HER2-Overexpressing/Amplified Breast Cancer As a Testing Ground for Antibody-Drug Conjugate Drug Development in Solid Tumors

Home , Atezolizumab, Cetuximab, Trastuzumab deruxtecan

Author Manuscript Published OnlineFirst on October 3, 2019; DOI: 10.1158/1078-0432.CCR-18-1976 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Title: HER2-overexpressing/amplified breast cancer as a testing ground for antibody-drug conjugate drug development in solid tumors

Mark D. Pegram1, David Miles2, C. Kimberly Tsui3, Yu Zong1

1. Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA 94305, USA 2. Mount Vernon Cancer Centre, Mount Vernon Hospital, Northwood, London, HA6 2RN, UK. 3. Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA

Corresponding author: Mark Pegram, MD Stanford Comprehensive Cancer Institute 265 Campus Drive West Office G2021B Lorry I. Lokey Building Stanford University School of Medicine Stanford, CA 94305-5456 E-mail: [email protected]

Conflict of Interest Declaration:

Mark Pegram: Roche/Genentech, Zymeworks, Astra-Zeneca/Daiichi Sankyo

David Miles: Roche, Inc., Seattle Genetics, Eisai, Genomic health, Inc.

Kimberly Tsui: No conflicts to declare

Yu Zong: No conflicts to declare

Running title: Drug development of HER2 antibody-drug conjugates

Acknowledgement: M.D. Pegram and Y. Zong are supported by the Breast Cancer Research Foundation (116453).

Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 3, 2019; DOI: 10.1158/1078-0432.CCR-18-1976 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Efficacy data from the KATHERINE clinical trial comparing ado-trastuzumab emtansine (T- DM1) to trastuzumab in patients with early-stage human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer with residual disease after neoadjuvant therapy (hazard ratio for invasive disease or death, 0.50; P<0.001). This establishes foundational precedent for antibody drug conjugates (ADCs) as effective therapy for treatment of subclinical micrometastasis in an adjuvant (or post-neoadjuvant) early-stage solid tumor setting. Despite this achievement, general principles from proposed systems pharmacokinetic modeling for intracellular processing of ADCs indicate potential shortcomings of T-DM1: 1) Cmax limited by toxicities, 2) slow internalization rate, 3) resistance mechanisms due to defects in intracellular trafficking (loss of lysosomal transporter solute carrier family 46 member 3, [SLC46A3]), and increased expression of drug transporters MDR1 and MRP1, and 4) lack of payload bystander effects limiting utility in tumors with heterogeneous HER2 expression. These handicaps may explain the inferiority of T-DM1-based therapy in the neoadjuvant and first-line metastatic HER2+ breast cancer settings, and lack of superiority to chemotherapy in HER2+ advanced gastric cancer. In this review, we discuss how each of these limitations are being addressed by manipulating internalization and trafficking using HER2:HER2 bispecific or biparatopic antibody backbones, using site-specific, fixed DAR conjugation chemistry, and payload swapping to exploit alternative intracellular targets and promote bystander effects. Newer HER2- directed ADCs have impressive clinical activity even against tumors with lower levels of HER2 expression. Finally, we highlight ongoing clinical efforts to combine HER2 ADCs with other treatment modalities, including chemotherapy, molecularly targeted therapies, and immunotherapy.

Introduction

To date, the human epidermal growth factor receptor 2 (HER2)-directed antibody-drug conjugate (ADC) ado-trastuzumab emtansine (T-DM1) is the sole ADC with regulatory approval for treatment of a solid tumor malignancy, namely HER2-overexpressing/amplified (HER2+) metastatic breast cancer after receiving prior trastuzumab and a taxane.1,2 T-DM1 is a non- reducible thioether heterogeneous lysine conjugate of trastuzumab with average drug:antibody ratio (DAR) ~3.5. A derivative of maytansine (DM1) payload binds tubulin, thereby disrupting microtubule assembly/disassembly, selectively killing HER2-overexpressing tumor cells.3 Recently, positive efficacy data from the KATHERINE clinical trial (NCT 01772472) were reported for T-DM1 in patients with early-stage HER2+ breast cancer.4 This large prospective randomized clinical trial compared T-DM1 to standard adjuvant trastuzumab in HER2+ primary breast cancers found to have residual invasive disease in the breast or axilla at surgery after receiving neoadjuvant therapy containing a taxane (with or without an anthracycline) and trastuzumab.4 In a planned interim analysis of 1,486 randomly assigned patients, the estimated percentage who were free of invasive disease at 3 years was 88.3% in the T-DM1 arm and 77.0% in the trastuzumab control group (HR for invasive disease or death, 0.50; 95% CI, 0.39 to 0.64; P<0.001).4 Albeit in a different clinical (post-neoadjuvant) setting, this result rivals that from the original adjuvant trastuzumab trials where, in the joint NSABP B-31/NCCTG N9831 analysis for example, the percentages of patients alive and disease-free at 3 years were 75.4 percent in the chemotherapy alone control group and 87.1 percent in the trastuzumab + chemotherapy group (HR 0.48; P<0.0001).5 Accordingly, the KATHERINE results are considered to be practice- changing, and on May 3, 2019, the U.S. Food and Drug Administration approved T-DM1 for the adjuvant treatment of patients with HER2-positive early breast cancer who have residual invasive disease after neoadjuvant taxane and trastuzumab-based treatment.6 It is important to note however that more serious adverse events occurred in patients who received T-DM1 than in those who received trastuzumab (12.7% vs. 8.1%), and more patients discontinued T-DM1 than trastuzumab (18.0% vs. 2.1%) before completion of the planned 14 post-surgical cycles.4 In particular, hepatotoxicity is one potentially serious adverse event that can be associated with T- DM1 therapy.6 Yan, et al. have shown that T-DM1 is internalized upon binding to cell surface HER2 in hepatocytes, and is colocalized with lysosomal-associated membrane protein 1, resulting in DM1-associated cytotoxicity, including disorganized microtubules, nuclear fragmentation/multiple nuclei, and cell growth inhibition.7 Thus, hepatic function must be monitored prior to initiation of, and during treatment with T-DM1, with dose modification(s) instituted according to published prescribing information.6 Thrombocytopenia was the adverse event defining dose-limiting toxicity in the phase I T-DM1 study.8 Consequently, measurement of the platelet count is also necessary prior to T-DM1 dosing; and depending on severity of thrombocytopenia, dose delay and/or reduction may be necessary.6 Remarkably, megakaryocytes internalize T-DM1 in a HER2-independent, FcγRIIa-dependent fashion, resulting in intracellular release of DM1, diminished megakaryocyte maturation, and disruption of cytoskeletal structure.9 Categorically, it is important to stress that HER2 may be a special ADC target by virtue of its high-level overexpression in tumor versus normal tissues (resulting in unique potential for high therapeutic index), and therefore lessons learned in development of HER2-directed ADCs may not be universal for all ADC targets. Nonetheless, we posit the KATHERINE trial results may establish an important foundational precedent of ADCs as adjuvant therapies for early-stage solid tumor malignancies.

Pharmacokinetic (PK) constraints, intra-tumoral heterogeneity, and lack of bystander effects pose challenges to T-DM1 in the clinic

