<i>In vivo</i>
            activity in a catalytic antibody-prodrug system: Antibody catalyzed etoposide prodrug activation for selective chemotherapy

Shabat, Doron; Lode, Holger N.; Pertl, Ursula; Reisfeld, Ralph A.; Rader, Christoph; Lerner, Richard A.; Barbas, Carlos F.

doi:10.1073/pnas.131187998

Cited by 99 publications

(47 citation statements)

References 25 publications

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“…Key to this assembly is antibody-catalyzed retro-aldolization to provide the vinyl ketone product that could then self-attach to 38C2. Our previous studies concerning 38C2-catalyzed prodrug activation in vivo provides precedence for this approach (18). To evaluate this, we carried out experiments using compound 2a and the conventional diketone 2c as a control.…”

Section: Resultsmentioning

confidence: 99%

Breaking the one antibody–one target axiom

Guo

Das

Mueller

et al. 2006

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

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Studies at the interface of chemistry and biology have allowed us to develop an immunotherapeutic approach called chemically programmed antibodies (cpAbs), which combines the merits of traditional small-molecule drug design with immunotherapy. In this approach, a catalytic antibody catalyzes the covalent conjugation of a small molecule or peptide to the active site of the antibody, effectively recruiting the binding specificity of the conjugated molecule to the antibody. In essence, this technology provides the tools for breaking the ''one antibody-one target axiom'' of immunochemistry. Our studies in this area have focused on using the chemistry of the well studied aldolase catalytic antibodies of which mAb 38C2 is a member. Previously, we explored reversible assembly of cpAbs available through diketone chemistry. In this article, we explore a unique proadapter assembly strategy wherein an antibody 38C2-catalyzed transformation unveils a reactive tag that then reacts to form a stable covalent bond with the antibody. An integrin ␣v␤3 antagonist was synthesized with the designed proadapter and studied using human breast cancer cell lines MDA-MB-231 and MDA-MB-435. We demonstrate that this approach allows for (i) the effective assembly of cpAbs in vitro and in vivo, (ii) selective retargeting of 38C2 to integrin ␣v␤3 expressing breast cancer cell lines, (iii) intracellular delivery of cpAbs into cells, (iv) dramatically increased circulatory half-life, and (v) substantial enhancement of the therapeutic effect over the peptidomimetic itself in animal models of breast cancer metastasis. We believe that this technology possesses potential for the treatment and diagnosis of disease.catalytic antibody ͉ chemical programming ͉ combinatorial antibody libraries M onoclonal antibodies are a rapidly growing class of therapeutics for a wide variety of diseases (1, 2). Some of the advantages of antibodies include their relative lack of nonspecific toxicity, long half-life, and ease of access from patient-derived or synthetic combinatorial antibody libraries. For certain diseases, such as cancer, that antibodies can carry their own effector functions is of prime importance because the antibody specificity directs the killing function endemic to the effector domain, the Fc. It has always been axiomatic in immunochemistry that even though one may desire one or more of the advantageous properties common to all antibodies, due to their clonal nature, each task requires a different antibody. A solution to this problem, namely chemically programmed antibodies (cpAbs), has emerged at the interface of chemistry and biology: One can use different low-molecular-weight targeting agents (programming agents or adapters) to selectively target the same antibody to different sites for different uses (3). This strategy has the advantage that only a single antibody is required for a multiplicity of tasks, and it taps into the unlimited chemical diversity and the specificity that can be engendered by organic synthesis (4). The antibody provides ...

