Rapid Development of Affinity Matured Monoclonal Antibodies Using RIMMS

Kilpatrick, Katherine E.; Wring, Stephen A.; Walker, David H.; Macklin, Michael D.; Payne, Janice; Su, Jing; Champion, Brian; Caterson, Bruce; McIntyre, Gordon D.

doi:10.1089/hyb.1997.16.381

Cited by 77 publications

(50 citation statements)

References 45 publications

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“…The CPZ-1 protein was then cleaved from the GST fusion peptide by thrombin according to the recommendation from the manufacturer. A mouse anti-serum was raised against the purified CPZ-1 recombinant protein using the repetitive immunizations/multiple sites strategy (23,24). Each mouse received a total of 10 g of CPZ-1 emulsified in Ribi's adjuvant on days 0, 3, 6, 8, and 10.…”

Section: Methodsmentioning

confidence: 99%

The Caenorhabditis elegans Cathepsin Z-like Cysteine Protease, Ce-CPZ-1, Has a Multifunctional Role during the Worms' Development

Hashmi¹,

Zhang²,

Oksov

et al. 2004

Journal of Biological Chemistry

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We have analyzed the expression and function of Cecpz-1, a Caenorhabditis elegans cathepsin Z-like cysteine protease gene, during development of the worm. The cpz-1 gene is expressed in various hypodermal cells of all developmental stages and is specifically expressed in the gonads and the pharynx of adult worms. Disruption of cpz-1 function by RNA interference or cpz-1(ok497) deletion mutant suggests that cpz-1 has a role in the molting pathways. The presence of the native CPZ-1 protein in the hypodermis/cuticle of larval and adult stages and along the length of the pharynx of adult worms, as well as the cyclic expression of the transcript during larval development, supports such function. We hypothesize that the CPZ-1 enzyme functions directly as a proteolytic enzyme degrading cuticular proteins before ecdysis and/or indirectly by processing other proteins such as proenzymes and/or other proteins that have an essential role during molting. Notably, an impressive level of the CPZ-1 native protein is present in both the new and the old cuticles during larval molting, in particular in the regions that are degraded prior to shedding and ecdysis. The similar localization of the related Onchocerca volvulus cathepsin Z protein suggests that the function of CPZ-1 during molting might be conserved in other nematodes. Based on the cpz-1 RNA interference and cpz-1 (ok497) deletion mutant phenotypes, it appears that cpz-1 have additional roles during morphogenesis. Deletion of cpz-1 coding sequence or inhibition of cpz-1 function by RNA interference also caused morphological defects in the head or tail region of larvae, improperly developed gonad in adult worms and embryonic lethality. The CPZ-1 native protein in these affected regions may have a role in the cuticular and the basement membrane extracellular matrix assembly process. The present findings have defined a critical role for cathepsin Z in nematode biology.Based on the identification and characterization of a cysteine protease in human brain, a new subfamily of cysteine proteases of the papain family, the cathepsin Z-like enzyme, was recently classified (1). The human brain cathepsin Z was, however, found to be widely expressed in many tissues, suggesting that this enzyme is possibly involved in the normal intracellular protein degradation that takes place in all cell types. Notably, the enzyme was also found in many cancer cell lines and in primary tumors from different sources, which also suggested a role for this enzyme in tumor progression (1). The human cathepsin Z contains distinctive features that separate it from other human cysteine proteases (2). Cathepsin Z is characterized by an unusual and unique 3-amino acid insertion (HIP) in the highly conserved region between the glutamine of the putative oxyanion hole and the active site cysteine, which might confer special properties to the enzyme (3). It functional diversity is generated by the addition of the HIP residues including His 23 in the mature enzyme that provides an anchor for the C terminus of a s...

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Section: Methodsmentioning

confidence: 99%

The Caenorhabditis elegans Cathepsin Z-like Cysteine Protease, Ce-CPZ-1, Has a Multifunctional Role during the Worms' Development

Hashmi¹,

Zhang²,

Oksov

et al. 2004

Journal of Biological Chemistry

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“…All mutants are therefore significantly more active than the previously characterized pNO 2 Phe 86 mTNF-␣, which has only 2% of the activity of the wild-type protein in this assay. To determine the immunogenicity of these pNO 2 Phe mTNF-␣ mutants, 14 C57BL/6 mice were randomized into 5 groups and injected with these mutants, or WT mTNF-␣ by the RIMMS (repetitive immunization at multiple sites) protocol (14). An ELISA analysis revealed no correlation between mTNF-␣ activity in the NF B-luciferase reporter gene assay and the ability to induce an antibody response, ruling out a direct effect on the immune system.…”

