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A new algorithm to convert a normal antibody into the corresponding catalytic antibody.

ABSTRACT: Over thousands of monoclonal antibodies (mAbs) have been produced so far, and it would be valuable if these mAbs could be directly converted into catalytic antibodies. We have designed a system to realize the above concept by deleting Pro95, a highly conserved residue in CDR-3 of the antibody light chain. The deletion of Pro95 is a key contributor to catalytic function of the light chain. The S35 and S38 light chains have identical amino acid sequences except for Pro95. The former, with Pro95 did not show any catalytic activity, whereas the latter, without Pro95, exhibited peptidase activity. To verify the generality of this finding, we tested another light chain, T99wt, which had Pro95 and showed little catalytic activity. In contrast, a Pro95-deleted mutant enzymatically degraded the peptide substrate and amyloid-beta molecule. These two cases demonstrate the potential for a new method of creating catalytic antibodies from the corresponding mAbs.

SUBMITTER: Hifumi E

PROVIDER: S-EPMC7096177 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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A new algorithm to convert a normal antibody into the corresponding catalytic antibody.

Hifumi Emi E Taguchi Hiroaki H Tsuda Haruna H Minagawa Tetsuro T Nonaka Tamami T Uda Taizo T

Science advances 20200325 13

Over thousands of monoclonal antibodies (mAbs) have been produced so far, and it would be valuable if these mAbs could be directly converted into catalytic antibodies. We have designed a system to realize the above concept by deleting Pro95, a highly conserved residue in CDR-3 of the antibody light chain. The deletion of Pro95 is a key contributor to catalytic function of the light chain. The S35 and S38 light chains have identical amino acid sequences except for Pro95 ...[more]

PMID: 32232151

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Project description:BackgroundCancer stem cells (CSCs) are likely critical to carcinogenesis, and, like normal stem cells (NSCs), are affected by microenvironmental factors. Malignant cells release extracellular factors, modifying tumor behavior. Inorganic arsenic, a human carcinogen, is associated with an overproduction of CSCs in various model systems of carcinogenesis.ObjectiveWe aimed to determine if NSCs are influenced by nearby arsenic-transformed malignant epithelial cells (MECs) as a possible factor in arsenic-associated CSC overabundance.MethodsTranswell noncontact co-culture allowed the study of the effects of non-contiguous, arsenic-transformed prostate MECs on the isogenic human prostate NSC line, WPE-stem. Cancer phenotype was assessed by evaluating secreted matrix metalloproteinases (MMPs), invasiveness, colony formation, and spheroid formation. Gene expression was assessed at the protein (Western blot) or mRNA (real-time reverse transcription-polymerase chain reaction) levels.ResultsNoncontact co-culture of MECs and NSCs rapidly (≤ 3 weeks) caused hypersecretion of MMPs and marked suppression of the tumor suppressor gene PTEN in NSCs. NSCs co-cultured with MECs also showed increased invasiveness and clonogenicity and formed more free-floating spheroids and highly branched ductal-like structures in Matrigel, all typical for CSCs. MEC co-culture caused dysregulated self-renewal and differentiation-related gene expression patterns and epithelial-to-mesenchymal transition in NSCs consistent with an acquired cancer phenotype. Interleukin-6 (IL-6), a cytokine involved in tumor microenvironment control, was hypersecreted by MECs, and IL-6 exposure of NSCs resulted in the duplication of several responses in NSCs of conversion to CSCs via MEC co-culture (e.g., MMP hypersecretion, decreased PTEN).ConclusionsArsenic-transformed MECs recruit nearby NSCs into a cancer phenotype, thereby potentially increasing CSC number. This may be a factor in arsenic-induced CSC overabundance seen in multiple model systems.

Project description:We have performed a systematic variability study of polyclonal antibody catalysis by using five rabbits immunized with the same hapten. Important results from this work are the following. (1) Similarities were observed in the catalytic polyclonal antibodies derived from all five rabbits. Four of the five rabbits produced polyclonal samples that were nearly the same in terms of catalytic activity, whereas the fifth rabbit, designated as rabbit 2, displayed a somewhat higher level of catalytic activity. The catalytic activities (as kcat/kuncat) of these polyclonal samples were similar to that from the best murine monoclonal antibody that had been previously elicited by the same hapten. (2) Titre was not an accurate indicator of polyclonal antibody catalytic activity. (3) A mathematical analysis to describe a distribution of Michaelis-Menten catalysts was performed to help interpret our results. (4) Kinetic analysis indicated that the binding parameters of the different samples were remarkably homogeneous, because one or two components were all that were required to fit the on-rate and off-rate data satisfactorily. Interestingly, the most active catalytic polyclonal sample, that from rabbit 2, displayed the slowest off-rate (so slow it could not be measured) and thus the highest overall affinity. (5) Catalytic analysis of eluted fractions of antibody from a substrate column indicated that each polyclonal sample was also relatively homogeneous in terms of catalytic parameters. The main conclusion of our study is that for this hapten-animal system, the overall catalytic immune response is relatively consistent at two levels. Consistent catalytic activity was observed between the polyclonal samples elicited in the different animals, and the elicited hapten-specific polyclonal antibodies were relatively homogeneous in terms of binding and catalytic parameters within each immunized animal. The observed similarities of the catalytic activity in the different animals is surprising, because the immune response is based on specific binding of antibodies to hapten. There is no known selective pressure to maintain consistent levels of catalytic activity. Our results can therefore be interpreted as providing evidence that for this hapten there is a fixed relationship between hapten structure and catalytic activity and/or consistent genetic factors that dominate the catalytic immune response.

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A new algorithm to convert a normal antibody into the corresponding catalytic antibody.

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A new algorithm to convert a normal antibody into the corresponding catalytic antibody.

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