Search

3051 Background: LMB-100 recombinant immunotoxin consists of a mesothelin-binding Fab for targeting a modified Pseudomonas exotoxin A payload to tumors. Previous clinical trials demonstrated that almost all patients formed anti-drug-antibodies (ADAs) to LMB-100 that made administration beyond cycle 2 ineffective. Tofacitinib is an oral JAK inhibitor that prevented formation of ADAs against a closely related immunotoxin in pre-clinical studies. The primary objective of the dose escalation cohort was assessment of safety and tolerability of LMB-100 given with tofacitinib to patients with mesothelin-expressing solid tumors. The primary objective of the expansion cohort was to determine whether co-administration of tofacitinib delays formation of neutralizing LMB-100 ADAs. Methods: Patients (n = 13) with pancreatic adenocarcinoma and other mesothelin-expressing solid tumors (n = 3; cholangiocarcinoma, appendix, cystadenocarcinoma) were treated for up to 3 cycles with LMB-100 as a 30-minute infusion on days 4, 6, and 8 at two dose levels (100 and 140 mcg/kg) and co-treated with oral tofacitinib for the first 10 days of the cycle (10 mg BID). Results: Dose level 1 of LMB-100 was started at 100 mcg/kg one dose level below the single agent MTD. Dose escalation to 140 mcg/kg (dose level 2) resulted in DLTs in 2 of the 3 patients treated: grade 3 cardiac toxicity and grade 4 hyponatremia, both attributed to capillary leak syndrome. Ultimately, 7 patients were treated at dose level 1 without DLTs and 100 mcg/kg was chosen as the LMB-100 dose for the expansion cohort. The last of 6 patients treated in the expansion cohort developed grade 4 pericardial effusion leading to early closure of the study for toxicity. No objective responses were seen. Of the 8 patients who received two cycles of treatment at MTD, 4 met prespecified criteria for ADA prevention, and 2 patients who went on to receive cycle 3 had detectable LMB-100 plasma drug levels after administration. Conclusions: LMB-100 was unable to be co-administered safely with tofacitinib. ADA formation was prevented in 2 patients through 3 cycles, a rare occurrence. Clinical trial information: NCT04034238.

7036 Background: Anti-CD22 recombinant immunotoxin moxetumomab pasudotox (Moxe) is FDA-approved for hairy cell leukemia (HCL) patients who have received at least two prior systemic therapies including a purine nucleoside analog. In phase 3 testing the complete remission (CR) rate was 41%, and response was higher in patients with lower tumor burden and lower titers of antidrug antibodies (ADA). Phase 1 testing indicated that most CRs were without minimal residual disease (MRD) and eradication of MRD was associated with prolonged CR duration. Monoclonal antibody (Mab) rituximab binds to CD20 on HCL cells and induces apoptosis or immune-mediated killing, but as a single-agent achieved only 13% CRs in relapsed HCL requiring therapy. In a phase 1 trial to determine safety, rituximab was combined with Moxe, with the goal to help reduce tumor burden and to prevent or delay ADA by killing normal B-cells. Methods: To allow rituximab sufficient time to accomplish both goals, it was infused 3 days before day 1 of cycle 1 at 375 mg/m2, and Moxe was given by 30-minute infusion on days 1, 3 and 5. On repeat cycles of Moxe days 1, 3 and 5, rituximab was given on day 1. Cycles were generally spaced 4 weeks apart. Moxe was begun at a lower dose, 30 rather than the 40 mcg/kg dose used in phase 3 in case the rituximab would increase its toxicity. Bone marrow aspirate flow cytometry, which can detect 0.002% HCL cells, was the most sensitive test used for MRD detection, much more sensitive than BRAF V600E digital droplet PCR (ddPCR) or bone marrow biopsy immunohistochemistry (IHC). Patients could receive 4 cycles past MRD-free CR, but not more than 8 cycles. Results: Three patients received Moxe at 30 mcg/Kg/dose and 6 received 40 mcg/Kg/dose, all without dose limiting toxicity (DLT). There was no evidence of hemolytic uremic syndrome or capillary leak syndrome. To prevent intravascular hypovolemia due to expected third spacing, patients were encouraged to drink one cup per hour of water or other fluid from days 1 to 8 and take dexamethasone 4 mg orally if headache or nausea prevented good oral hydration. Of the 9 patients, 7 (78%) achieved CR after 2 (n = 6) or 3 (n = 1) cycles, and achieved MRD-free CR after 2 (n = 3), 4 (n = 3) or 6 (n = 1) cycles. No patients became infected with COVID-19. Conclusions: This phase 1 trial met its primary endpoint of determining whether rituximab could be safely combined with Moxe and will enroll 4 additional patients to further access clinical activity. Further testing will determine whether addition of a CD20 Mab to Moxe significantly improves clinical outcome compared to Moxe alone, particularly long-term MRD-free CR rate. Clinical trial information: NCT03805932.

