Development of a novel small antibody that retains specificity for tumor targeting

Abstract

Background: For the targeted therapy of solid tumor mediated by monoclonal antibody (mAb), there have different models of rebuilding small antibodies originated from native ones. Almost all natural antibody molecules have the similar structure and conformation, but those rebuilt small antibodies cannot completely keep the original traits of parental antibodies, especially the reduced specificity, which gravely influences the efficacy of small antibodies.

Methods: In this study, authors developed a novel mimetic in the form of V(H)FR1(C-10-)V(H)CDR1-V(H)FR2-V(L)CDR3-V(L)FR4(N-10) for a parental mAb induced with human breast cancer, and the mimetic moiety was conjugated to the C-terminal of toxicin colicin Ia. The novel fusion peptide, named protomimecin (PMN), was administered to MCF-7 breast cancer cells to demonstrate its killing competency in vitro and in vivo.

Results: Compared with original antibody-colicin Ia (Fab-Ia) and single-chain antibody-colicin Ia (Sc-Ia) fusion proteins, PMN retained the targeting specificity of parental antibody and could specifically kill MCF-7 cells in vitro. By injecting intraperitoneally into BALB/c athymic mice bearing MCF-7 tumors, with reduced affinity, PMN significantly suppressed the growth of tumors compared with control mice treated by toxicin protein, Fab-Ia protein, Sc-Ia protein or by PBS (p < 0.05).

Conclusion: This novel mimetic antibody retained original specificity of parental antibody, and could effectively guide killer moiety to suppress the growth of breast cancer by targeted cell death.

Figure 1

Schematic diagram for the process of constructing the small antibody and the fusion peptide. (a) The small antibody (the mimetic moiety) was composed of V_HFR1_C-10-V_HCDR1-V_HFR2-V_LCDR3-V_LFR4_N-10. (b, c) The mimetic was conjugated to the C-terminal of wild-type colicin Ia to construct the conjugated peptide, named protomimecin (PMN). (d) The 15% SDS- PAGE migration map of the fusion peptide PMN.

Figure 2

In vitro killing activity assays of PMN. (a) Killing effects of PBS, non-relative LWMP, wt Ia, Fab-Ia, PMN and Sc-Ia on MCF-7, Zr-75-30 and Raji cells lines. LWMP, low molecular weight marker protein; wt Ia, wild-type colicin Ia; Fab-Ia, Fab segment from original antibody-colicin Ia fusion peptide; PMN, protomimecin; Sc-Ia, ScFv segment from original antibody-colicin Ia fusion peptide. (b) MCF-7 breast cancer cells were incubated with 75 μg/ml PMN for 24, 48 and 72 hrs, respectively. And tumor cells were stained with acridine orange (green color) and propidium iodide (red color). Red spots, dead cell mass; Green spots, live cell. After co-incubation for 72 hrs, approximately 80% of all MCF-7 cells had died (upper panel). T, PMN-treated group; C, control group treated with PBS. (c) Cytotoxicity of different concentration of PMN against MCF-7.

Figure 3

The competition ability of synthetic V_HFR1_C-10-V_HCDR1-V_HFR2-V_LCDR3-V_LFR4_N-10. (a) Fixed MCF-7 cells were incubated with PBS, LWMP, Fab, PMN and Sc-Fv peptides (50 μM) and DAPI (50 ng/ml) prior to flow cytometry. LWMP, low molecular weight marker protein; Fab, Fab segment from original antibody; PMN, protomimecin; Sc-Fv, ScFv segment from original antibody. (b) The relative affinity of V_HFR1_C-10-V_HCDR1-V_HFR2-V_LCDR3-V_LFR4_N-10 peptides and OAbs to antigen. OAb, original antibody. (c) Concentration-dependent inhibition of different concentration of synthetic mimetic antibody or OAb against 75 μg/ml PMN. (d) MCF-7 cell survival ratio of the inhibition activity of OAb against PMN (75 μg/ml). OAb, original mAb antibody A520C9.

Figure 4

In vivo killing competency and the biodistribution of PMN. In vivo killing competency was compared with PBS, wt Ia, Fab-Ia and Sc-Ia in BALB/c athymic immunocomposed mice bearing MCF-7 tumors. (a) The tumors of mice were collected after 2-week administration. (b) The weights of each individual tumor were added together and the total weights were compared between groups. Compared with PBS, wt Ia, Fab-Ia and Sc-Ia, PMN could significantly suppress the growth of MCF-7 tumors (p < 0.05). PMN, protomimecin; wt Ia, wild-type colicin Ia; Fab-Ia, Fab segment from original antibody-colicin Ia fusion peptide; Sc-Ia, ScFv segment from original antibody-colicin Ia fusion peptide. (c) Fluorescence images of tumor (white arrow) in BALB/c mice traced by FITC-labeled PMN. The green fluorescence represented the location of FITC-labeled PMN protein. (d) Fluorescence images of incised tumor and vital organs from BALB/c mice traced by ip injecting FITC-labeled PMN. The green fluorescence showed the biodistribution of FITC-labeled PMN. T, tumor; S, spleen; L, liver; B, brain; M, muscle; K, kidney; I, intestine.

Figure 5

Histopathological staining revealed numerous fibrous foci (black arrow) in the tumors from the treated group with PMN (b), which were not seen in the other control groups (a). PMN, protomimecin. Scale bar, 50 μm.