Antibody Directed Enzyme Prodrug Therapy (ADEPT): Trials and tribulations.

Antibody directed enzyme prodrug therapy has the potential to be an effective therapy for most common solid cancers. Clinical studies with CPG2 system have shown the feasibility of this approach. The key limitation has been immunogenicity of the enzyme. Technologies now exist to eliminate this problem. Non-immunogenic enzymes in combination with prodrugs that generate potent cytotoxic drugs can provide a powerful approach to cancer therapy. ADEPT has the potential to be non -toxic to normal tissue and can therefore be combined with other modalities including immunotherapy for greater clinical benefit.

The clinical efficacy of first-generation carcinoembryonic antigen (CEACAM5)-specific CAR T cells is limited by poor persistence and transient pre-conditioning-dependent respiratory toxicity.

The primary aim of this clinical trial was to determine the feasibility of delivering first-generation CAR T cell therapy to patients with advanced, CEACAM5+ malignancy. Secondary aims were to assess clinical efficacy, immune effector function and optimal dose of CAR T cells. Three cohorts of patients received increasing doses of CEACAM5+-specific CAR T cells after fludarabine pre-conditioning plus systemic IL2 support post T cell infusion. Patients in cohort 4 received increased intensity pre-conditioning (cyclophosphamide and fludarabine), systemic IL2 support and CAR T cells. No objective clinical responses were observed. CAR T cell engraftment in patients within cohort 4 was significantly higher. However, engraftment was short-lived with a rapid decline of systemic CAR T cells within 14 days. Patients in cohort 4 had transient, acute respiratory toxicity which, in combination with lack of prolonged CAR T cell persistence, resulted in the premature closure of the trial. Elevated levels of systemic IFNγ and IL-6 implied that the CEACAM5-specific T cells had undergone immune activation in vivo but only in patients receiving high-intensity pre-conditioning. Expression of CEACAM5 on lung epithelium may have resulted in this transient toxicity. Raised levels of serum cytokines including IL-6 in these patients implicate cytokine release as one of several potential factors exacerbating the observed respiratory toxicity. Whilst improved CAR designs and T cell production methods could improve the systemic persistence and activity, methods to control CAR T 'on-target, off-tissue' toxicity are required to enable a clinical impact of this approach in solid malignancies.

Translating antibody directed enzyme prodrug therapy (ADEPT) and prospects for combination.

INTRODUCTION: The generation of cytotoxic drugs, selectively within tumours, from non-toxic prodrugs by targeted enzymes provides a powerful system for cancer therapy. In the form of Antibody directed enzyme prodrug therapy (ADEPT), this approach has shown feasibility in the clinic. Areas covered: Although numerous enzyme prodrug combinations have been reported over the last two decades, only the CPG2 ADEPT system has progressed to clinical trials. Using readily available components such as chemical antibody enzyme conjugate or recombinant multifunctional fusion protein, delivery of a specific enzyme to tumours, its elimination from non-tumour sites and prodrug activation has been achieved with therapeutic benefit in the clinic. The challenge here is to overcome immunogenicity of CPG2. Technology exists to overcome this limitation together with prospects for rational design of combined therapy. Expert opinion: ADEPT has the potential to be an effective treatment for solid cancer. However, the system necessitates a multi-disciplinary and iterative approach. Although xenograft studies provide a consistent guide it is only through clinical studies that the real challenges can be identified. The emerging preclinical data with other enzyme prodrug systems may provide the opportunity to develop the next generation ADEPT comprising non-immunogenic enzymes to generate potent cytotoxic drugs within tumours.

Optic nerve involvement in a borderline lepromatous leprosy patient on multidrug therapy.