TPS452 Background: LMB-100 recombinant immunotoxin consists of a mesothelin-binding Fab for targeting with a modified Pseudomonas exotoxin A payload. Formation of anti-drug antibodies (ADAs) is thought to contribute significantly to limited clinical efficacy of LMB-100 seen in prior clinical trials. Most patients develop clinically meaningful ADAs after 1-2 cycles of LMB-100, resulting in rapid neutralization of LMB-100 during subsequent cycles and undetectable plasma drug levels. Tofacitinib is an oral Janus Kinase-1 and -3 (JAK) inhibitor approved by the FDA for the treatment of rheumatoid arthritis and ulcerative colitis. Pre-clinical studies have shown that tofacitinib can prevent the formation of ADAs against recombinant immunotoxin (Onda et al. Journal of Immunology 2014), and that co-administration of tofacitinib with LMB-100 increases immunotoxin serum half- life in mice and anti-tumor efficacy (Simon et al. JCI Insight 2019). We hypothesize that co-administration of tofacitinib with LMB-100 will prevent or delay the formation of high titer ADAs to LMB-100, such that 2 effective cycles of immunotoxin can be administered to patients. Methods: This phase I clinical trial consists of a dose escalation phase using 3+3 design to determine the maximum tolerated dose (MTD) of LMB-100 that can be administered with tofacitinib in participants (n = 18 max) with mesothelin-expressing solid tumors, followed by a dose expansion phase at the MTD for participants (n = 15) with pancreatic adenocarcinoma or extrahepatic cholangiocarcinoma. The primary objective of the expansion phase is to determine whether co-administration of tofacitinib delays formation of neutralizing anti-LMB-100 ADAs for cycle 2 of treatment as measured by LMB-100 plasma drug levels. A positive outcome will be reached if percent of participants achieving threshold LMB-100 drug levels during cycle 2 increases from 50% to 80% (83.6% probability if >10 of 15 evaluable participants meet this milestone). Plasma drug levels during cycle 3 will also be analyzed as a secondary endpoint. Key inclusion criteria include adults with histologically confirmed previously treated solid tumor malignancies. Participants will receive tofacitinib 10 mg twice daily on days 1-10 and LMB-100 at 65, 100, or 140 mcg/kg on days 4, 6 and 8 of a 21-day cycle. The dose escalation phase has been completed and enrollment onto dose expansion phase is ongoing. Clinical trial information: NCT04034238.

3553 Background: LMB-100 recombinant immunotoxin consists of a mesothelin-binding Fab for targeting a modified Pseudomonas exotoxin A payload. Previous Phase 1 clinical testing of a 30-minute LMB-100 “short” infusion format identified a serum half-life of ~1 hour. Pre-clinical data suggested that extending infusion time could improve anti-tumor efficacy by increasing tumor cell duration of exposure to LMB-100. The primary objective of this study was to determine the safety and tolerability of administering LMB-100 in a long infusion format over 24-48 hours alone or with nab-paclitaxel chemotherapy in patients with mesothelin-expressing solid tumors. Methods: Patients (n = 15) with pancreatic adenocarcinoma and other mesothelin-expressing solid tumors (n = 3; mesothelioma, colon, and ampullary cancers) treated on 3 dose levels received long infusion of LMB-100 (65 or 100 mcg/kg/day) for 24 hour on Days 1 and 4 (n = 6) or 48 hour on Day 1 (n = 9) with or without a loading dose (40 mcg/kg over 30 minutes) for up to 2 cycles. In the second arm, patients (n = 5) with pancreatic adenocarcinoma were treated with LMB-100 over 24 hours on Day 1 concurrently with nab-paclitaxel (125 mg/m2) for up to 3 cycles. Results: DLT of proteinuria (grade 3) in one patient and acute kidney injury (grade 1) in one patient were observed amongst patients receiving 100 mcg/kg/day over 48 hours and 24 hours, respectively. No objective responses were seen but all patients receiving nab-paclitaxel had > 50% decrease in CA 19-9. Patients at all single agent dose levels (8 of 10 evaluable) developed high titer anti-drug antibodies (ADAs) against LMB-100. Those with ADAs (8 of 8) had undetectable cycle 2 peak plasma LMB-100 concentration. Development of high titer ADAs occurred more frequently with long infusion than seen previously with “short” infusion LMB-100. Most long infusion patients (19 of 20) developed increased serum IL-6 within 24 hours of LMB-100 infusion. However, the systemic inflammatory response to LMB-100 (as measured by increased serum CRP) which occurs in most “short” infusion patients was not observed. Conclusions: Long infusion format LMB-100 is generally well tolerated but immunogenicity limits treatment to 1 effective cycle. No anti-tumor efficacy of the single agent was observed. Clinical trial information: NCT02810418 .

