Background Armed monoclonal antibody (mAb)-based therapeutics, e.g., antibody-drug conjugates (ADCs) or antibody-oligonucleotide conjugates (AOCs), can be highly effective for patients with gene-amplified or protein-overexpressing tumors. However, many tumor cells do not overexpress surface antigens recognized by mAbs and, for these patients, armed mAb therapies are often poorly effective. Additionally, expression of checkpoint molecules on tumor infiltrating lymphocytes (TILS) can vary with disease progression. ASOCs are a new class of modified mAb that enable delivery of oligonucleotides into targeted cells without requiring overexpression of the antigen or internalization of the mAb (figure 1).
Methods We have covalently modified mAbs such that they are linked to protease substrates specific for proteases that enable metastasis; the latter are also covalently linked to oligonucleotides bearing H-type excitonic dimers. The substrates can be cleaved by proteolytic enzymes on a tumor cell surface essential for metastasis, e.g., matrix metalloproteases (MMPs). Cleavage of the protease substrates results in localization of oligonucleotides at a targeted cell surface. Oligonucleotides (both single strand DNA and double strand RNA) are then able to diffuse into the tumor cell or lymphocyte and inhibit translation of targeted mRNAs.
Results Oligonucleotides cleaved from ASOCs by cell surface proteases enter cells via the delivery mechanism we have previously used to block HIV proliferation in CD4+ T cells.1 Thus, ASOCs differ from the current armed mAb-based therapies, e.g., antibody-drug conjugates (ADCs) or antibody-oligonucleotide conjugates (AOCs), in that delivery of the oligonucleotides into the cytoplasm does not require internalization of the mAb or endosomal processing; additionally, off-target toxicity will be reduced by release at the cell surface of the payload (the oligonucleotide). With this approach, using an MHC-Class I mAb (KE-2) or the antiCD20 mAb Rituxan we have delivered antisense oligonucleotides targeting β-Actin and KRAS into the following tumor cell lines: Raji (Burkitt’s lymphoma), A549 (NSCLC), COLO205 (colon), MB-MDA231 (breast), and Caki (kidney). Additionally, anti-PD-1 and anti-PD-L1 mAbs can be derivatized to block checkpoint inhibition. Proof of delivery of oligonucleotides into cells was confirmed by confocal imaging after binding of mAbs to the cell surface with quantitation by flow cytometry.
Conclusions ASOCs represent a unique immunotherapeutic structural class that overcomes the previous requirements of: (1) internalization of the mAb and (2) delivery of the payload (oligonucleotides) through intracellular degradative organelles, e.g., lysosomes. Additionally, off-target toxicity is minimized.
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