Section: Extension To Mutations At Other Surface Sites Within Mtnf-␣mentioning

confidence: 99%

“…To determine the immunogenicity of the pNO 2 Phe mRBP4 mutants, 9 Bcl-2 transgenic mice were randomized into 3 groups and injected with pNO 2 Phe 43 mRBP4, pNO 2 Phe 108 mRBP4 and WT mRBP4 by the RIMMS protocol (14). According to ELISA analysis, mice immunized with either W T mRBP4 or pNO 2 Phe 108 mRBP4 had insignificant serum IgG titers against WT mRBP4 (Fig.…”

Section: Extension To Mutations At Other Surface Sites Within Mtnf-␣mentioning

confidence: 99%

Mechanistic studies of the immunochemical termination of self-tolerance with unnatural amino acids

Grünewald¹,

Hunt²,

Dong

et al. 2009

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

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For more than 2 centuries active immunotherapy has been at the forefront of efforts to prevent infectious disease [Waldmann TA (2003) Nat Med 9:269 -277]. However, the decreased ability of the immune system to mount a robust immune response to selfantigens has made it more difficult to generate therapeutic vaccines against cancer or chronic degenerative diseases. Recently, we showed that the site-specific incorporation of an immunogenic unnatural amino acid into an autologous protein offers a simple and effective approach to overcome self-tolerance. Here, we characterize the nature and durability of the polyclonal IgG antibody response and begin to establish the generality of p-nitrophenylalanine (pNO 2Phe)-induced loss of self-tolerance. Mutation of several surface residues of murine tumor necrosis factor-␣ (mTNF-␣) independently to pNO2Phe leads to a T cell-dependent polyclonal and sustainable anti-mTNF-␣ IgG autoantibody response that lasts for at least 40 weeks. The antibodies bind multiple epitopes on mTNF-␣ and protect mice from severe endotoxemia induced by lipopolysaccharide (LPS) challenge. Immunization of mice with a pNO 2Phe 43 mutant of murine retinol-binding protein (RBP4) also elicited a high titer IgG antibody response, which was cross-reactive with wild-type mRBP4. These findings suggest that this may be a relatively general approach to generate effective immunotherapeutics against cancer-associated or other weakly immunogenic antigens.retinol-binding protein ͉ tumor necrosis factor ͉ vaccination ͉ p-nitrophenylalanine ͉ genetic code

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“…To determine the immunogenicity of the pNO 2 Phe 86 mTNF-␣ mutant, 32 C57BL/6 mice were randomized into three groups and injected with pNO 2 Phe 86 mTNF-␣, WT mTNF-␣, and PBS buffer by using the RIMMS (repetitive immunization at multiple sites) protocol (25). To avoid adverse effects due to cytotoxicity of mTNF-␣, a dose of 5 g of mTNF-␣ per injection was used throughout this study (26).…”

Section: Expression and Characterization Of Mutant Mtnf-␣ Proteinsmentioning

confidence: 99%

Immunochemical termination of self-tolerance

Grünewald

Tsao

Perera

et al. 2008

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

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The ability to selectively induce a strong immune response against self-proteins, or increase the immunogenicity of specific epitopes in foreign antigens, would have a significant impact on the production of vaccines for cancer, protein-misfolding diseases, and infectious diseases. Here, we show that site-specific incorporation of an immunogenic unnatural amino acid into a protein of interest produces high-titer antibodies that cross-react with WT protein. Specifically, mutation of a single tyrosine residue (Tyr 86 ) of murine tumor necrosis factor-␣ (mTNF-␣) to p-nitrophenylalanine (pNO2Phe) induced a hightiter antibody response in mice, whereas no significant antibody response was observed for a Tyr 86 3 Phe mutant. The antibodies generated against the pNO 2Phe are highly cross-reactive with native mTNF-␣ and protect mice against lipopolysaccharide (LPS)-induced death. This approach may provide a general method for inducing an antibody response to specific epitopes of self-and foreign antigens that lead to a neutralizing immune response.TNF-␣ ͉ unnatural amino acids ͉ vaccination ͉ immunogenicity

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Rapid Development of Affinity Matured Monoclonal Antibodies Using RIMMS

Cited by 77 publications

References 45 publications

The Caenorhabditis elegans Cathepsin Z-like Cysteine Protease, Ce-CPZ-1, Has a Multifunctional Role during the Worms' Development

The Caenorhabditis elegans Cathepsin Z-like Cysteine Protease, Ce-CPZ-1, Has a Multifunctional Role during the Worms' Development

Mechanistic studies of the immunochemical termination of self-tolerance with unnatural amino acids

Immunochemical termination of self-tolerance

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