Oncology Advances

Potent Promise

By Donald Bergstrom, Research into the use of antibody-drug conjugates as a means of targeted Timothy Lowinger and oncology treatment is off to a successful start, with both Adcetris and Peter Park at Mersana Therapeutics Kadcyla gaining approval. This should lead to further opportunities in developing this approach to target even more patients

For three decades, drug developers (gemtuzumab ozogamicin) was mechanistically similar to the vinca have attempted to combine the granted accelerated approval alkaloid class of chemotherapies, specificity of a monoclonal by the FDA for the treatment of but are several hundred-fold more antibody for tumour cells with acute myelogenous leukaemia in potent. In the case of Adcetris, the potency of drugs that kill elderly patients. Although Mylotarg auristatin is linked to cysteine rapidly dividing cancer cells was able to demonstrate clinical residues on an anti-CD30 antibody (see Figure 1). Within the past responses, the drug payload was by an enzyme-cleavable linker three years, two antibody-drug relatively quickly released from that is stable in plasma, but is conjugates (ADCs) – Adcetris® the antibody in circulation, leading cleaved to release the drug upon (brentuximab vedotin) and to a narrow therapeutic index. internalisation into a cell. Adcetris Kadcyla® (ado-trastuzumab As a result, the product was is approved in two types of emtansine) – have been approved, voluntarily withdrawn from the lymphoma where the tumour cells validating the approach. These market in 2010, after confirmatory express high levels of CD30. forerunners have highlighted trials revealed no significant the tremendous potential of clinical benefit, with an excess Genentech licensed technology ADCs, as well as opportunities number of deaths in Mylotarg- from ImmunoGen to develop to further improve this promising treated patients compared to Kadcyla. As with Adcetris, the therapeutic approach. chemotherapy control. drug payload here is a tubulin binder, of the maytansinoid class. First Steps Reaching the Market The maytansinoids are several hundred-fold more potent as The early ADCs employed The early experiences with ADCs anti-cancer drugs, compared to cytotoxic chemotherapies that highlighted the need for highly chemotherapies approved for were approved for systemic potent drug payloads, combined systemic administration. In Kadcyla, administration for the treatment with stable linkers, to achieve maytansine is conjugated to lysine of cancer, such as doxorubicin suitable therapeutic index. Two residues on the anti-HER2 antibody, and methotrexate. While antibody companies – Seattle Genetics and trastuzumab, using a non-cleavable conjugation of these drugs allowed ImmunoGen – have emerged with linker. This linker only releases the them to be tumour-targeted, ADC platforms that have addressed drug when the ADC is internalised they were not sufficiently potent these limitations, and they have led into a cell and the antibody is to provide clinical benefit, and to the development of currently subsequently degraded. Kadcyla development was discontinued. approved products – Adcetris and is approved for patients with Kadcyla –as well as to more than advanced breast cancer carrying Wyeth attempted 30 ADCs in clinical development. amplification of the HER2 gene Keywords to address this issue (HER2-positive breast cancer). by conjugating Adcetris, from Seattle Antibody-drug conjugates highly potent Genetics, uses a payload from New Opportunities DNA damaging agents DNA-damaging the auristatin class. These are Tubulin binder agents called antimitotic agents that bind to Adcetris and Kadcyla represent calicheamicins. tubulin and block microtubule significant advances in the Drug-to-antibody ratio In 2000, the polymerisation, leading to ability to target highly potent Potent drug payloads drug Mylotarg® cell death. The auristatins are chemotherapies specifically to

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cancer cells. However, they also highlight opportunities to further MTD improve ADCs to achieve greater clinical benefit.

