Recombinant Immunotoxin Research Articles

A single chain immunotoxin, targeting the melanoma-associated chondroitin sulfate proteoglycan, is a potent inducer of apoptosis in cultured human melanoma cells

A recombinant immunotoxin was constructed by fusing a single chain fragment variable antibody fragment, specific for the melanoma-associated chondroitin sulfate proteoglycan (MCSP), to a truncated variant of Pseudomonas exotoxin A (ETA'), carrying a C-terminal KDEL-peptide for improved retrograde intracellular transport. The resulting immunotoxin MCSP-ETA' was periplasmatically expressed in Escherichia coli and purified under native conditions by affinity chromatography resulting in a yield of approximately 30 mug/l bacterial culture. This immunotoxin induced antigen-specific apoptosis in the cultured human melanoma-derived cell lines A2058 and A375M, and treatment with a single dose of the agent eliminated up to 80% of these cells within 72 h. The dose needed for half-maximum killing (EC50) was approximately 1 nmol/l for both cell lines. MCSP-ETA' also displayed cytotoxic activity against cultured primary melanoma cells from patients with advanced disease (pathologic stages IIIC and IV), with net cell death reaching up to 70% within 96 h after treatment with a single dose of 14 nmol/l. MCSP-ETA' induced cell death synergistically with cyclosporin A, both in established human melanoma cell lines and cultured primary melanoma cells. The distinctive antigen-restricted induction of apoptosis and the synergy with cyclosporin A justify further evaluation of this novel agent with regard to its potential application for the treatment of malignant melanoma.

Molecular Targets and Targeted Therapies for Malignant Mesothelioma

Malignant mesothelioma is a highly invasive tumor originating from the mesothelial linings of the pleura, peritoneum and pericardium. It is seldom amenable to surgical intervention and poorly responsive to radiotherapy, leaving chemotherapy as the main therapeutic option for most patients. The development of effective drug regimens against mesothelioma has proven extremely difficult and a standard first-line treatment for patients with unresectable tumors has not been established until recently. Despite the benefits obtained with this newly validated standard of care, which is based on the combination of pemetrexed and cisplatin, the prognosis for mesothelioma patients remains poor, median survival is still less than two years and more active treatments are urgently needed. This article will focus on the molecular basis providing the rationale for targeted interventions against mesothelioma and will review targeted agents under evaluation as new potential therapeutic options for mesothelioma patients. Such agents include inhibitors of growth factor receptors, ligands and intracellular effectors. The agents targeting vascular endothelial growth factor signaling are of particular interest, due to the involvement of this pathway both in tumor angiogenesis and autocrine stimulation of mesothelioma cell growth. Alternative approaches are based on inhibitors of the ubiquitin-proteasome pathway and of histone deacetylases which, notwithstanding the functional divergence of the corresponding targets, share the ability to determine a wide modulation of the cancer cell phenotype that can lead to cell cycle arrest, apoptosis and sensitization to different antineoplastic treatments. A recombinant immunotoxin targeted to the membrane antigen mesothelin is an additional agent whose activity is being evaluated in mesothelioma patients.

Depletion of autoreactive B-lymphocytes by a recombinant myelin oligodendrocyte glycoprotein-based immunotoxin

We report the construction of a fusion protein comprising the extracellular domain of myelin oligodendrocyte glycoprotein (MOG) and a truncated version of Pseudomonas aeruginosa exotoxin A (ETA′). The chimeric immunotoxin targeted MOG-reactive B-lymphocytes by binding selectively to the appropriate receptors, leading to internalization and apoptosis of the target cells. The functionality of the immunotoxin was tested on a MOG-sensitive murine hybridoma cell line and ex vivo on freshly isolated splenocytes from transgenic IgH MOG mice. These data demonstrate, for the first time, the specific cytotoxicity of a MOG-containing recombinant immunotoxin expressed in bacteria towards MOG-reactive B-lymphocytes.

