Glen C. MacDonald

Engineering and biological characterization of VB6-845, an anti-EpCAM immunotoxin containing a T-cell epitope-depleted variant of the plant toxin bouganin.

The clinical development of immunotoxins in the treatment of solid tumors has been impeded in part, by the induction of an immune response directed primarily against the toxin moiety. Bouganin, a type I ribosome inactivating protein isolated from the leaf of Bougainvillea spectabilis Willd, was mutated to remove the T-cell epitopes while preserving the biological activity of the wild-type molecule. The T-cell epitope-depleted variant of bouganin (de-bouganin) was genetically linked to an anti-epithelial cell adhesion molecule (EpCAM) Fab moiety via a peptidic linker containing a furin proteolytic site to create the fusion construct VB6-845. To determine the optimal construct design for VB6-845, several dicistronic units where de-bouganin was genetically linked to either the N-terminal or C-terminal of either the heavy or light chain were engineered. Only the C-terminal variants expressed the full-length molecule. An in vitro assessment of the biological activity of VB6-845 showed that it bound and selectively killed EpCAM-positive cell lines with a greater potency than many commonly used chemotherapeutic agents. In vivo efficacy was demonstrated using an EpCAM-positive human tumor xenograft model in SCID mice with the majority of the mice treated being tumor free at the end of the study.

A Phase II Study of Oportuzumab Monatox: An Immunotoxin Therapy for Patients with Noninvasive Urothelial Carcinoma In Situ Previously Treated with Bacillus Calmette-Guérin

PURPOSE A phase II study was performed to assess the efficacy and tolerability of intravesical oportuzumab monatox in patients with urothelial carcinoma in situ of the bladder. Bacillus Calmette-Guérin treatment had previously failed in all patients. MATERIALS AND METHODS A total of 46 patients received 1 induction cycle of 6 (cohort 1) or 12 (cohort 2) weekly intravesical oportuzumab monatox (VB4-845) instillations of 30 mg, followed by up to 3 maintenance cycles of 3 weekly administrations every 3 months. RESULTS A complete response to oportuzumab monatox was seen in 9 of 22 patients (41%) in cohort 1 and 9 of 23 (39%) in cohort 2 at the 3-month evaluation. A total of 20 patients (44%) achieved a complete response. Two other patients without carcinoma in situ who achieved a complete response were not included in the study due to the development of noninvasive papillary (Ta) disease. Median time to recurrence in patients who achieved a complete response was 274 and 408 days in cohorts 1 and 2, respectively. Overall 7 patients (16%) remained disease-free. Post-study assessment demonstrated that these patients were still disease-free at last followup (18 to 25 months). The most common adverse events were mild to moderate reversible bladder symptoms. CONCLUSIONS Oportuzumab monatox was effective and well tolerated in patients with bacillus Calmette-Guérin refractory carcinoma in situ of the bladder. These results demonstrate the clinical benefit of oportuzumab monatox and support its continued development for the second line treatment of nonmuscle invasive bladder cancer.

A Phase I study of an intravesically administered immunotoxin targeting EpCAM for the treatment of nonmuscle-invasive bladder cancer in BCGrefractory and BCG-intolerant patients

Purpose A Phase I dose-escalation study was performed to determine the maximum tolerated dose (MTD) of the immunotoxin VB4-845 in patients with nonmuscle-invasive bladder cancer (NMIBC) refractory to or intolerant of bacillus Calmette–Guerin (BCG). Secondary objectives included evaluation of the safety, tolerability, pharmacokinetics, immunogenicity, and efficacy of VB4-845. Patients and methods Sixty-four patients with Grade 2 or 3, stage Ta or T1 transitional cell carcinoma or in situ carcinoma, either refractory to or intolerant of BCG therapy, were enrolled. Treatment was administered in ascending dose cohorts ranging from 0.1 to 30.16 mg. After receiving weekly instillations of VB4-845 to the bladder via catheter for 6 consecutive weeks, patients were followed for 4–6 weeks post-therapy and assessed at week 12. Results An MTD was not determined, as a dose-limiting toxicity was not identified over the dose range tested. VB4-845 therapy was safe and well tolerated with most adverse events reported as mild; as a result, no patients were removed from the study in response to toxicity. By the end of the study, the majority of patients had developed antibodies to the exotoxin portion of VB4-845. A complete response was achieved in 39% of patients at the 12-week time point. Conclusions VB4-845 dosed on a weekly basis for 6 weeks was very well tolerated at all dose levels. Although an MTD was not determined at the doses administered, VB4-845 showed evidence of an antitumor effect that warrants further clinical investigation for the treatment of NMIBC in this patient population.

A phase I clinical study of VB4-845: weekly intratumoral administration of an anti-EpCAM recombinant fusion protein in patients with squamous cell carcinoma of the head and neck.

