Aldolase Antibodies of Remarkable Scope

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An obstacle in the utilization of catalytic Abs for selective prodrug activation in cancer therapy has been systemic tumor targeting. Here we report the generation of catalytic Abs that effectively target tumor cells with undiminished prodrug activation capability. Ab conjugates were prepared by covalent conjugation of an integrin ␣v␤3-targeting antagonist to catalytic Ab 38C2 through either sulfide groups of cysteine residues generated by reduction of the disulfide bridges in the hinge region or surface lysine residues not involved in the catalytic activity. Using flow cytometry, the Ab conjugates were shown to bind efficiently to integrin ␣v␤3-expressing human breast cancer cells. The Ab conjugates also retained the retro-aldol activity of their parental catalytic Ab 38C2, as measured by methodol and doxorubicin (dox) prodrug activation. Complementing these Ab conjugates, an evolved set of dox prodrugs was designed and synthesized. Dox prodrugs that showed higher stability and lower toxicity were evaluated both in the presence and absence of the integrin ␣v␤3-targeting 38C2 conjugates for cell-killing efficacy by using human breast cancer cells. Our study reveals that cell targeting and prodrug activation capabilities can be efficiently combined for selective chemotherapy with novel dox prodrugs.aldolase Ab ͉ Ab conjugate ͉ doxorubicin ͉ integrin ␣v␤3

Section: Resultsmentioning

confidence: 99%

Synthesis of the next-generation therapeutic antibodies that combine cell targeting and antibody-catalyzed prodrug activation

Abraham

Guo

et al. 2007

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“…Analogous to the strategy used by type I aldolases (10), formation of the iminium intermediate with the enzymatic lysine side chain provides an electron sink, facilitating the retroaldol cleavage. Several catalytic antibodies and peptide systems have been developed that utilize this lysine iminium strategy (11)(12)(13)(14)(15)(16).…”

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confidence: 99%

Origins of catalysis by computationally designed retroaldolase enzymes

Lassila

Baker

Herschlag

2010

100

We have investigated recently reported computationally designed retroaldolase enzymes with the goal of understanding the extent and the origins of their catalytic power. Direct comparison of the designed enzymes to primary amine catalysts in solution revealed a rate acceleration of 10 5 -fold for the most active of the designed retroaldolases. Through pH-rate studies of the designed retroaldolases and evaluation of a Brønsted correlation for a series of amine catalysts, we found that lysine pK a values are shifted by 3-4 units in the enzymes but that the catalytic contributions from the shifted pK a values are estimated to be modest, about 10-fold. For the most active of the reported enzymes, we evaluated the catalytic contribution of two other design components: a motif intended to stabilize a bound water molecule and hydrophobic substrate binding interactions. Mutational analysis suggested that the bound water motif does not contribute to the rate acceleration. Comparison of the rate acceleration of the designed substrate relative to a minimal substrate suggested that hydrophobic substrate binding interactions contribute around 10 3 -fold to the enzymatic rate acceleration. Altogether, these results suggest that substrate binding interactions and shifting the pK a of the catalytic lysine can account for much of the enzyme's rate acceleration. Additional observations suggest that these interactions are limited in the specificity of placement of substrate and active site catalytic groups. Thus, future design efforts may benefit from a focus on achieving precision in binding interactions and placement of catalytic groups.amine | Brønsted correlation | catalytic mechanism | computational enzyme design | retroaldol reaction B ecause natural enzymes catalyze reactions with tremendous rate accelerations and specificities, a long-standing goal in enzymology and protein engineering has been to reliably design new enzyme catalysts for chemical reactions of interest. Computational protein design offers a promising tool for achieving this goal. Computational protein design methods use specialized potential energy functions in combination with search algorithms to optimize an amino acid sequence for a given protein structure and function (1-3). These methods have been adapted to the challenge of designing enzyme active sites (4-8).Although new enzymatic activities have been designed computationally, the resulting catalysts have catalytic efficiencies (k cat ∕K M values) of about 1-100 M −1 s −1 (4,7,8), considerably less than values of 10 5 -10 9 M −1 s −1 typical of natural enzymes, and similar to those of early catalytic antibodies. In contrast to catalytic antibody methods and other stochastic processes, however, the potential for success of computational enzyme design is tied to the predictive power of the computational model. Thus, future improvement of our ability to computationally design enzymatic activity will require ongoing rigorous assessment of the successes and failures of the design process.We therefore soug...

“…mAb 38C2 belongs to a group of catalytic antibodies that were generated by reactive immunization and mechanistically mimic natural aldolase enzymes (14,15). Through a reactive lysine, these antibodies catalyze aldol and retro-aldol reactions using the enamine mechanism of natural aldolases (14)(15)(16)(17)(18). In addition to their remarkable versatility and efficacy in synthetic organic chemistry (reviewed in ref.…”

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confidence: 99%

Chemically programmed monoclonal antibodies for cancer therapy: Adaptor immunotherapy based on a covalent antibody catalyst

Rader

Sinha

Popkov

et al. 2003

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118

Proposing that a blend of the chemical diversity of small synthetic molecules with the immunological characteristics of the antibody molecule will lead to therapeutic agents with superior properties, we here present a device that equips small synthetic molecules with both effector function and long serum half-life of a generic antibody molecule. As a prototype, we developed a targeting device that is based on the formation of a covalent bond of defined stoichiometry between a 1,3-diketone derivative of an integrin ␣v␤3 and ␣v␤5 targeting Arg-Gly-Asp peptidomimetic and the reactive lysine of aldolase antibody 38C2. The resulting complex was shown to (i) spontaneously assemble in vitro and in vivo, (ii) selectively retarget antibody 38C2 to the surface of cells expressing integrins ␣v␤3 and ␣v␤5, (iii) dramatically increase the circulatory half-life of the Arg-Gly-Asp peptidomimetic, and (iv) effectively reduce tumor growth in animal models of human Kaposi's sarcoma and colon cancer. This immunotherapeutic has the potential to target a variety of human cancers, acting on both the vasculature that supports tumor growth as well as the tumor cells themselves. Further, by use of a generic antibody molecule that forms a covalent bond with a 1,3-diketone functionality, essentially any compound can be turned into an immunotherapeutic agent thereby not only increasing the diversity space that can be accessed but also multiplying the therapeutic effect.