Resume

• 2000

  Doctor of Philosophy - PhD

  Biology

  The Johns Hopkins University

  Ph.D.

  Graduate Student in the CMDB Program

  Department of Biology

  Krieger School of Arts and Sciences\nThesis research under Robert F. Schleif \nBiology Student Seminar Series

  co-founder and co-chair

• 1996

  B.A.

  Biology

  SMCM Running Club\nTri-Beta Biological Honors Society

• 1992

  High School

  Georgetown Preparatory School

  Cell

  Developmental

  & Genetic Biology

  Methods in Molecular Biophysics

  Physical Biochemistry

  Topics in Biochemistry

  Molecular Biophysics

  Advanced Biochemistry

  Advanced Molecular Biology

• Cell Based Assays

  Tissue Culture

  Microsoft Office

  Teaching Experience

  Cell Culture

  Protein Purification

  Mentoring

  SDS-PAGE

  Column Chromatography

  GraphPad Prism

  Writing

  ELISA

  Protein Expression

  Site-directed Mutagenesis

  FPLC

  Protein Engineering

  PCR

  recombinant DNA technology

  Molecular Biology

  Western Blotting

  Recombinant immunotoxin against B-cell malignancies with no immunogenicity in mice by removal of B-cell epitopes

  Ira Pastan

  Robert J. Kreitman

  Byungkook Lee

  Laiman Xiang

  Masanori Onda**[primary author]

  Many nonhuman proteins have useful pharmacological activities

  but are infrequently effective in humans because of their high immunogenicity. A recombinant immunotoxin (HA22

  CAT8015

  moxetumomab pasudotox) composed of an anti-CD22 antibody variable fragment fused to PE38

  a 38-kDa portion of Pseudomonas exotoxin A

  has produced many complete remissions in drug-resistant hairy-cell leukemia when several cycles of the agent can be given

  but has much less activity when antibodies develop. We have pursued a strategy to deimmunize recombinant immunotoxins by identifying and removing B-cell epitopes. We previously reported that we could eliminate most B-cell epitopes using a combination of point mutations and deletions. Here we show the location and amino acid composition of all of the B-cell epitopes in the remaining 25-kDa portion of Pseudomonas exotoxin. Using this information

  we eliminated these epitopes to produce an immunotoxin (HA22-LR-8M) that is fully cytotoxic against malignant B-cell lines

  has high cytotoxic activity against cells directly isolated from patients with chronic lymphocytic leukemia

  and has excellent antitumor activity in mice. HA22-LR-8M does not induce antibody formation in mice when given repeatedly by intravenous injection and does not induce a secondary antibody response when given to mice previously exposed to HA22. HA22-LR-8M also has greatly reduced antigenicity when exposed to sera from patients who have produced antibodies to HA22. The properties of HA22-LR-8M make it an excellent candidate for further clinical development.

  Recombinant immunotoxin against B-cell malignancies with no immunogenicity in mice by removal of B-cell epitopes

  S. Patricia Becerra

  Elena M. Alberdi

  BACKGROUND: Pigment epithelium-derived factor (PEDF) has binding affinity for cell-surface receptors in retinoblastoma cells and for glycosaminoglycans. We investigated the effects of glycosaminoglycans on PEDF-receptor interactions.\r\n\r\nRESULTS: 125I-PEDF formed complexes with protease-resistant components of medium conditioned by human retinoblastoma Y-79 cells. Using specific glycosaminoglycan degrading enzymes in spectrophotometric assays and PEDF-affinity chromatography

  we detected heparin and heparan sulfate-like glycosaminoglycans in the Y-79 conditioned media

  which had binding affinity for PEDF. The Y-79 conditioned media significantly enhanced the binding of 125I-PEDF to Y-79 cell-surface receptors. However

  enzymatic and chemical depletion of sulfated glycosaminoglycans from the Y-79 cell cultures by heparitinase and chlorate treatments decreased the degree of 125I-PEDF binding to cell-surface receptors.\r\n\r\nCONCLUSIONS: These data indicate that retinoblastoma cells secrete heparin/heparan sulfate with binding affinity for PEDF

  which may be important in efficient cell-surface receptor binding.