VB4-845 is a scFv-Pseudomonas exotoxin A fusion construct that targets epithelial cell adhesion molecule (EpCAM). A phase I trial was conducted to determine the maximum tolerated dose (MTD) of VB4-845 when administered as weekly intratumoral (IT) injections to patients with squamous cell carcinoma of the head and neck (SCCHN). Secondary objectives included the evaluation of the safety, tolerability, pharmacokinetic profile, and immunogenicity, and a preliminary assessment of tumor response. Twenty patients with advanced, recurrent SCCHN were treated weekly for four weeks in ascending dose cohorts of 100, 200, 330, 500, 700, and 930 μg. The MTD was established as 930 μg with a dose limiting toxicity of elevated liver enzymes in two of five patients. VB4-845 therapy was well tolerated with common treatment-related adverse events of injection site reactions, fever, gastrointestinal disorders, and elevated liver enzyme levels. All patients developed antibodies to VB4-845 by the end of the study, but only seven patients had neutralizing antibodies. Preliminary efficacy data found 87.5% of EpCAM-positive patients had a positive response to VB4-845 therapy. Noninjected dermal metastases were also resolved in one patient. VB4-845 IT therapy is safe and feasible and warrants further clinical evaluation for the treatment of SCCHN.

Preclinical Evaluation of VB6-845: An Anti-EpCAM Immunotoxin with Reduced Immunogenic Potential

VB6-845 is a recombinant immunotoxin comprised of deBouganin (a de-immunized plant toxin) genetically linked to an epithelial cell adhesion molecule (EpCAM)-targeting humanized Fab fragment (4D5MOCB). EpCAM is highly expressed on a wide range of epithelial tumors but has limited expression on most normal epithelia and therefore represents an excellent target for immunotherapy. A comprehensive preclinical evaluation was performed to determine the safety and suitability of VB6-845 as a systemically administered drug for the treatment of solid tumors. Efficacy studies in mice demonstrated that VB6-845 specifically and potently targeted EpCAM-positive tumors. In a dose-ranging study in Sprague-Dawley rats, single doses of VB6-845 were well-tolerated resulting in a no-observable adverse effect level (NOAEL) of 100 mg/kg whereas repeated doses of VB6-845 resulted in vascular leak-associated symptoms particularly at higher dose levels. However, much higher doses in Cynomolgus monkeys were well-tolerated when given as a 3-hour infusion mimicking the intended route of administration in the clinic. In addition, VB6-845 proved to be minimally immunogenic in monkeys. The toxicological data obtained in Cynomolgus monkeys indicated an excellent safety profile with a NOAEL value of 30 mg/kg (equivalent to a 10 mg/kg dose in humans). These results are supportive of an exploratory Phase I trial.

Development of a GMP Phase III purification process for VB4-845, an immunotoxin expressed in E. coli using high cell density fermentation.

VB4-845 is a recombinant immunotoxin comprised of an anti-epithelial cell adhesion molecule (EpCAM) scFv fused to a truncated form of the bacterial toxin, Pseudomonas exotoxin A. VB4-845, purified from TB fed-batch fermentation, showed clinical efficacy when administered locally to treat non-muscle invasive bladder cancer (NMIBC) and squamous cell carcinomas of the head and neck (SCCHN). Here, we describe the implementation of an Escherichia coli high cell density (HCD) cultivation and purification process for VB4-845. HCD cultivation was a prerequisite for achieving higher yields necessary for Phase III clinical trials and commercialization. Using this process, the VB4-845 titer in the supernatant was increased by 30-fold over the original TB fed-batch cultivation. To obtain clinical grade material, a process involving a five-step column purification procedure was implemented and led to an overall recovery of ∼ 40%. VB4-845 purity of >97% was achieved after the first three columns following the removal of low-molecular weight product-related impurities and aggregates. Endotoxins were effectively separated from VB4-845 on the Q-columns and by washing the Ni-column with a detergent buffer while host cell proteins were removed using ceramic hydroxyapatite. Comparability studies demonstrated that the purified product from the Phase III process was identical to the Phase II reference standard produced using TB fed-batch fermentation.

Preclinical Assessment of an Anti-EpCAM Immunotoxin: Locoregional Delivery Provides a Safer Alternative to Systemic Administration

VB4-845 is a recombinant immunotoxin that is comprised of a truncated form of Pseudomonas exotoxin A (ETA) genetically-linked to a humanized scFv fragment, (4D5MOCB), specific to epithelial cell adhesion molecule (EpCAM). EpCAM is overexpressed on a wide variety of human tumors and thus represents a suitable target antigen for immunotoxin therapy. Preclinical studies were used to evaluate the benefit of locoregional administration of an ETA-based immunotoxin versus systemic delivery. Repeated subcutaneous (s.c.) administration of VB4-845 (up to 77.8 microg/kg) in rats resulted in minimal adverse effects, except for injection-site reactions, while repeated systemic administration elicited symptoms consistent with vascular leak syndrome. S.c. weekly doses of the drug in cynomolgus monkeys resulted in minimal adverse effects limited to injection-site reactions and a transient elevation of liver enzymes in 1 animal. Toxicokinetics showed rapid clearance of the drug, with the development of an immune response by day 14 following repeated injections. These results argue that the local administration of VB4-845 has advantages with respect to safety over systemic administration and may be an effective alternative method for targeting those cancers that are amenable to local routes of administration.

