Showing papers by "Ira Pastan published in 2005"
TL;DR: BL22 was well tolerated and highly effective in HCL, even after one cycle, and phase II testing is underway to define the efficacy with one cycle and to study safety when additional cycles are needed for optimal response.
Abstract: Purpose To conduct a phase I trial of recombinant immunotoxin BL22, an anti-CD22 Fv fragment fused to truncated Pseudomonas exotoxin. Patients and Methods Forty-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. Results BL22 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 hemo...
262 citations
TL;DR: A mouse model of acquired progressive glomerular sclerosis in which onset and severity can be preprogrammed by experimental maneuvers is established.
Abstract: This study aimed to generate a mouse model of acquired glomerular sclerosis. A model system that allows induction of podocyte injury in a manner in which onset and severity can be controlled was designed. A transgenic mouse strain (NEP25) that expresses human CD25 selectively in podocytes was first generated. Injection of anti-Tac (Fv)-PE38 (LMB2), an immunotoxin with specific binding to human CD25, induced progressive nonselective proteinuria, ascites, and edema in NEP25 mice. Podocytes showed foot process effacement, vacuolar degeneration, detachment and downregulation of synaptopodin, WT-1, nephrin, and podocalyxin. Mesangial cells showed matrix expansion, increased collagen, mesangiolysis, and, later, sclerosis. Parietal epithelial cells showed vacuolar degeneration and proliferation, whereas endothelial cells were swollen. The severity of the glomerular injury was LMB2 dose dependent. With 1.25 ng/g body wt or more, NEP25 mice developed progressive glomerular damage and died within 2 wk. With 0.625 ng/g body wt of LMB2, NEP25 mice survived >4 wk and developed focal segmental glomerular sclerosis. Thus, the study has established a mouse model of acquired progressive glomerular sclerosis in which onset and severity can be preprogrammed by experimental maneuvers.
234 citations
TL;DR: Results show that mesothelin is expressed in most epithelial ovarian cancers and that Mesothelin expression in ovarian cancers can be evaluated in archival material and could be eligible for participation in clinical trials of novel agents targeting mesothelins.
Abstract: Mesothelin is a cell surface glycoprotein that is present on normal mesothelial cells and overexpressed in several cancers On immunohistochemical examination of a limited number of ovarian tumors, increased mesothelin expression has been previously noted The authors evaluated mesothelin expression in 48 patients with ovarian cancer who were screened for participation in phase 1 studies of a recombinant immunotoxin targeting mesothelin Eligibility criteria for participation in the studies included mesothelin expression by more than 30% of accessible tumor cells Sections of formalin-fixed paraffin-embedded tumor specimens were evaluated for mesothelin expression by immunohistochemistry using the anti-mesothelin monoclonal antibody K1 Between September 2000 and January 2003, 48 ovarian tumors were analyzed for mesothelin positivity Mesothelin positivity was noted in 34 of the 48 cases evaluated (71%) These results show that mesothelin is expressed in most epithelial ovarian cancers and that mesothelin expression in ovarian cancers can be evaluated in archival material Patients whose tumors express mesothelin could be eligible for participation in clinical trials of novel agents targeting mesothelin
156 citations
TL;DR: The findings indicate that mesothelin is a new tumor antigen in patients with mesothelioma and ovarian cancer and the immunogenicity of mesothelins is associated with its high expression on the tumor cells.
Abstract: Purpose: Mesothelin is a glycosyl-phosphatidylinositol–anchored glycoprotein present on the cell surface. Mesothelin is a differentiation antigen that is highly expressed on mesothelioma, ovarian cancer, and pancreatic cancer. The existence of a spontaneous humoral immune response to mesothelin in humans has not been fully studied. Here we addressed the issue of whether mesothelin elicits a humoral immune response in patients with mesothelioma and ovarian cancer. Experimental Design: Using an ELISA, we analyzed immunoglobulin G antibodies specific for mesothelin in sera from patients with mesothelioma and epithelial ovarian cancer. Tumor specimens were examined by immunohistochemistry for mesothelin protein expression. Results: Elevated levels of mesothelin-specific antibodies were detected in the sera of 39.1% of patients with mesothelioma (27 of 69 patients) and 41.7% with epithelial ovarian cancer (10 of 24 patients) when compared with a normal control population (44 blood donors; P 2 test: P P = 0.025 for ovarian cancer). Conclusions: Our findings indicate that mesothelin is a new tumor antigen in patients with mesothelioma and ovarian cancer and the immunogenicity of mesothelin is associated with its high expression on the tumor cells. Mesothelin represents an excellent target for immune-based therapies.
