Targeting Multiple EGFR Expressing Tumors with a Highly Potent Tumor-Selective Antibody

Home , Depatuxizumab mafodotin, Monomethyl auristatin F

Author Manuscript Published OnlineFirst on August 26, 2020; DOI: 10.1158/1535-7163.MCT-20-0149 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Drug Conjugate

Mark G. Anderson1, Hugh D. Falls1, Michael J. Mitten1, Anatol Oleksijew1, Kedar S. Vaidya2, Erwin R. Boghaert1, Wenqing Gao1, Joann P. Palma1, Diana Cao3, Puey-Ling Chia3, Thomas John3, Hui K. Gan3, Andrew M. Scott3 and Edward B. Reilly1

Authors Affiliation

1 AbbVie Inc., 1 North Waukegan Rd., North Chicago, IL 60064 2 Former AbbVie employee 3Olivia Newton-John Cancer Research Institute, and School of Cancer Medicine, La Trobe University Austin Hospital, Heidelberg, Victoria, Australia

Running Title

Anti-EGFR PBD dimer ADC

Correspondence

Edward B. Reilly, Ph.D.

AbbVie

Oncology Discovery, R460

1 North Waukegan Road

North Chicago, IL 60064-6099

Phone: (847) 937-0815

Email: [email protected]

Authors Disclosure

The design, study conduct, and financial support for the study were provided by AbbVie. AbbVie participated in the interpretation of the data, review, and approval of the publication. MGA, HDF, MJM,

Downloaded from mct.aacrjournals.org on September 28, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 26, 2020; DOI: 10.1158/1535-7163.MCT-20-0149 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

AO, ERB, WG, JPP and EBR are employees of AbbVie. KSV is a former employee of AbbVie. PLC, DC, TJ, have nothing to disclose, and HKG has been a consultant for AbbVie

Abstract

ABBV-321 (serclutamab talirine), a next-generation epidermal growth factor receptor (EGFR)- targeted antibody-drug conjugate (ADC) incorporates a potent pyrrolobenzodiazepine (PBD) dimer toxin conjugated to the EGFR-targeting ABT-806 affinity matured AM1 antibody. ABBV- 321 follows the development of related EGFR targeted ADCs including depatuxizumab mafodotin (depatux-m, ABT-414), ABT-806 conjugated to monomethyl auristatin F (MMAF), and ABBV-221 (losatuxizumab vedotin), AM1 antibody conjugated to monomethyl auristatin E (MMAE). The distinct tumor selectivity of ABBV-321 differentiates it from many previous highly active antibody PBD conjugates that lack a therapeutic window. Potency of the PBD dimer, combined with increased binding of AM1 to EGFR-positive tumor cells, opens the possibility to target a wide array of tumors beyond those with high levels of EGFR overexpression or amplification, including those insensitive to auristatin-based ADCs. ABBV-321 exhibits potent anti-tumor activity in cellular and in vivo studies including xenograft cell line and patient- derived xenograft (PDX) glioblastoma (GBM), colorectal, lung, head & neck and malignant mesothelioma tumor models which are less sensitive to depatux-m or ABBV-221. Combination studies with ABBV-321 and depatux-m suggest a promising treatment option permitting suboptimal, and potentially better tolerated, doses of both ADCs while providing improved potency. Collectively, these data suggest that ABBV-321 may offer an extended breadth of efficacy relative to other EGFR ADCs while extending utility to multiple EGFR-expressing tumor indications. Despite its highly potent PBD dimer payload, the tumor selectivity of ABBV-321 - coupled with its pharmacology, toxicology and pharmacokinetic profiles - support continuation of ongoing Phase 1 clinical trials in patients with advanced EGFR-expressing malignancies.