Amidst the plethora of ocular complications of leprosy, involvement of the posterior segment or optic nerve is extremely rare. The mechanism of optic neuritis in leprosy is poorly understood. A 47 year-old man presented with a single lesion suggestive of mid-borderline (BB) leprosy over left periorbital region; the histology showed borderline lepromatous (BL) leprosy with a BI of 3+. After initial improvement with WHO MDT-MB and prednisolone (40 mg/d) he developed sudden and painless diminished vision in the left eye, about 3 weeks later. His visual acuity was 6/9 in the left and 6/6 in the right eye, and there was left optic disc edema, hyperemia and blurred disc margins. Treatment with prednisolone (60 mg/d) along with WHO MDT-MB continued. A month later he returned with painless diminished vision in the other eye as well. Visual acuity was 6/6 in the right and 6/12 in the left eye, and there was right optic disc edema and left optic disc atrophy. CT of the head and MRI of the brain were normal. Inflammatory edema of the orbital connective tissue or other surrounding structures, or direct infiltration of vasa nervosa with resultant vascular occlusion leading to optic nerve ischemia, seems the most plausible explanation of optic nerve involvement in this case.

Left ventricular diastolic dysfunction in COPD may manifest myocardial ischemia.

BACKGROUND: Left ventricular diastolic dysfunction (LVDD) is observed frequently in advanced COPD and it appears unrelated to the co-presence of its common risk factors as hypertension, diabetes, ischemic heart disease, etc. The patho-physiology of this association is yet unclear. We postulated that ischemia not apparent in the routine clinical evaluation could be the cause of such LVDD. METHOD: Advanced COPD (GOLD III & IV) patients being excluded of concomitant presence of hypertension, diabetes, ischemic heart disease, and hypothyroidism by conventional evaluations were looked for the presence of LVDD by tissue Doppler echocardiography. The subjects having LVDD were further evaluated with 99(m)Tc MIBI stress myocardial perfusion imaging to detect the presence of ischemia. RESULTS: 7 out of 14 patients of advanced COPD sufferers without any known risk factors for LVDD had reversible perfusion defect in the stress perfusion imaging predominantly involving the inferior wall myocardium (71.43%). Most of the defects, though significant, were not picked up by stress ECG. CONCLUSION: Reversible ischemic defects are quite common (50%) in advanced COPD patients with LVDD without the presence of common risk factors. This association needs further evaluation.

Antibody-enzyme fusion proteins for cancer therapy.

Advances in biomolecular technology have allowed the development of genetically fused antibody-enzymes. Antibody-enzyme fusion proteins have been used to target tumors for cancer therapy in two ways. In one system, an antibody-enzyme is pretargeted to the tumor followed by administration of an inactive prodrug that is converted to its active form by the pretargeted enzyme. This system has been described as antibody-directed enzyme prodrug therapy. The other system uses antibody-enzyme fusion proteins as direct therapeutics, where the enzyme is toxic in its own right. The key feature in this approach is that the antibody is used to internalize the toxic enzyme into the tumor cell, which activates cell-death processes. This antibody-enzyme system has been largely applied to deliver ribonucleases. This article addresses these two antibody-enzyme targeting strategies for cancer therapy from concept to (pre)clinical trials.

A Phase I Clinical Trial of CHT-25 a 131I-Labeled Chimeric Anti-CD25 Antibody Showing Efficacy in Patients with Refractory Lymphoma.

PURPOSE: There is a need for new treatments for Hodgkin and T-cell lymphoma due to the development of drug resistance in a proportion of patients. This phase I study of radioimmunotherapy used CHT-25, a chimeric antibody to the alpha-chain of the interleukin-2 receptor, CD25, conjugated to iodine-131 ((131)I) in patients with refractory CD25-positive lymphomas. EXPERIMENTAL DESIGN: Fifteen patients were treated (Hodgkin lymphoma, 12; angioimmunoblastic T-cell lymphoma, 1; adult T-cell leukemia/lymphoma, 2). Tumor was monitored by computed tomography and in all but two patients by (18)F-fluorodeoxyglucose positron emission tomography. RESULTS: There were no grade 3 or 4 infusion reactions. At the maximum tolerated dose of 1,200 MBq/m(2), the major side effect was delayed myelotoxicity with the nadir for platelets at 38 days and for neutrophils at 53 days. One patient treated with 2,960 MBq/m(2) developed prolonged grade 4 neutropenia and thrombocytopenia and died of Pneumocystis jiroveci pneumonia. Nonhematologic toxicity was mild. Single photon emission computer tomography imaging showed tumor-specific uptake and retention of (131)I and no excessive retention in normal organs. Of nine patients receiving >/=1,200 MBq/m(2), six responded (three complete response and three partial response); one of six patients with administered radioactivity of

A phase I trial of radioimmunotherapy with 131I-A5B7 anti-CEA antibody in combination with combretastatin-A4-phosphate in advanced gastrointestinal carcinomas.