Partial Reduction of Human FOXP3+ CD4 T Cells In Vivo After CD25-directed Recombinant Immunotoxin Administration

The regulation of tolerance to self-proteins and the suppression of T-cell responses have in part been attributed to the activity of CD25+CD4+ T regulatory (Treg) cells. Further, Treg cells can inhibit the antitumor effectiveness of adoptive immunotherapy and active immunization approaches in preclinical models. In an effort to selectively eliminate Treg cells from human peripheral blood mononuclear cell to potentially bolster antitumor responses, we have evaluated the Treg-cell depleting capacity of the CD25-directed immunotoxin, RFT5-SMPT-dgA. In preclinical studies, incubation of human peripheral blood mononuclear cell with RFT5-SMPT-dgA mediated a partial reduction in the levels of CD25+, Foxp3-expressing CD4+ T cells in vitro. Administration of RFT5-SMPT-dgA to 6 patients with metastatic melanoma induced a transient but robust reduction in the number of CD25high CD4 T cells in vivo (a 97.5% mean reduction at nadir; from 69.4 +/- 12.4 cells/miroL to 1.7 +/- 0.3 cells/microL). The reduction in FOXP3+ CD4 T-cell number was less comprehensive (a 71.3% mean reduction at nadir; from 66.6 +/- 16.5 cells/microL to 14.2 +/- 3.9 cells/tL). This resulted in the selective persistence of a stable number of CD25(low/neg) FOXP3+ CD4+ T cells in vivo. No objective antitumor responses were seen in any patient. Our results indicate that the CD25-directed, RFT5-SMPT-dgA immunotoxin can mediate a transient, partial reduction in Treg-cell frequency and number in vitro and in vivo and suggest that comprehensive eradication of human Treg cells in vivo may require the ability to target and eliminate FOXP3+ CD4+ T cells expressing both high and low levels of CD25.

Modeling Recombinant Immunotoxin Efficacies in Solid Tumors

Effectiveness of cancer therapy is improved by the use of recombinant immunotoxins (RITs) that target membrane proteins unique to malignant tumor cells. Although RIT antitumor activity in vivo can always be improved with larger doses, clinical restriction on the dose toleration makes it critical to explore how RIT antitumor activity can be maximized without resorting to dose elevation. In this work, a mathematical model was developed to explore functional correlations between the properties of several recombinant immunotoxins and their antitumor efficacies in vivo. Simulations were compared with experimental data of human tumor xenografts grown on nude mice to assess parameters critical to optimal antitumor activity. We dissected out or held constant as many parameters of the model as possible to investigate the effect of the remaining parameters on the behavior of the system as a whole. Empirical correlations between immunotoxin binding affinity and the target binding site density were obtained for several recombinant immunotoxins targeting either human A431 carcinoma or CD46 Burkitt's lymphoma. Simulations reinforced the idea of binding site barrier for drug diffusion and suggested that optimal antitumor activity was achieved when the binding affinity is logarithmically dependent on the target binding site density.

FCRL1 on chronic lymphocytic leukemia, hairy cell leukemia, and B-cell non-Hodgkin lymphoma as a target of immunotoxins

FCRL1 (Fc receptor-like 1) is a cell-surface membrane protein belonging to FCRL family and is preferentially expressed on B cells. To evaluate FcRL1 as an immunotherapy target for B-cell malignancies, we prepared anti-FCRL1 mAbs without cross-reactivity to other FCRL family proteins and analyzed FCRL1 protein expression on malignant cells from patients and on B-cell lines. Frequent FCRL1 expression was observed by flow cytometry on 12 B-cell non-Hodgkin lymphoma (B-NHL) cell lines and many patient samples: 12 of 14 chronic lymphocytic leukemia (CLL), 7 of 7 follicular lymphoma (FL), 13 of 17 hairy cell leukemia (HCL), and 2 of 3 mantle cell lymphoma (MCL). Two recombinant immunotoxins, E3(Fv)-PE38 and E9(Fv)-PE38, were constructed. Both immunotoxins bound to FCRL1-positive cells with similar affinities (3.4 and 3.2 nM) and were cytotoxic to cell lines, but E9(Fv)-PE38 was 4- to 20-fold more cytotoxic than E3(Fv)-PE38. The concentrations that inhibited response by 50% (IC(50)s) of E9(Fv)-PE38 on 11 different FCRL1-positive cell lines ranged from 1.0 ng/mL to 90 ng/mL and correlated with the FCRL1 expression levels. Our results suggest that anti-FCRL1 immunotoxin E9(Fv)-PE38 exhibits remarkably specific cytotoxicity and merits further evaluation for the treatment of FCRL1-positive malignancies, including CLL, HCL, FL, MCL, and other B-NHL.

