ALBERT

Description: Unconjugated monoclonal antibodies that target hematopoietic differentiation antigens have been developed to treat hematologic malignancies. Although some of these have activity against chronic lymphocytic leukemia and hairy cell leukemia, in general, monoclonal antibodies have limited efficacy as single agents in the treatment of leukemia. To increase their potency, the binding domains of monoclonal antibodies can be attached to protein toxins. Such compounds, termed immunotoxins, are delivered to the interior of leukemia cells based on antibody specificity for cell surface target antigens. Recombinant immunotoxins have been shown to be highly cytotoxic to leukemic blasts in vitro, in xenograft model systems, and in early-phase clinical trials in humans. These agents will likely play an increasing role in the treatment of leukemia.

Description: Abstract 2443 Background: Complete remissions have been observed in some but not all children in an ongoing Phase 1 trial of the anti-CD22 recombinant immunotoxin moxetumomab pasudotox (m. pasudotox, HA22, CAT-8015) for relapsed and refractory ALL and NHL. Objectives: To develop ALL/NHL cell lines resistant to m. pasudotox in attempt to uncover possible mechanisms of immunotoxin resistance and to use this information to try to improve clinical outcomes. Methods: Using the ALL cell line HAL-01 and the Burkitt NHL cell line CA46, we generated two cell lines (HAL-01-R and CA46-R, respectively) that were resistant to killing by m. pasudotox by repeated exposure to sub-lethal doses of m. pasudotox. We studied the basis of their resistance. Results: Cytotoxicity was markedly reduced in the resistant cell lines (Table) and m. pasudotox was unable to ADP-ribosylate and inactivate elongation factor-2 (EF2). In HAL-01-R this was due to a low level of DPH4 mRNA and protein, which prevented diphthamide biosynthesis and rendered EF2 refractory to m. pasudotox. Analysis of the promoter region of the DPH4 gene showed that the CpG island was heavily methylated. Incubation of sensitive cells with the methylation inhibitor 5-azacytidine prevented the emergence of resistant cells. A second resistance mechanism in the diphthamide pathway was identified in the CA46-R line. There was a small decrease in CD22 site density and internalization rate in CA46-R cells, which were insufficient to account for m. pasudotox resistance. In contrast, protein synthesis measured by 3H-leucine incorporation was not reduced in CA46-R cells suggesting a defect in diphthamide modification of EF2. Consistent with this hypothesis was the finding that m. pasudotox catalyzed the incorporation of ADP-ribose into EF2 in extracts of sensitive CA46 cells, but not in CA46-R cells, although EF2 protein levels were similar in the two cell lines. We examined the expression of the six genes required for diphthamide synthesis (DPH1–5 and WDR85). Only the levels of WDR85 mRNA and protein were substantially decreased in CA46-R cells. We used shRNA to knock down WDR85 mRNA and protein levels and tested the cells for sensitivity to m. pasudotox. The IC50 increased 10-fold from 0.6 ng/ml to 6.0 ng/ml. Conversely, we restored WDR85 protein expression into CA46-R cells by transduction of resistant cells with a WDR85 expressing vector, which partially restored sensitivity to m. pasudotox. Altogether, these findings show that a reduction in WDR85 protein is sufficient to cause m. pasudotox resistance. To study why WDR85 expression is reduced in CA46-R cells, a high resolution SNP array analysis of the WDR85 gene was performed. A homozygous deletion at Chr9:140,468,104–140,610,581 (Build: GRCh37/19) was detected, which accounts for the inability of the cells to make WDR85 protein. Conclusion: We isolated two immunotoxin-resistant ALL/NHL cell lines and found that resistance was associated with reduced expression of diphthamide synthesis genes required to correctly post-translationally modify EF2. As a consequence, m. pasudotox was unable to ADP-ribosylate and inactivate EF2. Reduced expression of DPH4 in HAL-01-R was related to hypermethylation and could be prevented by 5-azacytidine. Reduced expression of WDR85 in CA46-R was due to a deletion of the gene. These represent novel mechanisms of immunotoxin resistance that will be of interest to explore in future studies. This study was supported by the Intramural Research Program of the NIH, National Cancer Institute (NCI), Center for Cancer Research and by a Cooperative Research and Development Agreement between MedImmune, LLC and the NCI. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2488 Hairy cell leukemia (HCL) is a B-cell malignancy which is thought to originate in most patients after the B-cell contacts antigen. To determine whether certain HLA types are preferentially expressed in HCL, HLA class I and class II allele frequencies at low resolution were collected from 247 HCL patients including 233 Caucasian, 5 Black, 5 Hispanic, 2 Asian and 2 Hawaiian/Pacific Islanders. Out of 494 total alleles from the 247 patients, the most frequent were HLA-A*02 (145, 29%), HLA-B*07 (58, 12%), HLA-C*07 (141, 29%), and HLA-DRB1*11 (76, 15%). In comparison with normal donors, only HLA-DRB1*11 was preferentially expressed in HCL, with a population frequency among the 233 Caucasians of 70 (30%), compared to 17% of a database of USA Caucasians (n=61655, p

Description: Abstract 2611 CD22 is expressed on the surface of B cell hematologic malignancies such as acute lymphoblastic leukemia (ALL). CD22 is a Siglec family lectin present on B cells, starting at the pre-B cell stage of development, but is not expressed on plasma cells. CD22 consists of 7 extracellular Ig domains and is found in 2 isoforms, one of which is missing the second and third N-terminal Ig domains. We generated CAR modified T cells containing anti-CD22 extracellular binding motifs fused to intracellular signaling domains for T cells activation (CD3 zeta) or costimulation (CD28 or 4-1BB). The binding motifs were derived from scFvs that targeted a membrane distal epitope of CD22, Ig domain 3, (BL22 and a higher affinity HA22 motif) or that bound a more membrane proximal, Ig domains 5–7, of CD22 (m971). The CAR constructs we generated were second-generation (CD28 and CD3 zeta; or, 4-1BB and CD3 zeta) or third generation (CD28, 4-1BB and CD3 zeta signaling domains). A CH2CH3 spacer domain from IgG1 was added in some constructs to examine the impact of extending the scFv-derived binding domain away from the transduced T cell membrane. In vitro cellular cytotoxicity and cytokine release experiments with 4 B cell-ALL cell lines (REH, SEM, NALM6, KOPN8) as well as the CD22 (+)ve Daudi and Raji cell lines were performed. Our results demonstrate that addition of the CH2CH3 domain did not improve tumor lysis and that standard affinity BL22 and higher affinity HA22-derived scFv epitopes were equivalent. With regard to signaling domains, second generation constructs were better than third generation constructs both in vitro and in vivo. In comparison between second generation constructs, CD28 containing domains outperformed 4-1BB with regard to lytic activity and cytokine release. Most surprising was the activity of the m971-derived scFv binding epitope. m971-CAR had significantly higher killing activity, a far more robust cytokine release profile, and superior in vivo activity. NSG mice were injected i.v. with 0.5× 106 NALM6-GL cells (pre-B cell ALL line engineered to express luciferase). Three days later, when disease was evident, mice were treated with 1×107 CAR+ T cells, and then followed by bioluminescent imaging to measure disease burden. The m971 CAR was significantly more potent at tumor clearance than our previously developed most active construct expressing the HA22-derived scFv domain (Figure 1). Disease progressed rapidly when non-transduced T cells were used (mock). We are currently examining the activity of different signaling domains on m971 CAR efficacy in vivo and directly comparing the anti-CD22 m971 CAR to the CD19 CAR currently being evaluated in clinical trials. These studies will guide future anti-CD22 CAR-based anti-leukemia immunotherapy trials. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2896 Background: Moxetumomab pasudotox is an anti-CD22 recombinant immunotoxin containing truncated Pseudomonas exotoxin which was recently reported to achieve a complete remission rate of 46% in 28 patients with relapsed/refractory hairy cell leukemia (HCL). An additional 20 patients were treated at the highest dose level and are now fully evaluable for response and minimal residual disease (MRD) determinations. RQ-PCR using clone-specific primers and a clone-specific TaqMan probe is capable of detecting one HCL cell in 106normal cells. Recently reported methods to detect the HCL-associated BRAF V600E mutation include pyrosequencing (5–10% sensitivity) and PCR (0.1–0.23% sensitivity). Methods: Moxetumomab pasudotox was administered to 16 patients at 5–40 ug/Kg every other day for 3 doses (QODx3) and to 32 patients at 50 ug/Kg QODx3, via 1–16 (median 4) cycles per patient at 4-week intervals. Complete remission (CR) required resolution of cytopenias and elimination of HCL in the blood and marrow by standard microscopy, but MRD could be present by flow cytometry of blood or bone marrow aspirate (BMA) or immunohistochemistry (IHC) of the bone marrow biopsy (BMBx). Blood and marrow from patients were also tested by PCR using consensus primers. When immunoglobulin (Ig) rearrangements could be cloned, RQ-PCR using clone-specific primer and probe was performed. To detect MRD by the BRAF V600E mutation, BRAF quantitative PCR (BRAF-qPCR) was performed on cDNA samples, using mutant-specific primer, and SYBR-Green detection followed by melting point analysis. MRD testing for BRAF-qPCR, unlike clone-specific RQ-PCR, did not require prior cloning of the Ig rearrangement. Results: All 198 cycles of moxetumomab administered to 48 patients were evaluable for toxicity and response. No dose limiting toxicity was observed, although 2 patients as previously reported had a grade 2 hemolytic uremic syndrome with transient grade 1 platelet and creatinine abnormalities. Of the 48 HCL patients at all dose levels, there were 26 (54%) CRs, with an overall response rate (ORR) of 88%. Of 32 at 50 ug/Kg QODx3, there were 19 (59%) CRs with an ORR of 91%. Of these 19 CRs, 11 (58%) achieved MRD negativity by repeated flow cytometry of both BMA and blood and IHC of BMBx. Flow cytometry of the BMA was the most sensitive conventional test of MRD. Of the 9 CRs at 50 ug/Kg QODx3 evaluable by clone-specific RQ-PCR of blood, 5 negative were also flow-negative, and 4 positive were also flow-positive (p=0.008). BRAF-qPCR on cDNA from limiting dilutions of BRAF V600E+ Colo-205 cells into BRAF wild-type cells achieved consistent detection at 1:105dilution (0.001%). Of 10 flow-negative CRs at 50 ug/Kg QODx3 evaluated by BRAF-qPCR, all 10 (100%) were BRAF-qPCR negative, including 4 which were nonevaluable by RQ-PCR due to inability to clone the Ig rearrangements prior to treatment. Currently 12 (63%) of the 19 CRs at 50 ug/Kg QODx3 are ongoing at 6–47 (median 21) months, including 10 (91%) of 11 MRD-negative vs 2 (25%) of 8 MRD+ CRs (p=0.006). Conclusions: Moxetumomab pasudotox is active in relapsed and refractory HCL and has a safety profile supporting further development for this disease. Retreatment on this trial could not necessarily be extended to achieve MRD-negative BMAs or molecular remission by RQ-PCR using sequence-specific or BRAF primers. However, these tests might be useful in the future to guide retreatment, optimize CR durability and possibly eradicate the HCL clone in selected patients. This summary contains investigator reported data. This study was sponsored by MedImmune, LLC, and supported by NCI's Intramural Research Program and the Hairy Cell Leukemia Research Foundation. Disclosures: Kreitman: NIH: Co-inventor on the NIH patent for Moxetumomab Pasudotox, Co-inventor on the NIH patent for Moxetumomab Pasudotox Patents & Royalties. Off Label Use: Moxetumomab Pasudotox is an experimental agent for CD22+ hematologic malignancies. FitzGerald:NIH: Coinventor on the NIH patent for Moxetumomab Pasudotox, Coinventor on the NIH patent for Moxetumomab Pasudotox Patents & Royalties. Fei:AstraZeneca: Stock, Stock Other; MedImmune, LLC: Employment. Ibrahim:AstraZeneca: Stocks, Stocks Other; MedImmune: Employment. Pastan:NIH: Coinventor on NIH patent for moxetumomab pasudotox, Coinventor on NIH patent for moxetumomab pasudotox Patents & Royalties.