PURPOSE: In preclinical models, radioimmunotherapy with (131)I-A5B7 anti-carcinoembryonic antigen (CEA) antibody ((131)I-A5B7) combined with the vascular disruptive agent combretastatin-A4-phosphate (CA4P) produced cures unlike either agent alone. We conducted a phase I trial determining the dose-limiting toxicity (DLT), maximum tolerated dose, efficacy, and mechanism of this combination in patients with gastrointestinal adenocarcinomas. EXPERIMENTAL DESIGN: Patients had CEA of 10 to 1,000 microg/L, QTc < or =450 ms, no cardiac arrhythmia/ischaemia, and adequate hematology/biochemistry. Tumor was suitable for blood flow analysis by dynamic contrast enhanced-magnetic resonance imaging (MRI). The starting dose was 1,800 MBq/m(2) of (131)I-A5B7 on day 1 and 45 mg/m(2) CA4P given 48 and 72 hours post-(131)I-A5B7, then weekly for up to seven weeks. RESULTS: Twelve patients were treated, with mean age of 63 years (range, 32-77). Two of six patients at the first dose level had DLTs (grade 4 neutropenia). The dose was reduced to 1,600 MBq/m(2), and CA4P escalated to 54 mg/m(2). Again, two of six patients had DLTs (neutropenia). Of ten assessable patients, three had stable disease and seven had progressive disease. Single-photon emission computed tomography confirmed tumor antibody uptake in all 10 patients. DCE-MRI confirmed falls in kinetic parameters (K(trans)/IAUGC(60)) in 9 of 12 patients. The change of both pharmacokinetic parameters reached a level expected to produce efficacy in one patient who had a minor response on computed tomography and a reduced serum tumor marker level. CONCLUSIONS: This is believed to be the first trial reporting the combination of radioimmunotherapy and vascular disruptive agent; each component was shown to function, and myelosuppression was dose-limiting. Optimal dose and timing of CA4P, and moderate improvements in the performance of radioimmunotherapy seem necessary for efficacy.

Clearance mechanism of a mannosylated antibody-enzyme fusion protein used in experimental cancer therapy.

MFECP1 is a mannosylated antibody-enzyme fusion protein used in antibody-directed enzyme prodrug therapy (ADEPT). The antibody selectively targets tumor cells and the targeted enzyme converts a prodrug into a toxic drug. MFECP1 is obtained from expression in the yeast Pichia pastoris and produced to clinical grade. The P. pastoris-derived mannosylation of the fusion protein aids rapid normal tissue clearance required for successful ADEPT. The work presented provides evidence that MFECP1 is cleared by the endocytic and phagocytic mannose receptor (MR), which is known to bind to mannose-terminating glycans. MR-transfected fibroblast cells internalize MFECP1 as revealed by flow cytometry and confocal microscopy. Immunofluorescence microscopy shows that in vivo clearance in mice occurs predominantly by MR on liver sinusoidal endothelial cells, although MR is also expressed on adjacent Kupffer cells. In the spleen, MFECP1 is taken up by MR-expressing macrophages residing in the red pulp and not by dendritic cells which are found in the marginal zone and white pulp. Clearance can be inhibited in vivo by the MR inhibitor mannan as shown by increased enzyme activities in blood. The work improves understanding of interactions of MFECP1 with normal tissue, shows that glycosylation can be exploited in the design of recombinant anticancer therapeutics and opens the ways for optimizing pharmacokinetics of mannosylated recombinant therapeutics.

A phase I study of single administration of antibody-directed enzyme prodrug therapy with the recombinant anti-carcinoembryonic antigen antibody-enzyme fusion protein MFECP1 and a bis-iodo phenol mustard prodrug.

