Antibody drug conjugates are complex delivery systems for selective delivery of cytotoxic payloads. Yet despite the potential for this therapeutic platform and hundreds of clinical studies only three ADCs have been approved by regulatory agencies and only two are currently marketed. The difficulties for this class of compounds are both categorized and explored in this review, and potential solutions identified.

introduction

Antibody drug conjugates (ADC's), composed of an antibody, linker, and cytotoxic agent, are one of the most complex drug platforms in the oncology armamentarium [1]. They are a payload delivery system with major variables influencing their success including: (i) the rate of internalization of the payload; (ii) the expression of the target antigen on the tumor and normal tissues with the implications for both patient selection as well as therapeutic index; (iii) the linker chemistry and, implicit within the choice of chemistry, the extracellular as well as intracellular stability; and (iv) the selection of payload for the tumor indication [2]. It is therefore not surprising that we have only two commercially available agents despite over one hundred clinical trials evaluating this platform. In this review we are going to look at some of the lessons learned over the years to include the challenges with (i) tumor-associated versus tumor-specific antigens and consequent unintended bio-distribution and toxicity; (ii) current preclinical murine models; (iii); microtubule-destabilizing payloads; (iv) off-target toxicities of payloads; and (v) the lessons that curative chemotherapy from multi-agent combinations. The goal is to outline the challenges over 20 years of clinical development and help identify why this platform's potential is still not fully realized while also offering suggestions for future directions.

background

The modern era of ADC's began with the promising data from a study on BR-96 doxorubicin [3]. In experimental murine models bearing tumors from human breast cancer cell lines BR-96 doxorubicin demonstrated significant antitumor activity including complete eradication of the tumor and long-term cures. BR-96 doxorubicin's target antigen was the LewisY antigen, the antibody was an IgG1 with a cleavable linker to a doxorubicin payload, and a drug to antibody ratio that approximated of 4 [3].

The preclinical data from the aforementioned study generated considerable excitement for the use of cytotoxic chemotherapy payloads in the ADC field as previous attempts of employed payloads, such as ricin, and other biologic exotoxins that were associated with infusion reactions, vascular leak syndrome, and the development of neutralizing antibodies [48]. The concept that a platform could selectively deliver a known cytotoxic to tumor cells with increased activity and reduced normal tissue toxicity had considerable appeal and prompted human clinical trials [9]. Doxorubicin was selected as a suitable payload due to the activity of this agent against adjuvant and advanced breast cancer. However, systemically administered doxorubicin also possessed significant toxicities of neutropenia, alopecia, mucositis, and a cumulative dose-dependent risk of myocardial injury and congestive heart failure [10]. The avoidance of these toxicities with the promise of high antitumor activity from a doxorubicin payload was intriguing. However, the balance of ‘on-target’ toxicity between normal and tumor cells has largely been responsible for the failure of this, and many ADC's, despite promising preclinical data.

  • Lesson 1: Downside of tumor associated rather than tumor-specific antigens: unintended bio-distribution, toxicity, and implications for improving distribution

In an optimal situation, all normal cells that express the target antigen would be eliminated and the expression of the target antigen would largely be confined to the tumor population; making the target antigens tumor-specific [11]. However, most tumor antigens are not restricted to tumor cells alone resulting in the target antigens being tumor-associated and not tumor-specific [11]. The relative expression of the target antigen on the tumor, and also on normal tissues, hence becomes a critical factor in the therapeutic index of a particular ADC. This portends that lineage ablation by an ADC is not feasible in all but certain hematologic settings. Therefore, the relative bio-distribution of ADCs' to normal versus tumor tissue is both a critical factor in activity and toxicity. Since delivery of the payload from the ADC will occur to antigens whether they are expressed in the tumor or normal cells, the relative expression and distribution of this target antigen is critically important. An example would be a target antigen that is expressed strongly in a tumor population (1 × 1010 tumor cells) versus weak expression in the far larger number of normal cells within the gastrointestinal tract (1 × 1012 normal cells). The total body antigen expression thus may favor ADC distribution and binding to the normal tissue, rather than the tumor.