Anti‐PSMA immunotoxin as novel treatment for prostate cancer? High and specific antitumor activity on human prostate xenograft tumors in SCID mice

Expression of the prostate specific membrane antigen (PSMA) is highly restricted to prostate epithelial cells. Therefore, toxin-based immunotherapy against this antigen may represent an alternative therapeutic option for prostate cancer. For these purposes, the effects of the recombinant anti-PSMA immunotoxin A5-PE40 on prostate tumor growth were investigated in vitro and in vivo. The in vitro binding and cytotoxicity of A5-PE40 were tested on the PSMA-expressing prostate cancer cell line C4-2 and on the PSMA-negative cell line DU145 by flow cytometry and WST assays. The binding of the immunotoxin to SCID mouse xenografts and to various mouse organs was examined by Western blot analysis. In vivo, the antitumor activity of the immunotoxin was tested by injecting A5-PE40 in mice bearing C4-2 or DU145 xenografts. In vitro, a specific binding of A5-PE40 to C4-2 cells could be shown with a concentration-dependent cytotoxicity (IC(50) value=220 pM). In the next step, a specific binding of the immunotoxin to C4-2 xenografts could be demonstrated. In contrast, no binding on mouse organs expressing high homologous mouse PSMA was found. The treatment of mice with C4-2 tumors caused a significant inhibition of tumor growth in vivo, whereas DU145 xenografts remained totally unaffected. A5-PE40 represents a recombinant anti-PSMA immunotoxin with potent antitumor activity in mice bearing human prostate cancer xenograft tumors. Therefore, A5-PE40 could be a promising candidate for therapeutic applications in patients with prostate cancer.

Pre-Clinical Evaluation of the Anti-CD22 Immunotoxin CAT-8015 in Combination with Chemotherapy Agents for Childhood B-Precursor Acute Lymphoblastic Leukemia (Pre-B ALL).