307 Background: LMB-100 is a Pseudomonas exotoxin A-based immunotoxin that targets mesothelin (MSLN). MSLN is expressed by >75% of pancreatic adenocarcinomas (PDAC). LMB-100 kills MSLN-expressing cells by irreversibly modifying elongation factor-2 to halt protein synthesis. Phase I studies of LMB-100 defined the maximum tolerated dose (MTD) of 140 mcg/kg IV given on D1, 3 and 5 of a 21-day cycle. Development of anti-drug antibodies (ADAs) limited patient drug exposure beyond cycle 2. Our pre-clinical data showed that combination of LMB-100 with a taxane resulted in synergistic anti-tumor activity. Methods: We conducted a phase I single center study (standard 3+3 design) to determine the MTD of LMB-100 given with nab-paclitaxel in patients with previously treated advanced PDAC. LMB-100 was given on D1, 3 and 5 of a 21-day cycle, and nab-paclitaxel (125 mg/m2) on D1 and D8. Initial patients could receive a maximum of 4 cycles, but subsequently a 2-cycle maximum was employed. Results: Fourteen patients (median age 58) were enrolled. Two of 6 patients experienced DLTs at the 100 mcg/kg dose of LMB-100 (myalgia- 2 pts, fatigue- 1 pt, hypotension- 1 pt; all grade 3). One of 8 patients had DLT at the 65 mcg/kg dose (edema, urine output decrease- both grade 3). Other toxicities related to LMB-100 included hypoalbuminemia, edema-associated weight gain, hyponatremia, fatigue, drug fever, infusion-related reaction, hypophosphatemia, nausea and anorexia. One patient died on treatment from complications of bowel perforation attributed to cancer. All patients achieved detectable serum levels of LMB-100 during the first cycle, even those with pre-existing ADAs, and 5 of 8 did so during cycle 2. One patient receiving the 65 mcg/kg dose had a confirmed partial response, and CA 19-9 dropped by > 50% in 5 of 8 evaluable patients. Conclusions: MTD of LMB-100 is 65 mcg/kg given with nab-paclitaxel on this schedule. Anti-tumor activity was observed. A phase II cohort is currently being accrued. Clinical trial information: NCT02810418.

Purpose To conduct a phase I dose-escalation trial assessing safety and response of recombinant immunotoxin moxetumomab pasudotox (CAT-8015, HA22) in chemotherapy-resistant hairy cell leukemia (HCL). Patients and Methods Eligible patients had relapsed/refractory HCL after ≥ two prior therapies and required treatment because of abnormal blood counts. Patients received moxetumomab pasudotox 5 to 50 μg/kg every other day for three doses (QOD ×3), with up to 16 cycles repeating at ≥ 4-week intervals if patients did not experience disease progression or develop neutralizing antibodies. Results Twenty-eight patients were enrolled, including three patients each at 5, 10, 20, and 30 μg/kg, four patients at 40 μg/kg, and 12 patients at 50 μg/kg QOD ×3 for one to 16 cycles each (median, four cycles). Dose-limiting toxicity was not observed. Two patients had transient laboratory abnormalities consistent with grade 2 hemolytic uremic syndrome with peak creatinine of 1.53 to 1.66 mg/dL and platelet nadir of 106,000 to 120,000/μL. Drug-related toxicities in 25% to 64% of the 28 patients included (in decreasing frequency) grade 1 to 2 hypoalbuminemia, aminotransferase elevations, edema, headache, hypotension, nausea, and fatigue. Of 26 patients evaluable for immunogenicity, 10 patients (38%) made antibodies neutralizing more than 75% of the cytotoxicity of 1,000 ng/mL of immunotoxin, but this immunogenicity was rare (5%) after cycle 1. The overall response rate was 86%, with responses observed at all dose levels, and 13 patients (46%) achieved complete remission (CR). Only 1 CR lasted less than 1 year, with the median disease-free survival time not yet reached at 26 months. Conclusion Moxetumomab pasudotox at doses up to 50 μg/kg QOD ×3 has activity in relapsed/refractory HCL and has a safety profile that supports further clinical development for treatment of this disease.

7004Background: A pivotal multicenter, single-arm study evaluated moxetumomab pasudotox, a first-in-class recombinant immunotoxin, in patients (pts) with relapsed/refractory hairy cell leukemia (HC...