Historically, T-DM1 has not always enjoyed such robust results in the clinic. In the randomized, phase 3 first-line metastatic MARIANNE study (NCT01120184), 1,095 HER2+ (centrally assessed) breast cancer patients were randomly assigned to: 1) trastuzumab plus taxane control, 2) T-DM1 plus placebo, or 3) T-DM1 plus pertuzumab. Though T-DM1 (and pertuzumab plus T-DM1) showed noninferior PFS (median PFS range: 13.7 - 15.2 months, all 3 arms) compared with control, neither experimental arm showed PFS superiority to trastuzumab plus taxane.10 Moreover, during the conduct of the study, the standard of care in the first-line metastatic HER2+ setting changed to include pertuzumab plus taxane/trastuzumab, making the MARIANNE control arm redundant.11 The reason(s) for failure of T-DM1 in a first-line metastatic setting have been the subject of conjecture, ranging from simple play of chance (the trial had 80% power to detect a 33.3% improvement in PFS with statistical confidence for the T- DM1 arms), to the fact that the patients in the control arm received standard chemotherapy which may have theoretically been more effective against heterogeneous tumors containing “HER2 low-expressing” cells (as DM1 has no bystander effect), to mechanistic considerations of the lower “trastuzumab-equivalent dose” administered with T-DM1. The T-DM1 dose is 3.6mg/kg intravenously (IV) every 3weeks, compared to 6mg/kg IV every 3 weeks for free trastuzumab. And the serum half-life of T-DM1 (3.94 days) is far shorter than that of trastuzumab (18.3 days).12,13 These differences could be important, inasmuch as T-DM1 retains all of the known mechanisms of action of trastuzumab, including disruption of ligand-independent HER2:HER3 complexes, Fc receptor binding, ability to elicit antibody-dependent cell-mediated cytotoxicity, and inhibition of HER2 C-terminal fragment (p95) generation from proteolysis by ADAM family sheddases.14 If any of these mechanistic attributes make important contributions to the mechanism(s) of action of T-DM1, then T-DM1 may be handicapped simply by dose and PK. In support of this hypothesis, published exposure-response analysis indicates that higher T-DM1 exposure is associated with improved efficacy.15 Similarly, in the randomized phase 3 trial (NCT02131064; KRISTINE) of T-DM1 combined with pertuzumab vs. docetaxel/carboplatin combined with trastuzumab plus pertuzumab (6 cycles, both arms) in the treatment-naïve neoadjuvant early-stage HER2+ breast cancer setting, the pathologic complete response rate (ypT0/is, ypN0) was significantly inferior for T-DM1/pertuzumab (44.4% vs. 55.7% for the chemotherapy/trastuzumab/pertuzumab arm; p=0.0155).16 Moreover, with a median follow-up of 37 months, risk of an event free survival occurrence was higher with T-DM1+P (hazard ratio [HR], 2.61 [95% CI, 1.36 to 4.98]) with more locoregional progression events before surgery (15 [6.7%] v 0).17 Randomized trials of T-DM1 in gastric cancer also failed to demonstrate superior efficacy compared to taxane chemotherapy alone among 415 patients with previously treated HER2+ locally advanced or metastatic gastric or gastroesophageal junction adenocarcinoma treated in the second-line (NCT01641939; GATSBY trial).18 One possibility raised by these data is that the intrinsic sensitivity of gastric cancer cells to DM1 may differ from that of HER2+ breast cancer cells. For example, it has been proposed that there may be greater efflux of DM1 by ABC transporters in gastric cancer, internalization efficiency of the HER2/T-DM1 complex may differ, and mutation in 1 tubulin may lead to DM1 resistance in gastric cancer.18 To address these mechanisms, payload swapping and manipulation of ADC internalization (see HER2 internalization and trafficking discussion below) could theoretically rehabilitate HER2 ADC efficacy in this disease. Other mechanisms of resistance to T-DM1 in breast cancer may also apply to gastric cancer and warrant further investigation. For example alterations in CDK1/Cyclin B1 kinase activity -- a hallmark of mitotic catastrophe has been associated with resistance.19 Also Polo-like kinase 1 (PLK1) is a mitotic kinase whose genomic as well as 4

pharmacological inhibition restores T-DM1 sensitivity.20 It has been postulated that HER3 ligand, heregulin (NRG-1β), reduces the cytotoxic activity of T-DM1 in a subset of breast cancer lines; and that this effect is reversed by the addition of pertuzumab.21 This hypothesis could be tested in subset analysis from archival samples from clinical trials exploring the T- DM1/pertuzumab combination (i.e. MARIANNE, KRISTINE) by evaluating clinical efficacy endpoints in heregulin-(over)expressing tumors (using RNA in situ hybridization to capture expression of all heregulin isoforms), compared to those lacking heregulin transcript expression.22 Lastly, the expression of p185HER2 in gastric cancer is more heterogeneous than that seen in breast cancer, which could contribute to emergence of clinical resistance amongst lower HER2 expressing clones.23 Data in support of this argument have been presented from a single-arm phase II study enrolling 164 centrally confirmed HER2+ breast cancers, in which patients received 6 cycles of T-DM1 plus pertuzumab pre-operatively. The study met its primary objective by demonstrating a significant association between intra-tumoral heterogeneity of HER2 and pCR (stratified by ER status, p < 0.0001).24 Taken together, both the gastric and the breast cancer T-DM1 clinical data strongly suggest that the target antigen intra-tumoral heterogeneity problem must be taken into account when developing ADCs, at least for ADCs without significant bystander effects. The discordant outcomes from MARIANNE, KRISTINE and GATSBY compared to the KATHERINE data are also consistent with a hypothesis that there may be unique sensitivity of micrometastatic disease (in the post-surgical setting) to ADCs, as compared to bulk primary or metastatic tumor deposits. Upcoming data from the ongoing adjuvant T-DM1 trials may help to shed light on this issue. One example is the KAITLIN trial (NCT01966471), a study of T-DM1 plus pertuzumab in comparison with trastuzumab plus pertuzumab and a taxane, both arms following anthracyclines; although its statistical power is handicapped by accrual of just 1,846 of 2,500 planned (accrual being deliberately suspended in the wake of negative results from MARIANNE and KRISTINE). Another ongoing adjuvant T- DM1 trial (ATEMPT [sic]; NCT 01853748) randomizes (3:1) 500 stage I HER2+ patients to single-agent T-DM1 vs. trastuzumab + paclitaxel. If successful, the ATEMPT trial may, for the first time, allow for de-escalation to an adjuvant non-chemotherapy option for selected stage I HER2+ patients.

Shortcomings of T-DM1 in the clinic notwithstanding, based upon the success of T-DM1 against metastatic HER2+ disease (typically after the first line), and its newly discovered remarkable efficacy in the HER2+ early disease setting, enthusiasm for clinical use of T-DM1 is heightened. There is now eagerness to develop newer HER2-directed ADCs with different linker chemistries and payloads (with bystander effects), as well as novel bispecific/biparatopic ADC constructs targeting multiple HER2 epitopes to facilitate internalization (as well as endosomal/lysosomal trafficking), with the hope that further ADC optimization will result in improved therapeutic efficacy (and hopefully heightened therapeutic index).25

As an ADC target, HER2 is a remarkably internalization-resistant receptor

In contrast to other ADC targets, HER2 is known to largely remain at the plasma membrane after antibody or ligand binding.26 Through a combination of confocal microscopy, immunogold labeling electron microscopy, and biochemical techniques, Hommelgaard, et al. have shown that HER2 receptor is efficiently excluded from clathrin-coated pits and is not seen in transferrin receptor-containing endosomes (over experimental time scales of minutes to hours).26 This pattern is not changed after EGF, heregulin, or trastuzumab binding.26 Though HER2 may be relatively “internalization resistant”, obviously it is not internalization proof; and even if only a small fraction of HER2 molecules are undergoing receptor-mediated endocytosis over a given

time period, in cases of gene amplification where the number of HER2 receptors per cell may exceed ~106 (>10,000 fmol/mg)27, there are apparently ample internalization events to execute ADC cytotoxic action. But to what extent might HER2 internalization rate handicap T-DM1 (as well as other trastuzumab-based ADCs)? To address this potential shortcoming, Li et al. constructed a bivalent biparatopic antibody, targeting two distinct non-overlapping epitopes on HER2, that can induce HER2 receptor clustering, which in turn (and in contrast to trastuzumab) promotes robust internalization, lysosomal trafficking, and degradation.28 The biparatopic antibody construct contains the single-chain variable fragment (scFv) of trastuzumab fused to the amino terminus of the heavy chain of a fully human HER2 monoclonal antibody 39S IgG1 which binds to an epitope that includes part of subdomain 1 and the beginning of subdomain 2 of the HER2 extracellular domain (ECD), near the pertuzumab binding epitope (Figure 1A).28 Based on co-crystal structure of the 39S Fab-HER2 complex, the 39S and trastuzumab epitopes are located at opposite ends of HER2 ECD at a distance of more than 90 Å from each other.29 Accordingly, the C-terminal residue of trastuzumab scFv and N-terminal amino acid of 39S heavy chain are unable to bind simultaneously to the same HER2 receptor molecule.29 Rather, the biparatopic construct crosslinks adjacent HER2 receptors, resulting in receptor clustering at the cell surface.28,29 Such clustering results in rapid receptor internalization, inhibition of recycling, and promotes trafficking towards lysosomal degradation (Figure 1B). This biparatopic ADC (MEDI4276) addresses a number of theoretical shortcomings of T-DM1, and demonstrates superior anti-tumor activity over T-DM1 in various tumor models, including substantial growth inhibition of four T-DM1-resistant cell lines (with EC50 < 100 pM), and a T-DM1-resistant breast cancer xenograft model.28

An interim analysis of the first-in-human phase 1/2 multicenter, open-label, dose-escalation, and dose-expansion study of MEDI4276 in patients with HER2+ breast or gastric cancer has recently been presented.30 In the dose escalation sequence, dose-limiting toxicity was observed at doses of less than 1mg/kg, and at the maximum tolerated dose (MTD), even though anecdotal efficacy was observed (including in T-DM1 refractory disease), over half of patients experienced ≥ 1 serious and/or grade ≥ 3 severity event – namely hepatotoxicity and/or gastrointestinal toxicity.30 Since MEDI4276 has a number of structural/mechanistic differences compared to T-DM1 (namely biparatopic binding, site-specific DAR 4 conjugation, and potent bystander effect of the tubulysin payload), it was not possible (based on first-in-human clinical data alone) to dissect which attribute (or combination of attributes) was most responsible for toxicity to particular normal tissues. Moreover, though HER2 receptor may be relatively internalization resistant over a period of several hours in laboratory conditions, it is unclear what advantage a biparatopic HER2 construct may confer over clinical time scales (i.e. days to weeks to even months). To further explore manipulation of HER2 internalization events, other novel HER2-HER2 bispecific ADCs are currently in early clinical development (NCT03821233). Hamblett and colleagues published preclinical characterization of a new anti-HER2 biparatopic ADC, ZW49, which is generated from the conjugation of a novel N-acyl sulfonamide auristatin payload to the inter- chain disulfide bond cysteines of the bispecific anti-HER2 IgG1 antibody (ZW25), via a protease cleavable linker.31 ZW25 is a novel bispecific antibody, targeting HER2 ECD subdomain 2 and ECD subdomain 4, resulting in multiple differentiated mechanisms of action, including increased tumor cell binding, blockade of ligand-dependent and independent growth, and improved receptor internalization and downregulation relative to trastuzumab.32 Indeed in early phase human clinical trials, ZW25 as a single agent has already shown remarkable clinical activity against multiple HER2+ tumor types in heavily pre-treated patients.32 Based upon the ZW25 backbone, ZW49 displays potent in vitro cytotoxicity in multiple cancer cell lines expressing HER2, and is efficacious in multiple patient-derived xenograft models. Furthermore, in

nonhuman primates ZW49 administered intravenously every two weeks for four doses was well tolerated (highest non-severely toxic dose = 18mg/kg).31 Based on these findings, further development of ZW49 as a therapeutic candidate in HER2-expressing cancers is warranted. If ZW49 finds success in the clinic, it would suggest that the untoward toxicity observed with the MEDI4276 ADC is most likely a payload problem, rather than its biparatopic configuration.

