42 Pharmaceutical Technology January 2012 PharmTech .com

In the quest for more targeted thera-pies and potentially more clinically efficacious drugs, bio/pharmaceutical

companies are increasing their research and product development in biologics. Although the majority of this work is fo-cused on monoclonal antibodies (mAbs) and recombinant proteins, progress is being made in specialized drug types. Antibody–drug conjugates (ADCs), which consist of a mAb chemically linked to a small-molecule therapeutic, are a niche class of drugs that offer promise, particu-larly as oncology drugs. In August 2011, FDA approved Adcetris (brentuximab vedotin), codeveloped by Seattle Genetics and Millennium Pharmaceuticals (now part of Takeda Pharmaceutical), mak-ing it only the second ADC approved by FDA. With the approval of Adcetris, a drug for treating Hodgkins lymphoma

and systemic anaplastic large-cell lym-phoma and with a number of ADCs in clinical development, the key question is whether ADCs will be able to fill a role in biopharmaceutical development.

ADCs at workAdcetris consists of three parts: the chi-meric IgG1 antibody cAC10, specific for human CD30, the microtubule-disrupting agent monomethyl auristatin E (MMAE), and a protease-cleavable linker that co-valently attaches MMAE to cAC10 (1). Before the approval of Adcetris this year, the only other ADC approved by FDA was Mylotarg (gemtuzumab ozo-gamicin), approved more than 10 years ago in 2000. The drug, an anti-CD33 mAb conjugated to the cytotoxin ca-licheamicin, was developed by Wyeth (now part of Pfizer) and was granted

accelerated approval in 2000 but was voluntarily withdrawn by Pfizer in 2010 because a required Phase III trial failed to demonstrate a survival advantage for Mylotarg plus chemotherapy compared with chemotherapy alone. Despite this setback, there are several ADCs cur-rently in development, with more than 15 in Phase I development and several compounds from Roche and Pfizer in late-stage clinical trials. In the decade that has elapsed between the first ADC approval and the second, advances in the understanding of cancer biology, lessons learned from the development of mAbs as therapeutics, and better methods for linking small molecules to mAbs have coalesced to advance ADCs into the forefront of new therapies.

The most active area of development for this class of therapeutics has been oncology, where a mAb serves to target the therapy to cancer cells while a po-tent small-molecule chemotherapeutic provides the cell-killing efficacy. Both mAbs and small-molecule chemothera-peutics are used individually as cancer therapies, but an ADC is designed to overcome the limitations of each. MAbs are highly specific, but as therapeutics have demonstrated only modest ef-ficacy and often are used in combina-tion with a conventional chemotherapy. Chemotherapeutics are highly toxic, but nonspecific, and so suffer from poor side-effect profiles and dose-limiting toxicities. In combination, the ADC serves to keep the chemotherapuetic bound until it reaches the cancer cell, thereby limiting its ability to interact with nontargeted tissues and therefore limiting nonspecific toxicity (2).

The concept of an ADC is not a new one, but creating a clinically success-ful one has been challenging. For the therapeutic to work well, each of the parts—the antibody, the toxin, and the linker that holds them together—must be carefully considered.

Choosing the right antibodyIn general, mAbs as therapeutics are selected to have high affinity for the targeted antigen and high selectivity. Other desirable properties in an anti-

Antibody-Drug ConjugatesLooking Ahead to an Emerging Class of Biotherapeutic

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Cover Story: Antibody-drug conjugates

The cell-killing ability of a cytotoxin is joined with the specificity of a monoclonal antibody to produce the next generation of anticancer therapeutics. Creating a successful antibody-drug conjugate requires careful selection of the drug, antibody, and linker.

44 Pharmaceutical Technology January 2012 PharmTech .com

Cover Story: Antibody-drug conjugates

body include long circulation times, immune-effector functions, and tumor-suppressing activity (2). When choos-ing the antigen, it is important that it be expressed at high levels in the tissue of interest to maximize the amount of ADC bound by the tumor, but at low levels elsewhere in the body to mini-mize off-target toxicity. Moreover, it is thought that internalization of the ADC is important for its effectiveness. Many of the chemical-linking strategies used to construct ADCs rely on conditions found inside a cell, either in the cyto-plasm or in the lysosome, to release the active agent (3).