Despite advances in curative treatment, childhood ALL remains a leading cause of cancer-related mortality in pediatrics and survivors are at risk of multiple late effects. Targeted therapies that overcome chemotherapy resistance and decrease nonspecific toxicities are needed. The B-lineage differentiation antigen CD22 represents a relevant target for pre-B ALL. Monotherapy with the anti-CD22 immunotoxin RFB4(dsFv)-PE38 (CAT-3888 or BL22) has been well tolerated in a pediatric Phase I trial. To date however, only transient clinical activity has been observed with this agent in children with ALL. We conducted cytotoxicity studies to evaluate combination regimens of a higher affinity anti-CD22 immunotoxin (CAT-8015 or HA22) with standard ALL chemotherapeutic agents.Methods: CD22+ human malignant cell samples studied included cell lines of Burkitt lymphoma (CA46) and pre-B ALL (REH and KOPN-8), as well as primary lymphoblasts from 2 children with multiply relapsed pre-B ALL. Flow cytometry was used to determine CD22 expression and anti-CD22 antibody binding using the BD Quantibrite System. Asparaginase, cytarabine, doxorubicin, methotrexate, and vincristine were added to cells individually and in various combinations with CAT-8015. Total incubation time was 2 days for cell lines and 5 days for primary patient blasts. Cell viability was assessed using WST-8. The Chou-Talalay median-effect method was used for data analysis (Chou TC, Talalay P. Adv Enzyme Regul 1984; 22:27–55).Results: All samples were sensitive to CAT-8015 and to most chemotherapy agents tested. Simultaneous addition of a fixed concentration of CAT-8015 with varying concentrations of each chemotherapy drug led to a 2- to 4-fold reduction in IC50 in comparison to individual agents. Assays with simultaneous addition of equipotency ratios of CAT-8015 and chemotherapy agents had synergistic or additive interactions. These interactions were sequence dependent, and the addition of CAT-8015 followed by chemotherapy led to greater positive interactions than the reverse order. Further assays with simultaneous addition of CAT-8015 and doublet chemotherapy drugs showed enhanced synergistic and additive interactions compared to assays with only doublet agents.Conclusions: CAT-8015 combined with standard chemotherapy agents results in synergistic or additive cytotoxicity against CD22-positive Burkitt lymphoma and pre-B ALL cell lines and primary blasts from children with pre-B ALL. High affinity anti-CD22 recombinant immunotoxins have the potential to overcome chemotherapy resistance and to decrease nonspecific treatment-associated toxicities. In addition, CAT-8015 might synergistically enhance chemotherapy-induced cytotoxicity, thus improving efficacy and allowing the use of less toxic regimens in the future. Clinical trials of recombinant immunotoxins targeting CD22 in combination with standard ALL chemotherapy regimens are planned.

Immunotoxin and Taxol synergy results from a decrease in shed mesothelin levels in the extracellular space of tumors

Recombinant immunotoxins are chimeric proteins composed of the Fv portion of a tumor-specific antibody fused to a toxin. SS1P (CAT-5001) is an immunotoxin composed of an antimesothelin Fv fused to a 38-kDa portion of Pseudomonas exotoxin A. Immunotoxins have been shown to be active in lymphomas and leukemias, but are much less active against solid tumors. We recently reported that Taxol and other chemotherapeutic agents show striking synergistic antitumor activity in mice when immunotoxin SS1P, which targets the mesothelin antigen on solid tumors, is given with Taxol. Using a pair of Taxol-sensitive and Taxol-resistant KB tumors equally sensitive to immunotoxin SS1P, we examined the mechanism of synergy. We show that synergy is only observed with Taxol-sensitive tumors, ruling out an effect of Taxol on endothelial cells. We also show that the KB tumors have high levels of shed mesothelin in their extracellular space; these levels increase with tumor size and, after Taxol treatment, dramatically fall in the drug-sensitive but not the drug-resistant tumors. Because the mesothelin levels in the tumor exceed the levels of SS1P in the tumor, and because shed mesothelin is being continuously released into the circulation at a high rate, we propose that synergy is due to the Taxol-induced fall in shed antigen levels.

Mesothelin targeted cancer immunotherapy

Mesothelin is a tumour differentiation antigen that is normally present on the mesothelial cells lining the pleura, peritoneum and pericardium. It is, however, highly expressed in several human cancers including malignant mesothelioma, pancreatic, ovarian and lung adenocarcinoma. The normal biologic function of mesothelin is unknown but recent studies have shown that it binds to CA-125 and may play a role in the peritoneal spread of ovarian cancer. The limited mesothelin expression in normal tissues and high expression in many cancers makes it an attractive candidate for cancer therapy. Three mesothelin targeted agents are in various stages of clinical evaluation in patients. These include SS1P (CAT-5001) a recombinant immunotoxin targeting mesothelin, MORAb-009 a chimeric anti-mesothelin monoclonal antibody and CRS-207 a live-attenuated Listeria monocytogenes vector encoding human mesothelin. These ongoing clinical trials will help define the utility of mesothelin as a target for cancer therapy.

