-
[show abstract]
[hide abstract]
ABSTRACT: HA22 is a recombinant immunotoxin that kills CD22 expressing cells by ADP-ribosylating and inactivating elongation factor-2 (EF2). HA22 is composed of an Fv that binds to CD22 that is fused to a potion of Pseudomonas exotoxin A. HA22 is very active in drug resistant Hairy Cell Leukemia, but less active in children with acute lymphoblastic leukemia. To understand why some patients don't respond to HA22, we isolated a HA22 resistant lymphoma cell line and showed that resistance is due to the inability of HA22 to ADP-ribosylate and inactivate EF2. We analyzed the diphthamide synthesis genes and found that WDR85 gene was deleted. We showed that WDR85 knockdown confers HA22 resistance to sensitive cells and sensitivity is restored by introduction of a WDR85 cDNA into resistant cells. Analysis of EF2 in the mutant cells revealed a novel form of diphthamide with an additional methyl group that prevents ADP-ribosylation and inactivation of EF2. The abnormal methylation appears to be catalyzed by DPH5. Inactivation of the WDR85 gene could be a mechanism of immunotoxin resistance in patients undergoing immunotoxin therapy.
Journal of Biological Chemistry 03/2013; · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: SS1P is a recombinant immunotoxin composed of an anti-mesothelin Fv fragment fused to a truncated portion of Pseudomonas exotoxin (PE) A. SS1P targets and kills mesothelin-expressing tumors, which include mesothlioma as well as ovarian, lung and pancreatic cancers. SS1P is currently in clinical trials in mesothelioma. Because insulin (INS) acting through the insulin receptor (IR, INSR) is a survival factor for many cancer cell lines, we explored how lowering IR level would affect the cytotoxic action of SS1P. We show here that siRNA knock-down of the IR enhanced the cytotoxic action of native PE and enhanced SS1P toxicity on several human cell lines, but did not affect the response to other cytotoxic agents such as TRAIL, etoposide and cycloheximide. To determine how IR knock-down enhances SS1P action, we analyzed various steps involved in cell killing. We found that IR knock down increases the cleavage of SS1P by furin, which allows more toxin to reach the cytosol and inactivate elongation factor 2. These findings indicate that the IR negatively regulates immunotoxin action.
Cancer Research 01/2013; · 7.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Recombinant immunotoxins (RITs) are chimeric proteins that are being developed for cancer treatment. We have produced RITs that contain PE38, a portion of the bacterial protein Pseudomonas exotoxin A. Because the toxin is bacterial, it often induces neutralizing antibodies, which limit the number of treatment cycles and the effectiveness of the therapy. Because T cells are essential for antibody responses to proteins, we adopted an assay to map the CD4(+) T-cell epitopes in PE38. We incubated peripheral blood mononuclear cells with an immunotoxin to stimulate T-cell expansion, followed by exposure to overlapping peptide fragments of PE38 and an IL-2 ELISpot assay to measure responses. Our observation of T-cell responses in 50 of 50 individuals correlates with the frequency of antibody formation in patients with normal immune systems. We found a single, highly immunodominant epitope in 46% (23/50) of the donors. The immunodominant epitope is DRB1-restricted and was observed in subjects with different HLA alleles, indicating promiscuity. We identified two amino acids that, when deleted or mutated to alanine, eliminated the immunodominant epitope, and we used this information to construct mutant RITs that are highly cytotoxic and do not stimulate T-cell responses in many donors.
Proceedings of the National Academy of Sciences 12/2012; · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Background/Aim: To determine if early passage tumor cells obtained from patients with mesothelioma continue to express the tumor differentiation antigen mesothelin and their sensitivity to the anti-mesothelin immunotoxin SS1P.
Cell cultures were established from ascites or pleural effusion of 6 peritoneal and 3 pleural mesothelioma patients, respectively. These cells were evaluated for mesothelin expression by immunohistochemistry and flow cytometry.
Although mesothelin was highly expressed in tumor biopsies of all patients, only 3 out of 9 malignant effusions from these patients when grown in short-term culture showed strong mesothelin positivity by IHC. By flow cytometry, the number of mesothelin sites per cell was variable ranging from 580 to 210,000 sites/cell. Cells with strong mesothelin expression by IHC and increased number of mesothelin sites/cell were sensitive to SS1P.