Description: Abstract 2516 Currently no adequate therapies exist for patients with refractory hairy cell leukemia (HCL). The CD22 antigen is commonly expressed on the cell surface of B cells and B-cell malignancies. CD22 is highly expressed in HCL, even in chemoresistant disease, and is therefore a potential target for antibody-directed therapy in refractory/resistant HCL. We developed CD22-specific targeted immunotoxins composed of the variable domains of a monoclonal anti-CD22 antibody fused to a truncated form of a Pseudomonas exotoxin. The first anti-CD22 immunotoxin we studied was CAT-3888 (BL22), which demonstrated remarkable activity in patients with relapsed/refractory HCL. Moxetumomab pasudotox (also known as CAT-8015 or HA22) is a derivative of CAT-3888 with a higher affinity to CD22 and an improved cytotoxicity profile ex vivo against HCL and other B-cell malignancies. A phase 1 dose-escalation study is being conducted to determine the maximum tolerated dose (MTD) of moxetumomab pasudotox and to assess its safety and immunogenicity profiles and clinical activity in HCL. Preliminary safety data and antitumor activity from the study were previously reported (Kreitman et al, ASH 2009, ASCO 2010). Eligibility criteria include age ≥18 years, ≥2 previous systemic therapies (including purine analogs), ECOG performance status of 0–2, and the presence of cytopenia or symptomatic splenomegaly. A standard 3+3 dose-escalation design with an expanded cohort at the MTD or highest dose level is being used. Patients receive moxetumomab pasudotox as a 30-min IV infusion administered on days 1, 3, and 5 (QODx3) of a 28-day treatment cycle, up to a total of 10 cycles. Patients receive prophylaxis with low-dose aspirin (if platelets 〉100,000/μL) and IV hydration to prevent hemolytic uremic syndrome (HUS), the dose-limiting toxicity (DLT) in clinical studies with CAT-3888. Additionally, patients receive hydroxyzine, ranitidine, and acetaminophen to avoid infusion reactions. Response is evaluated before cycle 2 and thereafter before even-numbered cycles. Enrollment and treatment are ongoing. At the time of reporting, a total of 32 patients had received moxetumomab pasudotox at one of five dose levels: 5, 10, 20, and 30 μg/kg (n=3 in each cohort), and 40 μg/kg (n=4), and in an expanded cohort at the 50-μg/kg dose level (n=16). Median age was 59 years (range, 40–77 years). Patients were heavily pretreated; 19 (59%) were previously treated with rituximab. No DLTs have been observed, and an MTD has not been reached. Adverse events have been mostly mild and consistent with those seen in clinical studies with CAT-3888. The most frequently reported drug-related adverse events have been hypoalbuminemia (53%), peripheral edema (44%), pyrexia (34%), elevated ALT (34%) and AST (31%), fatigue (28%), headache (28%), localized edema (22%), and nausea (22%). Capillary leak syndrome (grade 2) has developed in 7 patients (22%); 5 patients were in the 50-μg/kg dose cohort. Reversible grade 2 HUS has been reported in 2 patients (6%; 30 μg/kg, n=1; 50 μg/kg, n=1); both began on day 8 of the treatment cycle. Treatment-related toxicities ≥grade 3 have been observed in 3 patients (30 μg/kg, n=1; 50 μg/kg, n=2) and include decreased haptoglobin levels, elevated gamma-glutamyltransferase, and lymphopenia. Moxetumomab pasudotox has shown clinical activity in 47% of recipients, and responses have been observed in both the dose-escalation and expansion cohorts. Overall response (OR), complete response (CR), and partial response (PR) rates observed to date are listed in the table. The median time to response is currently 2.79 months. Neutralizing antibodies have developed in 14 patients (44%). Moxetumomab pasudotox appears to have a safety profile similar to that of CAT-3888 (BL22), and with a lower incidence of HUS at the highest dose tested (50 μg/kg QODx3). Moxetumomab pasudotox has demonstrated antitumor activity in patients with relapsed/refractory HCL, including 44% of patients in the expansion cohort. Further studies in HCL and other B-cell malignancies expressing CD22 are warranted. Clinical Activity 5 μg/kg (n=3) 10 μg/kg (n=3) 20 μg/kg (n=3) 30 μg/kg (n=3) 40 μg/kg (n=4) 50 μg/kg (n=16) Total (N=32) OR 33% 67% 67% 33% 50% 44% 47% CR 0% 33% 67% 33% 50% 25% 31% PR 33% 33% 0% 0% 0% 19% 16% ClinicalTrials.gov Identifier: NCT00462189 This study was sponsored by MedImmune, LLC. Disclosures: Kreitman: NCI: Co-inventor on patents assigned to the NIH for the investigational product., Patents & Royalties. Robak: MedImmune, LLC: Research Funding. Noel: NIH: Patents & Royalties. FitzGerald: NCI: Co-inventor on patents assigned to the NIH for the investigational product., Patents & Royalties. Buzoianu: MedImmune, LLC: Employment. Lechleider: MedImmune, LLC: Employment. Pastan: NCI: Co-inventor on patents assigned to the NIH for the investigational product., Patents & Royalties.