On average, there are three to four chemotherapy drugs per antibody in these two agents, which exist Free ADC as heterogeneous mixtures of drug antibodies with a distribution of drug loads from zero to eight, and above. While higher drug loads would be expected to result in even more efficient drug delivery to tumours, it has been shown MED that an increase in drug load above four drugs per antibody leads to poor ADC exposure in vivo, decreased anti-tumour efficacy benefit of anti-tubulin-derived more defined, site-specific manner. Figure 1: The goal of and increased toxicity (1). ADCs, due to either acquired Genentech has developed a site- an ADC is to improve the therapeutic index resistance or intrinsic resistance. specific conjugation technology, by delivering the Adcetris and Kadcyla have For example, the tubulin binder THIOMAB, which introduces drug specifically to the tumour while achieved success in patient class is largely inactive in colorectal cysteine residues in the constant sparing normal populations with very high cancer, potentially restricting the regions of an antibody. Using the tissues, resulting in levels of target antigen utility of tubulin-binder ADCs for same linker and auristatin payload a potential increase in maximum tolerated expression. However, among colorectal cancer targets. found in brentuximab vedotin, dose (MTD) and the ADCs currently in clinical the company has optimised the a decrease in the development, there have been Diverse technologies and conjugation conditions for the minimally efficacious dose (MED) variable levels of clinical activity approaches are currently engineered antibody to produce a reported, especially for solid in development to address THIOMAB-drug conjugate (TDC) (2). tumour targets. One of the most these opportunities to further In the TDC, the drug payload is advanced solid tumour ADCs improve ADCs. conjugated to the engineered – an anti-CD56 maytansinoid cysteines, without the disruption from ImmunoGen – recently Site-Specific Technologies of interchain disulfide bonds reported negative results in a trial which occurs with the typical with small-cell lung cancer. One As previously discussed, while cysteine conjugation approach. hypothesis for the mixed results Kadcyla and Adcetris are the is that even with highly potent products of different conjugation The site-specific TDC displays payloads, ADCs with three to technologies, they have a similar a narrow distribution of drug four drugs per antibody are not average drug-to-antibody (DAR) conjugate species, with an average sufficiently potent to drive clinical ratio and, in reality, represent DAR of 1.8. When the TDC was benefit for antigens with lower mixtures of species with a compared with a standard ADC tumour expression. range of zero to more than eight (3.1 DAR), generated by inter- drugs per antibody. Higher drug chain cysteine conjugation, Both ADCs use natural product- loaded species are more rapidly the TDC showed similar anti- derived drug payloads from the cleared, and are hypothesised to tumour efficacy, but when the tubulin binder class, which interfere contribute to ADC toxicity, while toxicities of both conjugates were with microtubule formation. not improving anti-tumour efficacy. compared in rats and monkeys, This mechanism has the benefit the more homogeneous TDC was of preferentially killing rapidly To remove the undesirable higher better tolerated than the more proliferating cells, such as those DAR species and produce a more heterogeneous ADC. On balance, in the tumour, while having little homogeneous drug product, it was found that TDC improved the to no effect on non-dividing cells. various antibody technologies are therapeutic index by approximately However, the anti-tubulin class of being developed to conjugate the two-fold compared with the ADC. agents could also limit the clinical drug payload to an antibody in a It was also demonstrated that

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By generating a number of antibodies with specific Fleximer conjugation sites at different positions along the protein sequence, more homogenous ADCs can be prepared, and the influence of the conjugation site

Payload Very high aqueous solubility on parameters such as stability, Organic solvent Isolated, characterised and stored long-term efficacy and tolerability can be evaluated. Furthermore, the nnAAs provide the opportunity to Antibody explore alternative bioconjugation chemistries and linkers, beyond those typically used for cysteine Multiple standard and site-specific and lysine bioconjugation. bioconjugation chemistries available Lysine, cysteine or site-specific/engineered Water New Drug Payloads