Most mesothelioma tumors loose mesothelin when grown in vitro and the sensitivity of these cells to SS1P is dependent on the number of mesothelin sites/cell.
Anticancer research 12/2012; 32(12):5151-8. · 1.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: SS1P is a recombinant immunotoxin (RIT) engineered for the targeted elimination of malignant cells that express the tumor-associated antigen mesothelin. It is comprised of an anti-mesothelin antibody Fv linked to a cytotoxic fragment of Pseudomonas exotoxin A (PE) that includes domains II and III of native PE. The clinical utility of SS1P is limited by its propensity to induce neutralizing antibodies and to cause a dose-limiting capillary leak syndrome (CLS) in patients. In this paper we describe a reengineered SS1P with improved properties that overcome these deficits. The redesign of SS1P consists of [1] removing the bulk of PE domain II (residues 251-273 and 284-394 of native PE), leaving only an 11-residue furin cleavage site, [2] adding a Gly-Gly-Ser peptide linker after the furin cleavage site, and [3] replacing eight highly solvent-exposed residues in the catalytic domain of PE. The new molecule, SS1-LR/GGS/8M, has cytotoxic activity comparable to SS1P on several mesothelin-expressing cell lines, and remarkably improved activity on primary cells from patients with mesothelioma. In a mouse xenograft tumor model, high doses of SS1-LR/GGS/8M elicit antitumor activity superior to the activity of SS1P at its maximum tolerated dose. In addition, SS1-LR/GGS/8M has greatly decreased ability to cause CLS in a rat model, and reduced antigenicity, or reactivity with antibodies, to the sera of patients previously treated with SS1P.
Molecular Cancer Therapeutics 11/2012; · 5.23 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Recombinant immunotoxins (RITs) are hybrid proteins used to treat cancer. These proteins are composed of an Fv that reacts with cancer cells joined to a portion of Pseudomonas exotoxin A, which kills the cell. Because the toxin is a foreign protein, it can induce neutralizing antibodies and thereby limit the number of doses a patient can receive. We previously identified seven major mouse B-cell epitopes in the toxin, and subsequently silenced them using point mutations that converted large hydrophilic amino acids to alanine, yet retained full antitumor activity. Here we present results in which we identify and silence human B-cell epitopes in the RIT HA22. We obtained B cells from patients with antibodies to RITs, isolated the corresponding variable fragments (Fvs), and constructed a phage-display library containing Fvs that bind to the RITs. We then used alanine scanning mutagenesis to locate the epitopes. We found that human and mouse epitopes frequently overlap but are not identical. Most mutations that remove mouse epitopes did not remove human epitopes. Using the epitope information, we constructed a variant immunotoxin, HA22-LR-LO10, which has low reactivity with human antisera, yet has high cytotoxic and antitumor activity and can be given to mice at high doses without excess toxicity. The toxin portion of this RIT (LR-LO10) can be used with Fvs targeting other cancer antigens and is suitable for clinical development.
Proceedings of the National Academy of Sciences 07/2012; 109(29):11782-7. · 9.68 Impact Factor
-
Shutao Wang,
In Soo Shin,
Hilary Hancock,
Beom-su Jang,
Hyung-sub Kim,
Sang Myung Lee,
Vesna Zderic,
Victor Frenkel, Ira Pastan,
Chang H Paik,
Matthew R Dreher
[show abstract]
[hide abstract]
ABSTRACT: The success of radioimmunotherapy for solid tumors remains elusive due to poor biodistribution and insufficient tumor accumulation, in part, due to the unique tumor microenvironment resulting in heterogeneous tumor antibody distribution. Pulsed high intensity focused ultrasound (pulsed-HIFU) has previously been shown to increase the accumulation of (111)In labeled B3 antibody (recognizes Lewis(y) antigen). The objective of this study was to investigate the tumor penetration and therapeutic efficacy of pulsed-HIFU exposures combined with (90)Y labeled B3 mAb in an A431 solid tumor model. The ability of pulsed-HIFU (1 M Hz, spatial averaged temporal peak intensity=2685 W cm(-2); pulse repetition frequency=1 Hz; duty cycle=5%) to improve the tumor penetration and therapeutic efficacy of (90)Y labeled B3 mAb ((90)Y-B3) was evaluated in Le(y)-positive A431 tumors. Antibody penetration from the tumor surface and blood vessel surface was evaluated with fluorescently labeled B3, epi-fluorescent microscopy, and custom image analysis. Tumor size was monitored to determine treatment efficacy, indicated by survival, following various treatments with pulsed-HIFU and/or (90)Y-B3. The pulsed-HIFU exposures did not affect the vascular parameters including microvascular density, vascular size, and vascular architecture; although 1.6-fold more antibody was delivered to the solid tumors when combined with pulsed-HIFU. The distribution and penetration of the antibodies were significantly improved (p-value<0.05) when combined with pulsed-HIFU, only in the tumor periphery. Pretreatment with pulsed-HIFU significantly improved (p-value<0.05) survival over control treatments.