143 citations
TL;DR: It is demonstrated that germlinehot spots but not non-germline hot spots are effective for in vitro antibody affinity maturation.
115 citations
TL;DR: Protein engineering can be used to increase the efficacy of recombinant immunotoxins by improving the activity of HA22, a mutant of BL22 with mutations in heavy-chain CDR3 resulting in increased cytotoxic activity.
Abstract: Purpose: RFB4 (dsFv)-PE38 (BL22) is a recombinant immunotoxin containing an anti-CD22 (Fv) fused to truncated Pseudomonas exotoxin A, which induces a high complete remission rate in patients with purine analogue–resistant hairy cell leukemia. HA22 is a mutant of BL22 with mutations in heavy-chain CDR3 resulting in increased cytotoxic activity. Our goal was to improve the activity of HA22. Experimental Design: Arg 490 , which is located in the catalytic domain (III) of the immunotoxin HA22, was mutated to alanine. Purified immunotoxins were produced and tested for cytotoxic activity in cell culture and for antitumor activity and nonspecific toxicity in mice. ADP-ribosylation activity was also measured. Results: HA22 (R490A) is ∼2-fold more cytotoxic than HA22 on several CD22-positive cell lines. When injected i.v., HA22 (R490A) has more potent antitumor activity than HA22 against CA46 tumors in mice. HA22 and HA22 (R490A) have similar LD 50 s (∼1.3 mg/kg) and similar plasma half-lives. The R490A mutation also improved the cytotoxicity of the antimesothelin recombinant immunotoxin SS1 (dsFv)-PE38 (SS1P). In vitro ADP-ribosylation assays show that HA22 R490A has increased activity. Increased cytotoxic activity is probably related to this increase in ADP-ribosylation activity. Conclusion: Protein engineering can be used to increase the efficacy of recombinant immunotoxins. Because HA22 (R490A) has increased antitumor activity without increased animal toxicity, immunotoxins with this mutation are candidates for clinical development.
92 citations
TL;DR: Two newly established novel MAbs, MN and MB, have different and useful properties compared with current antibodies used for the detection of mesothelin by immunohistochemistry, fluorescence-activated cell sorting, ELISA, and Western blotting.
Abstract: Purpose: Mesothelin is a cell surface protein that is highly expressed in some malignant tumors, and is a promising target for immunotherapy. Recent data suggests that mesothelin is an adhesive protein and may have a role in the metastases of ovarian cancer. Although a few monoclonal antibodies (MAb) to mesothelin have been produced, they have limitations for the study of expression of native mesothelin because of their low affinity or reactivity only with denatured mesothelin protein. We have produced novel MAbs to mesothelin to help study mesothelin function and to develop improved diagnosis and immunotherapy of mesothelin-expressing tumors. Experimental Design: Mesothelin-deficient mice were immunized with plasmid cDNA encoding mesothelin, and boosted with a mesothelin-rabbit IgG Fc fusion protein prior to cell fusion. Hybridomas were screened by an ELISA using plates coated with mesothelin-Fc protein. Results: Seventeen hybridomas producing anti-mesothelin antibodies were established and shown to react with two epitopes on mesothelin. One group reacts with the same epitope as the low affinity antibody K1 that was originally used to identify mesothelin. The other is a new group that reacts with a new epitope. One antibody from each group was chosen for further study and shown to react strongly on ELISA, on immunohistochemistry, and by fluorescence-activated cell sorting on living cells. Conclusion: Our two newly established MAbs, MN and MB, have different and useful properties compared with current antibodies used for the detection of mesothelin by immunohistochemistry, fluorescence-activated cell sorting, ELISA, and Western blotting.
82 citations
TL;DR: When podocytes are severely injured, proliferating parietal epithelial cells migrate onto the visceral site, thereby mimicking proliferating podocytes, and the fate of injured podocytes was pursued.
Abstract: Injured podocytes lose differentiation markers. Therefore, the true identity of severely injured podocytes remains unverified. A transgenic mouse model equipped with a podocyte-selective injury induction system was established. After induction of podocyte injury, mice rapidly developed glomerulosclerosis, with downregulation of podocyte marker proteins. Proliferating epithelial cells accumulated within Bowman's space, as seen in collapsing glomerulosclerosis. In this study, the fate of injured podocytes was pursued. Utilizing Cre-loxP recombination, the podocyte lineage was genetically labeled with lacZ in an irreversible manner. After podocyte injury, the number of lacZ-labeled cells, which were often negative for synaptopodin, progressively declined, correlating with glomerular damage. Parietal epithelial cells, but not lacZ-labeled podocytes, avidly proliferated. The cells proliferating within Bowman's capsule and, occasionally, on the outer surface of the glomerular basement membrane were lacZ-negative. Thus, when podocytes are severely injured, proliferating parietal epithelial cells migrate onto the visceral site, thereby mimicking proliferating podocytes.