7061Background: Moxe is a recombinant immunotoxin that specifically binds to CD22 on the B cell surface and internalizes leading to cell death. The PK, PD, and immunogenicity of moxe were evaluated...

7060Background: Moxe is an immunotoxin targeting CD22 on B cells leading to cell death. The objectives of this analysis were to characterize the pharmacokinetics (PK) and evaluate the exposure-effi...

PurposeTo conduct a phase I trial of recombinant immunotoxin BL22, an anti-CD22 Fv fragment fused to truncated Pseudomonas exotoxin.Patients and MethodsForty-six pretreated patients with CD22+ non-Hodgkin's lymphoma (NHL; n = 4), chronic lymphocytic leukemia (CLL; n = 11), and hairy cell leukemia (HCL; n = 31) received 265 cycles at 3 to 50 μg/Kg every other day × 3 doses.ResultsBL22 was active in HCL, with 19 complete remissions (CRs; 61%) and six partial responses (PRs; 19%) in 31 patients. Of 19 CRs, 11 were achieved after one cycle and eight after two to 14 cycles. All 25 responders benefited clinically with one cycle. The CR rate was 86% in patients enrolled at ≥ 40 μg/Kg every other day × 3, and 41% at lower doses (P = .011). The median duration for CR was 36 months (range, 5 to 66 months), and eight patients remain in CR at 45 months (range, 29 to 66 months). Lower but significant activity occurred in CLL. Neutralizing antibodies occurred in 11 (24%) of 46 patients (all HCL). A reversible hemolytic uremic syndrome requiring plasmapheresis was observed in one patient with NHL during cycle 1 and in four patients with HCL during cycle 2 or 3. The maximum-tolerated dose (MTD) evaluated at cycle 1 was 40 μg/Kg IV. QOD × 3. The most common toxicities at 30 to 50 μg/Kg every other day × 3 included hypoalbuminemia, transaminase elevations, fatigue, and edema.ConclusionBL22 was well tolerated and highly effective in HCL, even after one cycle. Phase II testing is underway to define the efficacy with one cycle and to study safety when additional cycles are needed for optimal response.

102 Background: Immunotoxins can induce direct and rapid cytotoxicity by targeting specific tumor antigens. D2C7 is a unique recombinant immunotoxin targeting EGFRwt/EGFRvIII, two frequently overexpressed proteins on gliomas, and is currently being tested in a phase I/II clinical trial (NCT02303678) for recurrent malignant gliomas. Immunotoxins have also been shown to induce a secondary antitumor immune response via stimulation of cytotoxic T lymphocytes (CTLs). Immune checkpoint inhibitors, which have successfully treated several advanced tumors by promoting the antitumor function of CTLs, may further enhance this immunotoxin-induced antitumor response. Thus, we hypothesize that combining D2C7 with immune checkpoint inhibitors will promote long-term tumor regression due to primary cytotoxicity and enhanced anti-tumor immunity. Methods: We developed a CT2A-mD2C7 mouse glioma cell line with robust in vitro cytotoxicity of D2C7 (IC50= 0.47 ng/mL). In vivo anti-tumor efficacy was evaluated by intratumoral injection of D2C7 combined with intraperitoneal injection of anti-CTLA4 or anti-PD1 antibodies in single-side and bilateral subcutaneous (SC) CT2A-mD2C7 glioma models in C57BL/6 immunocompetent mice. Results: In the single-side model, D2C7 monotherapy and combinatorial therapy showed a significant tumor growth delay (P < 0.01). Complete regression ( ≥ 40%) was only observed in combinatorial therapy groups. All cured mice rejected the rechallenging of CT2A parental cells in the contralateral flank and the subsequent rechallenging of CT2A-mD2C7 cells in the brain. In the bilateral model, the larger right tumors were treated with D2C7/anti-CTLA4/anti-PD1 monotherapy or D2C7+anti-CTLA4/PD1 combinatorial therapy, while the left tumors were untreated by D2C7. In the groups where the right tumors were treated with monotherapy or combinatorial therapy, the left untreated tumors also grew much slower. Furthermore, the combinatorial therapy led to the most significantly delayed growth of the left untreated tumors (P < 0.05). Conclusions: Immune checkpoint inhibitors can enhance D2C7-induced anti-tumor immunity to achieve a synergistic long-term tumor regression.