Other HER2-targeting ADCs have also recently faced toxicity challenges in the clinic. A phase I trial of ADCT-502 (composed of trastuzumab with site-specific conjugation to the extremely potent pyrrolobenzodiazepine dimer-based linker-drug tesirine) was terminated in 2018 for toxicities of fluid retention and pulmonary edema, the latter presumed due to the extensive expression of HER2 in pulmonary tissue.33 Also, a partial temporary clinical hold (due to a grade 5 serious adverse event in dose level 7) was imposed (then subsequently reversed) by the FDA on a phase I dose-escalation study of XMT-1522, a high DAR  12 ADC targeting HER2 comprised of a proprietary HER2 antibody conjugated with Mersana’s Dolaflexin platform – a fleximer polymer linked with a proprietary auristatin payload.34 In an erudite review of ADC toxicology by the FDA, Saber and Leighton have observed that ADC toxicity is largely driven by linker/payload composition, rather than expression/anatomical distribution of the target antigen.35 Interestingly they noted that ADCs sharing the same linker/payload composition tend to reach the same MTD, even when their target antigens showed endogenous expression in completely different tissue/organ compartments.35

Potent new HER2 ADCs enable HER2 “low”-expressing tumors to be targets for HER2- directed therapy

Trastuzumab Deruxtecan (DS-8201a) and (vic-)trastuzumab duocarmazine (SYD985) are newer HER2-targeting ADCs with novel payloads (Figure 2). DS-8201a is composed of humanized anti-HER2 antibody trastuzumab, an enzymatically cleavable peptide-linker, and a novel topoisomerase I inhibitor payload (DAR 8), while SYD985 is a novel HER2-targeting ADC which has a cleavable linker-duocarmycin (DAR 2-4) payload conjugated to trastuzumab.37,41 The SYD985 payload is a minor groove DNA binder, leading to irreversible alkylation of DNA.40 Each of these HER2 ADCs has demonstrated impressive clinical activity against multiple HER2-positive disease states (33% overall response rate for SYD985 in heavily pretreated HER2+ advanced breast cancer, and 64.2% for DS-8201a, also in pre-treated patients).39,41 Further phase I studies of these two HER2 ADCs have recently been presented in heavily pretreated “HER2 low” (defined as IHC 1+ or 2+ and in situ hybridization-negative for amplification at the ERBB2/HER2 gene locus). Objective clinical response rates were 38.5% for DS-8201a and 27% (hormone receptor positive) to 40% (hormone receptor-negative) for SYD985 (Figure 2).37,39 Both ADCs are generally well tolerated, with the most common adverse drug reactions for SYD985 being fatigue, dry eyes, conjunctivitis and increased lacrimation. For DS-8201a, common adverse events included nausea 73.5% (3.5% grade ≥3), decreased appetite 59.5% (4.5% grade ≥3) and vomiting 39.5% (1.5% grade ≥3). However, a number of serious (grade 5) cases of interstitial lung disease (ILD) have been reported during the early clinical development of DS-8201a, prompting close monitoring and early clinical intervention for pulmonary toxicity for all ongoing and future DS-8201a clinical trials. As noted above, pulmonary toxicity may be an on-target toxicity.42 Indeed, as noted in the T-DM1 prescribing information, cases of ILD, including pneumonitis, some leading to acute respiratory distress syndrome or fatal outcome have been reported in clinical trials with T-DM1.6 Moreover, it is well established that trastuzumab itself can (rarely) be associated with serious (even fatal) pulmonary toxicity (garnering a “boxed warning” on the FDA label), including interstitial 7

pneumonitis, pulmonary infiltrates, pleural effusions, non-cardiogenic pulmonary edema, acute respiratory distress syndrome, and pulmonary fibrosis.43

Taken together, DS-8201a and SYD985, as well as other ADCs targeting lower expressing targets in breast cancer (sacituzumab govitecan [IMMU-132] targeting Trop-2, and ladiratuzumab vedotin targeting LIV-1 [SLC39A6]) all highlight that extreme target antigen overexpression, such as that seen with high-level HER2 overexpression (IHC 3+) in breast cancer (typically > 106 HER2 molecules per cell), is not a prerequisite for effective ADC internalization and cytotoxic payload delivery to therapeutic effect.44,45 This remains an area of high unmet need since adjuvant trastuzumab failed to achieve an efficacy signal in a large randomized phase III trial (N = 3,260) in a HER2 low, early stage breast cancer patient population.46 Perhaps reincarnation of therapy for this target population may be achieved by next generation HER2-directed (or other antigen-directed) ADCs.

General principles of ADC drug development gleaned from a proposed systems pharmacokinetic (PK) model for intracellular processing of ADCs

Singh and colleagues have proposed an elegant comprehensive systems PK model for ADCs based on current understanding about the disposition of an ADC molecule on a cellular level (Figure 3).47 The model includes parameters for ADC stability in the circulation and extracellular space, target antigen binding, internalization, endosomal/lysosomal trafficking (including futile endosomal recycling), unconjugated free drug pharmacological action, payload drug efflux via passive or active processes, and drug bystander effects48 – all of which are variables to be considered which may impact therapeutic index. Now that such sophisticated models for ADC cellular fate are developed and being refined, it should be possible to utilize these models as a framework to be systematic (rather than empiric) in considering the most desirable ADC attributes for a given target, linker and payload trinity. Moreover, a more fundamental understanding of absorption, distribution, metabolism/excretion and pharmacokinetic fates of both intact ADCs and their small molecule payloads is necessary in order to better predict clinical outcomes.49,50 Thus the detailed framework proposed by Singh, et al. is a roadmap for a more systematic approach to future ADC development.47 The model further raises the possibility of manipulating intracellular trafficking properties for optimization of ADCs, as further mechanistic insights into trafficking pathways from early to late endosomes to lysosomes come to light. Differing linker chemistries have already been shown to favor liberation of payloads in early versus late endosomes, versus lysosomes.47,51 One might envision antibody fusions for example, designed deliberately to shuttle ADCs to particular intracellular compartments for delivery of targeted payloads with unique mechanisms of action (even non-chemotherapeutic payloads).

Enhanced therapeutic index from fixed drug-to-antibody ratio (DAR) chemistries

Current bioconjugation methods that underpin ADC synthesis rely largely on nonspecific, promiscuous chemistries that exploit reactive amino acid side chains (Glu, Asp, Lys, or Cys residues), resulting in nonspecific conjugation at multiple sites, producing a mixture of ADC products with differing DARs ranging typically from 0 to ~8.52-54 These high DAR “contaminating” subpopulations tend to show enhanced toxicity relative to lower DAR constituents.55 Consequently, the past few years have seen the emergence of site-specific conjugation methods that promise to produce homogeneous products with controlled drug loading, and improved therapeutic index.56 One example is a novel site-specific conjugation

platform comprising linker payloads designed to selectively react with site-specifically engineered aldehyde tags on an antibody backbone, resulting in a stable C−C bond between the antibody and the cytotoxin payload, providing a uniquely stable connection with respect to the other linker chemistries used to generate ADCs.56 Use of such technology has resulted in a HER2 ADC (fixed DAR 2) with far higher therapeutic index in vivo, as compared to conventional α- HER2-DM1.56 Another advantage is that the aldehyde tag can be introduced at different locations on an IgG1 backbone, the site of conjugation having significant impact on in vivo efficacy and pharmacokinetic behavior in rodents.56 Currently, ADC clinical pipelines are still dominated by non-site specific heterogeneous (lysine) conjugation; however, product uniformity and potential safety advantages of DAR-controlled site-specific conjugation are becoming clear.57 Indeed, between 2013 and 2017, as the number of ADCs undergoing clinical investigation more than tripled, the fraction with site-specific conjugation chemistry increased to nearly 15%.58 Development of high DAR ADCs has been challenging given the first generation of extremely potent payloads. But high DAR chemistry may open new opportunities to reconsider less toxic payloads. It has been shown for example that a trastuzumab-paclitaxel ADC construct has robust in vivo efficacy against HER2+ human tumor xenografts with no observable toxicity.59 Enforced internalization techniques (as noted above) may also be able to rehabilitate consideration of less toxic conventional chemotherapeutic payloads.

