Development and Evaluation of PNU-159682 ADCs

Potential Advantagess of PNU-159682

PNU-159682 is a bioactive metabolite of Nemorubicin that holds significant promise within the realm of ADCs. Derived from human liver microsomes, PNU-159682 stands as the principal bioactive metabolite of Nemorubicin, belonging to the anthracycline analog naturals family. Remarkably, it surpasses its parent compound in potency (800-2400-fold more potent than MMDX and 2100-6400-fold more potent than doxorubicin, as illustrated in Fig. 1). Furthermore, PNU's distinct mechanism of action sets it apart from doxorubicin, minimizing the known dose-related cardiotoxicity. This distinctiveness positions PNU as a promising foundation for novel antibody-drug conjugates (ADCs). Despite this potential, none of the existing seven commercial ADCs have harnessed an anthracycline-based warhead sucha as PNU-159682 or its metabolic parent nemorubicin. Consequently, there exists a pressing need to delve deeper into the structure-activity relationship of PNU-159682 to foster the development of pioneering derivatives and linked drugs.

Fig. 1. Structures of Anthracyclines in the PNU-159682 (1) Synthetic Family (Holte D, et al., 2020)

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PNU-159682-Linker "Warhead " Preparation

The synthesis of PNU-159682-linker 19 embarks with the preparation of PNU-p-nitrocarbonate 20 via the reaction between PNU (1) and bis(4-nitrophenyl) carbonate under basic conditions. This reaction, swiftly executed and concentrated, precedes the interaction of compound 20 with N,N'-dimethylethylenediamine, yielding secondary amine 21. Subsequent coupling of secondary amine 21 with advanced linker 22, utilizing Hünig's base and 1-hydroxybenzotriazole (HOBt), results in fully protected linker drug 23. Prudent two-step deprotection eliminates all protective groups while hydrolyzing the methyl ester. The removal process entails initial elimination of Fmoc protecting group using excess diethylamine, succeeded by low-temperature lithium hydroxide hydrolysis of the methyl ester and acetates to yield deprotected penultimate amine 24. Concluding the process, the reactive maleimide is incorporated via peptide coupling using HOBt and Hünig, culminating in the formation of linker drug 19(see Fig. 2).

Fig. 2. Synthesis of Linker Drug 19 (Holte D, et al., 2020)

Similarly, five additional linker drugs (25, 26, 27, 28, and 29) were synthesized following analogous methods (see Fig. 3).

Fig. 3. Linker Drugs 25-29. (Holte D, et al., 2020)

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PNU-159682-Linker "Warhead" Evaluation

• Lipophilicity

During the progression of these compounds, encompassing conjugates 19,25, 26, 27, 28, and 29, through the selection funnel, conjugate 27 was discarded due to inadequate conjugation, yielding a low drug-to-antibody ratio (DAR) attributed to its relatively high lipophilicity. alculations involving cLogP showcased 27 > 26 and 29 > 28, consistent with the molecules' anticipated lipophilic nature. Compound 28 was excluded as it was isolated as an approximate 1:1 diastereomer mixture at that juncture.

• In Vitro Plasma Stability

ADCs 25, 26 and 29 were tested for in vitro plasma stability in both mouse and rat plasma. The result showed linker drugs 25 and 26 with less than 75% recovery at day 7 in both mouse and rat plasma. Conversely, ADC 29 passed the in vitro plasma stability screening funnel.

PNU-159682 ADCs Assessment

• In Vitro Cytotoxicity

High CD46 expression cell lines, MES SA, and Naïve 293T, were employed for in vitro cytotoxicity assays to assess the potency of several ADCs. Antibodies conjugated with linker drugs 19, 25, 26, and 29 assessed against anti-CD46 antibody (target) and HuIgG1 as an isotype control. A correlation surfaced between payload potency and corresponding ADC potency. Notably, the high potency payload PNU-159682 yielded the most potent ADC hCD46-19 (47 pM in HEK 293T cells), while the less potent thiazole analogue resulted in a relatively lower potency ADC hCD46-29 (1.6 nM in HEK 293T cells). These findings underscored the elevated potency of targeted antibodies compared to non-targeted isotype controls.

• In Vivo Efficacy

Evaluation encompassed patient-derived xenograft (PDX) models, featuring five non-small cell lung cancer (NSCLC) and six colorectal cancer (CR) PDX models, all characterized by elevated CD46 expression. Notably, Fig. 4 illustrates the in vivo efficacy within NSCLC LU253 and colorectal CR188 PDX subcutaneous models in NOD/SCID mice. Administration of a single dose of 0.5 or 1.0 mg/kg hCD46-19 ADC yielded enduring responses, with over 80 days of tumor regrowth absence observed in 10 out of 11 PDX models. Exceptions included the CR120 model (dosed at 1 mg/kg) and LU253 (dosed at 0.5 mg/kg), exhibiting tumor re-growth within 60 days post-dosing. These compelling results emphasize ADC 19's remarkable efficacy in NSCLC and CR PDX models. Notably, a single 1.0 mg/kg dose led to complete tumor regression and enduring responses in most models..

Fig. 4. In vivo efficacy of PNU-conjugated ADCs in NSCLC LU253 and colorectal CR188 PDX subcutaneous models in NOD/SCID mice (Holte D, et al., 2020)

For ADC development encompassing thorough in vitro and in vivo analyses, Creative Biolabs remains your unparalleled partner, offering one-stop ADC development services tailored to your needs.

Additionally, leveraging our years of extensive experience in ADC cytotoxic drug discovery, we offer clients a broad range of PNU-159682 derivative products.

• MA-PEG4-VC-PAB-DMAE-PNU159682 (ADC-S-039)
• DBCO-PEG4-VC-PAB-DMAE-PNU159682 (WJY-0423-LS59)
• anti-HIgG(Fc)Fab-C-PNU ADC (ADC-AA-014)
• anti-MIgG(Fc)-C-PNU ADC (ADC-AA-025)
• anti-HIgG(Fc)-C-PNU ADC (ADC-AA-007)
• VC-PAB-DMAE-PNU159682 (WJY-0423-LS8)
• GGVA-PAB-PNU (WJY-0423-LS22)

Reference

1. Holte D, Lyssikatos JP, Valdiosera AM, et al. Evaluation of PNU-159682 antibody drug conjugates (ADCs). Bioorg Med Chem Lett. 2020. 30(24):127640.

For Research Use Only. NOT FOR CLINICAL USE.