74 Background: Immunotoxins (ITs) are a class of bifunctional chimeric proteins composed of an antibody fragment linked to a toxin. When ITs internalize into target cells, they induce protein synthesis inhibition and apoptosis. While ITs are highly specific and potent, the efficacy of IT-based therapies in some tumor cells is limited by hyperactive anti-apoptotic pathways and inefficient translocation of ITs from the endoplasmic reticulum to the cytosol. Therefore, to improve the efficacy of IT-based therapies, we evaluated a dual-pathway therapy that combines an IT with the ABT-737, ABT-263, or ABT-199 small molecule Bcl-2 inhibitor. Methods: The immunotoxin 9.2.27-PE38KDEL (9.2.27-IT) was generated by fusing a truncated mutant form of Pseudomonas exotoxin A to a single-chain variable fragment antibody. It targets human chondroitin sulfate proteoglycan 4 (CSPG4), an antigen highly expressed in a variety of cancer cells. We screened and identified 3 human glioblastoma xenografts, 3 human melanoma cell lines, and 5 human breast cancer cell lines resistant to the 9.2.27-IT despite their high levels of cell surface expression of CSPG4 (IC50 of IT alone was >100 ng/ml in all cell lines except for one melanoma cell line). In vitro cytotoxicity of the 9.2.27-IT —alone or in combination with the individual Bcl-2 inhibitors ABT-737, ABT-263, or ABT199—was assessed. Concentrations of ABT analogues were chosen so that ABT alone did not induce cytotoxicity. Results: The treatment groups that responded to the combination therapy yielded IC50 values ranging from 0.04 – 9 ng/ml for glioblastoma xenografts, 0.21-15 ng/ml for melanoma cell lines, and 4.5-50 ng/ml for breast cancer cell lines. The most potent combination group showed >1000 fold improvement of IC50 compared to using the immunotoxin alone. ABT-737 produced the strongest synergistic effects among the ABT analogues. Preliminary results from in vivo studies further demonstrated that this approach engendered a synergistic response and delayed tumor growth in immunotoxin-resistant mouse tumor models. Conclusions: This new combinatorial approach will potentially help to overcome immunotoxin resistance in cancer patients and provide better therapeutic outcomes.

PURPOSE: To evaluate the toxicity, pharmacokinetics, immunogenicity, and antitumor activity of anti-Tac(Fv)-PE38 (LMB-2), an anti-CD25 recombinant immunotoxin that contains an antibody Fv fragment fused to truncated Pseudomonas exotoxin. PATIENTS AND METHODS: Patients with CD25+ hematologic malignancies for whom standard and salvage therapies failed were treated with LMB-2 at dose levels that ranged from 2 to 63 μg/kg administered intravenously over 30 minutes on alternate days for three doses (QOD × 3). RESULTS: LMB-2 was administered to 35 patients for a total of 59 cycles. Dose-limiting toxicity at the 63 μg/kg level was reversible and included transaminase elevations in one patient and diarrhea and cardiomyopathy in another. LMB-2 was well tolerated in nine patients at the maximum-tolerated dose (40 μg/kg QOD × 3); toxicity was transient and most commonly included transaminase elevations (eight patients) and fever (seven patients). Only six of 35 patients developed significant neutralizing antibodies after the first cycle. The median half-life was 4 hours. One hairy cell leukemia (HCL) patient achieved a complete remission, which is ongoing at 20 months. Seven partial responses were observed in cutaneous T-cell lymphoma (one patient), HCL (three patients), chronic lymphocytic leukemia (one patient), Hodgkin’s disease (one patient), and adult T-cell leukemia (one patient). Responding patients had 2 to 5 log reductions of circulating malignant cells, improvement in skin lesions, and regression of lymphomatous masses and splenomegaly. All four patients with HCL responded to treatment. CONCLUSION: LMB-2 has clinical activity in CD25+ hematologic malignancies and is relatively nonimmunogenic. It is the first recombinant immunotoxin to induce major responses in cancer. LMB-2 and similar agents that target other cancer antigens merit further clinical development.

