Chemically Programmed Bispecific Antibody Targeting Legumain Protease and αvβ3 Integrin Mediates Strong Antitumor Effects

Abstract

A chemically programmed bispecific antibody (cp-bsAb) that targeted cysteine protease legumain and αvβ3 integrin has been prepared using the aldolase antibody chemical programming (AACP) strategy. In vitro evaluation of the anti-legumain, anti-integrin cp-bsAb and its comparison with cpAbs targeting either integrin or legumain have shown that the former possesses superior functions, including receptor binding and inhibitory effects on cell proliferation as well as capillary tube formation, among all three cpAbs. The anti-legumain, anti-integrin cp-bsAb also inhibited growth of primary tumor more effectively than either anti-legumain or anti-integrin cpAb as observed in the MDA-MB-231 human breast cancer mouse model. The AACP-based cp-bsAb, which contains a generic aldolase antibody, can also serve as a suitable platform for combination therapy, where two equally potent compounds are used to target extracellular receptors.

Keywords: 38C2; aldolase; alpha(v)beta(3); antibody; bispecific; chemical; integrin; legumain; protease.

Figure 1.

Structure of legumain and αvβ3 integrin inhibitors, the antibody programming agents, and chemically programmed antibodies. Compounds 3–5 reacted with Ab 38C2 at 37 °C for 2–24 h affording anti-legumain, anti-integrin cpAbs (38C2-3, 38C2-4) and cp-bsAb (38C2-5). Compound [4a] is an intermediate produced by an in situ retro aldol reaction of compound 4 before the latter reacts with the Ab. Key: legumain and integrin inhibitors are portrayed in green and blue, respectively.

Figure 2.

In vitro cellular assays. Flow cytometry histograms showing binding of 38C2-3, 38C2-4, and 38C2-5 to (A) U87 glioblastoma, (B) Ln-CAP prostate cancer, and (C) MDA-MB-231 breast cancer cells. 38C2-5 has a stronger cell binding affinity than 38C2-3 and 38C2-4. The 38C2 Abs and FITC-labeled goat anti-mouse secondary Abs were used as negative controls. (D) ELISA histogram showing specific binding of 38C2-3, 38C2-4, and 38C2-5 to legumain and/or human integrin αvβ3 (*p < 0.001).

Figure 3.

In vitro cell proliferation and tube formation assays. (A) A line graph showing the proliferation of human U87 cells treated with Ab 38C2, cpAbs 38C2-3 and 38C2-4, and cp-bsAb 38C2-5 (left) (38C2-5 vs 38C2-3, p < 0.001; 38C2-5 vs 38C2-4, p < 0.001). Microscope image showing morphology of the human U87 cells on day 6 after cells were treated with Ab 38C2 or the chem-Abs (right). (B) 38C2-3, 38C2-4, and 38C2-5 inhibit HUVEC tube formation in vitro (left). Tube formation was quantified by comparing pixel tube number in each image by using the NIH Image program (right). The experiments were repeated three times. (38C2-5 vs 38C2-3, p < 0.001; 38C2-5 vs 38C2-4, p < 0.001.) Data are represented as mean ± SEM.

Figure 4.

In vivo tumor growth study. (A) Inhibition of primary MDA-MB-231 tumor growth by 38C2-5. Nude mice were treated with 125 μg of 38C2, 38C2-3, 38C2-4, or 38C2-5 on day 15, 20, 25, …, 50 after 15 days of MDA-MB-231 carcinoma challenge. Primary tumor volumes were measured by micro caliper measurements (volume = (1/2)(width)² × length) (n = 6 mice per group (38C2-5 vs 38C2-3, p < 0.01; 38C2-5 vs 38C2-4, p < 0.01)). (B) Immunofluorescence staining of MDA-MB-231 tumor tissue. Legumain and integrin αvβ3 are localized on MDA-MB-231 tumor cell surface. (C) Microvascular density quantified on tumor sections. Five sections from each mice and 4 mice from each treated group were examined. Number of microvessels per mm² was counted under a light microscope.

Scheme 1.. Production of a cpAb or cp-bsAb by Aldolase Ab Programming (AACP) Strategy Using a Fully Functionalized Antibody-Programming Agent (Ab-PA) or Precursors^a

^aAb PA or a functionalized linker (* stands for two different linker-functional groups) that can react covalently through a β-lactam (L) function with reactive lysine residues in the aldolase Ab binding sites yielding cpAb, cp-bsAb, and cpAb intermediates. CpAb intermediates further react with compounds (comp1/2) affording a cpAb or cp-bsAb. Linkers and Ab PAs can also react with Ab through the β-diketone (DK) or vinylketone (VK) function. (Ab, antibody. PA, programming agent: a compound fully functionalized with lactam, vinyl ketone, or diketone linker. LNK, linker. FG, functional group.).