  Glycosaminoglycans in human retinoblastoma cells: heparan sulfate

  a modulator of the pigment epithelium-derived factor-receptor interactions

  Ira Pastan

  Itai Benha

  David J. Venzon

  Richard Beers

  Jaime A. Eberle

  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.

  Identification and elimination of an immunodominant T-cell epitope in recombinant immunotoxins based on Pseudomonas exotoxin A

  Robert J. Kreitman

  Masanori Onda

  Ira Pastan**[primary author]

  Recombinant immunotoxins

  containing an Fv fragment and a bacterial toxin

  frequently elicit neutralizing antibodies

  nearly always against the toxin. Moxetumomab pasudotox (previously called CAT-8015 or HA22) contains an anti-CD22 Fv fused to PE38

  a truncated form of Pseudomonas exotoxin

  containing amino acids 253-364 and 381-613. One avenue to reducing immunogenicity is to identify B- and T-cell epitopes and remove them while retaining toxin activity. To determine B-cell epitopes on PE38

  60 monoclonal antibodies against PE38 were tested in a pairwise manner

  and seven major epitope groups with 13 subgroups were identified. The locations of many of these epitopes were identified by mutating large surface-exposed residues to alanine. A mutant of moxetumomab pasudotox containing eight epitope-eliminating mutations (HA22-8X) was prepared

  and greatly reduced immunogenicity in mice. In parallel

  two large sections of PE38 containing lysosomal protease cleavage sites were removed

  leaving only amino acids 274-284 and 394-613 of the toxin. The resulting molecule

  HA22-LR

  retained cytotoxicity toward CD22+ cell lines

  killed primary chronic lymphocytic leukemia cells more potently than moxetumomab pasudotox

  was much less toxic to mice

  and had significantly improved antitumor activity toward murine xenografts. The immunogenicity and activity of recombinant immunotoxins may be optimized by combinations of these approaches.

  Immunotoxins with decreased immunogenicity and improved activity

  Jianying Shen

  Jyothishmathi Swaminathan

  Christopher A. Dunn

  Joseph D. Walsh

  Maurice J. Bessman**[primary author]

  ygdP

  a gene associated with the invasion of brain microvascular endothelial cells by Escherichia coli K1 (Badger

  J. L.

  Wass

  C. A.

  and Kim

  K. S. (2000) Mol. Microbiol. 36

  174-182)

  the primary Gram-negative bacterium causing meningitis in newborns

  has been cloned and expressed in E. coli. The protein

  YgdP

  was purified to near homogeneity and identified as a member of the Nudix hydrolase subfamily of dinucleoside oligophosphate pyrophosphatases. It catalyzes the hydrolysis of diadenosine tetra-

  penta-

  and hexa-phosphates with a preference for diadenosine penta-phosphate

  from which it forms ATP and ADP. The enzyme has a requirement for a divalent metal cation that can be met with Mg2+

  Zn2+

  or Mn2+ and

  like most of the Nudix hydrolases

  has an alkaline pH optimum between 8.5 and 9. This is the second identification of a gene associated with the invasiveness of a human pathogen as a member of the Nudix hydrolase subfamily of dinucleoside oligophosphate pyrophosphatases

  and an examination of homologous proteins in other invasive bacteria suggests that this may be a common feature of cellular invasion.

  The gene ygdP

  associated with the invasiveness of Escherichia coli K1

  designates a Nudix hydrolase

  Orf176

  active on adenosine (5')-pentaphospho-(5')-adenosine (Ap5A)

  Ira Pastan

  Robert J. Kreitman

  Inger Margulies

  Oleg Chertov

  Laiman Xiang

  Immunotoxins based on Pseudomonas exotoxin A (PE) are promising anticancer agents that combine a variable fragment (Fv) from an antibody to a tumor-associated antigen with a 38-kDa fragment of PE (PE38). The intoxication pathway of PE immunotoxins involves receptor-mediated internalization and trafficking through endosomes/lysosomes

  during which the immunotoxin undergoes important proteolytic processing steps but must otherwise remain intact for eventual transport to the cytosol. We have investigated the proteolytic susceptibility of PE38 immunotoxins to lysosomal proteases and found that cleavage clusters within a limited segment of PE38. We subsequently generated mutants containing deletions in this region using HA22

  an anti-CD22 Fv-PE38 immunotoxin currently undergoing clinical trials for B-cell malignancies. One mutant