Introduction

Targeted therapies are commonly prescribed anti-neoplastic agents in the United States and worldwide. EGFR plays a causal role in the development and maintenance of many human carcinomas. EGFR is a well-validated oncology target, as EGFR-directed therapies [monoclonal antibodies (mAbs) and small molecules] have gained widespread use for a variety of cancer types, including lung, head & neck, colon, and pancreatic cancer. These EGFR-directed therapies have shown improvements in both progression-free survival and overall survival while preserving quality of life (1-8). Despite the enhancements, these approaches are limited by toxic side effects and development of resistance. The most common toxicity of these agents is a well characterized skin rash, similar in appearance to acne, usually limited to the face, upper chest and back. Other reported toxicities include diarrhea, constipation, stomatitis, fatigue, and electrolyte disturbances (9,10). These mechanism based therapies are further limited by narrow efficacy profiles because the mutational status of tumorigenic genes including EGFR, K- Ras, B-Raf, phosphoinositide 3-kinase and the phosphatase and tensin homolog gene each play a role in EGFR resistance mechanisms (11-13).

Oncology ADCs are a class of therapeutics that combine mAb specificity with cytotoxicity of potent anti-cancer small molecules. Significant advancements in linker stability and toxin potency are primarily responsible for the resurgence in ADC development. Recent examples of clinically relevant, approved ADCs include brentuximab vedotin/Adcetris®, an anti-CD30 ADC for Hodgkin's lymphoma and anaplastic large cell lymphoma, polatuzumab vedotin/Polivy®, an anti- CD79b ADC targeting the B-cell receptor for diffuse large B-cell lymphoma; trastuzumab emtansine/Kadcyla® , an anti-Her2 ADC for metastatic breast cancer; inotuzumab ozogamicin/Besponsa®, an anti-CD22 ADC for relapsed or refractory B-cell precursor acute lymphoblastic leukemia; gemtuzumab ozogamicin/Mylotarg® an anti-CD33 for acute myeloid leukemia; Trodelvy, an anti-Trop-2 ADC for metastatic TNB, and Enhertu, an anti-Her2 ADC for relapsed/recurrent metastatic Her2+ breast cancer (14-18). A distinct clinical advantage of ADCs is their ability to selectively deliver toxic loads to a tumor, bypassing downstream resistance mechanisms related to intracellular signaling (15,16).

The ABT-806 antibody is a humanized derivative of the murine anti-EGFR mAb (mAb806) that binds preferentially to tumors expressing EGFR or EGFR with a deletion of exons 2-7 (EGFRvIII); the latter predominantly exposes a unique epitope that is also exposed when EGFR is expressed above physiological levels (19). Depatux-m, an ADC comprised of ABT-806 and MMAF cytotoxin, has been in multiple clinical studies in glioblastoma patients with amplified EGFR although dosing has been limited by corneal side effects common to MMAF conjugates (20-23). The ABT-806 component of depatux-m was affinity matured using recombinant extracellular domain EGFRC271A,C283A (a derivative exposing the ABT-806 epitope) and wild-type EGFR extracellular domain proteins as selection antigens (24). The resulting affinity matured antibody AM1 contains only three amino acid differences in the complementarity-determining region relative to parental ABT-806 but displays increased binding to wild-type EGFR. Despite increased affinity for EGFR, AM1 competes with ABT-806 to bind the same epitope, suggesting that the unique tumor-selective properties of ABT-806 are retained in AM1. ABBV-221, a second-generation ADC, is comprised of AM1 conjugated to a AM1 different toxin, MMAE (25). ABBV-221 demonstrated clinical activity in select patients with advanced solid tumor types (25,26). Results from this study, however, have shown dose-related infusion reactions to be a frequently occurring adverse event, potentially limiting the maximum tolerated dose.

PBD dimers, DNA-crosslinking agents, represent an emerging class of warheads with enhanced potent anti-cancer activity compared to the clinically validated auristatins or maytansinoids (27). PBD dimers exhibit significant cell permeability, potentially enabling bystander killing of neighboring tumor cells (28). Multiple ADCs with PBD-dimer payloads and a drug antibody ratio (DAR) of 2 have advanced to the clinic although, at least partially owing to increased toxicities, there have been no approvals for clinical use (29). The highly tumor-selective nature of ABT-806 AM1 suggested that an AM1 PBD dimer conjugate may impart an expanded breadth of efficacy relative to depatux-m or ABBV-221, extending utility beyond tumors with amplified or highly expressed EGFR, while providing for an acceptable therapeutic window. To test this premise, an AM1 PBD dimer conjugate (ABBV-321; serclutamab talirine) was characterized and evaluated against EGFR positive tumor types in cellular and in vivo studies including PDX

models. ABBV-321 is currently under clinical investigation in patients with advanced solid tumors (30).

