Breaking the one antibody–one target axiom

Abstract

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 ...

Preparation of integrin α(v)β(3)-targeting Ab 38C2 constructs

“…However, a detailed protocol for the syntheses of all these compounds is beyond the scope of this article (one should refer to the original publications) 4,7 . Here, we provide methods for the transformation of a representative a v b 3 -and a v b 5 -targeting molecule possessing a linker arm to its diketone or provinylketone derivatives, for conjugate formation of the latter with Ab 38C2 and for analysis of the resultant constructs.…”

“…The conjugate formation was also established by ELISA experiments, and by flow cytometry to record the binding to cells expressing integrins a v b 3 and a v b 5 . These methods are described elsewhere [3][4][5]7,9 .…”

“…38C2 (Fig. 2) 4,7 . In the latter case, an aldol linker was used as a prolinker (i.e., an acetone aldol adduct of the vinylketone), because the vinylketone linker was highly reactive.…”

“…Notably, one common feature of Abs is that they are highly specific to a single target and, because of their clonal nature, a different Ab is required for each target. We have devised a new approach in which we modify the binding sites of a single Ab, 38C2, allowing it to be retargeted to more than one receptor [3][4][5][6][7] . Such Ab constructs are useful for adapter immunotherapy.…”

“…The above-mentioned cp38C2 constructs bound strongly to integrins a v b 3 -and a v b 5 -expressing cancer cells, such as human Kaposi's Sarcoma (SLK), human melanoma (M21) and human breast carcinoma (MDA-MB-231 and MDA-MB-435) [3][4][5]7 . The constructs inhibited the growth of the primary tumor and/or metastases of these human cancers as well as several others, such as human colon cancer (SW1222), which lack expression of a v b 3 .…”