79 citations
TL;DR: The identification of novel CTL agonist epitopes supports and extends observations that mesothelin is a potential target for immunotherapy of pancreatic and ovarian cancers, as well as mesotheliomas.
Abstract: Purpose: Mesothelin is overexpressed in many pancreatic and ovarian cancers, mesotheliomas, and other tumor types. Clinical trials are ongoing using immunotoxins to target mesothelin, and patients immunized with allogeneic pancreatic tumor cell lines have shown immune responses to previously defined mesothelin epitopes. The purpose of this study was to define novel mesothelin CTL epitopes and, more importantly, agonist epitopes that would more efficiently activate human T cells to more efficiently lyse human tumors. Experimental Design and Results: Two novel mesothelin HLA-A2 epitopes were defined. T-cell lines generated from one of these epitopes were shown to lyse pancreatic and ovarian tumor cells. Several agonist epitopes were defined and were shown to ( a ) have higher affinity and avidity for HLA-A2, ( b ) activate mesothelin-specific T cells from normal individuals or cancer patients to a greater degree than the native epitope in terms of induction of higher levels of IFN-γ and the chemokine lymphotactin, and ( c ) lyse several mesothelin-expressing tumor types in a MHC-restricted manner more effectively than T cells generated using the native peptide. External beam radiation of tumor cells at nontoxic levels was shown to enhance the expression of mesothelin and other accessory molecules, resulting in a modest but statistically significant increase in tumor cell lysis by mesothelin-specific T cells. Conclusions: The identification of novel CTL agonist epitopes supports and extends observations that mesothelin is a potential target for immunotherapy of pancreatic and ovarian cancers, as well as mesotheliomas.
71 citations
TL;DR: A dramatic and sustained clinical and radiographic response in a patient with a bifrontal glioblastoma multiforme treated with intratumoral infusion of a novel targeted toxin, TP-38 is described.
Abstract: Glioblastoma multiforme remains refractory to conventional therapy, and novel therapeutic modalities are desperately needed. TP-38 is a recombinant chimeric protein containing a genetically engineered form of the cytotoxic Pseudomonas exotoxin fused to transforming growth factor (TGF)-α. TGF-α binds with high affinity to the epidermal growth factor receptor, which is uniformly overexpressed in malignant gliomas, often because of gene amplification. Prior to therapy with TP-38, the patient described here was completely refractory to multiple other therapies, with radiographic and pathologic evidence of tumor progression. After therapy, she improved clinically, was weaned off steroids and anticonvulsants, and experienced a progressive decrease in enhancing tumor volume. Despite multiple prior recurrences, she has not progressed for >43 months after TP-38 therapy. Small remaining areas of enhancement demonstrate no evidence of tumor histologically and are hypometabolic on positron emission tomography. This report describes a dramatic and sustained clinical and radiographic response in a patient with a bifrontal glioblastoma multiforme treated with intratumoral infusion of a novel targeted toxin, TP-38.
53 citations
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TL;DR: The results provide the first evidence that IRTA2 is expressed on the surface of human lymphoma cell lines and HCL cells, and could be useful as a new target for immunotherapy.
Abstract: Purpose: The immunoglobulin superfamily receptor translocation associated 2 (IRTA2) gene encodes a cell surface receptor homologous to the family of Fc receptors. Because of the restricted expression of mRNA in B cell–lineage cells, IRTA2 is a new potential target for the immunotherapy of B cell malignancies. To study the expression of the IRTA2 gene product, we produced monoclonal antibodies (MAbs) specific to IRTA2. Experimental Design: A mouse used for cell fusion was DNA-immunized with an expression plasmid encoding the IRTA2 cDNA. The reactivity of MAbs secreted from the hybridomas were characterized with recombinant proteins of IRTA family members in an enzyme immunoassay and a fluorescence-activated cell sorter (FACS). Nineteen human lymphoma cell lines and blood cells from five patients with hairy cell leukemia (HCL) were analyzed with IRTA2 expression using FACS. Results: Three MAbs (F25, F56, and F119) were selected based on their specific reactivity with recombinant IRTA2 and lack of cross-reactivity with other IRTA family members. In a FACS analysis, MAbs F56 and F119 detected IRTA2 expression in six of seven B cell non–Hodgkin9s lymphoma and one of six Burkitt9s lymphoma cell lines. Reverse transcriptase-PCR experiments and Western blotting using MAb F25 confirmed the expression profile. We also found that HCL cells from five patients expressed IRTA2. Conclusions: Our results provide the first evidence that IRTA2 is expressed on the surface of human lymphoma cell lines and HCL cells. IRTA2 could be useful as a new target for immunotherapy.
