Rationally Engineered Therapeutic Proteins with Reduced Immunogenicity

Tangri, Shabnam; Mothé, Bianca R.; Eisenbraun, Julie; Sidney, John; Southwood, Scott; Briggs, Kristen; Zinckgraf, John W.; Bilsel, Pamuk; Newman, Mark J.; Chesnut, Robert W.; LiCalsi, Cynthia; Sette, Alessandro

doi:10.4049/jimmunol.174.6.3187

Cited by 160 publications

(180 citation statements)

References 44 publications

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“…The direct genetic modification used in this study eliminates the additional step of PEGylation and retains the full cytotoxic capability of parental molecule. Other studies have shown that using genetic engineering to mutate T-cell-specific epitopes and the proteolytic cleavage site can improve the pharmacokinetic profile and in vivo efficacy of the biological drug (Tangri et al, 2005;Weldon et al, 2008). Additional studies may show that incorporation of similar mutations into EGF4KDEL 7Mut can further enhance its already potent antitumour activity.…”

Section: Discussionmentioning

confidence: 99%

Design and modification of EGF4KDEL 7Mut, a novel bispecific ligand-directed toxin, with decreased immunogenicity and potent anti-mesothelioma activity

Stish

Chen

et al. 2009

Br J Cancer

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BACKGROUND: Potency, immunogenicity, and toxicity are three problems that limit the use of targeted toxins in solid tumour therapy. METHODS: To address potency, we used genetic engineering to develop a novel bispecific ligand-directed toxin (BLT) called EGF4KDEL, a novel recombinant anti-mesothelioma agent created by linking human epidermal growth factor (EGF) and interleukin-4 (IL-4) to truncated pseudomonas exotoxin (PE38) on the same single-chain molecule. Immunogenicity was reduced by mutating seven immunodominant B-cell epitopes on the PE38 molecule to create a new agent, EGF4KDEL 7Mut. RESULTS: In vitro, bispecific EGF4KDEL showed superior anti-mesothelioma activity compared with its monospecific counterparts. Toxicity in mice was diminished by having both ligands on the same molecule, allowing administration of a 10-fold greater dose of BLT than a mixture of monomeric IL4KDEL and EGFKDEL. EGF4KDEL 7Mut, retained all of its functional activity and induced about 87% fewer anti-toxin antibodies than mice given the parental, non-mutated form. In vivo, intraperitoneal (IP) injection of the BLT showed significant (Po0.01) and impressive effects against two aggressive, malignant IP mesothelioma models when treatment was begun 14-16 days post tumour innoculation. CONCLUSION: These data show that EGF4KDEL 7Mut is a promising new anti-mesothelioma agent that was developed to specifically address the obstacles facing clinical utility of targeted toxins.

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Section: Discussionmentioning

confidence: 99%

Design and modification of EGF4KDEL 7Mut, a novel bispecific ligand-directed toxin, with decreased immunogenicity and potent anti-mesothelioma activity

Stish

Chen

et al. 2009

Br J Cancer

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“…Alternative approaches are to identify and remove B-cell or T-cell epitopes (13)(14)(15)(16)(17). We have recently used a mouse model to identify the major B-cell epitopes in the PE38 portion of RITs made by our group (18).…”

mentioning

confidence: 99%

An immunotoxin with greatly reduced immunogenicity by identification and removal of B cell epitopes