Journal of Controlled Release 06/2012; 162(1):218-24. · 5.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Recombinant immunotoxins (RIT) are targeted anticancer agents that are composed of a targeting antibody fragment and a protein toxin fragment. SS1P is a RIT that targets mesothelin on the surface of cancer cells and is being evaluated in patients with mesothelioma. Mesothelin, like many other target antigens, is shed from the cell surface. However, whether antigen shedding positively or negatively affects the delivery of RIT remains unknown. In this study, we used experimental data with SS1P to develop a mathematical model that describes the relationship between tumor volume changes and the dose level of the administered RIT, while accounting for the potential effects of antigen shedding.
Cancer Research 05/2012; 72(13):3143-52. · 7.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The selective cell surface expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) has made ROR1 a novel and promising target for therapeutic monoclonal antibodies (mAbs). Four mouse mAbs generated by hybridoma technology exhibited specific binding to human ROR1. Epitope mapping studies showed that two mAbs (2A2 and 2D11) recognized N-terminal epitopes in the extracellular region of ROR1 and the other two (1A1 and 1A7) recognized C-terminal epitopes. A ROR1- immunotoxin (BT-1) consisting of truncated Pseudomonas exotoxin A (PE38) and the VH and VL fragments of 2A2-IgG was made recombinantly. Both 2A2-IgG and BT-1 showed dose-dependent and selective binding to primary CLL and MCL cells and MCL cell lines. Kinetic analyses revealed 0.12-nM (2A2-IgG) to 65-nM (BT-1) avidity/affinity to hROR1, depicting bivalent and monovalent interactions, respectively. After binding to cell surface ROR1, 2A2-IgG and BT-1 were partially internalized by primary CLL cells and MCL cell lines, and BT-1 induced profound apoptosis of ROR1-expressing MCL cell lines in vitro (EC 50 = 16 pM-16 nM), but did not affect ROR1-negative cell lines. Our data suggest that ROR1-immunotoxins such as BT-1 could serve as targeted therapeutic agents for ROR1-expressing B cell malignancies and other cancers.
mAbs 05/2012; 4(3):349-61.
-
[show abstract]
[hide abstract]
ABSTRACT: HA22 is a recombinant immunotoxin composed of an anti-CD22 Fv fused to a portion of Pseudomonas exotoxin A. HA22 produced a high rate of complete remissions in drug-resistant hairy cell leukemia and has a lower response rate in pediatric acute lymphoblastic leukemia (ALL). To understand why patients with ALL have poorer responses, we isolated an ALL cell line that is resistant to killing by HA22. The resistance is unstable; without HA22 the cells revert to HA22 sensitivity in 4 mo. We showed that in the resistant cell line, HA22 is unable to ADP ribosylate and inactivate elongation factor-2 (EF2), owing to a low level of DPH4 mRNA and protein, which prevents diphthamide biosynthesis and renders EF2 refractory to HA22. Analysis of the promoter region of the DPH4 gene shows that the CpG island was hypomethylated in the HA22-sensitive cells, heavily methylated in the resistant cells, and reverted to low methylation in the revertant cells. Our data show that immunotoxin resistance is associated with reversible CpG island methylation and silencing of DPH4 gene transcription. Incubation of sensitive cells with the methylation inhibitor 5-azacytidine prevented the emergence of resistant cells, suggesting that this agent in combination with HA22 may be useful in the treatment of some cases of ALL.