Materials and Methods

Antibodies and reagents

AM1, the affinity matured ABT-806 antibody variant, AbA (VH SEQ ID NO: 9; VL SEQ ID NO: 5) in patent US9493568B2 (31), and an isotype-matched control mAb recognizing tetanus toxin (huIgG1) were engineered to include an S238C point mutation [S239C based on Kabat numbering system (32) to permit site-specific DAR 2 conjugation to a PBD dimer (SGD-1882) as previously described (27). Recombinant forms of EGFR [soluble EGFR (sEGFR), wild-type extracellular domain (ECD), sEGFRde2-7 ECD and EGFRC271A,C283A ECD] were generated as previously described (19).

Cell culture

Tumor cell lines from ATCC (except where indicated) were maintained in media supplemented with 10% fetal bovine serum as follows: A172, A431, A549, LS174T, SW403, SW480, SW1463, U87MGvIII (The Ludwig Institute for Cancer Research), and HEK-293 in high glucose DMEM; COLO 201, COLO205, COLO 320DM, COLO 320HSR, DBTRG-05MG, DLD-1, HCT15, HCT-116, HT29, LS1034, PFSK-1, SF-264, SF-539, SNB19, SNB-75, SW48, SW620, SW1116, U-251MG, in RPMI-1640; CHLA-03-AA, LN-18, M059J, M059K, and T84 in DMEM/F12; RKO, SK-CO-1, T98G, U- 138 MG, U-87 MG (The Ludwig Institute for cancer Research), and WiDr in EMEM; and Caco-2 and LoVo in MEM. Upon receipt, all cell lines were authenticated by short tandem repeat authentication and confirmed as mycoplasma-negative with the MycoAlert assay (Lonza Inc). EGFR expression levels for cell lines not authenticated in the 6 months before use were confirmed by flow cytometry.

Western blot analysis

Cell lysates (5-10 µg) were resolved by SDS-PAGE on 4-12% NuPAGE Bis-Tris Midi gels and transferred to nitrocellulose membranes with the iBlot Dry Blotting System. Blots were blocked with 3% milk/PBS or 5% milk/Tween/Tris buffered saline (TTBS) for 1 hr at room temperature, washed three times with TTBS, and then incubated overnight with appropriate primary antibodies at 4°C. Blots were washed three times with TTBS for 5 min and incubated with either donkey anti-rabbit or donkey anti-mouse IgG-HRP secondary antibodies (Jackson Laboratories; 1:2000) for 1 hr at room temperature. Blots were washed three times with TTBS, treated with chemiluminescent substrate (Thermo Fisher Scientific, 34076) and visualized with an ImagQuant LAS 400 scanning system. Primary antibodies were as follows: rabbit anti-EGFR (Millipore Sigma, 06-847; 1:500 dilution) and rabbit anti-pan actin (Cell Signaling Technology, 4968; 1:1000 dilution).

Binding ELISA and FACS analysis

Binding ELISA and FACS analysis of antibodies and ADCs were performed as previously described (25,33).

Cytotoxicity assay

The cell lines were plated at 500-2000 cells/well in complete growth medium containing 10% FBS in 96 well plates. The following day, antibodies/ADCs were added in medium containing 0 10% FBS and cells were incubated for 144 hr at 37 C in a C02 incubator. Cell viability was assessed using an ATPlite assay (Perkin Elmer #6016739) according to the manufacturer’s instructions. A negative control ADC (HuIgG-PBD dimer) was included in all assays to confirm cell killing was antigen-dependent.

Surface plasmon resonance of antibodies

Surface plasmon resonance was evaluated on a Biacore T100 surface plasmon resonance instrument (Biacore Life Sciences) to measure binding kinetics of recombinant soluble EGFR proteins forms (analytes) binding to anti-EGFR mAbs (ligands) as previously described (19).