  HA22-LR

  lacks all identified cleavage sites

  is resistant to lysosomal degradation

  and retains excellent biologic activity. HA22-LR killed chronic lymphocytic leukemia cells more potently and uniformly than HA22

  suggesting that lysosomal protease digestion may limit immunotoxin efficacy unless the susceptible domain is eliminated. Remarkably

  mice tolerated doses of HA22-LR at least 10-fold higher than lethal doses of HA22

  and these higher doses exhibited markedly enhanced antitumor activity. We conclude that HA22-LR advances the therapeutic efficacy of HA22 by using an approach that may be applicable to other PE-based immunotoxins.

  A protease-resistant immunotoxin against CD22 with greatly increased activity against CLL and diminished animal toxicity

  Hong Zhou

  Ira Pastan

  Robert Kreitman

  Joshua Ostovitz

  Recombinant immunotoxins (RITs) are fusion proteins that join antibodies to protein toxins for targeted cell killing. RITs armed with Pseudomonas exotoxin A (PE) are undergoing clinical trials for the treatment of cancer. The current design of PE-based RITs joins an antibody fragment to the catalytic domain of PE using a polypeptide linker that is cleaved by the protease furin. Intracellular cleavage of native PE by furin is required for cytotoxicity

  yet the PE cleavage site has been shown to be a poor furin substrate. Here we describe the rational design of more efficiently cleaved furin linkers in PE-based RITs

  and experiments evaluating their effects on cleavage and cytotoxicity. We found that changes to the furin site could greatly influence both cleavage and cytotoxicity

  but the two parameters were not directly correlated. Furthermore

  the effects of alterations to the furin linker were not universal. Identical mutations in the anti-CD22 RIT HA22-LR often displayed different cytotoxicity from mutations in the anti-mesothelin RIT SS1-LR/GGS

  underscoring the prominent role of the target site in their intoxication pathways. Combining several beneficial mutations in HA22-LR resulted in a variant (HA22-LR/FUR) with a remarkably enhanced cleavage rate and improved cytotoxicity against five B cell lines and similar or enhanced cytotoxicity in five out of six hairy cell leukemia patient samples. This result informs the design of protease-sensitive linkers and suggests that HA22-LR/FUR may be a candidate for further preclinical development.

  Designing the Furin-Cleavable Linker in Recombinant Immunotoxins Based on Pseudomonas Exotoxin A

  Robert F. Schleif

  Michael E. Rodgers

  The arabinose-binding pockets of wild type AraC dimerization domains crystallized in the absence of arabinose are occupied with the side chains of Y31 from neighboring domains. This interaction leads to aggregation at high solution concentrations and prevents determination of the structure of truely apo AraC. In this work we found that the aggregation does not significantly occur at physiological concentrations of AraC. We also found that the Y31V mutation eliminates the self-association

  but does not affect regulation properties of the protein. At the same time

  the mutation allows crystallization of the dimerization domain of the protein with only solvent in the arabinose-binding pocket. Using a distance difference method suitable for detecting and displaying even minor structural variation among large groups of similar structures

  we find that there is no significant structural change in the core of monomers of the AraC dimerization domain resulting from arabinose

  fucose

  or tyrosine occupancy of the ligand-binding pocket. A slight change is observed in the relative orientation of monomers in the dimeric form of the domain upon the binding of arabinose but its significance cannot yet be assessed.