The clinical development of BR-96 doxorubicin represents an important example of this lesson and was complicated by significant on-target toxicities that differed considerably from what was seen with ‘free’ doxorubicin administered systemically [12]. In the Phase I, Study of BR-96 doxorubicin dose-limiting toxicities included nausea, vomiting, and hemorrhagic gastritis—the latter being dose limiting—and an upper endoscopy of the affected patients' gastric mucosa revealed significant inflammation and ulceration [13]. The underlying cause of the hemorrhagic gastritis ultimately was revealed to be a heretofore unrecognized expression of LewisY antigen on gastric mucosa cells. Attempts to ameliorate the nausea, vomiting, and gastritis, including the use of anti-emetics, antacids, and steroids, met with only limited success [13]. Antitumor activity was observed in this study including a partial response in a patient with metastatic breast cancer as well as a partial response in a patient with gastric carcinoma that prompted further development.

An important test with all antibody drug conjugates is whether the payload delivery of a cytotoxic using an ADC is superior, both in tolerability and antitumor activity, compared with cytotoxic chemotherapy alone. In the case of BR-96 doxorubicin, a randomized Phase II study was performed in metastatic breast cancer subjects [14]. Patients were treated with either BR-96 doxorubicin (700 mg/m2) or doxorubicin (75 mg/m2) alone. Once again, the adverse events of nausea, vomiting, and hematemesis were noted in patients treated with BR-96 Doxorubicin. Strikingly, the tolerability and antitumor activity was superior for the doxorubicin alone arm (1 CR, 3 PR) compared with BR-96 doxorubicin (1 PR). This randomized comparison is the most valid way to determine whether the antibody drug conjugate will live up to the expectations of greater antitumor activity with lower systemic toxicity. In these circumstances, one should always be prepared to evaluate, rigorously and objectively, whether the antibody drug conjugate meets this standard over the systemic chemotherapy alone. Furthermore, based on the activity in the original phase I study, as well as the apparent targeting of gastric mucosa, a single arm single agent study of BR-96 Doxorubicin was performed in metastatic gastric carcinoma, but it was inactive [15].

More extreme examples showing the potential consequences of unintended bio-distribution occurred with bivatuzumab mertansine, which targeted the CD44v6 antigen, and BAY794620, an ADC targeting CA9 antigen. In the first circumstance, the target antigen was expressed in the deep layers of skin and fatal exfoliate of skin toxicity was observed [16]. For BAY794620, CA9 expression within the gut resulted in gastrointestinal toxicity that led to death in two patients [17]. The striking conclusion from both of these agents was that mal-distribution of the high potency payload to normal tissues, even at low levels, can lead to tragic consequences.

  • Lesson 2: Current preclinical models fail us

Tumor-bearing murine models used to demonstrate preclinical antitumor activity are not adequate in predicting ADC's clinical activity and tolerability. The chief disadvantage is the target tumor antigen is not commonly expressed in the animal host murine tissues [18]. Because of this limitation, the ADC distributes exclusively to the tumor xenograft resulting in provocative antitumor activity and complete tumor regressions. Oftentimes ADC agents enter the clinic with strong preclinical activity that is sadly not observed in clinical studies; or on-target toxicity in normal tissue intervenes that restricts dose-escalation to levels not particularly active. For this platform to evolve successfully lessons from past ADC history and critical re-evaluation of preclinical and clinical results must be undertaken.

  • Lesson 3: The tyranny of the microtubule-destabilizing payload in ADC

The dilemma for early ADC's discovery and development was the limitation of linker chemistry to drug to antibody ratios (DAR) of 4 or less. This resulted in the selection of highly potent microtubule-destabilizing agents as payloads (sub-nanomolar IC50) to ensure adequate cell death with a limited number of payload molecules delivered. Unfortunately, the solubilizing antimicrotubule agents, such as the vinca alkaloids, maytansines, auristatins, and dolastatins have had a very limited spectrum of single agent antitumor activity clinically. Through a substantial number of clinical trials, over many decades, robust activity has only been seen in acute lymphocytic leukemia, Hodgkin's disease, and non-Hodgkin's lymphoma where the vincas became the standard of care in combination therapy [19]. In contrast, there have been rather modest response rates in solid tumors such as breast and lung cancer, and in most other solid tumors, such as prostate, colon, pancreatic, gastric, uterine, cervical, and hepatocellular cancer, these drugs are considered inactive [2026]. Based on hundreds of clinical trials, few tumors are intrinsically sensitive to solubilizing antimicrotubule agents, and yet the vast majority of ADC's utilize a solubilizing antimicrotubule agent such as maytansine and auristatin derivatives as the payload.