The Disease With Hope: Hairy Cell Leukemia

Hairy cell leukemia (HCL), comprising 2% of all leukemias, is a chronic disorder characterized by mononuclear cells with prominent cytoplasmic projections. For years, patients with HCL underwent splenectomies and then interferon alpha for treatment, which provided high response rates but low percentages of complete remission. More recent treatments with 2-chlorodeoxyadenosine result in 85%-90% complete remission, minimal toxicity, and lower rates of relapse using a single course of therapy. A second course of therapy can be administered if HCL continues to be resistant or recurs. New research using anti-CD22 recombinant immunotoxin BL22 is proving successful. With these latest chemotherapy options, patients' prognoses are optimistic.

Expression, purification, and characterization of an immunotoxin containing a humanized anti-CD25 single-chain fragment variable antibody fused to a modified truncated Pseudomonas exotoxin A

Vascular leak syndrome (VLS) is the major dose-limiting toxicity of immunotoxin therapy. In our previous study, a modified PE38KDEL, denoted PE38KDELKQK, was engineered to eliminate VLS. The PE38KDELKQK-based immunotoxin has been proved to retain potent anti-tumor activity but with a remarkable attenuation in VLS. In the present study, we have constructed and expressed a recombinant immunotoxin CD25-PE38KDELKQK containing humanized anti-CD25 single-chain antibody (scFv) genetically fused to PE38KDELKQK in Escherichia coli. After washing with buffer containing 2 M urea, the purity of inclusion body was about 82%. The denatured inclusion bodies were refolded on-column in Tris buffer (pH 8.0) containing 4 mM of GSH and 1 mM of GSSG using a gradient of decreasing urea. We found that the presence of GSH/GSSG (4:1) in the on-column refolding buffer was important for efficient refolding. In addition, slow flow rate was another important factor could increase refolding. Under these conditions, the activity of the refolded protein could reach about 90% of that of the native protein. The refolded proteins were purified to homogeneity (∼95% purity) by a combination of His-Ni 2+ metal affinity chromatography and gel filtration chromatography. The in vitro cytotoxicity assay indicated the purified immunotoxin CD25-PE38KDELKQK had specific cytotoxicity to CD25-positive leukemic cells comparable to wild-type CD25-PE38KDEL (wt). In contrast, CD25-PE38KDELKQK was shown to be much weaker in inducing VLS in mice than wt. The protein expression, purification, and refolding system established in this paper is important for further study on immunotoxin CD25-PE38KDELKQK.

Phase I Study of SS1P, a Recombinant Anti-Mesothelin Immunotoxin Given as a Bolus I.V. Infusion to Patients with Mesothelin-Expressing Mesothelioma, Ovarian, and Pancreatic Cancers

To determine the toxicities, maximum tolerated dose (MTD) and pharmacokinetics of the recombinant immunotoxin SS1P (anti-mesothelin dsFv-PE38) in patients with mesothelin-expressing cancers. SS1P given as a 30-min i.v. infusion every other day (QOD) for six or three doses was administered to 34 patients with advanced mesothelioma (n = 20), ovarian (n = 12), and pancreatic (n = 2) cancer. The initial cohort of 17 patients received SS1P QOD x 6 doses and the MTD was 18 microg/kg/dose. Dose-limiting toxicities (DLT) included grade 3 uticaria (one patient) and grade 3 vascular leak syndrome (two patients). To allow further SS1P dose escalation, 17 patients were treated on the QOD x 3 schedule and the MTD was 45 microg/kg/dose. The DLT was grade 3 pleuritis and was seen in two of two patients treated at a dose of 60 microg/kg and in one of nine patients treated at a dose of 45 microg/kg. At the MTD of 45 microg/kg, the mean C(max) of SS1P was 483 ng/mL and half-life was 466 min. Of the 33 evaluable patients treated, 4 had minor responses, 19 had stable disease (including 2 with resolution of ascites), and 10 had progressive disease. SS1P is well tolerated with pleuritis as the DLT at the highest dose level. Evidence of clinical activity was noted in a group of heavily pretreated patients. Phase II clinical trials of SS1P are being planned for malignant mesothelioma and other mesothelin-expressing malignancies.

