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

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...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 109; no. 51; pp. E3597 - E3603
Main Authors Mazor, Ronit, Vassall, Aaron N., Eberle, Jaime A., Beers, Richard, Weldon, John E., Venzon, David J., Tsang, Kwong Y., Benhar, Itai, Pastan, Ira
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 18.12.2012
National Acad Sciences
SeriesPNAS Plus
Subjects
ADP Ribose Transferases - chemistry
alanine
alleles
Amino acids
Antibodies - chemistry
antibody formation
Bacterial proteins
bacterial toxins
Bacterial Toxins - chemistry
Biological Sciences
CD4-Positive T-Lymphocytes - cytology
Correlation analysis
cytotoxicity
Enzyme-Linked Immunosorbent Assay - methods
Epitopes - chemistry
Epitopes, T-Lymphocyte - chemistry
Epitopes, T-Lymphocyte - immunology
exotoxins
Exotoxins - chemistry
Gene Deletion
Genetic Variation
Humans
Immune System
immunodominant epitopes
Immunotoxins
interleukin-2
Interleukin-2 - metabolism
Leukocytes, Mononuclear - cytology
Molecular Conformation
mutants
neoplasms
neutralizing antibodies
patients
Peptides - chemistry
PNAS Plus
Protein Binding
Protein Conformation
Protein Engineering - methods
Protein Structure, Tertiary
Proteins
Pseudomonas
Pseudomonas aeruginosa Exotoxin A
recombinant fusion proteins
T cell receptors
T-lymphocytes
therapeutics
Toxins
Virulence Factors - chemistry
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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.
AbstractList 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. [PUBLICATION 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. We have shown previously that many complete remissions were obtained in patients with refractory hairy cell leukemia; in this trial patients received a median of four cycles (12 doses) of immunotoxin HA22 ( 5 ). The patients’ immunogenicity was low because their immune systems were damaged by prior chemotherapy and therefore did not produce neutralizing antibodies to HA22. We anticipate that the immunotoxins lacking T-cell epitopes described here and B-cell epitopes previously described ( 3 ) can be given for four cycles to patients with normal immune systems, allowing better antitumor responses to be achieved. We also anticipate that the approaches described here to identify and remove T-cell epitopes can be used to deimmunize other foreign proteins and allow their use in humans. Depending on the type of assay used, it is possible to identify peptides that result in T-cell activation, but these peptides might never be formed in vivo. To ensure that the epitopes we identified were produced naturally by APCs, we adapted an assay developed by Sette and colleagues ( 4 ), in which we first incubated donor peripheral blood mononuclear cells (PBMCs) with an RIT for 14 d to allow the immunotoxin to be processed by donor APCs and relevant peptides to be presented to T cells. We subsequently exposed the activated T cells to overlapping synthetic peptides from the sequence of PE38 and used an ELISpot assay for IL-2 to measure T-cell activation. We analyzed samples from 50 normal donors with no recorded previous exposure to PE38 and with a broad distribution of HLA alleles and found that all 50 donors showed a significant response to at least one peptide, as would be expected from a highly immunogenic foreign protein. We identified one immunodominant epitope that was HLA class II DRB1-restricted and promiscuous because of the diversity of donors that responded to it. Using alanine-scanning mutagenesis, we identified single amino acid residues within PE38 that were responsible for the immunodominant epitope. We used this information to abolish T-cell responses by constructing two mutant RITs targeting CD22 in which amino acid residue leucine 297 or tyrosine 298 was converted to alanine. Both were highly active as shown in Fig. P1 . More importantly, we showed that T-cell responses were absent in 34% of donors and were diminished in an additional 42% with immunotoxin HA22-LR, which harbors a deletion of domain II of PE38 that removes the immunodominant epitope as well as other T-cell epitopes. We predict that mutating amino acid residues in epitopes identified in domain III will reduce immunogenicity further ( Fig. P1 ). Because of the concern that removal of major T-cell epitopes would allow the emergence of new (cryptic) epitopes, we examined the mutants with diminished immunogenicity for new epitopes and did not find any. To deimmunize PE38, we initially focused on identifying and removing B-cell epitopes. We recently reported the identification of the major human B-cell epitopes in PE38 and the production of a highly active recombinant immunotoxin with mutations that abolish many of the epitopes ( 3 ). This immunotoxin should be able to be given to many cancer patients for more than one treatment cycle but needs further improvement to allow even more treatment cycles to be given. T cells play a pivotal role in eliciting an antibody response. An early event in the development of antibodies is the antigen-specific activation of CD4 + T-helper cells. CD4 + T-cell support is initiated by antigen-presenting cells (APCs), which display peptide fragments derived from foreign proteins on MHC class II