Discovery and Preclinical Characterization of XMT-1660, an Optimized B7-H4-Targeted Antibody-Drug Conjugate for the Treatment of Cancer

Abstract

Antibody-drug conjugates (ADC) achieve targeted drug delivery to a tumor and have demonstrated clinical success in many tumor types. The activity and safety profile of an ADC depends on its construction: antibody, payload, linker, and conjugation method, as well as the number of payload drugs per antibody [drug-to-antibody ratio (DAR)]. To allow for ADC optimization for a given target antigen, we developed Dolasynthen (DS), a novel ADC platform based on the payload auristatin hydroxypropylamide, that enables precise DAR-ranging and site-specific conjugation. We used the new platform to optimize an ADC that targets B7-H4 (VTCN1), an immune-suppressive protein that is overexpressed in breast, ovarian, and endometrial cancers. XMT-1660 is a site-specific DS DAR 6 ADC that induced complete tumor regressions in xenograft models of breast and ovarian cancer as well as in a syngeneic breast cancer model that is refractory to PD-1 immune checkpoint inhibition. In a panel of 28 breast cancer PDXs, XMT-1660 demonstrated activity that correlated with B7-H4 expression. XMT-1660 has recently entered clinical development in a phase I study (NCT05377996) in patients with cancer.

Graphical abstract

Figure 1.

Development of DS and site-specific technology. A, DS ADCs (bottom structure) incorporate structural elements of the DF platform (top structure) within a fully synthetic, well-defined scaffold with a specific number of drugs per conjugated unit. B, The HIC of DS ADC indicates enhanced homogeneity over the DF ADC; C, Antitumor activity of DS and DF ADC following a single dose is comparable at equivalent drug dose and 2 dose levels; D, Trastuzumab ADCs made by four distinct approaches and two scaffold-linker payloads to generate DAR12 and DAR6 conjugates; E, HIC HPLC of trastuzumab ADCs; ADC3 and ADC4 show a fully homogeneous profile; F, Pharmacokinetics profile of ADC1–4 following a single intravenous bolus administration of ADC equivalent to a 0.199 mg/kg AF HPA dose to female CB.17 SCID mice bearing JIMT-1 human breast carcinoma xenograft tumors (6 mice per group) and samples collected at 10 min, 24 hours, 96 hours (ADC3 did not have the 96 hours timepoint due to operator error), 168 hours, and 336 hours. Graph depicts conjugated drug analyte concentration over the course of the study.

Figure 2.

In vitro characterization of B7-H4 ADCs. A, The anti–B7-H4 antibody does not block the functional activity of B7-H4 ligand. HEK293 or HEK293-B7-H4 cells were incubated with 10 nmol/L antibody (B7-H4 or nonbinding control) prior to the addition of CellTrace Violet-labeled CD3⁺ T cells and CD3/CD28 T-cell Activator. T cell proliferation was determined after 4-day incubation. Data represent the mean (± standard deviation) of triplicate samples. B, Schematics of the B7-H4–targeted ADCs evaluated in this study: site-specific DS DAR 2 and DAR 6, and DF DAR 12. C, Binding of ADCs to HEK293-B7-H4 cells and MX-1 breast cancer cells. Flow cytometry analysis was performed with B7-H4 ADCs, unconjugated antibody, and nonbinding control ADCs. D, Binding of ADCs to recombinant human B7-H4 protein. ELISA was performed with B7-H4 ADCs, unconjugated antibody, and non-binding control ADCs. E, B7-H4 ADCs elicit target-dependent cytotoxicity. HEK293-B7-H4 cells were incubated with test article for 3 days, and viability was measured with CellTiter-Glo.

Figure 3.

In vivo profile comparison of B7-H4 ADCs. A, Antitumor activity of B7-H4 ADCs in HBCx-24 PDX model. Inset, B7-H4 IHC. B, Antitumor activity of B7-H4 ADCs in MX-1 breast cancer cell line model. Inset, B7-H4 IHC. C, Plasma levels of conjugated drug in MX-1 tumor-bearing animals following a single administration of ADC at 0.15 mg/kg payload. Refer to legend in part B. D, Plasma levels of three analytes in cynomolgus monkeys following a single administration of ADC at 0.09 mg/kg payload. Blue, XMT-1660 DAR 6 DS; purple, DAR 2 DS.

Figure 4.

XMT-1660 activity in ovarian tumors and PD-1 refractory breast tumor. A, Antitumor activity of XMT-1660 in OV2423 ovarian cancer PDX. Inset, B7-H4 IHC. B, Antitumor activity of XMT-1660 in CTG-1692 ovarian cancer PDX. Inset, B7-H4 IHC. C, Antitumor activity of XMT-1660 in mBR9013 MMTV-ERBB2-derived syngeneic tumor in FVB/NJ. Anti–PD-1 immune checkpoint inhibitor was also evaluated (dosed BIWx3 (IP)). Inset, B7-H4 (top) and PD-L1 (bottom) IHC.

Figure 5.

XMT-1660 activity across a panel of breast cancer PDX. A, Waterfall plot of MBR among n = 3 per PDX model. Pink bars, TNBC. Green bars, ER+ breast cancer. B, The data from part A shown with B7-H4 IHC score in each model. Inset, responsive (MBR ≤ −0.3) and nonresponsive tumors when categorized by B7-H4 IHC as low TPS (< 75) or high TPS (≥75). C, Representative B7-H4 IHC images from pink (TNBC) and green (ER+) PDX models with low TPS (<75) and high TPS (≥75).