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TL;DR: The biodistribution and tumor-targeting ability of an antimesothelin tetravalent single-chain Fv-streptavidin fusion protein (SS1scFvSA) in mice was evaluated, resulting in a high accumulation of radiolabeled DOTA-biotin.
Abstract: Mesothelin is a glycoprotein that is overexpressed in several human tumors, including mesotheliomas and ovarian cancers, and has been identified as a potential target for therapy. We evaluated the biodistribution and tumor-targeting ability of an antimesothelin tetravalent single-chain Fv-streptavidin fusion protein (SS1scFvSA) in mice. Methods: SS1scFvSA was labeled with 125I or 111In for evaluation of internalization in vitro and for optimization of its biodistribution. The A431-K5 mesothelin transfected cell line was used as the target. We used a 3-step pretargeting approach consisting of injections of (i) SS1scFvSA, followed 20 h later by (ii) a synthetic clearing agent, and (iii) 4 h later, radiolabeled (111In, 88Y/90Y, or 177Lu) 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA)-biotin. To optimize the tumor uptake, the effect of the specific activity of 111In-DOTA-biotin was evaluated. Results: Approximately 60% of SS1sc FvSA internalized within 6 h. The optimal dose of SS1scFvSA for pretargeting was 600 μg. Decreasing the specific activity of DOTA-biotin by administering 0.1–5 μg of DOTA-biotin resulted in tumor uptake decreasing from 31.8 to 5.5 %ID/g (percentage injected dose per gram) at 2 h. Pretargeted therapy of A431-K5 tumor with 90Y doses of 11.1–32.4 MBq resulted in a dose-dependent tumor response. With 32.4 MBq, 86% of mice survived tumor free for 110 d. All nontreated mice died, with a median survival of 16 d. Conclusion: SS1scFvSA localized in the mesothelin-expressing tumor, resulting in a high accumulation of radiolabeled DOTA-biotin. The specific activity of DOTA-biotin had a significant effect on its tumor uptake. Therapeutic tumor doses were obtained without dose-limiting toxicity.
TL;DR: This study identified two epitopes on membrane-associated CD30 that are missing on soluble CD30 probably because of a conformational change upon shedding and suggests that it may be possible to find membrane-specific epitope on other immunotherapy target molecules.
Abstract: Because CD30 is highly expressed on Hodgkin's lymphoma and anaplastic large cell lymphoma, it is a promising target for immunotherapy. Soluble CD30, the extracellular domain of CD30 that is shed from the cells, can reduce the effects of CD30-targeting agents by competitive binding. In this study, we identified two epitopes on membrane-associated CD30 that are missing on soluble CD30 probably because of a conformational change upon shedding. These epitopes are potentially superior targets for immunotherapy because targeting them should be free from the competitive effects of soluble CD30. We studied 27 anti-native CD30 mAbs that were assigned to 8 different topographical epitopes. Soluble CD30 was prepared from culture supernatants of L540 cells or Karpas 299 cells. In an ELISA, the mAbs to two epitopes, Ep2 (amino acids 107–153) and Ep7 (amino acids 282–338), showed less than a 2% average cross-reactivity to soluble CD30 compared with a CD30-Fc fusion protein. In addition, these mAbs bound to CD30 on cells in the presence of an excess of soluble CD30. These epitopes (Ep2 and Ep7) are, therefore, more efficiently presented on cell-associated CD30 than on soluble CD30 (membrane-specific epitopes). Also, soluble CD30 in the sera of mice bearing L540 tumors did not form immune complexes with the membrane-specific mAbs analyzed by size-exclusion chromatography. In contrast, mAbs to the other epitopes reacted with both soluble CD30 and membrane CD30. Our results suggest that it may be possible to find membrane-specific epitopes on other immunotherapy target molecules.