IHC

Immunohistochemical analysis of xenograft models were evaluated for EGFR using the Dako Pharm Dx kit per manufacturer’s instructions. Briefly, 4 µm paraffin sections were routinely deparaffinized and rehydrated. Antigen retrieval was performed and endogenous peroxidase, non-specific protein binding sites were blocked (peroxidase blocking reagent, Dako S2001, Carpinteria, CA and Background Sniper, Biocare BS966G, Concord, CA) and subsequently incubated in primary antibodies for 1 hr at room temperature. The detection system used was EnVision+ polymer for mouse or rabbit with DAB as chromogen (Dako #K4007 or K4011) and counterstained with haematoxylin (Dako #S3301). IHC results were evaluated by a pathologist based on a semi-quantitative 0 to 3+ scale.

Pharmacokinetics

The pharmacokinetic profiles of antibody and released PBD dimer were characterized in a cynomolgus monkeys after intravenous administration of ABBV-321 at 25, 50, or 100 µg/kg IV. Comparable ABBV-321 binding, as assessed by FACS, was observed for cells overexpressing human and cynomolgus monkey wild-type EGFR. The antibody concentration was determined as total antibody (AM1 S238C antibody irrespective of the PBD dimer load) and conjugated antibody (ABBV-321 with at least one PBD dimer attached to the antibody and irrespective of the PBD dimer load); both were measured in serum by electrochemiluminescence-based ligand

binding assays. A high-performance liquid chromatography/mass spectrometry method was used to measure the released PBD dimer concentrations in samples.

In vivo studies For experiments with SNO199, SNO207 (The Jackson Laboratory), U87MG, SNO207,EBC-1, FaDu, A253, LoVo, SW48, HCT-116 and NCI-H1650, female SCID, SCID-Beige and nude mice were obtained from Charles River (Wilmington, MA). Eight to ten mice were housed per cage. The body weight upon arrival was 20-22 g. Food and water were available ad libitum. Mice were acclimated to the animal facilities for a period of at least one week prior to commencement of experiments. Animals were tested in the light phase of a 12-hr light:12-hr dark schedule (lights on at 06:00 hrs). All experiments were conducted in compliance with AbbVie's Institutional Animal Care and Use Committee and the National Institutes of Health Guide for Care and Use of Laboratory Animals Guidelines in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care.

To generate xenografts, a suspension of viable tumors cells mixed with an equal amount of Matrigel (BD Biosciences) was injected subcutaneously into the flank of 6- to 8-week old mice. The injection volume was 0.2 mL composed of a 1:1 mixture of S-MEM and Matrigel (BD Biosciences). Tumors were size matched at approximately 200-250 mm3. Therapy began the day of or 24 hrs after size matching the tumors. Mice weighed approximately 25 g at the onset of therapy. Each experimental group included 8-10 animals. Tumors were measured two to three times weekly. Measurements of the length (L) and width (W) of the tumor were obtained via electronic calipers and the volume was calculated according to the following equation: V = L x W2/2. Mice were euthanized when tumor volume reached a maximum of 3,000 mm3 or upon presentation of skin ulcerations or other morbidities, whichever occurred first. For all groups, tumor volumes were plotted only until the full set of animals remained on study. If animals had to be taken off study, the remaining animals were monitored for tumor growth until they reached defined endpoints. Mice that had no palpable tumor were maintained up to 16 weeks

to monitor for regrowth. Control arms for in vivo experiments included vehicle, huIgG and huIgG ADCs which are denoted in the figures.