OVB3-PE is an immunotoxin composed of a murine monoclonal antibody reactive with human ovarian cancer and conjugated to Pseudomonas exotoxin (PE). Twenty-three patients with refractory ovarian cancer were treated intraperitoneally (IP) with escalating doses of OVB3-PE to study toxicity, pharmacokinetics, antiimmunotoxin antibody formation, and antitumor response. Dose-limiting CNS toxicity occurred after repeated doses at 5 and 10 micrograms/kg. Other non-dose-limiting toxicities included transient elevation of liver enzymes, fever, and gastrointestinal toxicity. Pharmacokinetics of IP and serum OVB3-PE were determined in 16 patients. Peak peritoneal fluid levels exceeded the in vitro median effective dose at all doses tested. At doses of 1 to 2 micrograms/kg, the immunotoxin concentration in the peritoneal fluid remained constant for up to 8 hours and dropped to negligible levels after 12 hours. At the 5 and 10 micrograms/kg doses, levels remained high for up to 24 hours (greater than 100 ng/mL) and then gradually decreased and became undetectable (less than 4 ng/mL) after 72 hours. Serum levels of OVB3-PE were also analyzed in 16 patients. At doses of 1 micrograms/kg and 2 micrograms/kg, serum levels were not detectable (less than 5 ng/mL). However, after doses of 5 or 10 micrograms/kg, peak serum level occurred at 24 hours after each dose and dropped to negligible levels by 72 hours. Sera from 12 patients were analyzed for anti-PE antibodies and antibodies to mouse immunoglobulin (HAMA). All patients developed antibodies against PE within 14 days of therapy. Domain II of PE appeared to be the most immunogenic portion of the PE molecule. HAMA was detected on day 14 of therapy in nine patients, on day 21 in two, and on day 28 in one patient. No clinical antitumor responses were observed. We conclude that IP OVB3-PE at dose levels of 5 micrograms/kg (x 3) and 10 micrograms/kg (x 2) is accompanied by dose-limiting toxic encephalopathy. Neurologic toxicity is likely to be due to crossreactivity of OVB3 to normal human brain tissue, which was not appreciated during preclinical screening.

7079 Background: Moxetumomab pasudotox is a recombinant immunotoxin containing truncated Pseudomonas exotoxin fused to an anti-CD22 Fv, reported to achieve 46% complete remissions in 28 relapsed/re...

61 Background: Novel targets and therapeutics are of profound importance if we are to improve outcomes in gastric cancer. This study assessed the incidence of mesothelin expression in tumors of Western patients with gastric cancer. In addition, gastric cancer cell lines were then tested for their sensitivity to SS1(dsFv)PE38 (SS1P), a mesothelin-targeted immunotoxin. Methods: Tissue specimens from 127 patients with gastric or gastro-esophageal junction (GEJ) tumors were examined by immuno-histochemistry (IHC) for intensity of staining for mesothelin. Expression of HER2-neu, e-cadherin and c-Met were also assessed. Tumors were considered positive for mesothelin if moderate cytoplasmic/membranous or luminal staining was present in at least 10% of the cells. Gastric cancer cell lines were assessed for surface mesothelin expression by flow cytometry and the viability of cells treated with SS1P was measured. Results: Gastric and GEJ cancers were mesothelin positive in 64/127 tumors (50%) while only 9 tumors (7%) were Her2-neu IHC +3 positive and 8 tumors (6%) were c-Met positive (c-Met low 5% and c-Met high 1%). Mesothelin expression rates changed from stage I tumors to stage IV tumors (39% to 56% respectively). We found that both the AGS and MKN28 gastric cancer cell lines express mesothelin and are sensitive to the immunotoxin with EC50 values in the low picomolar range (0.4 and 0.3 ng/mL, respectively). Conclusions: Mesothelin is a cell surface glycoprotein that is expressed in 50% of resected gastric cancers. Gastric cancer cell lines were exquisitely sensitive to SS1P. The high expression of mesothelin and its sensitivity to the immunotoxin SS1P makes it an attractive tumor associated antigen for therapeutic targeting. Based on these observations clinical trials involving novel mesothelin targeted drugs in gastric cancer are currently in development.

2503^ Background: The anti-CD22 recombinant immunotoxin moxetumomab pasudotox, also known as HA22 or CAT-8015, was recently reported in phase I testing to achieve complete remissions (CRs) in 13 (46%) of 28 patients with relapsed/refractory hairy cell leukemia (HCL); 3 of 13 patients have relapsed. Methods: To complete this trial, 20 additional patients received the highest dose level (50 µg/Kg every other day x 3 doses); none of the 48 HCL patients had dose-limiting toxicity (DLT). Results: Of the first 42 patients with >6 mo of follow up off-treatment, 23 (55%) had CRs, with an overall response rate of 88%. Of the 23 CRs, 21 were evaluable for minimal residual disease (MRD) using flow cytometry of blood and immunohistochemistry of the bone marrow biopsy, and 17 (81%) were negative. Of these 17 patients, 11 (65%) were negative by bone marrow aspirate (BMA) flow cytometry. PCR using consensus primers for the heavy chain immunoglobulin (IgH) rearrangement was less specific than flow cytometry of blood, since IgH rearrangements of normal B cells, which recovered rapidly after immunotoxin treatment, were also amplified. For better MRD detection in blood, patient IgH sequences were cloned and sequence specific primers and probes designed for real-time quantitative PCR (RQ-PCR). RQ-PCR of blood was negative in 6 (100%) of 6 patients achieving flow-negativity in both blood and BMA and positive in 3 (100%) of 3 patients flow-negative in blood but not BMA (p=0.01). No relapses from CR have been observed in 10 patients who became RQ-PCR-negative in blood or flow-negative in BMA, with 5-38 (median 11) mo of follow-up. Conclusions: We conclude that clone-specific RQ-PCR is the most sensitive blood test for MRD in our HCL patients after moxetumomab pasudotox, and could be used to assess the possibility of long-term molecular remissions. We believe these results, including durable CRs without DLT, support a pivotal trial in which moxetumomab pasudotox is compared with alternative therapy. Note: this summary contains investigator reported data. This study was funded by MedImmune, LLC, and supported by NCI’s Intramural Research Program and the Hairy Cell Leukemia Research Foundation.