PURPOSE: Antibody-directed enzyme prodrug therapy is a two-stage treatment whereby a tumor-targeted antibody-enzyme complex localizes in tumor for selective conversion of prodrug. The purpose of this study was to establish optimal variables for single administration of MFECP1, a recombinant antibody-enzyme fusion protein of an anti-carcinoembryonic antigen single-chain Fv antibody and the bacterial enzyme carboxypeptidase G2 followed by a bis-iodo phenol mustard prodrug. MFECP1 is manufactured in mannosylated form to facilitate normal tissue elimination. EXPERIMENTAL DESIGN: Pharmacokinetic, biodistribution, and tumor localization studies were used to test the hypothesis that MFECP1 localizes in tumor and clears from normal tissue via the liver. Firstly, safety of MFECP1 and a blood concentration of MFECP1 that would avoid systemic prodrug activation were tested. Secondly, dose escalation of prodrug was done. Thirdly, the dose of MFECP1 and timing of prodrug administration were optimized. RESULTS: MFECP1 was safe and well tolerated, cleared rapidly via the liver, and was less immunogenic than previously used products. Eighty-fold dose escalation from the starting dose of prodrug was carried out before dose-limiting toxicity occurred. Confirmation of the presence of enzyme in tumor and DNA interstrand cross-links indicating prodrug activation were obtained for the optimal dose and time point. A total of 28 of 31 patients was evaluable for response, the best response being a 10% reduction of tumor diameter, and 11 of 28 patients had stable disease. CONCLUSIONS: Optimal conditions for effective therapy were established. A study testing repeat treatment is currently being undertaken.

Cyclooxygenase-2 expression increases with the stage and grade in transitional cell carcinoma of the urinary bladder.

OBJECTIVES: Experimental models of carcinogenesis show that non-steroidal anti-inflammatory drugs (NSAIDs) increase apoptosis, inhibit angiogenesis and reduce metastases. A linkage between the activity of prostaglandin synthase enzyme cyclooxygenase-2 (COX-2), a known mediator of inflammation, and cancer angiogenesis is implicated. We investigated the expression of COX-2 in bladder cancer tissue specimens using immunohistochemistry. METHODS: The immunohistochemical expression of COX-2 in bladder cancer was evaluated by scoring the intensity of immunoreactivity from 0 to 3. Further, the degree of COX-2 expression was correlated with the tumor grade and depth of invasion (T stage). RESULT: Fifty eight percent patients (n=22) had superficial bladder tumors, while 42% (n=16) were invasive bladder cancers. Overall, COX-2 immuno-positivity was seen in 84.2% (32/38) patients. COX-2 expression was positive in 76.4% (13/17) cases with pTa tumors, 100% (5/5) of pT1 tumors, 86.6% (13/15) of pT2 tumors and in 100% (1/1) of pT3 tumor. The higher stage tumors stained more intensely; this correlation was significant(p=0.01987; chi2=19.6977). With reference to the grade of tumors, a positive expression was seen in 81.25% (13/16) of the low-grade tumors and 89% (17/19) of the high-grade tumors. The differential COX-2 expression relative to the grade of tumor was found to be statistically significant (p=0.05; chi2=15.8612). CONCLUSION: The degree of COX-2 expression is significantly increased with advancing grade and T stage of disease (p < 0.05).

Advances in antibody-directed enzyme prodrug therapy.

Antibody-directed enzyme prodrug therapy has demonstrated feasibility as a treatment for cancer. Numerous prodrug/drug systems have been developed for activation by a variety of enzymes and although many have shown potential in preclinical studies, so far only one system has progressed to the clinic. Clinical studies have identified issues that were not readily apparent in xenograft models, however, these have not been addressed in the development and testing of new prodrugs. The issue of immunogenicity arising from the use of non-human enzymes has also been a major hurdle. The development of recombinant fusion proteins provides reproducible and effective antibody-enzyme products that retain the necessary specificity for prodrug activation. Advances in molecular, structural and systems biology, in combination with bioinformatics, have allowed these molecules to be readily manipulated to provide the desired characteristics.

Sustained tumor regression of human colorectal cancer xenografts using a multifunctional mannosylated fusion protein in antibody-directed enzyme prodrug therapy.

