Development and Validation of an Immuno-PET Tracer as a Companion Diagnostic Agent for Antibody-Drug Conjugate Therapy to Target the CA6 Epitope.

PURPOSE: To develop and compare three copper 64 ((64)Cu)-labeled antibody fragments derived from a CA6-targeting antibody (huDS6) as immuno-positron emission tomography (immuno-PET)-based companion diagnostic agents for an antibody-drug conjugate by using huDS6. MATERIALS AND METHODS: Three antibody fragments derived from huDS6 were produced, purified, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and evaluated in the following ways: (a) the affinity of the fragments and the DOTA conjugates was measured via flow cytometry, (b) the stability of the labeled fragments was determined ex vivo in human serum over 24 hours, and (c) comparison of the in vivo imaging potential of the fragments was evaluated in mice bearing subcutaneous CA6-positive and CA6-negative xenografts by using serial PET imaging and biodistribution. Isotype controls with antilysozyme and anti-DM4 B-Fabs and blocking experiments with an excess of either B-Fab or huDS6 were used to determine the extent of the antibody fragment (64)Cu-DOTA-B-Fab binding specificity. Immunoreactivity and tracer kinetics were evaluated by using cellular uptake and 48-hour imaging experiments, respectively. Statistical analyses were performed by using t tests, one-way analysis of variance, and Wilcoxon and Mann-Whitney tests. RESULTS: The antibody fragment (64)Cu-DOTA-B-Fab was more than 95% stable after 24 hours in human serum, had an immunoreactivity of more than 70%, and allowed differentiation between CA6-positive and CA6-negative tumors in vivo as early as 6 hours after injection, with a 1.7-fold uptake ratio between tumors. Isotype and blocking studies experiments showed tracer-specific uptake in antigen-positive tumors, despite some nonspecific uptake in both tumor models. CONCLUSION: Three antibody fragments were produced and examined as potential companion diagnostic agents. (64)Cu-DOTA-B-Fab is a stable and effective immuno-PET tracer for CA6 imaging in vivo.

[The growing field of immunoconjugates in oncology. A successful link(er) between antibodies and small cytotoxic molecules]. / La montée en puissance des immunoconjugués en oncologie - Une liaison réussie entre un anticorps et une petite molécule cytotoxique.

As evidenced by the recent FDA approvals of brentuximab vedotin (Adcetris(®)) and trastuzumab emtansine (Kadcyla(®)), antibody-drug conjugates (ADC) are becoming an important class of biotherapeutics in oncology. In addition, the strong pipeline of ADC in clinical development, bearing 38 ADC for the treatment of both solid and hematological malignancies, emphasizes the interest and confidence of clinicians and pharmaceutical industry in the area. Although the ADC concept looks pretty simple, it took decades before it turned out to be a successful therapeutic solution. Improving the understanding of ADC mechanism of action allowed identifying the most critical factors influencing ADC efficacy and toxicity. This led to still ongoing research efforts in antibody and alternative scaffold engineering, in linker and conjugation chemistry, as well as in the screening and selection of optimal target antigens and cytotoxics.

Advances in Antibody-Drug Conjugate Design: Current Clinical Landscape and Future Innovations.

The targeted delivery of potent cytotoxic molecules into cancer cells is considered a promising anticancer strategy. The design of clinically effective antibody-drug conjugates (ADCs), in which biologically active drugs are coupled through chemical linkers to monoclonal antibodies, has presented challenges for pharmaceutical researchers. After 30 years of intensive research and development activities, only seven ADCs have been approved for clinical use; two have received fast-track designation and two breakthrough therapy designation from the Food and Drug Administration. There is continued interest in the field, as documented by the growing number of candidates in clinical development. This review aims to summarize the most recent innovations that have been applied to the design of ADCs undergoing early- and late-stage clinical trials. Discovery and rational optimization of new payloads, chemical linkers, and antibody formats have improved the therapeutic index of next-generation ADCs, ultimately resulting in improved clinical benefit for the patients.

Antibody-drug conjugate (ADC) clinical pipeline: a review.

Biological therapies play an increasing role in cancer treatment, although the number of naked antibodies showing clinical efficacy as single agent remains limited. One way to enhance therapeutic potential of antibodies is to conjugate them to small molecule drugs. This combination is expected to bring together the benefits of highly potent drugs on the one hand and selective binders of specific tumor antigens on the other hand. However, designing an ADC is more complex than a simple meccano game, requiring thoughtful combination of antibody, linker, and drugs in the context of a target and a defined cancer indication. Lessons learned from the first-generation antibody-drug conjugate (ADC) and improvement of the technology guided the design of improved compounds which are now in clinical trials. Brentuximab vedotin (Adcetris(®)), an anti-CD30 antibody conjugated to a potent microtubule inhibitor for the treatment of Hodgkin's lymphoma and anaplastic large cell lymphomas, is the only marketed ADC today. A total of 27 ADC are currently undergoing clinical trials in both hematological malignancies and solid tumor indications. Among them, T-DM1 (trastuzumab emtansine), an ADC comprised of trastuzumab conjugated to DM1, via a non-cleavable linker, is showing very promising results in phase III for the treatment of HER2-positive refractory/relapsed metastatic breast cancer. Other compounds, such as CMC-544, SAR3419, CDX-011, PSMA-ADC, BT-062, and IMGN901 currently in clinical trials, targeting varied antigens and bearing different linker and drugs, contribute to the learning curve of ADC, as do the discontinued ADC. Current challenges include improvement of the therapeutic index, linked to a careful selection of the targets, a better understanding of ADC mechanism of action, the management and understanding of ADC off-target toxicities, as well as the selection of appropriate clinical settings (patient selection, dosing regimen) where these molecules can bring highest clinical benefit.

Analysis of the distribution of the kinetochore protein Ndc10p in Saccharomyces cerevisiae using 3-D modeling of mitotic spindles.

Ndc10p is one of the DNA-binding constituents of the kinetochore in Saccharomyces cerevisiae but light microscopy analysis suggests that Ndc10p is not limited to kinetochore regions. We examined the localization of Ndc10p using immunoelectron microscopy and showed that Ndc10p is associated with spindle microtubules from S-phase through anaphase. By serial section reconstruction of mitotic spindles combined with immunogold detection, we showed that Ndc10p interacts with microtubules laterally as well as terminally. About 50% of the gold label in serial section reconstructions of short mitotic spindles was associated with the walls of spindle microtubules. Interaction of kinetochore components with microtubule walls was also shown for kinetochore protein Ndc80p. Our data suggest that at least a subset of kinetochore-associated protein is dispersed throughout the mitotic spindle.