Description: Abstract 4313 CD22 is expressed on a number of hematologic malignancies. A recombinant immunotoxin composed of an anti-CD22 Fv fused to a 38 kDa fragment of Pseudomonas exotoxin A (Moxetumomab pasudodtox, HA22) is currently being evaluated in the treatment of B cell malignancies. We used the Fv sequence to construct a series of chimeric antigen receptors (CARs) to determine the optimal affinity, domain structure, and signaling required for optimal anti-leukemic activity. The original anti-CD22 binding domain, BL22, or a newer generation high-affinity domain, HA22, were fused to transmembrane and signaling sequences derived from the TCR zeta-chain, CD28, and CD137. In some constructs we extended the binding domain away from the membrane using constant domains from IgG (CH2CH3). Retroviral gene vectors were used to transduce activated primary T cells with CAR constructs. We found: HA22 Fv, 2 as opposed to 3 signaling motifs, and non-CH2CH3 containing CARs were superior in short-term in vitro tumor cell cytolysis assays, indicating that Fv affinity, signaling, and 3-D structure of the CAR all impact the anti-leukemic effectiveness of CARs. Finally, direct comparison to CD19-specific CAR showed an equivalent or superior killing activity in 2 out of 3 ALL lines tested. Using Scatchard analysis to define the number of CD22 moleules on the surface, the ALL line with the lowest number of CD22 molecules is also the least able to be killed by CAR-transduced T cells. The supeior lytic activity of CD19-CAR for this single cell line was due to preservation of high levels of CD19 at the cell surface. We are currently testing the in vivo activity of anti-CD22 CAR in an immunodeficient mouse model bearing human cell lines and primary patient-derived ALL. Our results indicate that CD22-CAR should be developed for the immunotherapy of CD22+ hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points We have created a new highly active chimeric antigen receptor (CAR) specific for CD22. The design of new CARs may benefit more from target antigen epitope selection than from optimizing affinity.