Proceedings of the National Academy of Sciences 04/2012; 109(18):6898-903. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mesothelin is a tumor differentiation antigen that is highly expressed in several malignant diseases in humans, including malignant mesothelioma and pancreatic, ovarian, and lung adenocarcinomas. The limited expression of mesothelin on normal human tissues and its high expression in many common cancers make it an attractive candidate for cancer therapy. Several agents, including an immunotoxin, monoclonal antibody, antibody drug conjugate, and tumor vaccine, are in various stages of development to treat patients with mesothelin-expressing tumors. This review highlights ongoing clinical trials, as well as other approaches to exploit mesothelin for cancer therapy, that are in preclinical development.
Molecular Cancer Therapeutics 03/2012; 11(3):517-25. · 5.23 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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).
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.
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.
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.
Journal of Clinical Oncology 02/2012; 30(15):1822-8. · 18.37 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We want to emphasize that tumor targeting is a complicated process. The modulation of mesothelin shedding could have a systemic influence on drug kinetics in both circulation and tumor tissue. Our in vitro assay did not take this into consideration, which could be essential when developing a therapeutic strategy. Besides shedding, mesothelin levels could be modulated like other antigens by trogocytosis [7] or antigen masking [8]. Whether they play a role in mesothelin targeted therapy remains to be evaluated. An understanding of possible mesothelin modulation will be instrumental to new targeting strategies.
Oncotarget 02/2012; 3(2):114-5. · 4.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The POTE gene family encodes very closely related proteins that are highly expressed in testis and in many cancers. Recent studies indicate that the POTE proteins have a pro-apoptotic function. To examine if POTE is associated with cells that are undergoing apoptosis in testis, we determined the cellular location of POTE and of Cleaved Caspase-3 in testicular tissues from 26 azoospermic men. We found intense expression of POTE in round spermatids that are undergoing apoptosis, which are positive for Cleaved Caspase-3. This study suggests POTE may have a role in apoptosis in the human testis.
Biochemical and Biophysical Research Communications 01/2012; 417(4):1271-4. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Partial inactivation of the Ankyrin repeat domain 26 (Ankrd26) gene causes obesity and diabetes in mice and increases spontaneous and induced adipogenesis in mouse embryonic fibroblasts. However, it is not yet known how the Ankrd26 protein carries out its biological functions. We identified by yeast two-hybrid and immunoprecipitation assays the triple functional domain protein (TRIO), the G protein pathway suppressor 2 (GPS2), the delta-interacting protein A (DIPA) and the hyaluronan-mediated motility receptor (HMMR) as ANKRD26 interacting partners. Adipogenesis of 3T3-L1 cells was increased by selective down-regulation of Ankrd26, Trio, Gps2, Hmmr and Dipa. Furthermore, GPS2 and DIPA, which are normally located in the nucleus, were translocated to the cytoplasm, when the C-terminus of ANKRD26 was introduced into these cells. These findings provide biochemical evidence that ANKRD26, TRIO, GPS2 and HMMR are novel and important regulators of adipogenesis and identify new targets for the modulation of adipogenesis.
PLoS ONE 01/2012; 7(5):e38130. · 4.09 Impact Factor
-
Viola Biberacher,
Thomas Decker,
Madlen Oelsner,
Michaela Wagner,
Christian Bogner,
Burkhard Schmidt,
Robert J Kreitman,
Christian Peschel, Ira Pastan,
Christian Meyer Zum Büschenfelde,
Ingo Ringshausen
[show abstract]
[hide abstract]
ABSTRACT: In spite of potent first-line therapies for chronic lymphocytic leukemia, treatment remains palliative and all patients frequently relapse. Treatment options for these patients are more limited. BL22 is a recombinant protein composed of the variable region of a monoclonal antibody that binds to CD22 and of PE38, a truncated Pseudomonas exotoxin. BL22 is a very potent drug already used in patients with hairy cell leukemia, whereas in chronic lymphocytic leukemia its cytotoxicity is limited by a lower expression of CD22. Here we demonstrate that this limitation can be overcome by pre-activation of chronic lymphocytic leukemia cells with bryostatin 1.