PURPOSE: Antibody-directed enzyme prodrug therapy (ADEPT) requires highly selective antibody-mediated delivery of enzyme to tumor. MFE-CP, a multifunctional genetic fusion protein of antibody and enzyme, was designed to achieve this by two mechanisms. First by using a high affinity and high specificity single chain Fv antibody directed to carcinoembryonic antigen. Second by rapid removal of antibody-enzyme from normal tissues by virtue of post-translational mannosylation. The purpose of this paper is to investigate these dual functions in an animal model of pharmacokinetics, pharmacodynamics, toxicity, and efficacy. EXPERIMENTAL DESIGN: MFE-CP was expressed in the yeast Pichia pastoris and purified via an engineered hexahistidine tag. Biodistribution and therapeutic effect of a single ADEPT cycle (1,000 units/kg MFE-CP followed by 70 mg/kg ZD2767P prodrug at 6, 7, and 8 hours) and multiple ADEPT cycles (9-10 cycles within 21-24 days) was studied in established human colon carcinoma xenografts, LS174T, and SW1222. RESULTS: Selective localization of functional enzyme in tumors and rapid clearance from plasma was observed within 6 hours, resulting in tumor to plasma ratios of 1,400:1 and 339:1, respectively for the LS174T and SW1222 models. A single ADEPT cycle produced reproducible tumor growth delay in both models. Multiple ADEPT cycles significantly enhanced the therapeutic effect of a single cycle in the LS174T xenografts (P = 0.001) and produced regressions in the SW1222 xenografts (P = 0.0001), with minimal toxicity. CONCLUSIONS: MFE-CP fusion protein, in combination with ZD2767P, provides a new and successful ADEPT system, which offers the potential for multiple cycles and antitumor efficacy. These results provide a basis for the next stage in clinical development of ADEPT.

Antibody-directed enzyme prodrug therapy (ADEPT) for cancer.

Antibody-directed enzyme prodrug therapy (ADEPT) aims to restrict the cytotoxic action to tumour sites. The obstacles to achieve this were recognised at the outset, but time and experience have given these better definition. The development of fusion proteins has provided the means of making consistent antibody-enzyme constructs on an adequate scale, and glycosylation has provided the means to control the clearance of enzyme from non-tumour sites. Human enzymes have yet to be tested in a clinical setting, and there are pointers indicating that the immunological response to foreign enzymes can be overcome. The relatively small number of purpose-designed prodrugs tested so far leaves this an area ripe for further development. The ongoing iterative process between preclinical and clinical studies is critical to achieving the objective.

Glycoforms obtained by expression in Pichia pastoris improve cancer targeting potential of a recombinant antibody-enzyme fusion protein.

MFE-CP is a recombinant antibody-enzyme fusion protein used for antibody-mediated delivery of an enzyme to cancer deposits. After clearance from normal tissues, the tumor-targeted enzyme is used to activate a subsequently administered prodrug to give a potent cytotoxic in the tumor. MFE-CP localizes to cancer deposits in vivo, but we propose that its therapeutic potential could be improved by N-glycosylation, obtained by expression in Pichia pastoris. Glycosylation could enhance clearance from healthy tissue and result in better tumor:normal tissue ratios. To test this, glycosylated MFE-CP was expressed and purified from P. pastoris. The resultant MFE-CP fusion protein was enzymatically active and showed enhanced clearance from normal tissues in vivo. Furthermore, it showed effective tumor localization. This favorable glycosylation pattern was analyzed by tandem mass spectrometry. High-resolution, high-detection sensitivity collision-induced dissociation experiments proved essential for this task. Results showed that of the three potential N-glycosylation sites only two were consistently occupied with oligomannose structures. Asn-442 appeared the most heterogeneously populated with oligomannose carbohydrates extending from 5 to 13 units in length. Asn-484 was found only in its nonglycosylated form. There was less heterogeneity at Asn-492, which was glycosylated with oligosaccharide structures ranging from 8 to 10 mannose units. Nonglycosylated forms of Asn-442 and Asn-492 were not observed.

A strategy for mapping and neutralizing conformational immunogenic sites on protein therapeutics.

