L-DOS47 Elevates Pancreatic Cancer Tumor pH and Enhances Response to Immunotherapy.

Acidosis is an important immunosuppressive mechanism that leads to tumor growth. Therefore, we investigated the neutralization of tumor acidity to improve immunotherapy response. L-DOS47, a new targeted urease immunoconjugate designed to neutralize tumor acidity, has been well tolerated in phase I/IIa trials. L-DOS47 binds to CEACAM6, a cell-surface protein that is highly expressed in gastrointestinal cancers, allowing urease to cleave endogenous urea into two NH4+ and one CO2, thereby raising local pH. To test the synergetic effect of neutralizing tumor acidity with immunotherapy, we developed a pancreatic orthotopic murine tumor model (KPC961) expressing human CEACAM6. Using chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) to measure the tumor extracellular pH (pHe), we confirmed that L-DOS47 raises the tumor pHe from 4 h to 96 h post injection in acidic tumors (average increase of 0.13 units). Additional studies showed that combining L-DOS47 with anti-PD1 significantly increases the efficacy of the anti-PD1 monotherapy, reducing tumor growth for up to 4 weeks.

L-DOS47 enhances response to immunotherapy in pancreatic cancer tumor.

Acidosis is an important immunosuppressive mechanism that leads to tumor growth. Therefore, we investigated the neutralization of tumor acidity to improve immunotherapy response. L-DOS47, a new targeted urease immunoconjugate designed to neutralize tumor acidity, has been well tolerated in phase I/IIa trials. L-DOS47 binds CEACAM6, a cell surface protein highly expressed in gastrointestinal cancers, allowing urease to cleave endogenous urea into two NH4+ and one CO2, thereby raising local pH. To test the synergetic effect of neutralizing tumor acidity with immunotherapy, we developed a pancreatic orthotopic murine tumor model (KPC961) expressing human CEACAM6. Our results demonstrate that combining L DOS47 with anti-PD1 significantly increases the efficacy of anti-PD1 monotherapy, reducing tumor growth for up to 4 weeks.

A Phase 1, Open-Label, Dose-Escalation Study of L-DOS47 in Combination With Pemetrexed Plus Carboplatin in Patients With Stage IV Recurrent or Metastatic Nonsquamous NSCLC.

Introduction: L-DOS47, a targeted urease-anti-CEACAM6 immunoconjugate, alters the acidity of the tumor microenvironment by increasing local ammonia production. In vitro, the cytotoxic effects of L-DOS47 were additive when combined with pemetrexed and carboplatin. Methods: This phase I, open-label, dose-escalation study evaluated the safety and tolerability of up to four cycles of L-DOS47 (administered on days 1, 8, and 15 of each cycle at doses ranging from 0.59 to 9.0 µg/kg) combined with pemetrexed and carboplatin in patients with stage IV nonsquamous NSCLC. Continued L-DOS47 treatment after the fourth cycle was allowed at the treating physicians' discretion. Results: A total of 14 patients received at least one dose of L-DOS47. Overall, L-DOS47 was well tolerated. Grade greater than or equal to 3 adverse events (AEs) were typically neutropenia related. Two grade greater than or equal to 3 AEs and no serious AEs were considered at least possibly related to L-DOS47. No dose-limiting toxicities were reported, so the maximum tolerated dose was not reached. The objective response rate was 41.7% with a median duration of response of 187 days. Clinical benefit was observed in 75.0% of the patients. After the first dose, L-DOS47 systemic exposure increased in a generally dose-proportional manner but decreased substantially with repeat dosing. Anti-L-DOS47 antibodies were detectable in 13 of 14 patients by cycle 2 with titers typically increasing with continued treatment. There was an apparent association between best overall response rate and highest anti-L-DOS47 antibody titer measured. Conclusions: L-DOS47 combined with standard pemetrexed and carboplatin chemotherapy is well tolerated in patients with recurrent or metastatic nonsquamous NSCLC at doses up to 9.0 µg/kg.

Production and characterization of a camelid single domain antibody-urease enzyme conjugate for the treatment of cancer.

A novel immunoconjugate (L-DOS47) was developed and characterized as a therapeutic agent for tumors expressing CEACAM6. The single domain antibody AFAIKL2, which targets CEACAM6, was expressed in the Escherichia coli BL21 (DE3) pT7-7 system. High purity urease (HPU) was extracted and purified from Jack bean meal. AFAIKL2 was activated using N-succinimidyl [4-iodoacetyl] aminobenzoate (SIAB) as the cross-linker and then conjugated to urease. The activation and conjugation reactions were controlled by altering pH. Under these conditions, the material ratio achieved conjugation ratios of 8-11 antibodies per urease molecule, the residual free urease content was practically negligible (<2%), and high purity (>95%) L-DOS47 conjugate was produced using only ultradiafiltration to remove unreacted antibody and hydrolyzed cross-linker. L-DOS47 was characterized by a panel of analytical techniques including SEC, IEC, Western blot, ELISA, and LC-MS(E) peptide mapping. As the antibody-urease conjugate ratio increased, a higher binding signal was observed. The specificity and cytotoxicity of L-DOS47 was confirmed by screening in four cell lines (BxPC-3, A549, MCF7, and CEACAM6-transfected H23). BxPC-3, a CEACAM6-expressing cell line was found to be most susceptible to L-DOS47. L-DOS47 is being investigated as a potential therapeutic agent in human phase I clinical studies for nonsmall cell lung cancer.