All experiments with the PDX GBM1 (an EGFR amplified GBM PDX cell line) and the PDX #1174 (an EGFR and mAb806 IHC positive mesothelioma PDX) model were conducted with female 4-6 weeks old (average 20-25g body weight) NSG (NOD-SCID-IL2R-/-) mice obtained from Biological Research facility, Austin Hospital, Victoria, Australia and Animal Research Centre, Western Australia. All experiments were approved by and conducted in compliance with the Austin Health Animal Ethics Committee and were performed as previously described (25). Tumors were established by subcutaneous placement of 2-3 mm3 tumor pellets. Treatment was given every 4th day by intraperitoneal injection. tumor measurements were taken two to three times per week until mice were culled for ill health, death or euthanasia as per our institutional ethics guidelines. For the PDX #1174 study, mice in the experimental treatment arm were dosed twice a week by intraperitoneal (IP) injection, mice in the cisplatin chemotherapy treatment arm received cisplatin at 3mg/kg once a week and mice in the PBS control arm received PBS (100µl per injection) IP injection twice a week. Treatments were given for a total duration of 3 weeks. Tumor measurements were taken two to three times per week until mice were culled for ill health, death or euthanasia as per our institutional ethics guidelines. Tumor volumes were measured by electronic calipers. Data analysis for TGI (%) were used Microsoft Excel and for in vivo using one-way ANOVA with Bonferroni and student t-test (GraphPad prism 8.2.0).

Control arms for in vivo experiments included vehicle, huIgG and huIgG ADC controls which are denoted in the figures.

Statistical analysis

IC50 and EC50 values were determined by nonlinear regression analysis of concentration response curves using GraphPad Prism 6.0 program. In vivo data were analyzed using two-way

ANOVA with post-hoc Bonferroni correction for TGImax, and the Mantel-Cox log-rank test for TGD (GraphPad Prism 6.0).

Results

Binding properties of ABBV-321 for EGFR

A series of binding assays were performed to determine whether the S238C mutation and subsequent conjugation to the PBD dimer influenced the binding properties of AM1. As measured by Biacore analysis, the affinities of AM1, AM1 S238C, and ABBV-321 to the recombinant forms of wild-type EGFR 1-501 ECD, EGFRvIII and EGFRC271A,C283A were comparable (Table 1).

Binding of AM1, AM1S238C and ABBV-321 to cell surface human EGFR was measured by fluorescence activated cell sorting (FACS). AM1, AM1 S238C, and ABBV-321, bind cells expressing human wild-type EGFR with similar apparent affinity. Furthermore, AM1, AM1 S238C, and ABBV-321 bind cells expressing human EGFRC271A,C283A with similar apparent affinity, with a higher apparent affinity than binding to the cells expressing wild-type EGFR (Figure 1). These results indicate that introduction of S238C mutation and subsequent conjugation to the PBD dimer did not alter the binding characteristics of AM1.

In vitro cytotoxicity of ABBV-321 on GBM tumor cell lines

Depatux-m and ABBV-221 have previously been shown to display significant cytotoxic activity against GBM tumor cells with the most sensitive cell lines being those with higher levels of EGFR overexpression. The activity of ABBV-321 was evaluated against a panel of human GBM cell lines with a wide range of EGFR expression as measured by Western blot analysis. As indicated in Table 2, whereas ABBV-221 was primarily active against the U-87 MGvlll tumor cell

line with amplified EGFR (EC50 0.06 nM), ABBV-321 showed significant improved potency in many of the non-EGFR amplified GBM cell lines (single digit nM EC50 values) suggesting that ABBV-321 may be broadly active in GBM and expand the breadth of activity beyond tumors with amplified EGFR.

In vivo efficacy of ABBV-321 in EGFR-expressing GBM xenograft and PDX tumor models

The efficacy of ABBV-321 was evaluated in GBM tumor models with different EGFR genotypes including amplified, mutant de2-7 or overexpressed wild-type EGFR. SNO199 PDX, a tumor model with amplified mutant de2-7 EGFR (EGFR IHC score 3+), is sensitive to treatment with both depatux-m and ABBV-221 (Figure 2A and (33)). ABBV-321 administered at 10-20-fold lower doses than ABBV-221 (0.2 mg/kg, Q7Dx3) was also effective in regressing these tumors (Figure 2A). In the U87MG cell line-derived xenograft (EGFR IHC score 3+) and the SNO207 PDX (EGFR IHC score 1+) models, both with overexpressed wild-type EGFR, ABBV-321 administered at 0.2 mg/kg on a Q7Dx3 dosing regimen was highly effective at inhibiting tumor growth including induction of regressions compared to higher doses of ABBV-221 (Figure 2B and 2C).