TL;DR: The data indicate that PATE is made by various sexual accessory tissues and secreted into the semen where it becomes associated with sperm, suggesting that Pate is a novel sperm-associated protein with a possible role in mammalian sperm maturation.
Abstract: The PATE gene is expressed in prostate and testis. To determine if PATE is expressed in other accessory tissues of the male genital tract, RT-PCR of the epididymis and seminal vesicle was performed. PATE mRNA was highly expressed in the epididymis and seminal vesicle. In situ hybridization of the testis showed PATE mRNA is strongly expressed in the spermatogonia. The PATE gene encodes a 14-kDa protein with a predicted signal sequence and a cleavage site between residues G21 and S22. To determine if PATE is a secreted protein, 293T cells were transfected with a pcDNA-PATE-myc-His plasmid and protein immunoprecipitated with anti-myc monoclonal antibody. Western blot analysis showed the presence of PATE-myc-His protein was in the medium and the cell lysate. Confocal microscopy demonstrated that PATE-myc-His protein is found in the endoplasmic reticulum. The polyclonal antibody SOL-1 was generated by immunization of rabbits with recombinant PATE protein expressed and purified from Escherichia coli. Western blots were performed on extracts of prostate, testis, seminal vesicle and ejaculated spermatozoa, but PATE protein was only detected in the spermatozoa. Immunostaining of sperm smears revealed that PATE is located in a band-like pattern in the sperm head. Our data indicate that PATE is made by various sexual accessory tissues and secreted into the semen where it becomes associated with sperm, suggesting that PATE is a novel sperm-associated protein with a possible role in mammalian sperm maturation.
TL;DR: Because TP-3-based immunotoxins have good antitumor activity in mice, these molecules merit additional development for possible treatment of osteosarcoma in humans.
Abstract: We measured the antitumor activity of two types of TP-3 immunotoxins that target an antigen expressed in tumors associated with osteosarcoma. Development of novel agents for treatment of patients with osteosarcoma is important. We previously described a monovalent-disulfide-stabilized recombinant im
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TL;DR: The combination of the Avastin and chemotherapy in nude mice with human cancer xenografts shows an increased activity compared with chemotherapy alone or antibody alone, and the effect of combiningAvastin with immunotoxins is investigated.
Abstract: 4989 Avastin (Bevacizumab; Genentech, South San Francisco, CA) is a human mAb against VEGF-A. Avastin slows or prevents blood vessel growth and thereby inhibits tumor growth. Avastin has been approved for the treatment of colon cancer and is being tested in other cancers. Additionally, the combination of the Avastin and chemotherapy in nude mice with human cancer xenografts shows an increased activity compared with chemotherapy alone or antibody alone. Because tumor penetration is an essential step in immunotoxin action and Avastin affects capillary density and function, we investigated the effect of combining Avastin with immunotoxins. Mice bearing human tumor xenografts (A431/K5 expressing mesothelin or CA46 Burkitt’s lymphoma expressing CD22) were treated with 1. Avastin (5 mg/ kg i.p. 2 times/week for 1 week), 2. immunotoxin (SS1P, 0.2 mg/ kg QOD x 3 for A431/K5 or HA22, 0.2 mg/ kg QOD x 3 for CA46, or 3. a combination of immunotoxin plus Avastin. Mice were implated with tumors subcutaneously and treated began on day 7. In the A431/K5 model, mice treated with the Avastin plus SS1P exhibited significantly reduced mean tumor volumes compared with mice treated with the control (88.8 mm3 vs. 1,715 mm3 at day 17, P
TL;DR: I was trained as a physician at Tufts Medical School and came to NIH in 1959 as a clinical associate in the Clinical Endocrinology Branch, and spent the next 10 years studying hormone action and how cyclic AMP works, which was the first example of positive control of gene expression.
01 Jan 2005
TL;DR: This work has utilized the EST and genome databases, different bioinformatics tools, and several experimental methods to identify tissue-specific genes for prostate and breast cancer.
Abstract: Completion of the human genome sequence has opened up an enormous opportunity to researchers all over the world. The Human Genome Project, which includes the expressed sequence tags (ESTs) database and the genome sequence database, provides a huge source of data that can be used to study and identify molecular targets for a wide range of diseases, including cancer. Major efforts must now be devoted to develop strategies by which these databases will be mined efficiently to identify the hidden therapeutic treasures. We have utilized the EST and genome databases, different bioinformatics tools, and several experimental methods to identify tissue-specific genes for prostate and breast cancer. The genes identified can be used as novel targets for the diagnosis and treatment of prostate and breast cancer.