Preclinical Evaluation of 9MW2821, a Site-Specific Monomethyl Auristatin E-based Antibody-Drug Conjugate for Treatment of Nectin-4-expressing Cancers

Abstract

Overexpression of nectin cell adhesion protein 4 correlates with cancer progression and poor prognosis in many human malignancies. Enfortumab vedotin (EV) is the first nectin-4-targeting antibody-drug conjugate (ADC) approved by the FDA for the treatment of urothelial cancer. However, inadequate efficacy has limited progress in the treatment of other solid tumors with EV. Furthermore, ocular, pulmonary, and hematologic toxic side effects are common in nectin-4-targeted therapy, which frequently results in dose reduction and/or treatment termination. Thus, we designed a second generation nectin-4-specific drug, 9MW2821, based on interchain-disulfide drug conjugate technology. This novel drug contained a site specifically conjugated humanized antibody and the cytotoxic moiety monomethyl auristatin E. The homogenous drug-antibody ratio and novel linker chemistry of 9MW2821 increased the stability of conjugate in the systemic circulation, enabling highly efficient drug delivery and avoiding off-target toxicity. In preclinical evaluation, 9MW2821 exhibited nectin-4-specific cell binding, efficient internalization, bystander killing, and equivalent or superior antitumor activity compared with EV in both cell line-derived xenograft and patient-derived xenograft (PDX) models. In addition, 9MW2821 demonstrated a favorable safety profile; the highest nonseverely toxic dose in monkey toxicologic studies was 6 mg/kg, with milder adverse events compared with EV. Overall, 9MW2821 is a nectin-4-directed, investigational ADC based on innovative technology that endowed the drug with compelling preclinical antitumor activity and a favorable therapeutic index. The 9MW2821 ADC is being investigated in a phase I/II clinical trial (NCT05216965 and NCT05773937) in patients with advanced solid tumors.

Graphical abstract

Figure 1.

Structure and characterization of 9MW2821 and EV. A, Chemical structure of 9MW2821 contains three key components, a nectin-4 specific antibody, a hydrolyzable di-substituted maleimide (IDM) with a cleavable valine-citrulline dipeptide, and a microtubule inhibitor MMAE. B, Chemical structure of EV. Images were created with Biorender.com.

Figure 2.

Antigen expression and in vitro cytotoxicity. A, FFPE samples (176) were analyzed by IHC for H-score. Most cancers were nectin-4-positive. Although only 10% of the samples exhibited an H-score greater than 200, in breast cancer, lung cancer, cervical cancer, or prostate cancer, 30% to 50% exhibited an H-score greater than 100. B, Bystander killing effect of 9MW2821 and EV. Nectin-4–negative PC-3 cells were unaffected by 9MW2821 and EV, whereas the negative PC-3 cells were inhibited when the PC-3/nectin-4 (nectin-4–positive cells) were gradually added. Each experiment was performed three times to calculate the mean cytotoxicity and SD. C, The relation between antigen expression and ADC efficacy. The pIC₅₀ are mean values of triplicate measurements of each cell line.

Figure 3.

Internalization and lysosomal trafficking. MW282 mAb, 9MW2821, and EV were labeled with Alexa Fluor 488 using a commercial Alexa Fluor 488 conjugation kit (Abcam, catalog No. ab236553). A, Internalization and lysosomal trafficking of 9MW2821 in PC-3/nectin-4 cells, stained for lysosomes (red) and 9MW2821 (green). Costaining of lysosomes and 9MW2821 observed as yellow. Scale bars, 100 μm. B and C, Fluorescence signal increased as drugs accumulated intracellularly. Alexa Fluor 488–labeled MW282 mAb, 9MW2821, and EV were internalized and stored continuously in PC-3/nectin-4 (left) and MDA-MB-468 (right). Fluorescence signals are mean values of triplicate measurements for each cell line.

Figure 4.

Pharmacokinetic profile of 9MW2821. A–C,In vitro serum stability of 9MW2821 and EV in monkey serum at 37°C from 0 to 14 days. A, Release rate of free MMAE of two ADC measured by LC/MS-MS. The LC/MS-MS method was validated for accuracy. B,C, Total antibodies and intact ADC were quantified by ELISA. The data are mean values for double measurements. D–H, Pharmacokinetic profiles of 9MW2821 and EV in MDA-MB-468 tumor-bearing mice (n = 54, each group). Six animals were included in each blood collection point. D and G, We compared the PK profile of 9MW2821 with EV for free MMAE in serum, intact ADC, total antibodies, and MMAE intra-tumors. E and H, We compared the ADC in mole numbers of free MMAE in serum, intact ADC in serum, and MMAE intratumors. F, We compared the free MMAE in serum and MMAE intratumors in one picture. I–K, Tissue distribution of 9MW2821 in tumor-bearing mice (n = 6). MIP images of PET/CT scanning of MDA-MB-468 tumor-bearing mice after a single intravenous administration of ⁸⁹Zr-9MW2821 at different timepoints. The average tumor-to-muscle ratio showed a rising trend, reaching a peak value of 7.48 at 240 h postdose. The absorption of radioactivity was significantly less in the heart, liver, lung, spleen, and kidney.

Figure 5.

Antitumor efficacy of 9MW2821 in CDXs. A–C, TGI evaluated in MDA-MB-468, NCI-H322M, HT1376 xenograft models. Xenograft mice (n = 8) were intravenously treated with 1, 3, or 10 mg/kg 9MW2821 or EV at 3 mg/kg or 10 mg/kg for one injection. All treatments were initiated on the day when the average tumor volume reached 100 to 150 mm³. The tumor inhibition rates are mean values of eight animals.

Figure 6.

Antitumor efficacy of 9MW2821 in PDX. Seven typical PDX models were evaluated for antitumor efficacy: bladder cancer (BL3578), cervical cancer (CV13641), lung cancer (LU3073), and four triple-negative breast cancers (BR9457, BR9479, BR1282, and BR1458). A, BL3578 xenograft mice were intravenously treated with three injections of 3 mg/kg 9MW2821 once a week. B, CV13641 xenograft mice were intravenously treated with three injections of 3 mg/kg 9MW2821 weekly. The vehicle group was administered in three repeat doses (n = 8, 3 mg/kg, weekly) after the tumor volumes reached 100 mm³. C, CV13641 xenograft mice were intravenously treated with 3 mg/kg 9MW2821 and EV respectively once a week for three injections. Then the vehicle group was repeat dosed (n = 8, 3 mg/kg, weekly) after the tumor volumes reached 100 mm³. D–G, Four typical triple-negative breast cancers with different nectin-4 expression were also treated with three injections of 3 mg/kg 9MW2821 weekly. For BR9457, the vehicle group was also administered three repeat doses (n = 8, 3 mg/kg, weekly) after the tumor volumes reached 100 mm³.