Zip-code drug conjugates (ZDCs): Ctdna-guided therapy shows strong efficacy and low toxicity in vitro and In Vivo

Abstract Multiple myeloma (MM) is a malignancy marked by the clonal expansion of plasma cells. Despite therapeutic advances, MM remains incurable, especially in high-risk patients with p53 deletions or mutations, 1q gain, or IGH, MMSET, FGFR3, MAFB, or MAF translocations. To address this, we developed a novel therapeutic platform called Zip-Code Drug Conjugates (ZDCs). This approach was based on our discovery that certain transposons within circulating tumor DNA (ctDNA) enable targeting of other plasma cells (REF). Using the features of these transposable elements, we screened several synthetically engineered elements—Zip-Codes (ZCs)—for specificity and efficiency in targeting plasma cells. High-throughput screening of MM cell lines and patient samples identified two key candidates matching ctDNA specificity. We selected AluSx (ZC2) as our lead ZC due to its easy production. ZC2 showed no effect on inflammatory cytokine expression and did not activate the cGAS-STING pathway or caspases, though it did activate DNA repair enzymes. We then conjugated ZC2 with various agents to assess selective cytotoxicity. Our lead compound, KTS002, consists of DM4 (a tubulin inhibitor) linked to ZcDNA via SPDB. Native PAGE confirmed its successful conjugation. Quality testing showed that KTS002 was endotoxin-free, sterile, low in beta-glucan, and consistent in concentration, hydrodynamic size, and zeta potential. Because premature plasma release contributes to ADC toxicity, we evaluated ZDC stability. HPLC-LC Orbitrap analysis showed that KTS002 remained stable in mouse and human plasma, with half-lives of 24 hours (0.5 μg/mL) to 58 hours (1 μg/mL). In contrast, glutathione and TCEP release controls had half-lives of 48 days and 27 hours, respectively. In blood and plasma assays, KTS002 did not cause hemolysis, platelet aggregation, complement activation, or coagulation disruption. In vitro, KTS002 exhibited similar IC50 values to DM4 in various MM cell lines, with only modest cytotoxicity in non-myeloma lines (A549, DU145, MIA, BT549, HCT116). Viability and LDH release assays using LLC-PK1 and Hep G2 cells showed minimal off-target effects, suggesting low renal and hepatic toxicity. Compared to Belantamab (an anti-BCMA MM ADC), KTS002 was 1000-fold more cytotoxic in cells with p53 deletions or mutations. It also overcame resistance due to BCMA mutations (e.g., R27P), which can impair activity of anti-BCMA bispecific or CAR T cell therapies. In vivo titration showed good tolerance up to 40 mg/kg. In MM1S xenograft models, KTS002 demonstrated dose-dependent tumor suppression, with high-dose treatment outperforming bortezomib. No significant changes in blood counts or liver enzyme levels were observed. A three-day regimen of 30 mg/kg led to 90% tumor growth suppression and doubled progression-free survival. KTS002 is a promising new therapy for MM, especially in high-risk patients, offering strong efficacy with minimal toxicity. Ongoing studies are focused on dose optimization, pharmacokinetics, and safety to support IND-enabling and clinical development.

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