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.

Development and Characterization of a Camelid Single Domain Antibody-Urease Conjugate That Targets Vascular Endothelial Growth Factor Receptor 2.

Angiogenesis is the process of new blood vessel formation and is essential for a tumor to grow beyond a certain size. Tumors secrete the pro-angiogenic factor vascular endothelial growth factor, which acts upon local endothelial cells by binding to vascular endothelial growth factor receptors (VEGFRs). In this study, we describe the development and characterization of V21-DOS47, an immunoconjugate that targets VEGFR2. V21-DOS47 is composed of a camelid single domain anti-VEGFR2 antibody (V21) and the enzyme urease. The conjugate specifically binds to VEGFR2 and urease converts endogenous urea into ammonia, which is toxic to tumor cells. Previously, we developed a similar antibody-urease conjugate, L-DOS47, which is currently in clinical trials for non-small cell lung cancer. Although V21-DOS47 was designed from parameters learned from the generation of L-DOS47, additional optimization was required to produce V21-DOS47. In this study, we describe the expression and purification of two versions of the V21 antibody: V21H1 and V21H4. Each was conjugated to urease using a different chemical cross-linker. The conjugates were characterized by a panel of analytical techniques, including SDS-PAGE, size exclusion chromatography, Western blotting, and LC-MSE peptide mapping. Binding characteristics were determined by ELISA and flow cytometry assays. To improve the stability of the conjugates at physiologic pH, the pIs of the V21 antibodies were adjusted by adding several amino acid residues to the C-terminus. For V21H4, a terminal cysteine was also added for use in the conjugation chemistry. The modified V21 antibodies were expressed in the E. coli BL21 (DE3) pT7 system. V21H1 was conjugated to urease using the heterobifunctional cross-linker succinimidyl-[(N-maleimidopropionamido)-diethyleneglycol] ester (SM(PEG)2), which targets lysine resides in the antibody. V21H4 was conjugated to urease using the homobifunctional cross-linker, 1,8-bis(maleimido)diethylene glycol (BM(PEG)2), which targets the cysteine added to the antibody C-terminus. V21H4-DOS47 was determined to be the superior conjugate as the antibody is easily produced and purified at high levels, and the conjugate can be efficiently generated and purified using methods easily transferrable for cGMP production. In addition, V21H4-DOS47 retains higher binding activity than V21H1-DOS47, as the native lysine residues are unmodified.

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.

Urease-induced alkalinization of extracellular pH and its antitumor activity in human breast and lung cancers.

Jack bean urease catalyzes the decomposition of urea into ammonia, which in turn increases the pH of the surrounding medium. Based on these two properties, we have investigated the antitumor effects of urease in vitro and in vivo on human lung and breast cancer cell lines either by the enzyme itself or in combination with other chemotherapeutic drugs. First, through the generation of toxic ammonia, urease exerted direct cytotoxicity on A549 and MDA-MB-231 tumor cells with LC50 of 0.22 and 0.45 U/ml, respectively. The cytotoxic effects could effectively be blocked using the reversible urease inhibitor acetohydroxamic acid. Complete protection was observed at dose > or = 2 mM. In addition, nude mouse xenograft models demonstrated that intratumoral urease injections (1 - 10 U/dose) inhibited A549 and MCF-7 tumor growth in vivo. Second, when combined with weak-base anticancer drugs, urease provided indirect antitumor effects via pH augmentation. Alkalinization of extracellular pH by urease (2 U/ml) and urea (> or = 2 mM) was found to enhance the antitumor efficacy of doxorubicin (50 microM) and vinblastine (100 microM) significantly.

Enhancement of the antagonistic potency of transforming growth factor-beta receptor extracellular domains by coiled coil-induced homo- and heterodimerization.

Transforming growth factor-beta (TGF-beta) plays a causal role in several human pathologies including fibrotic diseases and metastasis. TGF-beta signaling is mediated through its interaction with three types of cell surface receptors, RI, RII, and RIII. The soluble ectodomains of RII and RIII bind to TGF-beta, making them attractive candidates to sequester TGF-beta and inhibit its activity. To optimize the activity of the ectodomains, we studied the effect of artificially dimerizing them upon their kinetics of binding to TGF-beta using an optical biosensor and studied their antagonistic potencies using an in vitro signaling assay. We fused the RII ectodomain and the membrane-proximal ligand-binding domain of the RIII ectodomain to de novo designed heterodimerizing coil strands and demonstrated that the coil strands within the fusion proteins were capable of promoting the dimerization of the coil-tagged ectodomains. Our results indicate that coiled coil-induced dimerization of the ectodomains stabilized their interaction with TGF-beta as compared with the monomeric ectodomains. Also, in contrast to the monomeric ectodomains, which did not block signaling, the coiled coil-induced dimers were characterized by antagonistic potencies in the low nanomolar range.