New Immunotoxins Targeting CD123, a Stem Cell Antigen on Acute Myeloid Leukemia Cells

The specific alpha subunit of the interleukin-3 receptor (IL-3Ralpha, CD123) is strongly expressed in various leukemic blasts and leukemic stem cells and seems to be an excellent target for the therapy of leukemias. In this study, immunotoxins were developed to target CD123 only, which bypasses the dependence on other subunits to form intact IL-3R. Three anti-CD123 hybridomas (26292, 32701, and 32716) were selected on the basis of their affinity for CD123. Total RNAs were extracted from the 3 anti-CD123 hybridomas and used to clone the fragment of variable region (Fvs). The Fvs were assembled into single chain Fvs and fused to a 38-kd fragment of Pseudomonas exotoxin A to make recombinant immunotoxins. 26292(Fv)-PE38 was found to have the highest cytotoxic activity on the CD123 expressing leukemia cell line TF-1. It bound the cells with a kd of 3.5 nM. Another immunotoxin, 32716(Fv)-PE38, belonging to a different epitope group, had a similar binding ability but was less active, demonstrating the role of epitope selection in immunotoxin action. The cytotoxic activity of 26292(Fv)-PE38 was increased from 200 to about 40 ng/mL by mutating the REDLK sequence at the C terminus to KDEL. 26292(Fv)-PE38-KDEL was specifically cytotoxic to several CD123 expressing cell lines (TF-1, Molm-13, and Molm-14) with good CD123 expression but not to ML-1 or U937 with low or absent expression. In conclusion, 26292(Fv)-PE38-KDEL shows good cytotoxic activity against CD123 expressing cell lines, and merits further development for the possible treatment of acute myeloid leukemia and other CD123 expressing malignancies.

ChFRP5-ZZ-PE38, a large IgG-toxin immunoconjugate outperforms the corresponding smaller FRP5(Fv)-ETA immunotoxin in eradicating ErbB2-expressing tumor xenografts

As therapeutics, antibodies can be used “un-armed” or as immunoconjugates to direct cytotoxic moieties to tumor cells. Immunoconjugates are made by attaching chemotherapy drugs, radioisotopes or toxins to the antibody. Small recombinant antibody fragments fused to cytotoxic moieties, termed recombinant immunotoxins are also being developed as an additional approach for a targeted cancer therapy. Key parameters in determining the therapeutic potential of such targeted therapies are target specificity, affinity, stability and size. With regard to treating solid tumors, tumor penetration (which is inversely proportional to size) is currently regarded as the prime factor for efficacy, while parameters such as binding affinity and residence time in the body are thought to contribute to a lesser extent. When comparing recombinant immunotoxins and antibody-toxin immunoconjugates that target ErbB2/HER2, here we found that a bivalent antibody-toxin immunoconjugate (200 kDa) was superior to the corresponding recombinant monovalent immunotoxin (69 kDa) in killing ErbB2-expressing tumor cells in culture and as xenografts in nude mice, suggesting that higher avidity and longer residence time may outweigh tumor penetration. Our study suggests that the re-valuation of currently neglected, large IgG-effector molecule conjugates for anti-cancer therapy may be justified.

A fold-back single-chain diabody format enhances the bioactivity of an anti-monkey CD3 recombinant diphtheria toxin-based immunotoxin