Description: Background Moxetumomab pasudotox, an anti-CD22 recombinant immunotoxin, contains truncated Pseudomonas exotoxin and is currently undergoing pivotal phase III testing for relapsed and refractory hairy cell leukemia (HCL). The dose-escalation portion of the phase I trial was reported to achieve an overall response rate (ORR) of 86% including 46% complete remissions (CRs) in 28 HCL patients. A completely reversible form of grade 2 hemolytic uremic syndrome (HUS), based on laboratory changes but lacking clinical signs, was observed in 2 patients, and no dose-limiting toxicity (DLT) was observed. To determine the relationship between response and CD22 antigen ‘sink’ from high CD22 density on HCL cells, plasma levels were studied. Those analyzed included the original 28 in the dose-escalation arm, an additional 20 at the highest dose level enrolled prior to 2012, and one additional patient enrolled prior to initiation of the pivotal trial. Methods Moxetumomab pasudotox was administered to 49 patients at 5, 10, 20, and 30 µg/kg every other day for 3 doses (QODx3, n=3 each dose level), and then 4 and 33 patients received 40 and 50 µg/kg QODx3, respectively. Patients received retreatment cycles at 4 week intervals, with retreatment halted after progressive disease, high levels of neutralizing antibodies, HUS, or 2 cycles after achievement of MRD-free CR. MRD was studied using bone marrow biopsy (BMBx) immunohistochemistry (IHC), and flow cytometry of blood or bone marrow aspirate (BMA). Pharmacokinetics was determined from cytotoxicity assays to quantify active moxetumomab pasudotox in the plasma. Results There were 28 (57%) CRs and 15 (31%) PRs for an ORR of 88% out of 49 patients. While CRs were insignificantly more frequent at 50 µg/kg QODx3 vs lower doses (64% of 33 vs 44% of 16, p=0.23), the difference in MRD-free CR rate was significant (39% vs 6%, p=0.02). Of the 21 CRs at 50 µg/kg QODx3, 13 (62%) remain in CR at a median relapse-free follow-up time of 32 months, and relapse occurred in 7 (88%) of 8 with MRD vs 1 (8%) of 13 without MRD (p

Description: Abstract 2575 Background: Adult T-cell leukemia (ATL) is a highly aggressive CD25+ HTLV-1-associated malignancy with median survivals of 4 and 9 months for leukemic and lymphomatous forms, respectively. There is no standard therapy capable of altering median survival by more than a few months. The anti-CD25 recombinant immunotoxin LMB-2 as a single agent was previously reported in phase I testing to have activity, but limited by rapid tumor growth between cycles and immunogenicity. Human CD25+ xenografts in mice contained soluble CD25 (sCD25) levels 〉100-fold higher than in serum, which were rapidly depleted by chemotherapy. Treatment with fludarabine was previously reported to be associated with lower immunogenicity to murine antibodies (HAMA), and the fludarabine-cyclophosphamide (FC) combination was reported associated with reductions in normal T- and B-cells. Methods: To prevent the formation of neutralizing antibodies and enhance anti-tumor activity, leukemic or lymphomatous ATL patients received FC chemotherapy with fludarabine 25 mg/m2 and cyclophosphamide 250 mg/m2 on days 1, 2, and 3, and 2 weeks later began cycles every 3 weeks in which FC was administered on days 1, 2 and 3, followed by LMB-2 30–40 ug/Kg on days 3, 5 and 7. Formation of neutralizing antibodies and clinical response were determined with each cycle. Results: Interim results with 8 evaluable patients receiving 28 cycles (2–5 each) include an ORR of 50% with 2 CRs and 2 PRs. In 6 patients with circulating ATL cells detectable in the blood, reductions of ATL cells varied from 90.8 to 100% compared to baseline. CRs were associated with complete resolution of chest wall and pelvic masses, circulating ATL cells, and bone marrow involvement as assessed by immunohistochemistry and flow cytometry. One of 2 PRs is ongoing for nearly 1 year after complete resolution of skin involvement and partial decrease in circulating ATL cells. One of 2 CRs recurred after 6 months only in a sanctuary site (testis). The toxicity of LMB-2 was not increased by FC and 40 ug/Kg of LMB-2 days 3, 5 and 7 was not associated with dose-limiting toxicity (DLT). While the original doses of FC used were also without DLT, escalation to 30 and 300 mg/m2/day x3 resulted in severe thrombocytopenia in 1 of 2 patients. The most common toxicities, based on percent of cycles observed, were neutropenia (69%), lymphopenia (52%), leukopenia and nausea (48%), and anemia, thrombocytopenia and fever (41%). With FC, normal T-cells were reduced 0–95% (median 70%), but normal B-cells were reduced 91–100% (median 96%). Two (25%) patients made high levels of neutralizing antibodies after 3–4 cycles of LMB-2. Conclusions: LMB-2 is effective in ATL when preceded by FC, and can achieve major responses including CR. Additional patients will be needed to determine if FC can delay immunogenicity significantly, and allow enough cycles to result in long-term remission from this disease (Supported in part by NCI, intramural research program, NIH). Disclosures: Kreitman: NIH: Patents & Royalties. Off Label Use: Use of investigational agent and FC chemotherapy for treatment of ATL. Pastan:NIH: Patents & Royalties.