molecules that bind T-cell receptors. Recombinant immunotoxins (RITs) are chimeric proteins being developed for the targeted therapy of cancer. RITs are composed of the variable fragment (Fv) of an antibody specific for a tumor-associated cell-surface antigen joined to a fragment of Pseudomonas exotoxin A (PE38) ( 1 ). We are developing RITs that target CD22 for treating B-cell malignancies (HA22) or mesothelin for treating mesothelioma and other epithelial malignancies expressing mesothelin (SS1P). When used as therapeutic agents in humans, foreign proteins commonly induce the formation of neutralizing antibodies, and the more foreign the protein, the more likely it is that a rapid immune response will be generated. Ninety percent of patients with mesothelioma treated with one cycle (three doses) of SS1P rapidly produced neutralizing antibodies to PE38, preventing additional treatment. Therefore, most patients received only a single cycle of treatment ( 2 ). In this paper we describe how to identify human T-cell epitopes in the PE38 portion of recombinant immunotoxins and the identification of a dominant promiscuous epitope. Using this information, we show that a highly active immunotoxin in which domain II is deleted is missing the dominant T-cell epitope and therefore should have low immunogenicity in many patients, allowing more treatment cycles to be administered and better antitumor activity to be achieved. We also have identified additional T-cell epitopes in domain III that can be removed, further decreasing immunogenicity.
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.
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.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.
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.
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.
Author Ronit Mazor
Aaron N. Vassall
Jaime A. Eberle
Richard Beers
Ira Pastan
John E. Weldon
David J. Venzon
Itai Benhar
Kwong Y. Tsang
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  surname: Vassall
  fullname: Vassall, Aaron N.
  organization: Laboratory of Molecular Biology
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  givenname: Jaime A.
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  organization: Laboratory of Molecular Biology
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  surname: Weldon
  fullname: Weldon, John E.
  organization: Laboratory of Molecular Biology
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  givenname: David J.
  surname: Venzon
  fullname: Venzon, David J.
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  givenname: Kwong Y.
  surname: Tsang
  fullname: Tsang, Kwong Y.
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  surname: Benhar
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  organization: Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv 69978, Israel
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  givenname: Ira
  surname: Pastan
  fullname: Pastan, Ira
  organization: Laboratory of Molecular Biology
BackLink https://www.ncbi.nlm.nih.gov/pubmed/23213206$$D View this record in MEDLINE/PubMed
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Copyright Copyright National Academy of Sciences Dec 18, 2012
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Author contributions: R.M., A.N.V., K.Y.T., and I.P. designed research; R.M., A.N.V., J.A.E., and R.B. performed research; R.M., J.E.W., D.J.V., I.B., and I.P. analyzed data; and R.M. and I.P. wrote the paper.
1Present address: Yale Medical School, New Haven, CT 06511.
2Present address: Columbia University Medical Center, College of Physicians and Surgeons, New York, NY 10032.
Contributed by Ira Pastan, October 17, 2012 (sent for review August 9, 2012)
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Snippet Recombinant immunotoxins (RITs) are chimeric proteins that are being developed for cancer treatment. We have produced RITs that contain PE38, a portion of the...
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SubjectTerms ADP Ribose Transferases - chemistry
alanine
alleles
Amino acids
Antibodies - chemistry
antibody formation
Bacterial proteins
bacterial toxins
Bacterial Toxins - chemistry
Biological Sciences
CD4-Positive T-Lymphocytes - cytology
Correlation analysis
cytotoxicity
Enzyme-Linked Immunosorbent Assay - methods
Epitopes - chemistry
Epitopes, T-Lymphocyte - chemistry
Epitopes, T-Lymphocyte - immunology
exotoxins
Exotoxins - chemistry
Gene Deletion
Genetic Variation
Humans
Immune System
immunodominant epitopes
Immunotoxins
interleukin-2
Interleukin-2 - metabolism
Leukocytes, Mononuclear - cytology
Molecular Conformation
mutants
neoplasms
neutralizing antibodies
patients
Peptides - chemistry
PNAS Plus
Protein Binding
Protein Conformation
Protein Engineering - methods
Protein Structure, Tertiary
Proteins
Pseudomonas
Pseudomonas aeruginosa Exotoxin A
recombinant fusion proteins
T cell receptors
T-lymphocytes
therapeutics
Toxins
Virulence Factors - chemistry
Title Identification and elimination of an immunodominant T-cell epitope in recombinant immunotoxins based on Pseudomonas exotoxin A
URI http://www.pnas.org/content/109/51/E3597.abstract
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Volume 109
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