T-cell depleting anti-CD3 immunotoxins have utility in non-human primate models of transplantation tolerance and autoimmune disease therapy. We recently reported that an affinity matured single-chain (scFv) anti-monkey CD3 antibody, C207, had increased binding to T-cells and increased bioactivity in a diphtheria toxin (DT)-based biscFv immunotoxin compared with the parental antibody, FN18. However, FN18 scFvs and their mutant derivatives such as C207 did not exhibit robust bivalent character in the biscFv format. We now report that C207 in a diabody format exhibits a 7-fold increase in binding to T-cells over scFv (C207) indicating considerable divalent character for the diabody. This construct was formed by reducing the V(L)/V(H) linker to five residues and was secreted from Pichia pastoris as the non-covalent dimer. An immunotoxin based on this diabody format was secreted as a non-covalent dimer but was devoid of bioactivity and failed to bind T-cells, suggesting steric hindrance from the two large closely positioned truncated DT moieties. We constructed a single-chain diabody immunotoxin by fusing to the truncated DT C-terminus L1-VL-L1-VH-L2-VL-L1-VH where L1 is a five-residue linker and L2 is the longer (G4S)3 linker permitting interactions between the distal and proximal VL/VH domains. This 'fold-back' immunotoxin was secreted predominantly as the monomer and exhibited a 5- to 7-fold increase in bioactivity over DT390biscFv(C207) and depleted monkey T-cells in vivo.

Mesothelin, a possible target for immunotherapy, is expressed in primary AML cells

Mesothelin is a promising candidate for tumor-specific therapy because of its limited expression in normal tissues and high expression in several cancers. The expression of the protein mesothelin in hematological malignancies has not yet been analyzed. SS1(dsFv)PE38 is a recombinant anti-mesothelin immunotoxin which is undergoing clinical evaluation in patients with mesothelin-expressing tumors. In this study we show that the mesothelin protein is expressed in leukemic cells from children with acute myeloid leukemia (AML). This finding was confirmed by western blot, immunocytochemistry and real time polymerase chain reaction (PCR). Despite the expression of mesothelin, the patient samples were not sensitive to immunotoxin SS1(dsFv)PE38 in MTT assays. Primary AML cells express mesothelin but SS1(dsFv)PE38 is not active in killing these cells. Other approaches that utilize mesothelin as a target might be more effective and should be tested against AML cells.

Phase I study of SS1P, a recombinant anti-mesothelin immunotoxin for targeted therapy of mesothelin expressing mesotheliomas, ovarian and pancreatic cancer

3553 Background: Mesothelin, a cell surface glycoprotein, is an attractive candidate for targeted cancer therapy given its limited expression on normal mesothelial cells and high expression in several human cancers including mesothelioma, ovarian and pancreatic cancer. We conducted a phase I study to determine the maximum tolerated dose (MTD), toxicity and pharmacokinetics of SS1P, a recombinant anti-mesothelin immunotoxin. Methods: SS1P given as a 30 minute intravenous infusion was administered to 34 patients (pts) with advanced mesothelioma (n = 19), ovarian (n = 13) and pancreatic (n = 2) cancer. SS1P was administered every other day (QOD) for 6 doses to 17 pts and then QOD for 3 doses to 17 other pts. Results: The initial cohort of 17 pts received SS1P QOD x 6 doses and the MTD was 18 μg/kg/dose. Dose-limiting toxicity (DLT) included grade 3 urticaria (1 pt) and grade 3 vascular leak syndrome (2 pts). To allow further SS1P dose escalation 17 pts were treated on the QOD x 3 schedule and the MTD was 45 μg/kg/dose. The DLT was grade 3 pleuritis and was seen in 2/2 pts treated at a dose of 60 μg/kg and in 1/9 pts treated at a dose of 45 μg/kg. In all cases the pleuritis resolved completely. No grade 4 toxicity was observed. The most common grade 1/2 toxicities included hypoalbuminemia (21 pts), fatigue (17 pts), fever (9 pts) and edema (9 pts). At the MTD of 45 μg/kg the mean Cmax of SS1P was 483 ng/ml and half-life was 466 minutes. Of the 33 evaluable pts treated, 4 had minor response, 18 had stable disease and 11 had progressive disease. Two pts with stable disease had complete resolution of their ascites. A pt with peritoneal mesothelioma had complete resolution of her abdominal ascites, required no further therapy and died of unrelated causes 5 years after SS1P treatment. In the other pt with ovarian cancer there was resolution of abdominal and pelvic ascites that lasted 6 months. Conclusion: SS1P is well tolerated with pleuritis as the DLT at the highest dose level. Anti-tumor activity was noted in a group of heavily pretreated patients. Based on pre-clinical studies that show marked synergy between SS1P and chemotherapy, phase II clinical trials of SS1P in combination with chemotherapy for the treatment of mesothelin expressing cancers will open in 2007. No significant financial relationships to disclose.