In some instances, developers have been able to leverage experience gained through the development of mAb ther-apies to create their ADC. Trastuzumab emtansine (T-DM1) is an ADC in Phase III, which combines trastuzumab, (Her-ceptin), which targets human epidermal growth factor receptor 2 (HER2) receptors in breast and stomach cancer, with a may-tansine derivative DM1, a small-molecule cytotoxin that binds to tubulin to prevent microtubule formation, through a nonre-ducible bis-maleimido-trixyethylene gly-col linker (4). Trastuzumab was developed by Genentech (now part of Roche) and was approved by FDA in 1998 for use in women with metastatic breast cancer who have tumors that overexpress the HER2 protein. The maytansine derivative DM1 and linking technology were developed by ImmunoGen. In the case of the ADC trastuzumab emtansine, developers were able to use a target that had already been validated and a well-characterized anti-body with a known safety and efficacy profile as the starting point for an ADC.

Choosing the right cytotoxic small moleculeThe earliest versions of ADCs used stand-alone chemotherapeutics such as doxo-rubicin, 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).

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 in-terfere with tubulin polymerization. Ca-licheamicin, used in Mylotarg and in Pfiz-er’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. Duocar-mycins, isolated originally from Strepto-myces bacteria, are DNA minor-groove binding alkylating agents (2). Fully syn-thetic 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 repre-sented by two major classes: maytans-inoids and auristatins. Maytansinoids are deriviatives of maytansine, a natu-ral product originally isolated from the shrub Maytenus serrata. ImmunoGen has focused on development of this class of cytotoxic small molecules and associ-ated linker technologies and has been dev-loping maytansinoid ADC compounds singularly and in partnership with other companies. In addition to trastuzumab emtansine, which is being codeveloped by Roche and ImmunoGen, another ex-ample of a maytansinoid ADC being de-veloped 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 auricu-laria. Like the maytansinoids, auristatins are microtubule disruptors. Millennium and Seattle Genetics’ ADC Adcetris is a conjugate of an anti-CD30 mAb to mono-methyl auristatin E (MMAE). Seattle Genetics focuses on the development of auristatin-conjugated ADCs, using the auristatins MMAE and monomethyl au-ristatin F (MMAF) and proprietary linkers.

Choosing the right linkerDeveloping 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 manufactur-ing and their application has certainly contributed perhaps the most in mov-ing 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 re-sulted 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 pro-files similar to that of an unconjugated chemotherapeutic. The current genera-tion of linkers is more resistant to deg-radation 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 in-side 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 cata-bolic 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. Impor-tantly, 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, includ-ing mAbs, that are available for chemi-cal 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 het-

46 Pharmaceutical Technology January 2012 PharmTech .com

erogeneous mixture of conjugated spe-cies, but the proportion of each species in the mixture is reproducible from batch-to-batch and quantifiable.

Putting it all togetherProducing the ADC requires both biologic-based and small-molecule manufacturing. “One of the biggest challenges in manufacturing ADCs is controlling all the components that go into the final conjugation step,” says Boldt. “Namely, the three main com-ponents that make up an ADC (e.g., antibody, linker, and payload) are all manufactured in very different ways. For example, it is not uncommon for these components to be manufactured by synthetic chemistry and mamma-lian cell culture. Thus, there presents a challenge in ensuring all these compo-nents have been manufactured under cGMP, and subsequently bringing them all together to generate the final ADC under cGMP, as well.”

The biologics portion of the ADC and the high-potency API require very different handling methods, and man-ufacturers must make sure that han-dling requirements for both are met. “It is imperative that manufacturers emphasize the protection of the prod-uct from workers as well as the protec-tion of workers from the product,” says Jason Brady, head of business develop-ment, conjugates and cytotoxics at the CMO Lonza. Clinical ADC manufac-turing is executed in an aseptic bio-logical manufacturing environment to protect the product from contamina-tion, explains Brady. Once conjugated with the high-potency API (which is manufactured in a high-containment environment), the resulting ADC also is handled under high-containment conditions. The level of containment is determined by occupational exposure limits for the high-potency API and resulting ADC. The environment must provide manufacturing personnel with isolation from cytotoxic chemicals in the occupational exposure range of 5 ng/m3 of air. Also important is that facility de-sign includes design of equipment and process contact surfaces that permit

Although antibody-drug conjugates (ADCs) offer promise for delivering a drug payload—often a cytotoxic small molecule—with greater specificity through its attachment to a monoclonal antibody, one challenge is to create the link between the antibody and the drug molecule that remains stable until reaching the target cell but that does not affect the mechanism of action of the cytotoxic agent. Meeting both needs has presented a stumbling block for several ADCs in development. In these cases, inappropriate drug choice or unstable linking technologies have resulted in clinical-trial failures (1).