Trastuzumab-deBouganin Conjugate Overcomes Multiple Mechanisms of T-DM1 Drug Resistance.

The development of antibody drug conjugates has provided enhanced potency to tumor-targeting antibodies by the addition of highly potent payloads. In the case of trastuzumab-DM1 (T-DM1), approved for the treatment of metastatic breast cancer, the addition of mertansine (DM1) to trastuzumab substantially increased progression-free survival. Despite these improvements, most patients eventually relapse due to complex mechanisms of resistance often associated with small molecule chemotherapeutics. Therefore, identifying payloads with different mechanisms of action (MOA) is critical for increasing the efficacy of targeted therapeutics and ultimately improving patient outcomes. To evaluate payloads with different MOA, deBouganin, a deimmunized plant toxin that inhibits protein synthesis, was conjugated to trastuzumab and compared with T-DM1 both in vitro and in vivo. The trastuzumab-deBouganin conjugate (T-deB) demonstrated greater potency in vitro against most cells lines with high levels of Her2 expression. In addition, T-deB, unlike T-DM1, was unaffected by inhibitors of multidrug resistance, Bcl-2-mediated resistance, or Her2-Her3 dimerization. Contrary to T-DM1 that showed only minimal cytotoxicity, T-deB was highly potent in vitro against tumor cells with cancer stem cell properties. Overall, the results demonstrate the potency and efficacy of deBouganin and emphasize the importance of using payloads with different MOAs. The data suggest that deBouganin could be a highly effective against tumor cell phenotypes not being addressed by current antibody drug conjugate formats and thereby provide prolonged clinical benefit.

Fusogenics: a recombinant immunotoxin-based screening platform to select internalizing tumor-specific antibody fragments.

Antibody-based therapeutics play a vital role in the treatment of certain cancers; however, despite commercial success, various strategies are being pursued to increase their potency and hence improve patient outcomes. The use of antibodies to deliver a cytotoxic payload offers a promising alternative for more efficacious therapies. Immunotoxins are composed of an internalizing antibody fragment linked to a bacterial or plant toxin. Once internalized, the payload, such as Pseudomonas exotoxin A (PE), blocks protein synthesis and induces apoptosis. Typically, immunotoxins are developed by first isolating a tumor-specific antibody, which is then either chemically linked to a toxin or reengineered as a fusion protein. Here, the authors describe the development of Fusogenics, an immunotoxin-based screening method that selects internalizing tumor-specific antibodies using a functional assay. Selected immune library clones were characterized and shown to be selective against normal tissues and specific to tumor tissues. In summary, the Fusogenics immunotoxin platform represents a unique, single-step selection approach combining specificity and functionality to isolate novel internalizing tumor-specific antibody fragments with potential for direct clinical application in the treatment of cancer.

DeBouganin Diabody Fusion Protein Overcomes Drug Resistance to ADCs Comprised of Anti-Microtubule Agents

Antibody drug conjugates (ADC), comprised of highly potent small molecule payloads chemically conjugated to a full-length antibody, represent a growing class of therapeutic agents. The targeting of cytotoxic payloads via the specificity and selectivity of the antibody has led to substantial clinical benefits. However, ADC potency can be altered by mechanisms of resistance such as overexpression of efflux pumps or anti-apoptotic proteins. DeBouganin is a de-immunized variant of bouganin, a ribosome-inactivating protein (RIP) that blocks protein synthesis, thereby leading to apoptosis. When conjugated to trastuzumab (T-deB), deBouganin was more potent than ado-trastuzumab-emtansine (T-DM1) and unaffected by resistance mechanisms to which DM1 is susceptible. To further highlight the differentiating mechanism of action of deBouganin, HCC1419 and BT-474 tumor cells that survived T-DM1 or trastuzumab-MMAE (T-MMAE) treatment were treated with an anti-HER2 C6.5 diabody–deBouganin fusion protein or T-deB. C6.5 diabody–deBouganin and T-deB were potent against HCC1419 and BT-474 cells that were resistant to T-DM1 or T-MMAE killing. The resistant phenotype involved MDR pumps, Bcl-2 family members, and the presence of additional unknown pathways. Overall, the data suggest that deBouganin is effective against tumor cell resistance mechanisms selected in response to ADCs composed of anti-microtubule payloads.