Despite macromolecular size, HER2-directed ADCs can localize to HER2+ brain metastasis

Another interesting attribute of T-DM1 is that despite its macromolecular structure, T-DM1 has demonstrated clinically meaningful activity against HER2+ breast cancer brain metastasis (a common complication affecting up to ~50% of HER2+ metastatic patients).60-64 Indeed, there appears to be blood brain barrier (BBB) disruption in metastatic tumor microenvironments sufficient to facilitate positron emission tomographic imaging of HER2+ breast cancer brain metastasis with 89-zirconium conjugated trastuzumab, despite its high molecular weight.65 Remarkably, T-DM1 apparently shares this same property. Fabi, et al., reported objective responses to T-DM1 among 53 patients with HER2+ breast cancer brain metastasis: 2 patients achieved a complete response (3.8%), 11 patients obtained partial response (20.7%; overall response rate [ORR]: 24.5%), and 16 patients had a stable disease (30.1%).53 Indeed, ORR (35.1% vs. 38.3%) and median PFS (7 vs. 8 months) were similar in this study for both the brain metastasis group and the non-brain metastasis group, respectively.61 It is to be noted that the majority of patients enrolled in HER2+ brain metastasis T-DM1 trials had prior neurosurgery and/or ionizing radiation, which could result in further BBB disruption. However, in the study published by Bartsch et al., anecdotally, two asymptomatic patients received T-DM1 as primary systemic therapy without prior local treatment. One of these cases experienced objective partial response and one case enjoyed disease stabilization for 5 months.64 Moreover, 30% of patients with brain metastasis in the EMILIA trial had no prior whole brain radiotherapy or local brain surgical therapy.62 Prior local treatment(s) for brain metastasis notwithstanding, in our opinion these results establish an important precedent to not overlook ADC technologies in consideration of future drug development of treatment approaches for solid tumor brain metastasis (and by extrapolation, primary brain tumors).

Future directions informed by the HER2 ADC clinical experience

Future attention must be payed to ADC resistance mechanisms in order to engineer ADCs to bypass/overcome resistance pathways. For example, acquired resistance to T-DM1 has been evaluated in several preclinical studies, and due to the complexity of this molecule, several

potential mechanisms have come to light (summarized by Kinneer, et al., in reference 66 as follows): (i) antigen loss and/or downregulation, (ii) increased expression of drug transporters MDR1 (ABCB1) and MRP1 (ABCC1), (iii) defects in ADC trafficking, and/or (iv) changes in receptor and signaling pathways.66,67 In addition, aberrations in lysosomal pH and proteolytic activity68 and loss of the lysosomal transporter solute carrier family 46 member 3 (SLC46A3) have been observed in T-DM1–resistant cell lines.69,70

A general theorem of ADC action is that these drugs remain chemotherapeutics at their core, and therefore payload toxicity coupled with target antigen expression in normal tissues remain critical considerations to successful clinical development. Moreover, a corollary to this theorem predicts that for optimal efficacy, ADC cytotoxic payloads should be of a drug class to which the tumor target is known to be generally highly sensitive. Expecting monoclonal antibody delivery/targeting alone to overcome intrinsic drug resistance mechanism(s) seems unlikely. Another general principle of chemotherapy treatment is that for most common solid tumors, when treating with curative intent, chemotherapy combinations are, with rare exception, superior to single agents (e.g. testicular cancer, early stage breast cancer, early stage colorectal cancer, ovarian cancer, lymphomas, etc.). Extending this fundamental principle to ADC drug development, though challenging, should not be overlooked. Krop et al. have attempted to combine the ADC T-DM1 with paclitaxel (given in conventional dose and schedule). However, this approach proved quite toxic.71 Alternatively, one possibility is to design ADCs with multiple cytotoxic payloads.72,73 Another possibility is to target different shared tumor antigens simultaneously with ADCs from different drug classes for tumor types with more than one potential ADC target. Another opportunity is to target multiple non-competing epitopes on the same antigenic target – a paradigm now already routinely used in clinical practice with naked HER2 antibodies (pertuzumab targeting HER2 ECD subdomain 2 in combination with trastuzumab targeting HER2 ECD subdomain 4).11 Therefore, it should be possible to consider different non-cross resistant payloads on each of these antibody backbones to effectively deliver synergistic cytotoxic combinations via multiple ADCs against the same target. Moreover, in breast cancer, in both the adjuvant and metastatic disease settings, it has been shown that sequential use of single-agent chemotherapeutics is equally efficacious as simultaneous administration of the same drugs in combination.74,75 Sequential ADC dosing schedules could follow this same paradigm in future studies. Clinicians will ideally need a tool kit of ADCs against the same targets with unique payload/linker options most suitable for the desired clinical indication, just as is the case in the modern era for clinical application of chemotherapeutic agents. Preclinical data emerging from the next-generation of ADCs will provide important insight into the mechanistic basis of ADC design, and how changes in ADC properties impact therapeutic activity and safety. For example, Tsui et al. have demonstrated the use of in vitro CRISPR screens with ADCs bearing different types of linkers can be used to systematically identify genetic regulators of ADC intracellular trafficking and processing, revealing how ADC designs may influence efficacy.76 For example, they identified and characterized Regulator of MON1-CCZ1 Complex (RMC1, formerly C18ORF8) as a regulator of ADC toxicity through its role in endosomal maturation. Through comparative analysis of screens with ADCs bearing different linkers, they showed that a subset of late endolysosomal regulators selectively influence toxicity of noncleavable linker ADCs.76 Lastly, they found that inhibition of sialic acid biosynthesis enhances lysosomal delivery and sensitizes cells to multiple ADCs, including T- DM1, illustrating the power of CRISPR screens in identifying combination therapy targets and uncovering how ADC designs may influence efficacy.76 Further evolution of systems pharmacokinetic model(s) for intracellular processing of ADCs will be refined as mechanisms responsible for intracellular trafficking pathways are further elucidated (and then, exploited).

Once fully developed, the cellular level systems model for ADC action and fate can be utilized to identify the most rate-limiting intracellular processing pathways, evaluate the effect of different linker types, and develop strategies for successful intracellular drug release for future ADC clinical campaigns.47 Future directions in ADC clinical development will include expansion of clinical use of ADCs in combination with other treatment modalities. Clinical trials are already ongoing combining HER2-directed ADCs with HER2-direct tyrosine kinase inhibitors such as tucatinib (NCT01983501)77 or neratinib (NCT022360000)78, which like lapatinib may induce accumulation of HER2 at the cell surface.79 Use of other targeted small molecule payloads is also being explored80. And finally, integration with immune acting agents has strong preclinical scientific rationale, and is the focus of ongoing clinical research (Table 1).81,82

References:

1Verma S, Miles D, Gianni L, Krop IE, Welslau M, Baselga J, et al.. Trastuzumab emtansine for HER2-positive advanced breast cancer. N Engl J Med 2012;367:1783-91.

2Diéras V, Miles D, Verma S, Pegram M, Welslau M, Baselga J, et al. Trastuzumab emtansine versus capecitabine plus lapatinib in patients with previously treated HER2-positive advanced breast cancer (EMILIA): a descriptive analysis of final overall survival results from a randomised, open-label, phase 3 trial. Lancet Oncol 2017;18:732-742.

3Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E, et al. Targeting HER2- positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res 2008;68:9280-90.

4von Minckwitz G, Huang CS, Mano MS, Loibl S, Mamounas EP, Untch M, et al. Trastuzumab Emtansine for Residual Invasive HER2-Positive Breast Cancer. N Engl J Med 2019;380:617- 628.

5Romond EH, Perez EA, Bryant J, Suman VJ, Geyer CE Jr, Davidson NE, et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 2005;353:1673-84.

6https://www.accessdata.fda.gov/drugsatfda_docs/label/2019/125427s105lbl.pdf

7Yan H, Endo Y, Shen Y, Rotstein D, Dokmanovic M, Mohan N, et al. Ado-Trastuzumab Emtansine Targets Hepatocytes Via Human Epidermal Growth Factor Receptor 2 to Induce Hepatotoxicity. Mol Cancer Ther. 2016;15(3):480-90.

8Krop IE, Beeram M, Modi S, Jones SF, Holden SN, Yu W, et al. Phase I study of trastuzumab- DM1, an HER2 antibody-drug conjugate, given every 3 weeks to patients with HER2-positive metastatic breast cancer. J Clin Oncol. 2010;28(16):2698-704.

9Uppal H, Doudement E, Mahapatra K, Darbonne WC, Bumbaca D, Shen BQ, et al. Potential mechanisms for thrombocytopenia development with trastuzumab emtansine (T-DM1). Clin Cancer Res. 2015;21(1):123-33.

