cytotoxic effects of cyclic and linear mAb A4 H3 in B16F10-Nex2 cells. Black bar, untreated cells (*P < .001). (E) Addition of cyclic-extended mAb A4 H3 peptide (50 µg) abrogates mAb A4 binding to B16F10-Nex2 cells (blue peak). Negative control, cells incubated with anti-IgG FITC alone. Positive control, mAb A4 alone (red peak).

Figure 7

MAb A4M V_L CDR1 and 2 (L1, L2) are cytotoxic to B16F10-Nex2 melanoma cells (A) and induce DNA degradation in these cells (B). STD, 1 Kb Plus DNA Ladder. In human leukemia HL-60 cells, mAb A4M L2 peptide induces a dose- and time-dependent DNA degradation, which is blocked in HL-60 cells overexpressing antiapoptotic molecules (*P < .001, **P < .01) (C). Inhibition by mAb A4M CDRs L1 and L2 of HUVEC sprouting on Matrigel to form closed proangiogenic structures; *P < .001 relative to untreated control (-) (D). The mAb A4M cyclic-extended H3 inhibited mAb A4M binding to B16F10-Nex2 as shown by FACS (E) and by CL-ELISA (F). Negative control, cells incubated only with anti-IgM FITC. Positive control, mAb A4M alone (red).