Antibodies are highly specific recognition molecules which are increasingly being applied to target therapy in patients. One type of developmental antibody-based therapy is antibody directed enzyme prodrug therapy (ADEPT) for the treatment of cancer. In ADEPT, an antibody specific to a tumor marker protein delivers a drug-activating enzyme to the cancer. Subsequent intravenous administration of an inactive prodrug results in drug activation and cytotoxicity only within the locale of the tumor. Pilot clinical trials with chemical conjugates of the prodrug activating enzyme carboxypeptidase G2 (CPG2) chemically conjugated with an antibody to and carcinoembryonic antigen (CEA), have shown that CPG2-mediated ADEPT is effective but limited by formation of human antibodies to CPG2 (HACA). We have developed a recombinant fusion protein (termed MFE-CP) of CPG2 with an anti-CEA single chain Fv antibody fragment and we have developed methods to address the immunogenicity of this therapeutic. A HACA-reactive discontinuous epitope on MFE-CP was identified using the crystal structure of CPG2, filamentous phage technology and surface enhanced laser desorption/ionization affinity mass spectrometry. This information was used to create a functional mutant of MFE-CP with a significant reduction (range 19.2 to 62.5%, median 38.5%) in reactivity with the sera of 11 patients with post-therapy HACA. The techniques described here are valuable tools for identifying and adapting undesirable immunogenic sites on protein therapeutics.

Reactions of Dimethyl Sulfite with Diorganotin Oxides. One-Pot Synthesis of Methoxydiorganotin Methanesulfonates through the Arbuzov Rearrangement, Spectroscopic Characterization of These Compounds and Their Derivatives, and X-ray Crystal Structures of n-Bu(2)Sn(X)OS(O)(2)Me (X = acac, bzbz, OH).

One-pot reactions of diorganotin oxides, R(2)SnO, with dimethyl sulfite under reflux conditions (125-127 degrees C) proceed via the Arbuzov rearrangement at the sulfur center, yielding the corresponding methoxydiorganotin methanesulfonates, R(2)Sn(OMe)OS(O)(2)Me [R = n-Pr (1), n-Bu (2), i-Bu (3), c-Hx (4)], as white, hygroscopic solids. These compounds react with beta-diketones [acetylacetone (Hacac), benzoylacetone (Hbzac), and dibenzoylmethane (Hbzbz)] to afford mixed-ligand organotin derivatives, R(2)Sn(X)OS(O)(2)Me [X = acac, R = n-Pr (5), n-Bu (6); X = bzac, R = n-Pr (7), n-Bu (8); X = bzbz, R = n-Pr (9), n-Bu (10), i-Bu (11)]. Selective hydrolysis of the Sn-OMe bond in 1-3 occurs, resulting in the isolation of (&mgr;-hydroxo)diorganotin methanesulfonates, R(2)Sn(OH)OS(O)(2)Me [R = n-Pr (12), n-Bu (13), i-Bu (14)]. All the compounds are characterized by elemental analyses and IR, multinuclear ((1)H, (13)C, and (119)Sn) NMR, and mass spectra. Unequivocal evidence of the presence of the methanesulfonate group is provided by the X-ray crystal structures of 6, 10, and 13. [For 6: trigonal space group R&thremacr; (No. 148), a = 28.664(1) Å, c = 13.056(1)Å, Z = 18. For 10: triclinic space group P&onemacr; (No. 2), a = 13.056(3) Å, b = 14.062(3) Å, c = 16.282(3) Å, Z = 4. For 13: triclinic space group P&onemacr; (No. 2), a = 9.089(2) Å, b = 12.040(2) Å, c =13.894(2) Å, Z = 2]. For 6 and 10, the solid-state structural analyses reveal dimeric structures with a bridging bidentate methanesulfonate group forming a centrosymmetric eight-membered ring. Compound 13 possesses a polymeric sheet structure with repeating 20-membered macrocycles (including two four-membered [Sn(OH)](2) rings) by virtue of the bridging bidentate methanesulfonate groups. A search for a possible pathway to give Arbuzov-rearranged products 1-4 leads us to speculate that there is an initial catalytic transformation of dimethyl sulfite to methyl methanesulfonate via intermediate compounds, Bu(2)Sn(OMe)(2) (A) and [Bu(2)SnOMe](2)O (B). A and B subsequently react with methyl methanesulfonate to give 1-4.