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Section: Resultsmentioning

confidence: 99%

Breaking the one antibody–one target axiom

Guo

Das

Mueller

et al. 2006

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

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“…32 Finally, although CD44 was used as an example, there are numerous other genes that appear to be differentially spliced within tumor cells. 33 -44 Incorporation of such alternatively spliced exons into minigene constructs may further expand the potential usefulness of alternative splicing as a novel means of targeting gene expression to tumor cells in vivo.…”

Section: Discussionmentioning

confidence: 99%

Alternative splicing as a novel of means of regulating the expression of therapeutic genes

Hayes¹,

Carpenito²,

Davis³

et al. 2002

Cancer Gene Ther

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In order to determine the potential of alternative splicing as a means of targeting the expression of therapeutic genes to tumor cells in vivo, a series of episomal plasmid -based ''splice -activated gene expression'' ( pSAGE ) vectors was generated, which contain minigene cassettes composed of various combinations of the three alternatively spliced exons present in the differentially expressed adhesion protein CD44R1 ( v8, v9, and v10 ) with or without their corresponding intronic sequences, positioned in -frame between the CD44 leader sequence and a ''leaderless'' human liver / bone / kidney alkaline phosphatase ( ALP ) cDNA. Because both the v8 -v9 and v9 -v10 introns contain multiple in -frame stop codons, the expression and enzymatic activity of ALP are dependent upon the accurate removal of intronic sequences from the pre -mRNA transcripts encoded by these constructs. The various pSAGE constructs were introduced into CD44H -positive ( T24 ) and CD44R1 -positive ( PC3 ) target cells by electroporation and transfectants selected in hygromycin B. ALP expression was determined by staining with the ALP substrate, BCIP / INT, and the transfected cells tested for their sensitivity to the inactive prodrug, etoposide phosphate. ALP -mediated dephosphorylation of etoposide phosphate generates the potent topoisomerase II inhibitor etoposide. The data obtained indicate that whereas the v8 -v9 intron is spliced in both CD44H -and CD44R1 -positive cells, the v9 -v10 intron is efficiently and accurately removed only in CD44R1 -positive cells. Furthermore, only CD44R1 -positive cells were sensitized to etoposide phosphate when transfected with the v9 -v10.ALP construct. These data emphasize the potential usefulness of alternative splicing as a novel means of targeting gene expression to tumor cells in vivo. Cancer Gene Therapy ( 2002 ) 9, 133 -141 DOI: 10.1038 / sj / cgt / 7700427 Keywords: CD44; alternative splicing; cancer gene therapy G ene therapy shows great promise as a modality in the treatment of cancer.1 As the result of progress made in recent years, there is now no shortage of candidate genes which, if expressed at a high -enough level, can either directly kill tumor cells or sensitize them to the cytotoxic effects of ionizing radiation and/ or chemotherapeutic agents.2 A major outstanding challenge is to develop a safe, effective, and practical means of targeting such genes to tumor cells in vivo while minimizing as much as possible the damage inflicted on normal tissues.3 In this regard, promising results have been obtained using engineered viral vectors that exhibit restricted cellular tropism. 4 Similarly, a number of promoter /enhancer elements have been identified, which can be used to control gene expression in particular cell types or tissues. 5,6 Although little explored, there are additional ways in which gene expression could potentially be targeted to particular cell types in vivo. Specifically, because tumor cells and normal cells differ in their ability to alternatively splice certain pre -mRNA t...

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“…The excellent retro-aldolase activity (2, 3) of Abs 38C2 (4) and 93F3 (5) has allowed us to design, synthesize, and evaluate prodrugs of various chemotherapeutic agents that can be activated by retro-aldol reactions (6)(7)(8)(9)(10). In a syngeneic mouse model of neuroblastoma, systemic administration of an etoposide prodrug and intratumor injection of Ab 38C2 inhibited tumor growth (11). However, to evaluate the utility of these Abs for selective chemotherapy in a systemic setting, it will be necessary to go one step further and equip the Ab with a tumor-recognition device to target the catalytic Ab to the malignant cells.…”

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confidence: 99%

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

Abraham

Guo

et al. 2007

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

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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

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In vivo activity in a catalytic antibody-prodrug system: Antibody catalyzed etoposide prodrug activation for selective chemotherapy

Cited by 99 publications

References 25 publications

Breaking the one antibody–one target axiom

Breaking the one antibody–one target axiom

Alternative splicing as a novel of means of regulating the expression of therapeutic genes

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

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