  Structure and properties of a truely apo form of AraC dimerization domain

  Ira Pastan

  Masanori Onda

  Laiman Xiang

  Johanna K. Hansen**[primary author]

  Recombinant immunotoxins (RITs) are genetically engineered proteins designed to kill cancer cells. The RIT HA22 contains the Fv portion of an anti-CD22 antibody fused to a 38 kDa fragment of Pseudomonas exotoxin A (PE38). As PE38 is a bacterial protein

  patients frequently produce antibodies that neutralize its activity

  preventing retreatment. We have earlier shown in mice that PE38 contains 7 major B-cell epitopes located in domains II and III of the protein. Here we present a new mutant RIT

  HA22-LR-6X

  in which we removed most B-cell epitopes by deleting domain II and mutating 6 residues in domain III. HA22-LR-6X is cytotoxic to several lymphoma cell lines

  has very low nonspecific toxicity

  and retains potent antitumor activity in mice with CA46 lymphomas. To assess its immunogenicity

  we immunized 3 MHC-divergent strains of mice with 5 microg doses of HA22-LR-6X

  and found that HA22-LR-6X elicited significantly lower antibody responses than HA22 or other mutant RITs with fewer epitopes removed. Furthermore

  large (50 microg) doses of HA22-LR-6X induced markedly lower antibody responses than 5 microg of HA22

  indicating that high doses can be administered with low immunogenicity. Our experiments show that we have correctly identified and removed B-cell epitopes from PE38

  producing a highly active immunotoxin with low immunogenicity and low animal toxicity. Future studies will determine if these properties carry over to humans with cancer.

  A recombinant immunotoxin targeting CD22 with low immunogenicity

  low nonspecific toxicity

  and high antitumor activity in mice

  Robert F. Schleif

  Deletion of the regulatory N-terminal arms of the AraC protein from its dimerization domain fragments increases the susceptibility of the dimerization domain to form a series of higher order polymers by indefinite self-association. We investigated how the normal presence of the arm inhibits this self-association. One possibility is that arms can act as an entropic bristles to interfere with the approach of other macromolecules

  thereby decreasing collision frequencies. We examined the repulsive effect of flexible arms by measuring the rate of trypsin cleavage of a specially constructed ubiquitin-arm protein. Adding an arm to ubiquitin or increasing its length produced only a modest repulsive effect. This suggests that arms such as the N-terminal arm of AraC do not reduce self-association by entropic exclusion. We consequently tested the hypothesis that the arm on AraC reduces self-association by binding to the core of the dimerization domain even in the absence of arabinose. The behaviors of dimerization domain mutants containing deletions or alterations in the N-terminal arms substantiate this hypothesis. Apparently

  interactions between the N-terminal arm and the dimerization domain core position the arm to interfere with the protein-protein contacts necessary for self-association.

  Specific interactions by the N-terminal arm inhibit self-association of the AraC dimerization domain

  Ira Pastan

  Byungkook Lee

  Laiman Xiang

  Changhoon Kim

  Masanori Onda

  Wenhai Liu**[primary author]

  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.

  A recombinant immunotoxin engineered for increased stability by adding a disulfide bond has decreased immunogenicity

  Ira Pastan

  Pseudomonas exotoxin A (PE) is a highly toxic protein secreted by the opportunistic pathogen Pseudomonas aeruginosa. The modular structure and corresponding mechanism of action of PE make it amenable to extensive modifications that can redirect its potent cytotoxicity from disease to a therapeutic function. In combination with a variety of artificial targeting elements

  such as receptor ligands and antibody fragments

  PE becomes a selective agent for the elimination of specific cell populations. This review summarizes our current understanding of PE

  its intoxication pathway

  and the ongoing efforts to convert this toxin into a treatment for cancer.