10Edith A. Perez, Carlos Barrios, Wolfgang Eiermann, Masakazu Toi, Young-Hyuck Im, Pierfranco Conte, et al. Trastuzumab Emtansine With or Without Pertuzumab Versus Trastuzumab Plus Taxane for Human Epidermal Growth Factor Receptor 2–Positive, Advanced Breast Cancer: Primary Results From the Phase III MARIANNE Study. J Clin Oncol 2017;35: 141–148.

11Swain SM, Baselga J, Kim SB, Ro J, Semiglazov V, Campone M, et al. Pertuzumab, trastuzumab, and docetaxel in HER2-positive metastatic breast cancer. N Engl J Med 2015;372:724-34.

12Girish S, Gupta M, Wang B, Lu D, Krop IE, Vogel CL, et al. Clinical pharmacology of trastuzumab emtansine (T-DM1): an antibody-drug conjugate in development for the treatment of HER2-positive cancer. Cancer Chemother Pharmacol 2012;69:1229-40.

13Leyland-Jones B, Gelmon K, Ayoub JP, Arnold A, Verma S, Dias R, et al. Pharmacokinetics, safety, and efficacy of trastuzumab administered every three weeks in combination with paclitaxel. J Clin Oncol 2003;21:3965-71.

14Junttila TT, Li G, Parsons K, Phillips GL, Sliwkowski MX. Trastuzumab-DM1 (T-DM1) retains all the mechanisms of action of trastuzumab and efficiently inhibits growth of lapatinib insensitive breast cancer. Breast Cancer Res Treat 2011;128:347-56.

15Wang J, Song P, Schrieber S, Liu Q, Xu Q, Blumenthal G, et al. Exposure-response relationship of T-DM1: insight into dose optimization for patients with HER2-positive metastatic breast cancer. Clin Pharmacol Ther. 2014;95(5):558-64.

16Hurvitz SA, Martin M, Symmans WF, Jung KH, Huang CS, Thompson AM, et al. Neoadjuvant trastuzumab, pertuzumab, and chemotherapy versus trastuzumab emtansine plus pertuzumab in patients with HER2-positive breast cancer (KRISTINE): a randomised, open-label, multicentre, phase 3 trial. Lancet Oncol 2018;19:115-126.

17Hurvitz SA, Martin M, Jung KH, Huang CS, Harbeck N, Valero, et al. Neoadjuvant Trastuzumab Emtansine and Pertuzumab in Human Epidermal Growth Factor Receptor 2- Positive Breast Cancer: Three-Year Outcomes From the Phase III KRISTINE Study. J Clin Oncol. 2019 Jun 3:JCO1900882. doi: 10.1200/JCO.19.00882. [Epub ahead of print].

18Thuss-Patience PC, Shah MA, Ohtsu A, Van Cutsem E, Ajani JA, Castro H,et al. Trastuzumab emtansine versus taxane use for previously treated HER2-positive locally advanced or metastatic gastric or gastro-oesophageal junction adenocarcinoma (GATSBY): an international randomised, open-label, adaptive, phase 2/3 study. Lancet Oncol 2017;18:640-653.

19Sabbaghi MA, Gil-Gómez G, Guardia C, Servitja S, Arpi O, García-Alonso S, et al. Defective cyclin B1 induction in trastuzumab-emtansine (T-DM1) acquired resistance in HER2-positive breast cancer. Clin Cancer Res. 2017;23(22):7006-7019.

20Saatci Ö, Borgoni S, Akbulut Ö, Durmuş S, Raza U, Eyüpoğlu E, et al. Targeting PLK1 overcomes T-DM1 resistance via CDK1-dependent phosphorylation and inactivation of Bcl-2/xL in HER2-positive breast cancer. Oncogene 37, 2251–2269 (2018).

21Phillips GD, Fields CT, Li G, Dowbenko D, Schaefer G, Miller K, et al. Dual targeting of HER2-positive cancer with trastuzumab emtansine and pertuzumab: critical role for neuregulin blockade in antitumor response to combination therapy. Clin Cancer Res. 2014;20(2):456-68.

22Wang F, Flanagan J, Su N, Wang L-C, Bui S, Nielson A, et al. RNAscope A Novel in Situ RNA Analysis Platform for Formalin-Fixed, Paraffin-Embedded Tissues. J Mol Diagn. 2012;14(1): 22–29.

23Shah MA, Kang YK, Thuss-Patience PC, Ohtsu A, Ajani JA, Van Cutsem E, et al. Biomarker analysis of the GATSBY study of trastuzumab emtansine versus a taxane in previously treated HER2-positive advanced gastric/gastroesophageal junction cancer. Gastric Cancer. 2019 Jan 31. doi: 10.1007/s10120-018-00923-7. [Epub ahead of print]

24Metzger Filho O, Viale G, Trippa L, Li T, Yardley DA, Mayer IA, et al. HER2 heterogeneity as a predictor of response to neoadjuvant T-DM1 plus pertuzumab: Results from a prospective clinical trial. J Clin Oncol. 2019 37:15_suppl, 502-502.

25Pegram MD, Zong Y, Yam C, Goetz MP, Moulder SL. Innovative Strategies: Targeting Subtypes in Metastatic Breast Cancer. Am Soc Clin Oncol Educ Book 2018;38:65-77.

26Hommelgaard AM, Lerdrup M, van Deurs B. Association with Membrane Protrusions Makes ErbB2 an Internalization-resistant Receptor. Mol Biol Cell 2004;15:1557–67.

27Konecny G, Pauletti G, Pegram M, Untch M, Dandekar S, Aguilar Z, et al. Quantitative Association Between HER-2/neu and Steroid Hormone Receptors in Hormone Receptor-Positive Primary Breast Cancer. Journal of the National Cancer Institute, 2003;95(2):142–153.

28Li JY, Perry SR, Muniz-Medina V, Wang X, Wetzel LK, Rebelatto MC, et al. A Biparatopic HER2-Targeting Antibody-Drug Conjugate Induces Tumor Regression in Primary Models Refractory to or Ineligible for HER2-Targeted Therapy. Cancer Cell 2016;29:117-29.

29Oganesyan V, Peng L, Bee JS, Li J, Perry SR, Comer F, et al. Structural insights into the mechanism of action of a biparatopic anti-HER2 antibody. J Biol Chem 2018;293:8439-48. 30Pegram M, Hamilton E, Tan AR, Storniolo AM, Elgeioushi N, Marshall S, et al. Phase 1 Study of Bispecific HER2 Antibody-Drug Conjugate MEDI4276 in Patients with Advanced HER2- Positive Breast or Gastric Cancer. Annals of Oncology 2018;29: iii7-iii9.

31Hamblett KJ, Hammond PW, Barnscher SD, Fung VK, Davies RH, Wickman GR, et al. ZW49, a HER2-targeted biparatopic antibody-drug conjugate for the treatment of HER2-expressing cancers [abstract]. Proceedings of the American Association for Cancer Research Annual Meeting 2018. Cancer Res 2018;78(13 Suppl):Abstract nr 3914.

32Funda Meric-Bernstam, Murali Beeram, Jose Ignacio Mayordomo, Diana L. Hanna, Jaffer A. Ajani, Mariela A. Blum Murphy, et al. Single agent activity of ZW25, a HER2‑targeted bispecific antibody, in heavily pretreated HER2‑expressing cancers. J Clin Oncol 36;2018: (suppl; abstr 2500).

33MEDIA RELEASE: ADC Therapeutics Announces the Termination of its ADCT-502 Program Targeting HER2 Expressing Solid Tumors. Data on safety, tolerability, pharmacokinetics and

efficacy did not demonstrate sufficient patient benefit to justify further development. Lausanne, Switzerland, April 25th, 2018. https://www.businesswire.com/news/home/20180425005853/en/ADC-Therapeutics-Announces- Termination-ADCT-502-Program-Targeting.

34Mersana Announces FDA Lifts Partial Clinical Hold for XMT-1522. September 17, 2018. Phase 1 Clinical Trial to Resume Enrollment of New Patients. CAMBRIDGE, Mass., Sept. 17, 2018 (GLOBE NEWSWIRE) -- Mersana Therapeutics, Inc. http://ir.mersana.com/news- releases/news-release-details/mersana-announces-fda-lifts-partial-clinical-hold-xmt-1522.

35Saber H, Leighton JK. An FDA oncology analysis of antibody-drug conjugates. Regul Toxicol Pharmacol. 2015; 71:444–52.

36Burris HA 3rd, Rugo HS, Vukelja SJ, Vogel CL, Borson RA, Limentani S, et al. Phase II study of the antibody drug conjugate trastuzumab-DM1 for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer after prior HER2-directed therapy. J Clin Oncol 2011;29(4):398-405.

37Modi S, Tsurutani J, Tamura K, Park H, Sagara Y, Murthy RK, et al. Trastuzumab deruxtecan (DS-8201a) in subjects with HER2-low expressing breast cancer: Updated results of a large phase 1 study. Cancer Res. 2019(79)(4 Supplement)P6-17-02.