The flawed premise is that all mitotically active cells will be sensitive to a particular potent cytotoxic if delivered by an ADC. This neglects 50 years of experience with cytotoxic therapy where chemotherapy agents evolved for the treatment of certain indications based on the presence or absence of robust antitumor activity at concentrations that could be delivered. This ‘intrinsic sensitivity’ to particular agents was borne from thousands of clinical trials and explains why we treat, for example, small cell lung cancer and not breast cancer with etoposide. Although our current understanding of cytotoxic chemotherapy agents has not discerned which tumors are intrinsically sensitive to one class of cytotoxic versus another, we have enjoyed the benefit of significant clinical trial experience to know that antimicrotubule agents are not active in colon, and most other, gastrointestinal tumors.

Based upon this history, doubt should be cast upon the relevance of, and clinical application of, agents that demonstrate activity of maytansine and auristatin payload ADC's in preclinical models where antimicrotubule agents have no clinical activity. This is borne out clinically with mertansine-based ADC directed to CanAg, highly expressed in colon cancer, where no antitumor activity was observed [24]. Historically, despite many attempts, colon cancer has never been sensitive to antimicrotubule agents. Recent examples include SAR566658, an anti CA6 DM4 ADC that demonstrated no activity in pancreatic cancer despite high expression of the target antigen. [27]. The continued exploration of mertansine and auristatin payloads in tumor indications that are not intrinsically sensitive to antimicrotubule agents is therefore intellectually suspect, and reminds one of the quote from Abraham Maslow, ‘When all you have is a hammer – everything looks like a nail.’

  • Lesson 4: Dose-limiting off-target toxicities of the payload

Another challenge of ADC using solubilizing antimicrotubule agents, in particular DM4 and MMAF payloads, is off-target ocular toxicity [28]. The appearance of side-effects can be both acute as well as a cumulative toxicity. The constellation of signs and symptoms includes dry eyes, diminution of visual acuity, and under slit lamp assessment, corneal keratitis with marked corneal inclusion cysts [29]. Although reversible, it can be devastating with the most severe cases requiring corneal bandages. Attempts to mitigate this toxicity have included lubricating eye drops, alternate schedules of ADC administration (weekly), and dose reductions [30].

One can try to deconstruct the corneal toxicity to suggest that there may be a role for the payload, and linker, and not the target antigen (see Table 1). First, the toxicity is observed across multiple diverse target antigens suggesting that this is not related to on-target corneal ADC binding. Secondly, no keratitis was seen with cantuzumab mertansine using a DM1 payload. However, the linker was unstable and there was rapid dissolution of the antimicrotubule from the antibody. When the same payload (DM1) is used with a stable linker such as ado-trastuzumab mertansine, a minor incidence of keratitis is reported. Finally, when cantuzumab mertansine was modified with the new payload DM4 keratitis became the primary dose-limiting toxicity. Modified linker chemistry to abrogate this toxicity associated with mertansine-based-ADCs' is suggested by recent patent activity. To date, there does not appear to be a successful strategy that reduces the incidence without evidence of lowered dose and perhaps efficacy.

Table 1

Keratitis reported as an adverse event from ADCs across diverse antigen targets indicating that the etiology is the antimicrotubule payload

ADC name Antigen target Payload 
Cantuzumab mertansine CanAg DM4 
AVE 9633 CD33 DM4 
ABT 414 EGFR MMAF 
BAY-949343 Mesothelin DM4 
SAR566658 CA6 DM4 
SGN CD19A CD19 MMAF 
SAR3419 CD19 DM4 
ADC name Antigen target Payload 
Cantuzumab mertansine CanAg DM4 
AVE 9633 CD33 DM4 
ABT 414 EGFR MMAF 
BAY-949343 Mesothelin DM4 
SAR566658 CA6 DM4 
SGN CD19A CD19 MMAF 
SAR3419 CD19 DM4 

In addition, neutropenia, diarrhea, and hepatotoxicity have been seen with various ADC molecules [1, 31]. These are largely due to linker instability and represent off target toxicities. The appearance of cytotoxic payload toxicities defies the whole premise for ADC's being selective payload delivery systems, and implies that a comparator in clinical studies should be the cytotoxic agent alone.

  • Lesson 5: Curative chemotherapy came from multi-agent combinations

Although groundbreaking as the first ADC achieving regulatory approval in a solid tumor (Her2+ metastatic breast cancer) the proof of concept study of ado-trastuzumab mertansine in a molecularly selected patient population (HER2 amplified) had only a 30% response rate and no complete responses [32]. The absence of clinical complete responses in this disease, with an ideally selected patient population and cures in preclinical models, infers we are far from perfecting this platform. These unsurprising results reflect the historical lessons from single agent cytotoxic agent development that frequently failed to induce complete responses, and were certainly not curative [33, 34]. Historically, curative chemotherapy evolved through combinations of cytotoxic agents with non-overlapping toxicities and non-cross resistance mechanisms [3537].