  A guide to taming a toxin - recombinant immunotoxins constructed from Pseudomonas exotoxin A for the treatment of cancer

  Joe G. Hollyfield

  Vicente Notario

  Luigi Notari

  Preenie Senanayake

  Silvia Locatelli-Hoops

  L. Alberto Perez-Mediavilla

  S. Patricia Becerra**[primary author]

  Pigment epithelium-derived factor (PEDF) is a multifunctional serpin with antitumorigenic

  antimetastatic

  and differentiating activities. PEDF is found within tissues rich in the glycosaminoglycan hyaluronan (HA)

  and its amino acid sequence contains putative HA-binding motifs. We show that PEDF coprecipitation with glycosaminoglycans in media conditioned by human retinoblastoma Y-79 cells decreased after pretreatments with hyaluronidase

  implying an association between HA and PEDF. Direct binding of human recombinant PEDF to highly purified HA was demonstrated by coprecipitation in the presence of cetylpyridinium chloride. Binding of PEDF to HA was concentration-dependent and saturable. The PEDF-HA interactions were sensitive to increasing NaCl concentrations

  indicating an ionic nature of these interactions and having affinity higher than PEDF-heparin. Competition assays showed that PEDF can bind heparin and HA simultaneously. PEDF chemically modified with fluorescein retained the capacity for interacting with HA but lacked heparin affinity

  suggesting one or more distinct HA-binding regions on PEDF. The HA-binding region was examined by site-directed mutagenesis. Single-point and cumulative alterations at basic residues within the putative HA-binding motif K189A/K191A/R194A/K197A drastically reduced the HA-binding activity without affecting heparin- or collagen I binding of PEDF. Cumulative alterations at sites critical for heparin binding (K146A/K147A/R149A) decreased HA affinity but not collagen I binding. Thus these clusters of basic residues (BXBXXBXXB and BX3AB2XB motifs) in PEDF are functional regions for binding HA. In the spatial PEDF structure they are located in distinct areas away from the collagen-binding site. The HA-binding activity of PEDF may contribute to deposition in the extracellular matrix and to its reported antitumor/antimetastatic effects.

  Pigment epithelium-derived factor binds to hyaluronan. Mapping of a hyaluronan binding site

  Jed

  John

  National Cancer Institute

  Foundation for Advanced Education in the Sciences

  Towson University

  National Eye Institute

  NIH

  Bethesda

  MD

  Postdoctoral Fellow working on recombinant immunotoxins with Ira Pastan in the Laboratory of Molecular Biology

  Center for Cancer Research

  NCI

  NIH (http://ccr.cancer.gov/labs/lab.asp?labid=99)

  Research Fellow

  National Cancer Institute

  Bethesda

  MD

  Biochemistry Lecture Course Instructor

  Instructor

  Foundation for Advanced Education in the Sciences

  Baltimore

  Maryland Area

  Molecular Biology (BIOL 409)\nBiochemistry I (CHEM 351)\nBiochemistry Laboratory (CHEM 356)

  Associate Professor

  Towson University

  Bethesda

  MD

  Summer research on pigment epithelium-derived factor (PEDF) with Joyce Tombran-Tink

  Gerald J. Chader

  and S. Patricia Becerra in the Laboratory of Retinal Cell and Molecular Biology

  NIH

  Summer Student

  National Eye Institute

  NIH

  The longstanding NIGMS PRAT Program is a competitive postdoctoral fellowship program to pursue research in one of the laboratories of the National Institutes of Health (NIH) or the Food and Drug Administration (FDA). The program was initiated to address a national need for well-trained pharmacologists

  and as the field of pharmacology has matured and broadened

  the program has followed suit. To reflect this shift

  in 2012 the P in the program’s acronym changed from “Pharmacology” to “Postdoctoral.” The PRAT acronym remains the same. \n\n\nhttp://www.nigms.nih.gov/Training/PRAT.htm

  National Institute of General Medical Sciences (NIGMS)

  Federal Technology Transfer Award (2009-2011)

  A cash award that recognizes scientific

  engineering and technical personnel for inventions

  innovations

  computer software

  or other outstanding scientific or technological contributions of value to the United States due to commercial application or contributions to the missions of NIH

  HHS and/or the Federal Government

  or for exemplary activities that promote the domestic transfer of science and technology development within the Federal Government resulting in use by American industry or business

  universities

  State or local governments

  or other non-Federal parties.

  National Institutes of Health

  Fellows Award for Research Excellence (FARE) 2009

  Recognition for outstanding scientific research performed by NIH intramural postdoctoral fellows.\n\nhttps://www.training.nih.gov/felcom/fare\n

  National Institutes of Health (NIH)