Onda

Beers

Xiang

et al. 2008

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

153

164

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Recombinant immunotoxins are hybrid proteins composed of an Fv that binds to a tumor antigen fused to a bacterial or plant toxin. Immunotoxin BL22 targets CD22 positive malignancies and is composed of an anti-CD22 Fv fused to a 38-kDa fragment of Pseudomonas exotoxin A (PE38). BL22 has produced many complete remissions in drug-resistant Hairy cell leukemia, where many treatment cycles can be given, because neutralizing antibodies do not form. In marked contrast, only minor responses have been observed in trials with immunotoxins targeting solid tumors, because only a single treatment cycle can be given before antibodies develop. To allow more treatment cycles and increase efficacy, we have produced a less immunogenic immunotoxin by identifying and eliminating most of the B cell epitopes on PE38. This was accomplished by mutation of specific large hydrophilic amino acids (Arg, Gln, Glu, Lys) to Ala, Ser, or Gly. The new immunotoxin (HA22-8X) is significantly less immunogenic in three strains of mice, yet retains full cytotoxic and anti-tumor activities. Elimination of B-cell epitopes is a promising approach to the production of less immunogenic proteins for therapeutic purposes.antibody engineering ͉ BL22 ͉ HA22 ͉ immunotherapy ͉ Pseudomonas exotoxin A I mmunotoxins (ITs) are hybrid proteins that are composed of a cancer-specific antibody attached to a bacterial or plant toxin (1). Initially ITs were made by chemically coupling toxins to whole antibodies. Now they are made using a combination of antibody and protein engineering (2, 3). ITs kill cells by binding to a cell surface protein, being internalized by endocytosis and eventually reaching the cytosol, where they arrest protein synthesis by inactivating EF2 or ribosomes (4, 5). Our laboratory has developed recombinant immunotoxins (RITs) in which the Fv portion of an antibody is directly fused to a 38-kDa portion of the bacterial toxin Pseudomonas exotoxin A (PE). Three RITs are currently in clinical trials and all three have shown anti-tumor activity in phase 1 trials. LMB-2 [anti-Tac-(Fv)-PE38] targets CD25 expressed on many T cell malignancies and some B cell malignancies (6). BL22 [anti-CD22-(Fv)-PE38] targets CD22 expressed on most B cell malignancies (7), and SS1P antimesothelin-(Fv)-PE38 targets the mesothelin antigen expressed on mesotheliomas and on ovarian, lung, pancreatic, and gastric cancers (8). Because these ITs contain a portion of a bacterial protein, they can induce the formation of neutralizing antibodies, hindering their efficacy. In patients with B-and T-cell malignancies the formation of neutralizing antibodies is infrequent because of the immune-suppressed state of patients with these malignancies (6, 7). However, in patients with solid tumors treated with SS1P and other ITs, antibody formation was very frequently detected 21 days after the first treatment cycle, preventing readministration of the IT (9).Previous studies have shown that the formation of antibodies to foreign proteins can be prevented by coupling the protein to high-mol...

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“…In a study by Tangri et al, rationally designed and engineered erythropoietin, with epitopic regions modulated for HLA affinity, were found to be both bioactive and less immunogenic (78).…”

Section: Current Approaches For Reducing Alloimmunization Against Promentioning

confidence: 99%

Auditing Protein Therapeutics Management by Professional APCs: Toward Prevention of Immune Responses against Therapeutic Proteins

Dasgupta

Bayry²,

André³

et al. 2008

The Journal of Immunology

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Alloimmunization is a crippling concern in the management of patients undergoing administration of protein therapeutics as evidenced in replacement therapy and other treatment procedures. Several issues in the genesis and modulation of such deleterious immune responses have been studied. While authors have focused on the downstream events of the specific immune response and suggested modification of protein therapeutics to eliminate epitopes that interact with B cell receptors, T cell receptors, or MHCII molecules, the mechanisms underlying Ag interaction with APCs, a step upstream of immune effectors, have been grossly neglected. We wish to emphasize that the recent knowledge in understanding the capacities of an APC to handle an Ag and the importance of the surrounding microenvironment in this process are crucial for designing novel protein therapeutics with reduced immunogenicity.

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Rationally Engineered Therapeutic Proteins with Reduced Immunogenicity

Cited by 160 publications

References 44 publications

Design and modification of EGF4KDEL 7Mut, a novel bispecific ligand-directed toxin, with decreased immunogenicity and potent anti-mesothelioma activity

Design and modification of EGF4KDEL 7Mut, a novel bispecific ligand-directed toxin, with decreased immunogenicity and potent anti-mesothelioma activity

An immunotoxin with greatly reduced immunogenicity by identification and removal of B cell epitopes

Auditing Protein Therapeutics Management by Professional APCs: Toward Prevention of Immune Responses against Therapeutic Proteins

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