One area of increasing interest in the ADC field is the identification payload – is currently being and validation of drugs beyond Figure 2: A Fleximer® ADC is prepared in a two-step process. First, the payload of interest is conjugated to the Fleximer polymer in an organic evaluated in a Phase 1 trial for auristatins and maytansines that solvent optimally suited to the payload class. The resulting highly water- the treatment of acute myeloid can be effectively incorporated soluble Fleximer-drug conjugate can then be readily conjugated to the antibody via a variety of conjugation approaches including cysteine, lysine leukaemia (AML). into ADCs. Because of the typical or site-specific/engineered limited capacity of three to four In addition to the site-specific drugs per antibody when directly the variation of the specific site cysteine incorporation approach, conjugated, there has been a for conjugation could impact the companies such as Ambrx, focus on identifying more potent stability and characteristics of Sutro and Redwood Biosciences drugs, as well as drugs that provide the resultant ADC – not only the are using non-natural amino alternative mechanisms of action number, but the location of sites acid (nnAA) technology with – including different resistance could influence the performance orthogonal conjugation profiles – in comparison to the anti- of the conjugate. chemistries to facilitate site- tubulin agents widely used to date. specific ADC approaches. While the Taking it Further details of the specific approaches Pyrrolobenzodiazepines differ, the respective technologies One such class of drugs While Genentech has not provide alternatives to producing concerns the DNA-damaging publically disclosed clinical data antibodies which incorporate agents represented by Spirogen’s with a TDC, Seattle Genetics nnAAs into the antibody, at specific (recently acquired by MedImmune) recently announced that locations in the protein sequence. pyrrolobenzodiazepines (PBDs), SGN-CD33A – a site-specific The nnAAs so far incorporated which are highly cytotoxic minor anti-CD33 ADC designed using have reactive groups not found groove DNA cross-linking agents similarly engineered cysteine in natural amino acids, and can derived from the anthramycin methods and conjugated with be used to conjugate the drug class of natural products. In vitro, a pyrrolobenzodiazepine dimer payload in a chemospecific manner. these drugs typically demonstrate

Because of the typical limited capacity of three to four drugs per antibody when directly conjugated, there has been a focus on identifying more potent drugs, as well as drugs that provide alternative mechanisms of action, in comparison to the anti-tubulin agents widely used to date

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BT474 – High expressing HER-2 +++ Model 1,200 500,000 copies of HER-2/cell Vehicle

1,000 Trastuzumab- Dolaflexin single ) 3 800 dose 5mg/kg

10/10 tumour-free 600 survivors

400 Tumour volume (mm Tumour

200

0 0 10 20 30 40 50 60

Single intravenous Days injection day 1

Figure 3: A single dose of trastuzumab- IC50 values in the low to mid in preclinical models, including as safety data in cynomolgus Dolaflexin in a mouse picomolar range in a variety of cell multi-drug resistant models, and monkeys. Based on these results, model of HER2+ types, and unlike the anti-tubulin was advanced into clinical trials it plans to initiate clinical trials breast cancer results in complete and agents, they can induce cell death in towards the end of 2013. with a trastuzumab biosimilar – durable tumour both dividing and non-dividing cells. a duocarmycin ADC – in the regressions Duocarmycins latter half of 2014. The most advanced ADC A second class of DNA-damaging incorporating a PBD is that of agents includes duocarmycins, As duocarmycins are highly Seattle Genetics, which partnered which are analogs of the natural hydrophobic molecules with with Spirogen in the development product, CC-1065. Like the PBDs, a propensity to cause protein of this payload class (3). Seattle these molecules also irreversibly aggregation, companies such as Genetics explored the creation bind to DNA in the minor groove, Nerviano Medical Sciences have of ADCs targeted to the myeloid resulting in highly potent cell sought to identify more aqueous differentiation antigen CD33 for killing of both dividing and non- soluble analogs better suited the treatment of AML. As reported dividing cells. A Phase 1 clinical trial for incorporation into ADCs. By by the American Association for of the duocarmycin ADC MDX-1203, including undisclosed solubilising Cancer Research in 2013, initial targeted to the CD70 antigen, was moieties into their derivatives, efforts to conjugate three to four completed in November 2012 they have demonstrated the PBDs per antibody via reduced by Medarex/Bristol-Myers Squibb preparation of ADCs with interchain disulfides led to in patients with advanced/ trastuzumab, which lacked problems with aggregation, recurrent renal cell carcinoma or aggregation, and demonstrated due to the highly hydrophobic relapsed/refractory non-Hodgkin’s efficacy in HER-2 positive models. nature of the PBD payload. lymphoma, although results have However, by utilising a site- not been reported to date. Amanitin specific, engineered antibody Another highly potent payload approach and conjugating two Synthon has also reported class derived from a natural PBDs per antibody, the issue of preclinical results with a HER-2 product is the RNA polymerase aggregation could be addressed. targeted duocarmycin ADC, which inhibitor, amanitin, which is The resulting ADC showed showed efficacy in a number of also being investigated for high potency and tolerability HER-2 positive models, as well incorporation into ADCs.