Combination of even lower doses of ABBV-321 (0.1 mg/kg) and depatux-m (1.4 mg/kg) inhibited growth of the GBM1 PDX model with EGFR overexpression, whereas either monotherapy treatment was much less effective (Figure 2D). These results suggest that combination of suboptimal doses of ABBV-321 with depatux-m may be an effective strategy to enhance potency. Non-targeting PBD dimer ADC control exhibited activity at higher doses in this model, likely driven by tumor related enhanced permeability and retention effects as previously reported (Figure 2D, S1, and (33-36)).

ABBV-321 in vitro cytotoxicity against colorectal tumor cell lines

The cytotoxic activity of ABBV-321 was evaluated against a panel of colorectal tumor cell lines since colorectal tumor cells generally have lower EGFR expression compared to EGFR amplified or overexpressed tumors. ABBV-321 exhibits significant cytotoxic activity against colorectal cancer cell lines expressing over a 250-fold difference in levels of EGFR as measured by Western blot analysis (Table 3). The most sensitive cell lines are killed by ABBV-321 at sub-nanomolar to single-digit nanomolar concentrations whereas most of the cell lines tested are largely insensitive to the MMAE ADC, ABBV-221 (Table 3), confirming that activity of ABBV-321 may extend to low and moderate- EGFR-expressing colorectal tumors which are largely insensitive to auristatin-based ADCs.

ABBV-321 in vivo efficacy against lung, colorectal, head & neck and mesothelioma xenograft and PDX tumor models

In vivo efficacy of ABBV-321 was evaluated in human xenograft tumor models representing lung, colon and head & neck cancers with varying expression levels of EGFR. Dosing regimens initially were based on previous ABBV-221 and depatux-m studies, however with the demonstration of enhanced potency of ABBV-321, less frequent dosing regimens were also implemented. ABBV-321 administered at a single dose of 0.3 mg/kg was highly effective in inducing sustained regressions and, in some cases, complete regressions in xenografts of EBC-1, a lung squamous cell carcinoma cell line (EGFR IHC score of 2+) and head and neck cancer cell lines FaDu (EGFR IHC score of 3+) and A253 (EGFR IHC score of 2+) (Figure 3A-C). At an equivalent dose, the non-targeted PBD dimer ADC, huIgG-PBD, exhibited minimal tumor inhibitory effect, confirming antigen-mediated ADC activity.

Efficacy of ABBV-321 was also determined in additional human xenograft models of colorectal adenocarcinoma xenograft models including LoVo (EGFR IHC score 3+), SW48 (EGFR IHC score 3+) and HCT-116 (EGFR IHC score 1+). In the LoVo tumor model, ABBV-321 administered at 0.5 mg/kg on a Q7D×6 regimen induced complete responses (Figure 3D). Specificity of the anti- EGFR conjugates was demonstrated by the increased durability of response compared to the non-targeted control conjugate, huIgG-PBD dimer. The SW48 model was evaluated in dose titration studies to further establish the potency of ABBV-321. A single dose of 0.0125 mg/kg, the lowest dose tested, demonstrated tumor growth delay (Figure 3E). ABBV-321 administered at Q7Dx3 also inhibited tumor growth in the HCT-116 colorectal model with low EGFR expressing (Figure 3F). In all colorectal tumor models tested, ABBV-321 was more effective at inhibiting tumor growth than ABBV-221 administered at ten-fold higher doses.

Efficacy of ABBV-321 in suppressing tumor growth was evaluated in PDX #1174, a malignant mesothelioma PDX tumor model (EGFR IHC score of 3+). Significant tumor growth suppression was demonstrated with ABBV-321 dosed at 0.15 mg/kg 2Q7Dx3 compared to groups treated with either non-targeted ADC or cisplatin (Figure 3G). These results suggest a novel therapeutic approach for treatment of malignant mesothelioma with EGFR overexpression.