  • Lessons Learned and a Reason for Optimism: New Linker Technology

Contemporary ADC chemistry limits the number of payload molecules on any antibody scaffold. However, new technologies entering the clinic such as the Fleximer® platform aim to substantially increase the number of payload molecules that can be added to the antibody creating several opportunities including: (i) greater delivery of the payload increasing antitumor activity; (ii) the possibility of a greater spectrum of cytotoxic agents since the higher DAR will permit lower potency of agents; and (iii) theoretically permitting combinations of agents to be added to same ADC molecule using site-specific conjugation. These opportunities, however, are not trivial issues. Increasing the number of payload molecules to an antibody can decrease stability, promote antibody aggregation, and paradoxically diminish antitumor activity [38, 39].

The first use of Fleximer®-based ADCs technology to enter the clinic in the next year is a polymer-based antibody-vinca drug trastuzumab conjugate named that permit stoichiometric ratios of 20:1 versus the 4:1 of antibody to drug with ado-trastuzumab emtansine. The polymer-based drug engages the hydrophilic nature of the polymer and may help counterbalance the hydrophobicity of higher DAR's [40]. The implications for this platform, if successful, may be enhanced antitumor activity of ADCs' and an expanded spectrum of cytotoxic agents that can be incorporated into the ADC strategy.

new payloads

As described previously, the solubilizing antimicrotubule agents limit the spectrum of antitumor activity. There are a number of new payloads, summarized in Table 2, that indicate innovative strategies that marry intrinsic chemosensitivity to target indication. Given the broad spectrum of cytotoxic, biologic, and targeted agents currently available this should propel creative thinking in targeted payload selection and delivery as well as reinforcing the concept of target delivery of payloads when systemic delivery is not currently feasible.

Table 2

Examples of novel payload agents for ADC technology

Payload Payload mechanism of action Company Patent # or reference 
Duocarmycin DNA minor groove alkylating agent ASANA Patent # WO 2015/038426 A1 
KSP Kinesin motor spindle protein Bayer Patent # WO 2015/096982 A1 
De-Bouganin Type 1 ribosome inactivating protein Viventia [41
IGN DNA Alkylator Immunogen/Takeda Patent #US 8426402 B2
Patent #WO 2011/130613 A1 
Payload Payload mechanism of action Company Patent # or reference 
Duocarmycin DNA minor groove alkylating agent ASANA Patent # WO 2015/038426 A1 
KSP Kinesin motor spindle protein Bayer Patent # WO 2015/096982 A1 
De-Bouganin Type 1 ribosome inactivating protein Viventia [41
IGN DNA Alkylator Immunogen/Takeda Patent #US 8426402 B2
Patent #WO 2011/130613 A1 

finally, we must raise our expectations

The paucity of complete responses, and the prevalence of off-target dose-limiting toxicities from the current ADCs' in clinical development should be a concern for all investigators. If ADCs' cannot achieve the anticipated role of selective payload delivery of cytotoxic agents to tumors, the argument can be made they are an expensive formulation of systemic chemotherapy. I propose the following stringent criteria be addressed before an ADC transitions from early clinical trials to late development:

  1. In patients selected for target tumor antigen expression, complete responses must be observed in those patients with high expression/gene amplification

  2. Dose-limiting off-target toxicities must be substantially less than what is expected from cytotoxic payload

  3. Ensure the cytotoxic payload is appropriate for the indication

Failure to achieve complete responses reflects the failure of preclinical models to predict sensitivity; or the selection of the indication. This situation creates significant risk in later studies with an overall survival endpoint. For a platform that routinely induces cures in preclinical models, accepting partial responses without evidence significant antitumor activity may represent the self-deception sometimes referred to the ‘sunk cost fallacy’. We should not abandon 50 years of clinical experience with cytotoxic therapies and use payloads for indications where there is no evidence of intrinsic sensitivity to that particular class of cytotoxic as this defies the rationale for therapeutic index. I propose a principal to guide further development:

Match the target to the indication and match the indication to the payload

funding

No funding was received.

disclosure

All honoraria are paid to START, but I am a cofounder and co-owner of START.

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