Primary malignant B cells from chronic lymphocytic leukemia and mantle cell lymphoma patients were used in vitro to assess the therapeutic impact of drug combinations using BL22 and bryostatin 1.
We demonstrate that bryostatin 1 sensitizes chronic lymphocytic leukemia cells for the cytotoxic effects of BL22 through activation of protein kinase C and subsequently increased CD22 surface expression. Dose and time response analysis reveals that activation of protein kinase C further activates an autocrine feedback loop degrading protein kinase C-βII protein. Depletion of protein kinase C-βII and upregulation of CD22 persist for several days following pre-stimulation with bryostatin 1. Therefore, our data provide a rationale for the sequential administration of BL22 following bryostatin 1 treatment. In addition to primary chronic lymphocytic leukemia cells, bryostatin 1 also sensitizes diffuse large B-cell lymphoma and mantle cell lymphoma cells to BL22 induced apoptosis.
Our data suggest that the combination of bryostatin 1 with antibodies directed against CD22 is a potent drug combination for the treatment of low- and high-grade B-cell lymphoma.
Haematologica 12/2011; 97(5):771-9. · 6.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: This study was undertaken to investigate the effect of paclitaxel and bevacizumab on the therapeutic efficacy of (90)Y-labeled B3 monoclonal antibody, directed against Le(y) antigen, for the treatment of Le(y)-positive A431 tumors implanted subcutaneously in the right hind flank of nude mice.
When the tumor size reached ~200 mm(3), the mice received a single dose of intravenous (iv) (90)Y-labeled B3 (60 μCi/150 μg or 100 μCi/150 μg B3), intraperitoneal paclitaxel (40 mg/kg) or iv bevacizumab (5 mg/kg) for monotherapy. To investigate the effect of combined therapies on survival, the mice were treated with two or three agents in the following combinations: (90)Y-B3 on day 0 and paclitaxel on day 1; bevacizumab on -1 day and (90)Y-B3 on day 0; bevacizumab on -1 day and paclitaxel on day 1; bevacizumab, (90)Y-B3 and paclitaxel each at 1-day intervals. The mice with no treatment were used as a control. The tumor volume at 1000 mm(3) was used as a surrogate end point of survival.
Compared to control animals, paclitaxel delayed tumor growth with a significantly longer median survival time (P<.001), whereas bevacizumab alone showed a less pronounced effect on a median survival time (P=.18). (90)Y-B3 increased the median survival time in a dose-dependent manner (P<.05). The combined therapy of bevacizumab with paclitaxel produced a trend toward an increase of the median survival time compared to paclitaxel alone (P=.06), whereas bevacizumab combined with (90)Y-B3 showed a statistically insignificant increase in the median survival time compared to (90)Y-B3 alone (P=.25). The tumor sizes of all animals in these groups reached the surrogate end point of survival by day 35. In contrast, the combined therapy involving (90)Y-B3 with paclitaxel showed a striking synergistic effect in shrinking tumors and prolonging the survival time (P<.001); on day 120, three of nine mice (33%) and six of six mice (100%) were alive without tumor when treated with 60 μCi (90)Y-B3 and 100 μCi (90)Y-B3, respectively. The addition of bevacizumab treatment 1 day before the combined therapy of 60 μCi (90)Y-B3 with paclitaxel did not produce a statistically significant increase in survival when compared to the (90)Y-B3 with paclitaxel (P>.10). Fluorescence microscopy analysis indicated that paclitaxel increased, whereas bevacizumab decreased, the accumulation and penetration of Alexa Fluor 647-B3 into tumor microenvironment compared to the control (P<.05).
Our findings on the paclitaxel effect support a hypothesis that the increased tumor accumulation and penetration of (90)Y-B3 as well as the high radiosensitization of tumor cells by paclitaxel may be the major factors responsible for the synergistic effect of the combined therapy involving (90)Y-B3 with paclitaxel.
Nuclear Medicine and Biology 12/2011; 39(4):472-83. · 3.02 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Tumor mesothelin overexpression is present in different malignancies, including the majority of patients with pancreatic or biliary cancers. The objective of this study was to evaluate the use of shed serum mesothelin and megakaryocyte potentiating factor (MPF) concentrations as biomarkers for these cancers.
