Most FDA-approved therapeutic antibodies for cancer treatment are naked antibodies, which activate the host defenses to react against the cancer cell. Although these agents produce clinically significant responses in patients, they rarely result in a permanent remission of the tumor and the patients invariably relapse.
While unconjugated antibodies are a more "natural" approach to treatment, there is increasing interest in the use of conjugated antibodies for situations in which the natural host defenses mobilized by the naked antibody are insufficient to overcome the malignancy.
In principle, the concept is simple: the antibody targets a cancer cell, delivering a drug payload. This approach has proved difficult to realize in actuality, yet there are a number of conjugates in development or in clinical trials and one (Mylotarg) that has gained approval (Table 2).
Paul Carter, Ph.D., vp for antibody technologies of Seattle Genetics (Bothell, WA), discussed his company's efforts to target CD30 using a conjugated antibody. This attractive target for Hodgkin's disease and T and B cell lymphomas is expressed in up to 600,000 copies per cell, while it shows very limited expression on normal tissues. Some monoclonal antibodies against this marker are efficiently internalized in tissue culture model systems.
The Seattle Genetics team conjugated monomethyl auristatin E, a powerful dolastatin 10 derivative to their antibodies, through protease cleavable linkers. The auristatin derivative is released inside the cell where it inhibits tubulin formation.
In animal experiments, the anti-CD30 auristatin conjugate proved to be highly efficacious against Hodgkins disease and anaplastic large cell lymphoma tumor cell lines. The antibody-drug conjugated was well tolerated by the animals and cleared slowly, in some cases still detectable after 50 days in the circulation, with a half life of ~6 days in cynomolgus monkeys. The drug conjugates displayed significant antitumor activity with a broad therapeutic window in mice.
Seattle Genetics scientists embarked upon a program to produce an optimized antibody-drug conjugate. They used chemical modifications to reduce different cysteines and generated a family of molecules with different levels of conjugation.
These forms could be separated by hydrophobic interaction chromatography, reducing heterogeneity and yielding families of molecules with varying numbers of auristatin molecules conjugated to them. The in vivo therapeutic window was increased by ~twofold by tailoring the drug loading stoichiometry.
In addition, Carter and colleagues modified the amino acid sequences of the antibodies, replacing cysteines in the hinge region with serines, which do not form disulfide bonds. These mutational modifications did not alter the antigen binding of the antibody. This antibody engineering strategy was used to define the stoichiometry and site of drug loading to reduce conjugate heterogeneity and/or improve yield. In vivo antitumor efficacy and toxicity of engineered antibody-drug conjugates are comparable to partially loaded parent conugates.
The strides that have been made in the past year in engineering protein therapeutics have addressed issues of immunogenicity, toxicity, and aggregation. Variations on display technology including yeast and ribosomal display have allowed optimization of protein drugs by improving their stability and ease of delivery.
With the present flourishing pipeline and many more items in the laboratory the field will continue to overshadow low molecular weight drug development.