ABBV-321 is broadly active in still other EGFR-expressing solid tumor, including lung and head & neck, PDX models. Efficacy was compared across a panel of 18 PDX models with a single dosing regimen of either ABBV-321 (0.6 mg/kg, Q7Dx6) or ABBV-221 (6 mg/kg, Q7Dx6). Tumor growth inhibition data are shown in (Table 4). Consistent with in vivo results shown in Figures 2 and 3, ABBV-321 was in most cases significantly more effective in inhibiting tumor growth than ABBV- 221 at a ten-fold higher dose. These in vivo results demonstrate the potential of ABBV-321 to extend anti-tumor activity in multiple indications and beyond EGFR amplified and overexpressed tumors.

Pharmacokinetics

The pharmacokinetic profiles of ABBV-321 in cynomolgus monkeys were characterized at doses

ranging from 25 to 100 μg/kg. The Cmax values were similar between total antibody and conjugated antibody, showing dose-proportional increase. After a single dose of 100 μg/kg, the

AUC0-168 hr values for total antibody and conjugated antibody ranged between 70 and 80 μg•hr/mL, respectively, with conjugated antibody half-life around 2.7 days. Quantifiable concentrations of released PBD were not detected in any of the dose groups tested (lower limits of quantitation 50 pg/mL).

Discussion

ABBV-321 is a next generation EGFR-targeted ADC that incorporates potent PBD dimer cytotoxin molecules conjugated via a mc-Val-Ala linker to the AM1 S238C antibody. Similar to its predecessors, depatux-m and ABBV-221, ABBV-321 binds a cryptic EGFR epitope on the cell surface, exposed primarily in tumors when the receptor is activated, thereby providing tumor selectivity. ABBV-321 is differentiated from earlier generation ADCs, in that it targets a wide array of tumors beyond those with high levels of EGFR overexpression or amplification, including those insensitive to auristatin-based ADCs. ABBV-321 exhibits potent anti-tumor activity in cellular and in vivo assays including xenograft cell line and patient-derived xenograft (PDX) glioblastoma (GBM), colorectal, lung, and head & neck tumor models. Additionally, the effect of ABBV-321 on a malignant mesothelioma PDX tumor model suggests further evaluation of ABBV-321 for this indication may be warranted since EGFR is known to be overexpressed in mesothelioma (37). Of note, ABBV-321 is also active in select colorectal tumor models with variable EGFR expression levels which are less sensitive to depatux-m and ABBV-221 treatment. Microtubule inhibitors, such as MMAF and MMAE comprising depatux-m and ABBV-221, respectively, have not demonstrated clinical efficacy in colorectal cancer (38-40). In contrast, PBD dimers act by binding within the minor groove of the DNA helix and creating inter-chain crosslinks, DNA damage and ultimately cell apoptosis (41,42). DNA damaging agents with mechanisms of action similar to PBD dimer toxins have demonstrated clinical utility in colorectal cancers, providing additional support for the potential applicability of ABBV-321 in this indication (40,43). The anti-tumor activity of ABBV-321 also extends to GBM tumor models with high EGFR expression levels resulting from gene amplification. Unlike depatux-m and ABBV-221, however, ABBV-321 exhibits potent anti-tumor activity in GBM cell lines and PDX models with EGFR expression levels below the threshold required for sensitivity to depatux-m or ABBV-221. Since an intact blood-brain barrier represents a challenge in delivery of large molecule therapeutics to patients with GBM, ultimately demonstration of ABBV-321 efficacy in GBM orthotopic models may provide further preclinical validation for this indication. In this context it is important to note that objective responses have been observed with depatux-m in

GBM patients with amplified EGFR suggesting that delivery of an ADC to the brain is feasible at least in some instances (20-23).

