Choosing the right cytotoxic small molecule
The earliest versions of ADCs used stand-alone chemotherapeutics such as doxorubicin, methotrexate, or vinca alkyloids as
the cytotoxic arm of the conjugate. Clinical-trial results using these ADCs were disappointing, and it is thought that part
of the problem was the relatively low potency of the toxins used (2). The newer classes of cytotoxins are at least 100-fold
more potent than the older molecules, with in vitro potency against tumor cell lines of 10–9 to 10–11 M (5).
The importance of linker technology
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There are only a few major chemical classes of toxins being explored. They can be divided into two types, those that cause
damage to DNA and those that interfere with tubulin polymerization. Calicheamicin, used in Mylotarg and in Pfizer's inotuzumab
ozogamicin, an ADC in Phase III trials, binds to the minor groove of DNA and induces double-strand DNA breaks that result
in cell death. Duocarmycins, isolated originally from Streptomyces bacteria, are DNA minor-groove binding alkylating agents (2). Fully synthetic duocarmycin derivatives are being used by the
biopharmaceutical company Syntarga (acquired by the pharmaceutical company Synthon in June 2011) for ADC constructs (see sidebar).
Microtubule disruptors are represented by two major classes: maytansinoids and auristatins. Maytansinoids are deriviatives
of maytansine, a natural product originally isolated from the shrub Maytenus serrata. ImmunoGen has focused on development of this class of cytotoxic small molecules and associated linker technologies and has
been devloping maytansinoid ADC compounds singularly and in partnership with other companies. In addition to trastuzumab emtansine,
which is being codeveloped by Roche and ImmunoGen, another example of a maytansinoid ADC being developed by ImmunoGen is the
company's IMGN901, which uses the maytansinoid DM4. Auristatins are synthetic analogs of dolostatin 10, a natural product
derived from a marine mollusk, Dolabela auricularia. Like the maytansinoids, auristatins are microtubule disruptors. Millennium and Seattle Genetics' ADC Adcetris is a conjugate
of an anti-CD30 mAb to monomethyl auristatin E (MMAE). Seattle Genetics focuses on the development of auristatin-conjugated
ADCs, using the auristatins MMAE and monomethyl auristatin F (MMAF) and proprietary linkers.
Choosing the right linker
Developing the right linker and method of attachment is a crucial part ADC development. "Many areas around the process have
improved, however, the linker strategy for ADC manufacturing and their application has certainly contributed perhaps the most
in moving the field forward," says Grant Boldt, director of business development at the CMO SAFC. The creation of linkers
that are stable in circulation but labile upon binding of the ADC to its target has resulted in the current generation of
ADCs having better stability and lower systemic toxicity than earlier ADCs, according to Boldt. Early versions of ADCs, including
Mylotarg, suffered from instability while in circulation. The linkage between the mAb and the cytotoxic small molecule were
destroyed by endogenous proteases in the blood, and the premature release of the cytotoxin resulted in side-effect profiles
similar to that of an unconjugated chemotherapeutic. The current generation of linkers is more resistant to degradation in
the blood while still allowing release of the payload at the target. Choice of a linker is influenced by which toxin is used,
as each toxin has different chemical constraints (6).
Linkers can be divided into two broad categories: cleavable and noncleavable. Cleavable linkers rely on processes inside the
cell to liberate the toxin, such as reduction in the cytoplasm, exposure to acidic conditions in the lysosome, or cleavage
by specific proteases within the cell. Noncleavable linkages require catabolic degradation of the conjugate for release of
the cytotoxic small molecule. The released cytotoxic small molecule will retain the linker and the amino acid by which it
attached to the mAb. Importantly, both classes are designed to release the cytotoxic small molecule only after the ADC has
reached the interior of the cancer cell (2).
There are a limited number of chemical moities on proteins, including mAbs, that are available for chemical modification.
Linkers can attach to the mAb through the amino groups of lysine residues, or by the thiol groups on cysteine residues. Attachment
is a pseudorandom process: in theory, any of the targeted amino acids within the mAb, either cysteine or lysine, can be modified
(3). According to Boldt, the conjugation reaction results in a heterogeneous mixture of conjugated species, but the proportion
of each species in the mixture is reproducible from batch-to-batch and quantifiable.
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