Abstract
BackgroundRanpirnase (Rap) is an amphibian ribonuclease with reported antitumor activity, minimal toxicity, and negligible immunogenicity in clinical studies, but the unfavorable pharmacokinetics and suboptimal efficacy hampered its further clinical development. To improve the potential of Rap-based therapeutics, we have used the DOCK-AND-LOCK™ (DNL™) method to construct a class of novel IgG-Rap immunoRNases. In the present study, a pair of these constructs, (Rap)2-E1-(Rap)2 and (Rap)2-E1*-(Rap)2, comprising four copies of Rap linked to the CH3 and CK termini of hRS7 (humanized anti-Trop-2), respectively, were evaluated as potential therapeutics for triple-negative breast cancer (TNBC).MethodsThe DNL-based immunoRNases, (Rap)2-E1-(Rap)2 and (Rap)2-E1*-(Rap)2, were characterized and tested for biological activities in vitro on a panel of breast cancer cell lines and in vivo in a MDA-MB-468 xenograft model.Results(Rap)2-E1-(Rap)2 was highly purified (>95%), exhibited specific cell binding and rapid internalization in MDA-MB-468, a Trop-2-expressing TNBC line, and displayed potent in vitro cytotoxicity (EC50 ≤ 1 nM) against diverse breast cancer cell lines with moderate to high expression of Trop-2, including MDA-MB-468, BT-20, HCC1806, SKBR-3, and MCF-7. In comparison, structural counterparts of (Rap)2-E1-(Rap)2, generated by substituting hRS7 with selective non-Trop-2-binding antibodies, such as epratuzumab (anti-CD22), were at least 50-fold less potent than (Rap)2-E1-(Rap)2 in MDA-MB-468 and BT-20 cells, both lacking the expression of the cognate antigen. Moreover, (Rap)2-E1-(Rap)2 was less effective (EC50 > 50 nM) in MDA-MB-231 (low Trop-2) or HCC1395 (no Trop-2), and did not show any toxicity to human peripheral blood mononuclear cells. In a mouse TNBC model, a significant survival benefit was achieved with (Rap)2-E1*-(Rap)2 when given the maximal tolerated dose.ConclusionsA new class of immunoRNases was generated with enhanced potency for targeted therapy of cancer. The promising results from (Rap)2-E1-(Rap)2 and (Rap)2-E1*-(Rap)2 support their further investigation as a potential treatment option for TNBC and other Trop-2-expressing cancers.
Highlights
Ranpirnase (Rap) is an amphibian ribonuclease with reported antitumor activity, minimal toxicity, and negligible immunogenicity in clinical studies, but the unfavorable pharmacokinetics and suboptimal efficacy hampered its further clinical development
Representative results on the SDS-PAGE, size exclusion high-performance liquid chromatography (SEHPLC) and dynamic light scattering analyses demonstrating the molecular purity and integrity of DNL-Rap conjugates are shown for (Rap)2-E1-(Rap)2, (Rap)2-E1*(Rap)2, (Rap)2-22-(Rap)2, and (Rap)2-22*-(Rap)2 (Additional file 1: Figure S1A-E), and summarized below
CH3-AD2-IgG-hRS7 (Additional file 1: Figure S1A; lane 5); under reducing conditions, only three bands (Additional file 1: Figure S1A; lane 3) were evident, which correspond to the three constituents of (Rap)2-E1-(Rap)2, namely, Rap-DDD2 (Additional file 1: Figure S1A; lane 1), and the heavy (H) and light (L) chains from CH3-AD2-IgGhRS7 (Additional file 1: Figure S1A; lane 2), indicating the absence of non-product proteins
Summary
Ranpirnase (Rap) is an amphibian ribonuclease with reported antitumor activity, minimal toxicity, and negligible immunogenicity in clinical studies, but the unfavorable pharmacokinetics and suboptimal efficacy hampered its further clinical development. A pair of these constructs, (Rap)2-E1-(Rap) and (Rap)2-E1*-(Rap), comprising four copies of Rap linked to the CH3 and CK termini of hRS7 (humanized anti-Trop-2), respectively, were evaluated as potential therapeutics for triple-negative breast cancer (TNBC). Rap was shown to enhance both the in vitro and in vivo antitumor activity of vincristine against HT-29 human colorectal cancer cells that had been rendered multidrug-resistant by overexpressing the mdr gene [18], and induce caspase-independent cell death in both drug-sensitive and -resistant neuroblastoma cells and tumor xenografts [19]. An earlier Phase I trial of Rap in patients with solid cancers reported a lack of untoward immune response upon repeated weekly injections [22]
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