38Ogitani Y, Hagihara K, Oitate M, Naito H, Agatsuma T. Bystander killing effect of DS-8201a, a novel anti-human epidermal growth factor receptor 2 antibody-drug conjugate, in tumors with human epidermal growth factor receptor 2 heterogeneity. Cancer Sci. 2016;107(7):1039-46.

39Saura C, Thistlethwaite F, Banerji U, Lord S, Moreno V, MacPherson I, et al. A phase I expansion cohorts study of SYD985 in heavily pretreated patients with HER2-positive or HER2- low metastatic breast cancer. J Clin Oncol 2018;36:(suppl; abstr 1014).

40van der Lee MM, Groothuis PG, Ubink R, van der Vleuten MA, van Achterberg TA, Loosveld EM, et al. The Preclinical Profile of the Duocarmycin-Based HER2-Targeting ADC SYD985 Predicts for Clinical Benefit in Low HER2-Expressing Breast Cancers. Mol Cancer Ther. 2015;14(3):692-703.

41Iwata H, Tamura K, Doi T, Tsurutani J, Modi S, Park H, et al. Trastuzumab deruxtecan (DS- 8201a) in subjects with HER2-expressing solid tumors: Long-term results of a large phase 1 study with multiple expansion cohorts. J Clin Oncol 2018;36:(suppl; abstr 2501).

42Press MF, Cordon-Cardo C, Slamon DJ. Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues. Oncogene 1990;5:953-62.

43HERCEPTIN® (trastuzumab) for injection, for intravenous use -- full prescribing information. Genentech, Inc. A Member of the Roche Group, 1 DNA Way, South San Francisco, CA 94080- 4990. Revised: 10/2018.

44Bardia A, Mayer IA, Diamond JR, Moroose RL, Isakoff SJ, Starodub AN, et al. Efficacy and Safety of Anti-Trop-2 Antibody Drug Conjugate Sacituzumab Govitecan (IMMU-132) in

Heavily Pretreated Patients With Metastatic Triple-Negative Breast Cancer. J Clin Oncol 2017; 35: 2141–48.

45Modi S, Pusztai L, Forero A, Mita M, Miller KD, Weise A, et al. Phase 1 study of the antibody-drug conjugate SGN-LIV1A in patients with heavily pretreated triple-negative metastatic breast cancer. Proceedings of the San Antonio Breast Cancer Symposium, 2017, abstr. PD3-14.

46Fehrenbacher L, Jeong J-H, Rastogi P, Geyer CE, Paik S, Ganz PA, et al. NSABP B-47 (NRG Oncology): Phase III randomized controlled trial comparing adjuvant chemotherapy with adriamycin and cyclophosphamide followed by weekly paclitaxel, or docetaxel and cyclophosphamide with or without a year of trastuzumab in women with node-positive or high- risk node-negative invasive breast cancer expressing HER2 staining intensity of IHC 1+ or 2+ with negative FISH. 2017 San Antonio Breast Cancer Symposium. Abstract GS1-02. Presented December 6, 2017.

47Singh AP, Maass KF, Betts AM, Wittrup KD, Kulkarni C, King LE, et al. Evolution of Antibody-Drug Conjugate Tumor Disposition Model to Predict Preclinical Tumor Pharmacokinetics of Trastuzumab-Emtansine (T-DM1). AAPS J 2016;18:861-75.

48Singh AP, Sharma S, Shah DK. Quantitative characterization of in vitro bystander effect of antibody-drug conjugates. J Pharmacokinet Pharmacodyn 2016;43:567-582.

49Han TH, Zhao B. Absorption, distribution, metabolism, and excretion considerations for the development of antibody-drug conjugates. Drug Metab Dispos 2014;42:1914-20.

50Eugenia Kraynov, Amrita V. Kamath, Markus Walles, Edit Tarcsa, Antoine Deslandes, Ramaswamy A. Iyer, et al. Current Approaches for Absorption, Distribution, Metabolism, and Excretion Characterization of Antibody-Drug Conjugates: An Industry White Paper. Drug Metab Dispos 2016;44:617–23.

51Drake P, Rabuka D, ADCs – The Dawn of a New Era? (2018), DOI: 10.14229/jadc.2018.08.27.001.

52Sletten EM, and Bertozzi CR. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew. Chem. Int. Ed. Engl. 2009;48:6974–6998.

53Stephanopoulos N, Francis MB. Choosing an effective protein bioconjugation strategy. Nat Chem Biol. 2011;7(12):876-84.

54Kim MT, Chen Y, Marhoul J, Jacobson F. Statistical modeling of the drug load distribution on trastuzumab emtansine (Kadcyla), a lysine- linked antibody drug conjugate. Bioconjug. Chem. 2014;25:1223–1232.

55Hamblett KJ, Senter PD, Chace DF, Sun MMC, Lenox J, Cerveny CG, et al. Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin. Cancer Res. 2004;10:7063–7070.

56Drake PM, Albers AE, Baker J, Banas S, Barfield RM, Bhat AS, et al. Aldehyde Tag Coupled with HIPS Chemistry Enables the Production of ADCs Conjugated Site-Specifically to Different Antibody Regions with Distinct in Vivo Efficacy and PK Outcomes. Bioconjugate Chem. 2014;25(7):1331-1341.

57Drake PM, Rabuka D. An emerging playbook for antibody-drug conjugates: lessons from the laboratory and clinic suggest a strategy for improving efficacy and safety. Curr Opin Chem Biol 2015;28:174–80.

58Beck A, Goetsch L, Dumontet C, Corvaia N. Strategies and challenges for the next generation of antibody–drug conjugates. Nat Rev Drug Discov 2017;16:315–37.

59Gilbert CW, McGowan EB, Seery GB, Black KS, Pegram MD. Targeted prodrug treatment of HER-2-positive breast tumor cells using trastuzumab and paclitaxel linked by A-Z-CINN Linker. J Exp Ther Oncol 2003;3:27-35.

60Gebhart G, Lamberts LE, Wimana Z, Garcia C, Emonts P, Ameye L, et al. Molecular imaging as a tool to investigate heterogeneity of advanced HER2-positive breast cancer and to predict patient outcome under trastuzumab emtansine (T-DM1): the ZEPHIR trial. Annals of Oncology 2016;27: 619–624.

61Fabi A, Alesini D, Valle E, Moscetti L, Caputo R, Caruso M, et al. T-DM1 and brain metastases: Clinical outcome in HER2-positive metastatic breast cancer. The Breast 2018;41:137–43.

62Krop IE, Lin NU, Blackwell K, Guardino E, Huober J, Lu M, et al. Trastuzumab emtansine (T- DM1) versus lapatinib plus capecitabine in patients with HER2-positive metastatic breast cancer and central nervous system metastases: a retrospective, exploratory analysis in EMILIA. Ann Oncol 2015;26:113-19.

63Keith KC, Lee Y, Ewend MG, Zagar TM, Anders CK. Activity of trastuzumab-emtansine (TDM1) in HER2-positive breast cancer brain metastases: a case series. Cancer Treat Commun. 2016;7:43-46.

64Bartsch R, Berghoff AS, Vogl U, Rudas M, Bergen E, Dubsky P, et al. Activity of T-DM1 in HER2-positive breast cancer brain metastases. Clin Exp Metastasis 2015;32:729-37.

65Dijkers EC, Oude Munnink TH, Kosterink JG, Brouwers AH, Jager PL, de Jong JR, et al. Biodistribution of 89Zr-trastuzumab and PET imaging of HER2-positive lesions in patients with metastatic breast cancer. Clin Pharm Ther 2010;87:586.

66Kinneer K, Meekin J, Tiberghien AC, Tai YT, Phipps S, Kiefer CM, et al. SLC46A3 as a Potential Predictive Biomarker for Antibody-Drug Conjugates Bearing Noncleavable Linked Maytansinoid and Pyrrolobenzodiazepine Warheads. Clin Cancer Res 2018 Aug 21. doi: 10.1158/1078-0432.CCR-18-1300. [Epub ahead of print]

67Loganzo F, Sung M, Gerber HP. Mechanisms of resistance to antibody-drug conjugates. Mol Cancer Ther 2016;15:2825–34.

68Rios-Luci C, Garcia-Alonso S, Diaz-Rodriguez E, Nadal-Serrano M, Arribas J, Ocana A, et al. Resistance to the antibody-drug conjugate T-DM1 is based in a reduction in lysosomal proteolytic activity. Cancer Res 2017;77:4639–51.

69Hamblett KJ, Jacob AP, Gurgel JL, Tometsko ME, Rock BM, Patel SK, et al. SLC46A3 is required to transport catabolites of noncleavable antibody maytansine conjugates from the lysosome to the cytoplasm. Cancer Res 2015;75:5329–40.

70Li G, Guo J, Shen BQ, Bumbaca Yadav D, Sliwkowski MX, Crocker LM, et al. Mechanisms of acquired resistance to trastuzumab emtansine in breast cancer cells. Mol Cancer Ther 2018;17:1441–53.

71Krop IE, Modi S, LoRusso PM, Pegram M, Guardino E, Althaus B, et al. Phase 1b/2a study of trastuzumab emtansine (T-DM1), paclitaxel, and pertuzumab in HER2-positive metastatic breast cancer. Breast Cancer Res 2016;18:34.

