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

Mazor, Ronit; Vassall, Aaron; Eberle, Jaime A.; Beers, Richard; Weldon, John E.; Venzon, David; Tsang, Kwong Y.; Benhar, Itai; Pastan, Ira

doi:10.1073/pnas.1218138109

Cited by 85 publications

(97 citation statements)

References 43 publications

(43 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These results are consistent with the effects of PEGylation on other therapeutic proteins (32)(33)(34). Other approaches to limit immunogenicity have also been proposed (35)(36)(37)(38)(39)(40).…”

Section: Studies In Macaquessupporting

confidence: 85%

Design andIn VivoCharacterization of Immunoconjugates Targeting HIV gp160

Pincus

Song

Maresh

et al. 2017

J Virol

View full text Add to dashboard Cite

The envelope (Env) glycoprotein of HIV is expressed on the surface of productively infected cells and can be used as a target for cytotoxic immunoconjugates (ICs), in which cell-killing moieties, including toxins, drugs, or radionuclides, are chemically or genetically linked to monoclonal antibodies (MAbs) or other targeting ligands. Such ICs could be used to eliminate persistent reservoirs of HIV infection. We have found that MAbs which bind to the external loop of gp41, e.g., MAb 7B2, make highly effective ICs, particularly when used in combination with soluble CD4. We evaluated the toxicity, immunogenicity, and efficacy of the ICs targeted with 7B2 in mice and in simian-human immunodeficiency virus-infected macaques. In the macaques, we tested immunotoxins (ITs), consisting of protein toxins bound to the targeting agent. ITs were well tolerated and initially efficacious but were ultimately limited by their immunogenicity. In an effort to decrease immunogenicity, we tested different toxic moieties, including recombinant toxins, cytotoxic drugs, and tubulin inhibitors. ICs containing deglycosylated ricin A chain prepared from ricin toxin extracted from castor beans were the most effective in killing HIVinfected cells. Having identified immunogenicity as a major concern, we show that conjugation of IT to polyethylene glycol limits immunogenicity. These studies demonstrate that cytotoxic ICs can target virus-infected cells in vivo but also highlight potential problems to be addressed. IMPORTANCE It is not yet possible to cure HIV infection. Even after years of fully effective antiviral therapy, a persistent reservoir of virus-infected cells remains. Here we propose that a targeted conjugate consisting of an anti-HIV antibody bound to a toxic moiety could function to kill the HIV-infected cells that constitute this reservoir. We tested this approach in HIV-infected cells grown in the lab and in animal infections. Our studies demonstrated that these immunoconjugates are effective both in vitro and in test animals. In particular, ITs constructed with the deglycosylated A chain prepared from native ricin were the most effective in killing cells, but their utility was blunted because they provoked immune reactions that interfered with the therapeutic effects. We then demonstrated that coating of the ITs with polyethylene glycol minimized the immunogenicity, as has been demonstrated with other protein therapies.

show abstract

Section: Studies In Macaquessupporting

confidence: 85%

Design andIn VivoCharacterization of Immunoconjugates Targeting HIV gp160

Pincus

Song

Maresh

et al. 2017

J Virol

View full text Add to dashboard Cite

show abstract

“…Previously, we identified an immunodominant epitope in PE38 (12). Here, we identify the other seven epitopes in the toxin.…”

Section: Discussionmentioning

confidence: 75%

“…To identify the T-cell epitopes, we assessed T-cell responses using an ELISpot assay, which measured IL-2 production (12,21). We observed a significant T-cell response in all 50 PBMC samples, demonstrating the robustness of the assay.…”

Section: Discussionmentioning

confidence: 85%

“…To circumvent the immunogenicity of PE38, we previously used peptide pools to map the approximate location of the T-cell epitopes and found an immunodominant and promiscuous epitope that stimulated T cells in 42% of all donors (12). Here, we have done high-resolution mapping of the epitopes and used this information to mutate and suppress seven additional epitopes.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes

Mazor

Eberle

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

101

109

View full text Add to dashboard Cite

Nonhuman proteins have valuable therapeutic properties, but their efficacy is limited by neutralizing antibodies. Recombinant immunotoxins (RITs) are potent anticancer agents that have produced many complete remissions in leukemia, but immunogenicity limits the number of doses that can be given to patients with normal immune systems. Using human cells, we identified eight helper T-cell epitopes in PE38, a portion of the bacterial protein Pseudomonas exotoxin A which consists of the toxin moiety of the RIT, and used this information to make LMB-T18 in which three epitopes were deleted and five others diminished by point mutations in key residues. LMB-T18 has high cytotoxic and antitumor activity and is very resistant to thermal denaturation. The new immunotoxin has a 93% decrease in T-cell epitopes and should have improved efficacy in patients because more treatment cycles can be given. Furthermore, the deimmunized toxin can be used to make RITs targeting other antigens, and the approach we describe can be used to deimmunize other therapeutically useful nonhuman proteins.deimmunization | protein engineering I mmunotoxins are chimeric proteins that combine the "magic bullet" specificity of an antibody with the high potency of a toxin. The high specificity of recombinant immunotoxins (RITs) leads to a dramatic decrease in side effects compared with chemotherapy. Moxetumomab Pasudotox (MP) is an RIT that consists of PE38, a fragment of Pseudomonas exotoxin A, fused to an anti-CD22 Fv (1). In a phase I trial for refractory hairy-cell leukemia (HCL), MP had an 86% response rate (2), with 46% complete remissions, and is now in phase III clinical trials (3).Immunogenicity is a stumbling block in the clinical success of many therapeutic proteins (4). Formation of neutralizing antidrug antibodies (5) inactivates the therapeutic agent and can cause serious adverse effects. Although MP had low immunogenicity in the immune-suppressed patients of the HCL trial, some patients did eventually develop antibodies. Consequently, fewer doses could be given to these patients, leading to a reduced response rate. Additionally, RITs targeting solid tumors are less effective than MP because of their high immunogenicity in patients with normal immune systems (6, 7).The role of helper T cells in mounting an immune response is well-established (8, 9). It was previously shown that elimination of murine T-cell epitopes reduced neutralizing antibody formation in mice (10), leading us to the hypothesis that reduction of human T-cell epitopes in the bacterial moiety of RITs would diminish its immunogenicity in humans, allowing more treatment cycles and better antitumor responses, as previously attempted for other therapeutic proteins like erythropoietin (11).To circumvent the immunogenicity of PE38, we previously used peptide pools to map the approximate location of the T-cell epitopes and found an immunodominant and promiscuous epitope that stimulated T cells in 42% of all donors (12). Here, we have done high-resolution mapping of the epitopes ...

show abstract

“…One of the potential disadvantages that has emerged during systemic administration of immunotoxin for treatment of cancer is the development of an antibody response against the immunotoxin, which reduces its therapeutic efficacy (42)(43)(44). Several different approaches have been tried to reduce the immunogenicity of the exotoxin component while maintaining its efficacy (28,30,(45)(46)(47).…”

Section: Discussionmentioning

confidence: 99%

Antiviral Activity of a Single-Domain Antibody Immunotoxin Binding to Glycoprotein D of Herpes Simplex Virus 2

Geoghegan

Zhang

Desai

et al. 2015

Antimicrob Agents Chemother

View full text Add to dashboard Cite

c Despite years of research dedicated to preventing the sexual transmission of herpes simplex virus 2 (HSV-2), there is still no protective vaccine or microbicide against one of the most common sexually transmitted infections in the world. Using a phage display library constructed from a llama immunized with recombinant HSV-2 glycoprotein D, we identified a single-domain antibody VHH, R33, which binds to the viral surface glycoprotein D. Although R33 does not demonstrate any HSV-2 neutralization activity in vitro, when expressed with the cytotoxic domain of exotoxin A, the resulting immunotoxin (R33ExoA) specifically and potently kills HSV-2-infected cells, with a 50% neutralizing dilution (IC 50 ) of 6.7 nM. We propose that R33ExoA could be used clinically to prevent transmission of HSV-2 through killing of virus-producing epithelial cells during virus reactivation. R33 could also potentially be used to deliver other cytotoxic effectors to HSV-2-infected cells.

show abstract

Identification and elimination of an immunodominant T-cell epitope in recombinant immunotoxins based onPseudomonasexotoxin A

Cited by 85 publications

References 43 publications

Design andIn VivoCharacterization of Immunoconjugates Targeting HIV gp160

Design andIn VivoCharacterization of Immunoconjugates Targeting HIV gp160

Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes

Antiviral Activity of a Single-Domain Antibody Immunotoxin Binding to Glycoprotein D of Herpes Simplex Virus 2

Contact Info

Product

Resources

About