7030 Background: Amatuximab (MORAb-009) is a chimeric monoclonal antibody to mesothelin, a cell surface glycoprotein highly expressed in many cancers including malignant mesothelioma (MM). Based on safety of amatuximab in phase I clinical trial and pre-clinical studies showing synergy in combination with chemotherapy, a single arm phase II study of amatuximab plus pemetrexed (P) and cisplatin (C) was initiated in pts. with MM. Methods: Eligibility criteria included pts. with unresectable epithelial or biphasic pleural MM, no prior chemotherapy and KPS > 70%. Pts. received amatuximab 5 mg/kg on days 1 and 8 with P 500 mg/m2 and C 75 mg/m2 (PC) given on day 1, of each 21 day cycle for 6 cycles. Pts. with objective response or stable disease received amatuximab monotherapy until disease progression. Primary endpoint was progression-free survival (PFS) at 6 months. Secondary endpoints were overall survival (OS), objective response rate and safety of amatuximab with PC. Results: From 2/2009 to 10/2010, 89 pts. were enrolled at 26 sites. Pt. characteristics: median age 67 yrs. (range 46-80), 78% male, 70% with KPS >90%, 89% epithelial MM, 11% biphasic MM and 88% had stage III/IV disease. Median number of PC plus amatuximab cycles was 5 (range 1-6) and 56 (63%) pts. received single agent amatuximab. In addition to the expected toxicity from PC, hypersensitivity reactions (12.4%; Grade 3/4=4.5%) from amatuximab were noted. By independent radiological review 30 (39%) pts. had partial response and 39 (51%) had stable disease. PFS at 6 months was 52% (95% CI: 39.5-63.5) with a median PFS of 6.1 months (95% CI: 5.4-6.5). As of 1/10/12 the median OS was 14.5 months (95% CI: 12.4-18.5) with 31 pts. still alive and 7 pts. receiving maintenance amatuximab. Conclusions: Amatuximab in combination with PC was generally well-tolerated in this study with 90% of pts. having an objective tumor response or stable disease by independent radiological review. Although PFS is not significantly different from historical results of PC alone, the median OS is 14.5 months with 35% of pts. still alive. Updated OS and biomarker data will be presented at the meeting.

7026 Background: SS1P is an immunotoxin targeting mesothelin, a tumor antigen highly expressed in malignant mesothelioma (MM). Preclinical studies showed marked synergy between SS1P and chemotherap...

6523 Background: CAT-8015 (HA22) contains the Fv domains of the anti-CD22 Mab RFB4 and truncated pseudomonas exotoxin, inducing cell death after binding, internalization, and ADP-ribosylation of elongation factor 2. CAT-8015 is a high-affinity mutant of BL22 (CAT-3888), with increased cytotoxicity toward chronic lymphocytic leukemia (CLL) and hairy cell leukemia (HCL) cells ex vivo. Methods: To determine the clinical activity of CAT-8015, separate phase I trials were initiated in CLL, HCL, and non-Hodgkin lymphoma, and the HCL trial completed accrual. Eligible patients had several prior therapies including purine analogs and needed treatment for cytopenias or symptoms. CAT-8015 was administered over 30 min iv days 1, 3 and 5 (QODx3) at 28-day intervals for up to 10 cycles. Results: 28 HCL patients received CAT-8015: 3 each at 5, 10, 20, and 30 ug/kg QODx3, 4 at 40 and 12 at 50 ug/kg QODx3. Patients had 2-7 (median 3) prior therapies. Patients received 1-9 (median 4) cycles of CAT-8015 each. Dose-limiting ...

e17518 Background: SS1P is a recombinant immunotoxin, composed of an antimesothelin Fv fused to a 38 kDa fragment of Pseudomonas exotoxin A, targeting mesothelin, a cell surface glycoprotein that i...