One company active in ADC linker technology is Synthon through its acquisition in June 2011 of Syntarga, a company specializing in antibody payload chemistries. “We have a new family of duocarmycin derivatives—our warhead molecules—that we link to antibodies,” says Vincent de Groot, former CEO of Syntarga and vice-president of ADCs at Synthon. Duocarmycins are small-molecule DNA minor groove binding alkylating agents with potency in the subnanomolar to picomolar range, according to the company.

While this class of drug has been around for some time, says de Groot, Phase II clinical trials using the unconjugated compound proved to be too potent to offer a therapeutic window with adequate safety. However, given its mechanism of action—interacting with DNA—duocarmycin can kill tumor cells in all phases of the cell cycle, not just in the mitosis phase. Therefore, it offers great potential in treating solid tumors, where cells are dividing slowly or not at all, providing its cytotoxicity en route to the target cell can be limited, according to de Groot.

There are two important requirements for ADC linkers: stability in the blood and ADC lability inside the target cell for release of the active species (2). Synthon has three linker chemistries; its SpaceLink technology reversibly links the drug by means of a linear releasable linker, and the MultiLink technology allows linkage of multiple drugs to the antibody. The company’s AbLoad technology offers a synthetic approach to introduce the chemical group that reacts with the antibody (i.e., Ab-reactive group) in the final step of the linker-drug construct synthesis. The key to Synthon’s SpaceLink technology is that the linker molecule can reversibly bind to a drug molecule’s hydroxyl group, which is particularly

complementary to the duocarmycin class of drugs because the hydroxyl group is an essential part of a precursor molecule that rearranges into the active species spontaneously.

Synthon’s chief scientific officer Ian Anderson provides the following example. “The antibody directs the ADC to the cell of interest, for example an anti-HER2 would go to HER2 on the breast-cancer cell, where it would then be internalized as the ADC. There is then a lysosomal protease cleavage within the cell to liberate the duocarmycin moiety. The drug, once cleaved, spontaneously turns into the active species.” Essentially, the linker–drug undergoes spontaneous electronic cascade self-elimination followed by spontaneous cyclization elimination and finally spontaneous rearrangement into the active species, at which point duocarmycin is free to bind to and alkylate DNA and thus exerting its cytotoxic effect.

The next part of the conjugation involves attaching the linker–drug molecule to the antibody, and there are two main methods, according to de Groot. The first method is to attach the linker–drug molecule to free cysteine residues after antibody reduction, and the second method is to attach the linker–drug molecule to lysine residues. Lysines are positioned throughout the antibody molecule, so there is little control over where the linker–drug positions itself, but if cysteine attachment distribution can be narrowed, the number of species variants produced can be controlled. Both approaches are being pursued by the industry according to de Groot.

Looking ahead, the next generation of ADCs will likely use site-directed coupling of the linker–drug to the antibody, which is an area of active ADC research, says Anderson. Having more control over the conjugation process, through the use of nonnatural amino acids, for example, will enable pharmaceutical companies to develop better-characterized, more homogenous ADCs, and the potential for increased efficacy and safety.

References1. S. Webb, “Pharma Interest Surges in Anti-body Drug Conjugates,” Nat. Biotechnol. online, DOI:10.1038/nbt0411-297, Apr. 8, 2011.2. V. de Groot, “Novel ADC Linker–Drug Tech-nology for Next Generation ADC Products,” pre-sented at Peptalk–Protein Science Week (Cam-bridge Health Institute, San Diego, Jan. 10–14, 2011).

The importance of linker technology by Rich Whitworth

Cover Story: antibody-drug conjugates

Pharmaceutical Technology January 2012 47

clean-in-place and steam-in-place to remove minute traces of residual drug contamination during both interbatch and product changeover cleaning, ac-cording to Brady.

Room for improvementAs ADCs advance in the clinical pipeline so does the technology to manufacture ADCs to control certain product and process conditions. “New technology that can limit the hetero-geneity of ADC products is something that will be important in the future,” says Brady. “ADCs made via current technologies are heterogenous mix-tures. Heterogeneity can be controlled and measured by robust and repro-ducible manufacturing processes and proper analytics, but new technolo-gies will likely emerge to inf luence and improve ADC manufacturing,” he explains. Some fraction of the finished drug product consists of unconjugated antibody. The remaining portion of the finished drug product contains conju-gated antibody with a variable number of the cytotoxic small molecules conju-gated at different sites on the antibody. Controlling the number and location of cytotoxic molecules conjugated to the antibody is being pursued as a means to create a more uniform product and as a way of being able to explore structure–function relationships by varying the site of attachment of the cyotoxin.