72Tang Y, Tang F, Yang Y, Zhao L, Zhou H, Dong J, et al. Real-Time Analysis on Drug- Antibody Ratio of Antibody-Drug Conjugates for Synthesis, Process Optimization, and Quality Control. Sci Rep 2017;7:7763.

73Levengood MR, Xinqun Zhang, Kim M Kissler, Joshua H Hunter, Peter D. Senter. Development of homogeneous dual-drug ADCs: Application to the co-delivery of auristatin payloads with complementary antitumor activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 982.

74Sledge GW, Neuberg D, Bernardo P, Ingle JN, Martino S, Rowinsky EK, et al. Phase III trial of doxorubicin, paclitaxel, and the combination of doxorubicin and paclitaxel as front-line chemotherapy for metastatic breast cancer: an intergroup trial (E1193). J Clin Oncol 2003;21:588-92.

75Citron ML, Berry DA, Cirrincione C, Hudis C, Winer EP, Gradishar WJ, et al. Randomized trial of dose-dense versus conventionally scheduled and sequential versus concurrent combination chemotherapy as postoperative adjuvant treatment of node-positive primary breast cancer: first report of Intergroup Trial C9741/Cancer and Leukemia Group B Trial 9741. J Clin Oncol 2003;21:1431-9.

76Tsui CK, Barfield RM, Fischer CR, Morgens DW, Li A, Smith BAH, et al. CRISPR-Cas9 screens identify regulators of antibody–drug conjugate toxicity. Nature Chem Biol. (published online 26 August 2019).

77Borges VF, Ferrario C, Aucoin N, Falkson C, Khan Q, Krop I, et al. Tucatinib Combined With Ado-Trastuzumab Emtansine in Advanced ERBB2/HER2-Positive Metastatic Breast Cancer: A Phase 1b Clinical Trial. JAMA oncology 2018;4(9),1214–1220.

78Abraham J, Puhalla S, Sikov WM, et al. NSABP FB-10: Phase Ib dose-escalation trial evaluating trastuzumab emtansine (T-DM1) with neratinib (N) in women with metastatic HER2+ breast cancer (MBC). J Clin Oncol 2018 36:15_suppl, 1027.

79Scaltriti M, Verma C, Guzman M, Jimenez J, Parra JL, Pedersen K, et al. Lapatinib, a HER2 tyrosine kinase inhibitor, induces stabilization and accumulation of HER2 and potentiates trastuzumab-dependent cell cytotoxicity. Oncogene. 2009;28(6):803-14.

80Traila PA, Dubowchikb GM, Lowingerc TB. Antibody drug conjugates for treatment of breast cancer: Novel targets and diverse approaches in ADC design. Pharmacology and Therapeutics 2018;181:126–142.

81Müller P, Kreuzaler M, Khan T, Thommen DS, Martin K, Glatz K, et al. Trastuzumab emtansine (T-DM1) renders HER2+ breast cancer highly susceptible to CTLA-4/PD-1 blockade. Science Translational Medicine 2015;7(315):315ra188.

82D’Amico L, Menzel U, Prummer M, Müller P, Buchi M, Kashyap A, et al. A novel anti-HER2 anthracycline-based antibody-drug conjugate induces adaptive anti-tumor immunity and potentiates PD-1 blockade in breast cancer. J Immunother Cancer. 2019;7(1):16. https://doi.org/10.1186/s40425-018-0464-1.

83Patel TA, Ensor J, Rodriguez AA, Belcheva A, Darcourt JG, Niravath PA, et al. Phase Ib study of trastuzumab emtansine (TDM1) in combination with lapatinib and nab-paclitaxel in metastatic HER2-neu overexpressed breast cancer patients: Stela results. J Clin Oncol 2018 36:15_suppl, 1035.

84Emens LA, Esteva F, Beresford M, Saura C, De Laurentis M, Kim S-B, et al. Results from KATE2, a randomized phase 2 study of atezolizumab (atezo)+trastuzumab emtansine (T-DM1) vs placebo (pbo)+T-DM1 in previously treated HER2+ advanced breast cancer (BC). Cancer Res 2019 79: 4_Suppl, PD3-01.

85Knudsen ES, Frankel A, Chang J, Witkiewicz A, Haley B. Palbociclib in combination with TDM1 for metastatic HER2+ breast cancer. Ann Oncol 2017 28:1_Suppl, mdx137.002.

86Goel S, Pernas S, Tan-Wasielewski Z, Barry WT, Bardia A, Rees R, et al. Ribociclib Plus Trastuzumab in Advanced HER2-Positive Breast Cancer: Results of a Phase 1b/2 Trial. Clin Breast Cancer. 2019 May 30.[Epub ahead of print].

87Jain S, Santa-Maria CA, Rademaker A, Giles FJ, Cristofanilli M, Gradishar WJ. Phase I study of alpelisib (BYL-719) and T-DM1 in HER2-positive metastatic breast cancer after trastuzumab and taxane therapy. J Clin Oncol 2017 35:15_suppl, 1026.

88Filho OM, Goel S, Barry WT, Hamilton EP, Tolaney SM, Yardley DA, et al. A mouse-human phase I co-clinical trial of taselisib in combination with TDM1 in advanced HER2-positive breast cancer (MBC). J Clin Oncol 2017 35:15_suppl, 1030.

89Abraham J, Puhalla S, Sikov WM, Montero AJ, Salkeni MA, Razaq W, et al. NSABP FB-10: Phase Ib dose-escalation trial evaluating trastuzumab emtansine (T-DM1) with neratinib (N) in women with metastatic HER2+ breast cancer (MBC). J Clin Oncol 2018 36:15_suppl, 1027.

90Bhat G, Potter D, Bharadwaj J, et al. A phase 1b study of poziotinib in combination with T- DM1 in women with advanced or metastatic HER2-positive breast cancer. Cancer Res 2019 79: 4_Suppl, OT2-07-04.

91Freedman RA, Gelman RS, Anders CK, Melisko ME, Parsons HA, Cropp AM, et al. TBCRC 022: A Phase II Trial of Neratinib and Capecitabine for Patients With Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer and Brain Metastases. J Clin Oncol. 2019;37(13):1081-9.

Table 1. Clinical trials of antibody-drug conjugate combined with other therapies in HER2 positive metastatic breast cancers. Sample NCI identifier Phase Status Population Regimen size

ADC+ chemotherapy MBC with CNS metastasis T-DM1 vs T-DM1 + NCT03190967 I/II Recruiting treated with stereotactic radiation 125 Metronomic Temozolomide or surgery MBC following at least 2 lines of T-DM1 + Lapatinib + Nab- NCT0207391683 I Completed 24 therapy paclitaxel Received up to 2 lines of T-DM1 + Non-pegylated NCT02562378 I Completed chemotherapy for MBC; prior 15 Liposomal Doxorubicin Trastuzumab and a Taxane

ADC+ immunotherapy

Cohort 1: prior trastuzumab and a Cohort 1: T-DM1+ taxane Utomilumab NCT03364348 Ib Recruiting 79 Cohort 2: at least 2 lines of prior Cohort 2: Trastuzumab + therapy for MBC. Utomilumab Prior therapy with trastuzumab NCT03032107 Ib Recruiting and a taxane; 1 prior line of T-DM1+ Pembrolizumab 27 therapy for MBC Arm 1: Pembrolizumab+ Trastuzumab Arm 2: Pembrolizumab + T- NCT02318901 Ib/II Terminated HER2+ MBC 16 DM1 Arm 3: Pembrolizumab + Cetuximab Cohort 1A: Atezolizumab + Trastuzumab + Pertuzumab Cohort 1B: Atezolizumab + T- DM1 (3.6 mg/kg) Cohort 1C : Atezolizumab + NCT02605915 Active, not Ib HER2+ MBC T-DM1 (3.0 mg/kg) 98 recruiting Cohort 1D : Atezolizumab + T-DM1 (2.4 mg/kg) Cohort 1F: Docetaxel + Trastuzumab + Pertuzumab + Atezolizumab

Active, not Locally advanced/MBC; prior T-DM1 + Atezolizumab vs T- NCT0292488384 II 202 recruiting trastuzumab and a taxane DM1 + Placebo

Cohort 1: HER2+ MBC have Cohort 1: DS-8201a + NCT03523572 Ib Recruiting 30 received prior T-DM1 Nivolumab

ADC+ CDK 4/6 inhibitors

RB-proficient (RB normal and NCT0197616985 Ib Recruiting T-DM1+ Palbociclib 33 p16ink4a low by IHC) MBC;

prior trastuzumab or other HER2 targeted therapies. T-DM1+ Palbociclib vs T- NCT03530696 II Recruiting HER2+ MBC 132 DM1 Cohort A: At least one previous line of anti-HER2 treatment Cohort A: Ribociclib + T- including containing Trastuzumab DM1 or and taxane; Cohort B: Ribociclib + NCT0265734386 Ib/II Recruiting Cohort B: prior trastuzumab, 86 Trastuzumab or pertuzumab and T-DM1 Cohort C: Ribociclib + Cohort C: prior Trastuzumab, Trastuzumab+ Fulvestrant pertuzumab, and T-DM1 and up to five lines of therapy for MBC