3001 Background: BL22 is a 63 kDa anti-CD22 recombinant immunotoxin containing truncated Pseudomonas exotoxin. In phase I testing, BL22 induced 61% CRs and 19% PRs in 31 with cladribine (CdA)-pretr...

3578 Background: MORAb-009 is a monoclonal antibody that targets mesothelin, a cell surface adhesion protein over-expressed in pancreatic cancer, mesothelioma and other malignancies, with minimal e...

3039 Background: HA22 is a 63 kDa recombinant anti-CD22 immunotoxin containing the variable domains of the anti-CD22 Mab RFB4 connected to truncated Pseudomonas exotoxin. HA22 is a mutant form of B...

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.

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.

9560 Background: ALL is the most common childhood malignancy. Although highly curable, therapy is associated with multiple toxicities and ALL remains the most frequent cause of pediatric cancer mortality. Most ALL is CD22+ and we evaluated the anti-CD22 immunotoxin RFB4(dsFv)-PE38 (BL22 or CAT-3888) in the treatment of pediatric ALL. Methods: In vitro cytotoxicity was assessed on blasts from 42 children with CD22+ ALL. In vivo studies were performed using a xenograft model (SCID mouse - EU1 human ALL cell line). A pediatric phase I trial of CAT-3888 was initiated. Results: CAT-3888 induced in vitro cytotoxicity against most ALL samples (median IC50 9.8 ng/ml). A dose response was observed in murine xenografts with significant prolongation of leukemia free survival (p1/2 showed an inverse relationship to marrow blasts (r2=0.5) consistent with rapid drug binding by CD22+ cells. All subjects were heavily pre-treated and had progressive high burden disease at the time of treatment. Transient clinical activity was observed in 16 of 18 subjects as evidenced by decreased peripheral blast count (8), decreased marrow infiltration (3), decreased extramedullary disease (2), increased platelet count (2), increased neutrophil count (1), increased reticulocyte count (1), improved PET scan (1), decreased tumor-associated pain (2), and/or peripheral blast count stabilization (3). A dose response was apparent with 4 of 9 (44%) achieving stable disease at or above doses of 30 mcg/kg. Conclusions: The majority of pediatric ALL blasts are highly sensitive in vitro to CAT-3888 at concentrations far below clinically achievable levels. CAT-3888 is well tolerated in pediatric patients and activity has been seen in most subjects treated on the phase I trial. CD22 represents a relevant target for pediatric ALL. A new higher affinity anti-CD22 immunotoxin (HA22 or CAT-8015) is in development. No significant financial relationships to disclose.

6624 Background: Hairy cell leukemia (HCL) is effectively treated although not cured with cladribine and the likelihood and durability of response decrease after each relapse. BL22 is a recombinant anti-CD22 immunotoxin containing a truncated form of Pseudomonas exotoxin which kills CD22 positive cells. Methods: A total of 36 patients with cladribine-resistant hairy cell leukemia (HCL) have received BL22, 31 on a phase I trial, 2 patients by single patient exemption and 3 in a recently opened phase II trial. Results: In the phase I trial, complete remission (CR) was observed in 19 patients, with CR rates of 43% (6/14), 69% (9/13), and 100% (3/3) in patients enrolled at 30, 40 and 50 ug/Kg every other day for 3 days (QOD x3), respectively. CR was achieved after cycle 1 in 11 and after cycles 2–14 in 8 patients. Of the 19 CRs, 8 (42%) have remained in CR for 14–47 (median 27) months, and the median CR duration is 26 months. Hematologic remission (HR), defined as minimum platelets 100,000/ul, absolute neutro...

The cloning of the cDNA for the mdr1 gene, whose expression is associated with the development of multidrug-resistance in cultured cells, has made it possible to explore the mechanism of multidrug resistance in human tumors. We have found that normal human kidney, six of eight adenocarcinomas of the kidney, and four cell lines derived from kidney adenocarcinomas express high levels of mdr1 mRNA. Two criteria suggest that primary multidrug resistance in human adenocarcinomas of the kidney results, at least in part, from expression of the mdr1 gene: (1) mdr1 mRNA levels are elevated in four unselected kidney adenocarcinoma cell lines that show a multidrug-resistant phenotype; and (2) multidrug resistance in these kidney cancer cell lines is reversed by verapamil and quinidine, agents known to reverse mdr1-associated drug resistance in cell lines selected for multidrug resistance in vitro. These results suggest that appropriate pharmacological intervention to reverse multidrug resistance might make adenocarcinomas of the kidney more sensitive to chemotherapy with agents such as Adriamycin (Adria Laboratories, Columbus, OH) and the vinca alkaloids.