One strategy for controlling the site of attachment has been developed by researchers from Genentech, a member of the Roche Group. They describe pre-cise site-specific conjugation of human IgG1 to MMAE by replacing Ala114 at the junction of the CH1 and the vari-able heavy-chain domain with cysteine to create an engineered antibody called a THIOMAB. This site was chosen be-cause it does not participate in antigen binding or effector functions. Accord-ing to Jagath Reddy Junutula, senior scientist at Genentech, the process for creating a THIOMAB differs only slightly from that of a conventional mAb. The THIOMAB is subjected to partial reduction to remove cysteine and glutathione adducts. The partial

reduction also breaks interchain disul-fide bonds, which must be reformed by a reoxidation step. After reoxidation, the engineered cysteine residues are available for conjugation.

Genentech researchers used this process to conjugate MMAE to a THI-OMAB version of an antibody against MU16, a cell-surface protein expressed in ovarian cancer cells. The THIOMAB conjugate was shown to be homogenous and to contain a single drug molecule attached to each heavy chain, for a total of two MMAE molecules per ADC. The THIOMAB–MUC16 was found to have comparable efficacy to a conventionally produced ADC and to be better tolerated in two preclinical species (7). In a subse-quent study, a different cytotoxin, DM1, was conjugated to a THIOMAB version of trastuzumab. Results were similar, with the THIOMAB T–DM1 displaying comparable efficacy and better tolerabil-ity in preclinical species than its conven-tionally produced counterpart (8).

According to Junutula, the reoxidation step is the only thing that distinguishes manufacture of a THIOMAB drug con-jugate from that of a conventional ADC. “We can make up to grams scale without any difficulty. And the results are huge—you have a homogenously conjugated cy-totoxic drug to the antibody,” he says.

While the THIOMAB uses the substitution of one amino acid for another to control the site of conjuga-tion, several groups are working to-ward incorporating nonnatural amino acids into the mAb for to control the site of conjugation and also to provide an expanded repertoire of functional groups that could be used for linker chemistry. The biopharmaceutical company Ambrx has developed ex-pression systems in E. coli, yeast, and Chinese hamster ovary (CHO) cells that can be used for such substitu-tions and which can be scaled up to volumes required for commercial manufacturing. Ambrx’s expression systems contain engineered transfer RNAs that will read through a stop codon called amber, as well as engi-neered tRNA synthetases that will aminoacylate the orthoganal tRNA

with an Ambrx nonnatural amino acid. The expression system will insert a nonnatural amino acid whenever the amber stop codon is encountered (9).

Sutro Biopharma, a provider of protein-synthesis technology, also is developing a platform for introducing nonnatural amino acids, but in a cell-free translation system that is reported to be scalable to commercial production volumes (10). The system is based on an extract of E. coli, and because it is an open system, the tRNA charged with a nonnatural amino acid can be added directly to the reaction mix as a reagent.

Looking aheadThe future of ADCs in the biophar-maceutical market will ultimately de-pend on their clinical success. Com-panies and researchers are seeking to meet that challenge by optimizing the selection of all the components in the ADC—the antibody, linker, and cytotoxin—and successfully combin-ing manufacturing techniques for both high- potency APIs and biolog-ics. ADCs are sometimes described as armed antibodies, and their cytotoxic components as warheads. Whether ADCs will prove to be an effective weapon against cancer or other dis-eases has yet to be seen as more are tested in the clinic.

References 1. FDA, “Label for Adcetris, BLA 125338,”

FDA Approved Drug Products: Drugs@FDA, accessed Dec. 20, 2011.

2. V.S. Goldmacher and Y.V. Kovtun, Ther. Deliv. 2 (3), 397–416 (2011).

3. F. Dosio, P. Brusa and L. Cattel, Toxins 3, 848–883 (2011).

4. H.A. Burris, Expert Opin. Biol. Ther. 11 (6), 807–819 (2011).

5. A. Beck et al., Discov. Med. 10 (53), 329–359 (2010).

6. S.V. Govindan and DM Goldenberg, Scientific World Journal 10, 2070–2089 (2010).

7. Junutula et al., Nat. Biotechnol. 26 (8), 925-932 (2008).

8. Junutula et al., Clin. Canc. Res. 16, 4769–4778 (2010).

9. A. Ritter Pharm. Tech. 35 (6), 36–39 (2011).

10. Zawada et al., Biotech. Bioeng. 108 (7), 1570–1578 (2011). PT