Phase II trial of CAT-3888 (BL22) in chemo-resistant hairy cell leukemia

7095 Background: CAT-3888 (BL22) is a recombinant anti-CD22 immunotoxin which showed phase I activity in chemo-resistant hairy cell leukemia (HCL). Methods: Eligible patients had relapsed HCL < 4-years after prior cladribine (CdA) and needed additional treatment based on blood counts. Patients were stratified into 2 groups by most recent CdA response of < 1 year or 1–4 years. A 3rd group was added for patients with uncontrolled infection. The study used a 2-stage design of 10 patients each for the first two strata with expansion to 17–30 if hematological remission (HR, i.e. ANC, platelets and Hgb at least 1.5, 100, and 11, respectively) was achieved in at least 3/10. The third strata allowed up to 5 patients. Patients received 40 ug/Kg every other day (QOD) ×3 on cycle 1. Those achieving HR were observed. Patients without a HR and no neutralizing antibodies were retreated at 30 ug/Kg QOD ×3 every 4 weeks beginning at least 8 weeks after cycle 1. Results: 35 patients were enrolled including 25, 9 and 1 in groups 1–3, respectively. CR (44% vs 48%) and overall response (ORR)(64% vs 89%) were similar in strata 1 and 2 with 16 (47%) CRs and 8 (24%) partial responses (PRs) overall. Response was particularly high in 22 with baseline spleen size < 200 mm (95% ORR; 59% CR, 36% PR) compared to patients with spleen height > 200 (ORR 20%, p = 0.001) or patients post-splenectomy (ORR 25%, p = 0.0003). The only serious toxicity was completely reversible grade 3 hemolytic uremic syndrome, not requiring plasmapheresis, in 1 (3%) patient on cycle 2. Neutralizing antibodies which prevented additional cycles were observed in 4 (11%) patients. Conclusions: The activity of CAT-3888 (BL22) in HCL is confirmed in this phase II trial. Toxicity may be limited by retreating only those who do not respond to 1 cycle. Optimal response to CAT-3888 (BL22) following CdA failure may be achieved before the occurrence of massive splenomegaly or splenectomy. No significant financial relationships to disclose.

Anti-CD25 Targeted Killing of Bicistronically Transduced Cells: A Novel Safety Mechanism Against Retroviral Genotoxicity

Gene therapy for Fabry disease, a deficiency in alpha-galactosidase A (alpha-gal A) activity, has the potential to provide a cure for the disorder with a single treatment. Despite modifications to existing vectors, concerns have arisen regarding the risk of genotoxicity associated with the use of retroviruses. To address safety concerns, we propose that expression of a cell surface protein, human CD25 (huCD25) in a bicistronic format, with any therapeutic gene such as alpha-gal A can provide a target that can be used to kill transduced cells selectively should transformative events occur. We show that an anti-CD25 antibody and immunotoxin can specifically target and eliminate transduced leukemia cells expressing CD25. In a murine leukemia model, antibody treatment reduced tumor burden 32-fold and increased survival compared with untreated mice. Furthermore, after a bone marrow transplant of therapeutically transduced cells into Fabry mice, antibody treatment reduced the number of retrovirally transduced huCD25-expressing cells in the peripheral blood. A systemic loss of transduced cells with functional consequences was also evident in the liver and spleen. This proof-of-principle study demonstrates that a targeted antibody can reduce tumor burden and selectively clear bicistronically transduced hematopoietic cells that express a target antigen, thus acting as a built-in safety mechanism.