A total of 151 individuals, divided into five groups, were retrospectively analyzed: healthy donors (n=15), patients with benign non-pancreatic conditions (n=52), benign pancreatic conditions (n=33), biliary carcinoma (n=9), and pancreatic ductal adenocarcinoma (n=42). Mesothelin and MPF concentrations were measured in serum with the Mesomark™ and Human MPF ELISA, respectively.
Mesothelin and MPF concentrations did not significantly differ among the five individual participant groups (p=0.34, p=0.33, respectively), nor did any other combination and pair-wise comparison of the participant groups demonstrated a significant difference in biomarker concentrations. In patients with pancreatic cancer, mesothelin or MPF concentrations were not associated with tumor stage (p=0.87, p=0.48, respectively) or differentiation grade (p=0.73, p=0.52, respectively).
Serum mesothelin and MPF concentrations, measured with standard available ELISAs, were not specific for benign or pancreatic disease. Both biomarkers were not elevated in patients with pancreatic or biliary cancers, and consequently do not appear to be useful biomarkers for these malignancies.
Clinical Chemistry and Laboratory Medicine 12/2011; 50(4):721-5. · 2.15 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Recombinant immunotoxins (RITs) are anti-cancer agents that combine the Fv of an antibody against cancer cells with a protein toxin from bacteria or plants. Since RITs contain a non-human protein, immunogenicity can be an obstacle in their development. In this study, we have explored the hypothesis that increasing stability can reduce the immunogenicity of a RIT using HA22-LR, which is composed of an anti-CD22 Fv fused to domain III of Pseudomonas exotoxin A. We introduced a disulfide bond into domain III by identifying and mutating two structurally adjacent residues to cysteines at sites suggested by computer modeling. This RIT, HA22-LR-DB, displays a remarkable increase in thermal stability and an enhanced resistance to trypsin degradation. In addition, HA22-LR-DB retains cytotoxic and anti-tumor activity, while exhibiting significantly lower immunogenicity in mice. This study demonstrates that it is possible to design mutations in a protein molecule that will increase the stability of the protein and thereby reduce its immunogenicity.
Protein Engineering Design and Selection 11/2011; 25(1):1-6. · 2.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Although anti-CD25 recombinant immunotoxin LMB-2 is effective against CD25(+) hairy cell leukemia, activity against more aggressive diseases such as adult T-cell leukemia (ATL) is limited by rapid disease progression between treatment cycles. Our goal was to determine in vivo whether rapid growth of CD25(+) tumor is associated with high levels of tumor interstitial soluble CD25 (sCD25) and whether chemotherapy can reduce tumor sCD25 and synergize with LMB-2.
Tumor xenografts expressing human CD25 were grown in mice, which were then treated with LMB-2 and chemotherapy either alone or in combination, and sCD25 level and antitumor activity were measured.
CD25(+) human xenografts growing rapidly in nude mice had intratumoral sCD25 at levels that were between 21- and 2,200 (median 118)-fold higher than in serum, indicating that interstitial sCD25 interacts with LMB-2 in tumors. Intratumoral sCD25 levels were in the range 21 to 157 (median 54) ng/mL without treatment and 0.95 to 6.1 (median 2.6) ng/mL (P < 0.0001) 1 day after gemcitabine administration. CD25(+) xenografts that were too large to regress with LMB-2 alone were minimally responsive to gemcitabine alone but completely regressed with the combination. Ex vivo, different ratios of gemcitabine and LMB-2 were cytotoxic to the CD25(+) tumor cells in an additive, but not synergistic, manner.
Gemcitabine is synergistic with LMB-2 in vivo unrelated to improved cytotoxicity. Synergism, therefore, appears to be related to improved distribution of LMB-2 to CD25(+) tumors, and is preceded by decreased sCD25 within the tumor because of chemotherapy. To test the concept of combined treatment clinically, patients with relapsed/refractory ATL are being treated with fludarabine plus cyclophosphamide before LMB-2.
Clinical Cancer Research 11/2011; 18(1):152-60. · 7.74 Impact Factor