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An alternative approach to ADCs, designed to address and overcome several of the limitations inherent in directly linking a payload to an antibody, is currently in development

This bicyclic octapeptide, hydrophobic. Heidelberg Pharma with no aggregation. When which is isolated from the has reported the incorporation evaluated in vivo, a single dose extremely toxic Green Deathcap of amanitin into ADCs targeting of the trastuzumab-Dolaflexin mushroom, interferes with the the prostate-specific membrane ADC resulted in complete and cellular transcription machinery, antigen, and demonstrated sustained tumour regressions resulting in cell death of both efficacy both in vitro and in vivo. (see Figure 3, page 19). dividing and quiescent cells at Furthermore, in contrast to very low concentrations. Unlike Alternative Approaches the findings with conventional the majority of payloads utilised ADCs, the trastuzumab-Dolaflexin for ADCs, amanitin is not highly Mersana Therapeutics is ADC maintained excellent developing an alternative pharmacokinetic properties approach to ADCs, designed to and tolerability, despite the Dr Donald Bergstrom is Chief Medical Officer of Mersana. He was previously address and overcome several of significantly higher drug load. Associate Vice President and Global the limitations inherent in directly Head of Translational and Experimental linking a payload to an antibody. Summary Medicine at Sanofi Oncology and also The approach relies on Fleximer® spent six years at Merck Research Labs – a highly water-soluble polymer Kadcyla and Adcetris have where he held senior roles in the clinical molecular derived from naturally occurring established the viability of ADCs profiling, oncology clinical research and experimental carbohydrates. The specific as a therapeutic modality for the medicine oncology groups. Donald completed his MD properties of Fleximer allow for treatment of cancer. Building on degree at the University of Washington, and his PhD a wide variety of drug payload this foundation, next-generation and postdoctoral training at the Fred Hutchinson Cancer classes to be readily conjugated technologies hold the promise of Research Center. Email: [email protected] to the polymer with high drug realising ADCs that are even more loading, while maintaining efficacious, better tolerated, and Dr Timothy Lowinger, Chief Scientific Officer of Mersana, is currently excellent physicochemical relevant for a broader population responsible for all drug discovery properties. These resulting of patients. and chemistry manufacturing control Fleximer-drug conjugates, which activities. Prior to this, he held a number can be fully isolated, characterised References of positions at Bayer Pharmaceuticals and stored for prolonged periods, 1. Hamblett et al, Effects where he contributed to the discovery of more than can be efficiently conjugated of drug loading on the 15 preclinical and clinical candidates in a variety of to antibodies under aqueous anti tumour activity of a indications. Timothy has a BSc Hons in Chemistry and a conditions via lysine, cysteine monoclonal antibody drug PhD in Organic Chemistry from the University of British or site-specific bioconjugation conjugate, Clin Cancer Res Columbia. Email: [email protected] methods (see Figure 2, page 18). 10: pp7,063-7,070, 2004 2. Junutula et al, Site-specific Dr Peter Park, Vice President of Biology, brings to Mersana significant Mersana has also disclosed the conjugation of a cytotoxic experience in the discovery and development of an auristatin- drug to an antibody development of ADC therapies. Fleximer drug conjugation improves the therapeutic Prior to joining Mersana in 2013, he platform, termed DolaflexinTM, index, Nat Biotechnol 26: was a co-founder and Chief Executive and demonstrated its advantages pp925-932, 2008 Officer of Habgen – a start-up focused on antibody in the context of a trastuzumab- 3. Sutherland et al, SGN-CD33A: therapeutics – and also worked at ImmunoGen for Dolaflexin ADC. By utilising a A novel CD33-targeting 10 years. Peter is the author on many patents and has cysteine bioconjugation approach, antibody-drug conjugate contributed to numerous scientific publications. He a trastuzumab-Dolaflexin ADC using a pyrrolobenzodiazepine earned his PhD and BS in Biology from Massachusetts with a DAR of 20 was prepared, dimer is active in models of Institute of Technology. Email: [email protected] and shown to maintain excellent drug-resistant AML, Blood physicochemical properties 122: pp1,455-1,463, 2013

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