ADC+ PI3K inhibitors

Active, not Prior trastuzumab and a taxane NCT0203801087 I T-DM1+ BYL719 17 recruiting for MBC Arm A: Taselisib + T-DM 1 or Arm B: Taselisib + T-DM 1+ Pertuzumab or NCT0239042788 HER2+ MBC with any previous Arm C: Taselisib + Ib Recruiting 76 anti-HER2 therapy Trastuzumab +Pertuzumab or Arm D: Taselisib + Trastuzumab+ Pertuzumab + Paclitaxel or At least 1 prior line of therapy for T-DM1 + GDC-0941 vs NCT00928330 Ib Completed 57 MBC containing trastuzumab Trastuzumab + GDC-0941

ADC+ HER2 tyrosine kinase inhibitors

Prior treatment with trastuzumab NCT0223600089 Ib/II Recruiting and pertuzumab; failed up to 1 T-DM1+Neratinib 63 line of therapies for MBC Active, not Prior treatment with a taxane and NCT0198350177 Ib T-DM1+ Tucatinib 57 recruiting trastuzumab Not yet Prior treatment with a taxane and T-DM1+ Tucatinib vs NCT03975647 III 406 recruiting trastuzumab T-DM1+ Placebo

Active, not Must have at least 1 line of anti- NCT0342910190 Ib T-DM1 + Poziotinib 6 recruiting HER2 therapy HER2+ MBC with any previous anti-HER2 therapy Cohort 1: must have new or Cohort 1: Neratinib or progressive CNS lesions Cohort 2: Neratinib then Cohort 2: must have operable surgical resection or NCT0149466291 II Recruiting CNS disease 168 Cohort 3: Neratinib+ Xeloda Cohort 3: must have measurable or CNS disease with or without prior Cohort 4: Neratinib+ T-DM1 lapatinib therapy Cohort 4: must have measurable, new or progressive CNS disease

Figures:

Figure 1. Biparatopic HER2:HER2 Antibody-Drug Conjugate MEDI4276 A) The biparatopic antibody blocks ligand-induced HER2-HER3 receptor dimerization. 39S antibody functions synergistically with trastuzumab in inhibiting cell proliferation in vitro. Two engineered cysteine residues per heavy chain (S239C and S442C) enable site-specific DAR 4 conjugation of a tubulysin payload with a potent bystander effect. L234F reduces Fc gamma receptor binding. B) Confocal microscopy images of HER2+ BT474 cells showing HER2 internalization and lysosomal trafficking induced by the biparatopic antibody or trastuzumab (50 nM, each antibody). HER2-antibody complexes are stained by AF488-labeled antibody (green), lysosomes are stained by AF647-labeled antibody (red), and nuclei are stained by DAPI (blue).28 Figure 1B reprinted from Cancer Cell, vol. 29, Li JY and colleagues, A Biparatopic HER2-Targeting Antibody-Drug Conjugate Induces Tumor Regression in Primary Models Refractory to or Ineligible for HER2-Targeted Therapy, 117-29, copyright 2016, with permission from Elsevier.

Figure 2. Antibody-drug conjugates (ADCs) in HER2-“low” MBC patients. MBC, metastatic breast cancer; IHC, immunohistochemistry staining; FISH, fluorescent in-situ hybridization; ORR, objective response rate; CI, confidence interval; HR, hormone receptor; PFS, progression-free survival; mo, month; NA, not available; AEs, adverse events, scissor symbol – site of linker cleavage.

Figure 3. Disposition of an ADC molecule on a cellular level ADCs bind to cell surface antigens, followed by antigen-mediated internalization. Following internalization, ADCs undergo intracellular trafficking from early to late endosomes, ultimately to the lysosomal compartment where release of cytotoxic payload induces cell killing. Linker chemistry attributes dictate deconjugation of drug in the circulation and in the extracellular compartment, as well as generation of unconjugated drug in different endosomal/lysosomal subcellular compartments. ADC molecules bound to target/FcRn receptors undergo a futile recycling pathway back to the extracellular space via recycling endosome.28,47 Adapted from Cancer Cell, vol. 29, Li JY and colleagues, A Biparatopic HER2-Targeting Antibody-Drug Conjugate Induces Tumor Regression in Primary Models Refractory to or Ineligible for HER2-Targeted Therapy, 117-29, copyright 2016, with permission from Elsevier; and adapted with permission from Springer Nature: The AAPS Journal, Evolution of Antibody- Drug Conjugate Tumor Disposition Model to Predict Preclinical Tumor Pharmacokinetics of Trastuzumab-Emtansine (T-DM1), Singh AP and colleagues, copyright 2016.

Figure 1: A Trastuzumab scFv (binding to domain IV of HER2)

39S (binding to domain II of HER2)

H2N

O O H O N HN N N O S N O N O O H O O N H NH S O O

Linker Tubulysin warhead (AZ13599185) B T = 0 T = 30 min T = 2 hr T = 4 hr T = 6 hr

IgG1 CTRL

Trastuzumab

Biparatopic antibody

HER2+ BT474 BC cells Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 3, 2019; DOI: 10.1158/1078-0432.CCR-18-1976 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 2:

T-DM1 (36) DS-8201a (37) SYD985 (39)

O HN O O O O O CI O H H N OH S N N N O O N N N N O N S H H H N O O O NH N O Me O Characteristics CI Me O O O N Cleavable peptide MeΟ N H O O Antibody O O GGFG linker N O O F N N OH O O O O N O O H O N N O N O N N OH H H H MeΟ OH O O O Self-elimination module S DM1 MCC Trastuzumab NH DXd H2N O

Trial phase Phase II Phase I Phase I

Antibody Trastuzumab Trastuzumab Trastuzumab Maytansinoids Deruxtecan Duocarmycin Payload (mitotic inhibitor) (topoisomerase I inhibitor) (alkylating agents) Noncleavable thioether Cleavable dipeptide valine-citrulline Linker Cleavable tetrapeptide linker linker (MCC) linker

Drug-antibody 3.5 8 2.8 ratio Bystander killing No bystander killing effect Potent bystander killing (38) Potent bystander killing (40) effect in vitro ≥One chemotherapy agent Population Heavily pretreated MBC Heavily pretreated MBC for MBC

HER2 low IHC 0–2+ and/or FISH- IHC 1+ or IHC 2+/ISH- IHC 1+/2+/ISH- deﬁnition All 19/43 (44.2%) IHC 1+ 7/21 (33.3%) HR+ 8/30 (27%) ORRs (%) 1/21 (4.8%) IHC 2+ 12/22 (54.5%) HR- 6/15 (40%) HR+ 18/38 (47.4%) Prior CDK4/6 inhibitor 4/12 (33.3%)

All 7.6 (4.9, 13.7) IHC 1+ 5.7 (1.4, 7.9) PFS, median HR+ 4.1 (2.4, 5.4) 2.6 (1.4, 3.9) IHC 2+ 13.6 (NA) (95% CI), mo HR- 4.4 (1.0, 7.1) HR+ 7.9 (4.4, 13.7) Prior CDK4/6 inhibitor 7.1 (NA)

Anemia, neutropenia, thrombocytopenia, leukopenia, G3-5 AEs Thrombocytopenia, nausea, vomiting, diarrhea, Fatigue, dry eyes, conjunctivitis occurring >2% fatigue, hypokalemia, constipation, decreased appetite, No ≥ G4 AEs observed population anemia, neutropenia fatigue, pyrexia, hypokalemia, hypoalbuminemia, headache, weight loss, hyponatremia, pneumonitis

Pneumonitis Dose-limiting Thrombocytopenia (8) Pneumonitis (1 G5 event at 5.4 mg/kg; 1 G5 at 6.4 toxicity (2 G4 events at 4.8 mg/kg) (1 G5 event at 2.4 mg/kg) mg/kg) Maximum 3.6 mg/kg 5.4 mg/kg 1.2 mg/kg tolerated dose

ADC K Free 1. Clustering

KADC Bound KADC

Downloaded from Int

HER2 Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. Author ManuscriptPublishedOnlineFirstonOctober3,2019;DOI:10.1158/1078-0432.CCR-18-1976

Tumor cell 2. Internalization

ADC clincancerres.aacrjournals.org K Recycle endo

KADC Early endo 4. Lysosomal degradation KADC 3. Early Late endo endosome

Tub KDrug K on out

KTub • Mitotic arrest off • Apoptosis • Disrupted intracellular Bystander trafficking effect Author Manuscript Published OnlineFirst on October 3, 2019; DOI: 10.1158/1078-0432.CCR-18-1976 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

HER2-overexpressing/amplified breast cancer as a testing ground for antibody-drug conjugate drug development in solid tumors

Mark D Pegram, David Miles, C. Kimberly Tsui, et al.

Clin Cancer Res Published OnlineFirst October 3, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-1976

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2019/10/03/1078-0432.CCR-18-1976. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.