The enhanced potency of ABBV-321 is driven by both increased affinity of the AM1 antibody compared to ABT-806 and increased potency of the PBD dimer toxin. Activity is primarily dependent on the antibody-payload combination, as decoupled antibody and payload are not efficacious in vivo at doses comparable to those included in the ADC (Figure S1). To date, anti- cancer drugs comprised of a PBD dimer face considerable challenges in achieving an acceptable therapeutic window in the clinic although several PBD dimer-containing ADC clinical programs continue to advance for both solid and hematological tumor indications (29). In contrast, owing to its highly tumor selective binding properties, ABBV-321 may be a more attractive clinical candidate than many of these previous PBD drug conjugates. The highly tumor-selective nature of ABBV-321 also suggests that on target toxicities may be limited thereby providing the basis for development of a novel anti-neoplastic agent with an acceptable risk for cancer patients who have no alternative treatment options. This is consistent with the general PBD-related toxicity findings observed in non-clinical studies which were supportive of advancing ABBV-321 to investigational studies in humans with advanced cancers that are likely to express EGFR. In this trial the dose escalation phase has been completed and a tolerated dosing regimen has been identified for the expansion phase of the study (30). Provided clinical efficacy is maintained at safely tolerated doses, ABBV-321 could also provide added benefits compared to earlier generation ADCs. For example, ABBV-321 may mitigate the risks of corneal side effects observed following depatux-m dosing. As demonstrated in tumor models, combination strategies in which depatux-m and ABBV-321 are administered at suboptimal doses may also prove an effective therapeutic strategy to maintain efficacy while mitigating both dose- dependent severe ADC toxicities and tumor regrowth driven by resistance to a single mechanism of action (22,23).

In summary, the cumulative preclinical results presented herein support continuation of investigational studies with ABBV-321 in patients with EGFR-positive tumors.

Acknowledgements

For this manuscript, Julie J. Purkal, Kelly J. Doyle and Nadezda Klunder assisted with in vivo studies; Sally Schlessinger assisted with IHC, assisted with the and Hao Xiong contributed to PK analysis. HKG research time is supported by the Victorian Cancer Agency in conjunction with the Melbourne Academic Centre for Health.

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Table 1. Affinity of AM1, AM1 S238C, and ABBV-321 to Recombinant Human EGFR Biacore Analysis

Affinity (KD, nM) AM1 AM1 S238C ABBV-321 EGFR 1-501 ECD 230 240 240 EGFRvIII ECD 7.5 7.9 6.8 EGFRC271A,C283A ECD 24 26 24 Affinity of parental ABT-806 and affinity matured AM1 variant to recombinant EGFR was assessed by surface plasmon resonance.

KD = dissociation constant

Table 2. ABBV-321 Cytotoxicity in Brain Cancer Cell Lines

Brain Cancer Cell a b Line EGFR Expression ABBV-221 (IC50 nM) ABBV-321 (IC50 nM) U-87 MGvIIIc > 1.9 0.06 0.23 A172 1.71 59.2 23.2 T98G 1.65 28.1 14.5 MO59J 1.55 > 133 2.8 MO59K 1.43 > 133 1.5 LN-18 1.38 Not Tested 8.4 SF264 1.33 > 133 1.4 SF539 1.2 > 133 2.5 SNB-19 1.15 75 3.7 DBTRG-05MG 1.05 > 133 18 U-87 MG 1 29.3 4.6 U251 0.86 123 0.7 U-138 MG 0.79 14.5 16.1 SNB-75 0.49 > 133 10.6 CHLA-03-AA 0.46 > 133 4.8 PFSK-1 0.01 58.6 1.9

IC50 = half maximal inhibitory concentration aRelative protein expression determined by Western blot analysis with an anti-EGFR antibody and normalized to Caco-2 cells How? bCell viability was determined following incubation with ABBV-221 or ABBV-321 for 144 hr. The values represent the IC50 for ATPlite cell proliferation assays performed in triplicate. cEGFR-amplified cell line

Table 3. ABBV-321 Cytotoxicity in Colorectal Cancer Cell Lines

Colorectal Cancer a b Cell Line EGFR Expression ABBV-221 (IC50 nM) ABBV-321 (IC50 nM) SW48 2.84 8.6 0.015 DLD-1 2.16 > 133 9 HCT-116 2.09 > 133 12 COLO 201 2.00 81.6 2.6 LoVo 1.99 63 0.7 SW1116 1.96 > 133 4.6 WiDR 1.91 90.6 5.5 HT-29 1.88 56 1.5 SW480 1.76 > 133 8 HCT-15 1.64 > 133 28.4 LS 174T 1.63 > 133 1 SK-CO-1 1.52 23.9 1.1 T84 1.47 > 133 11.8 Caco-2 1.00 > 133 13.9 RKO 0.62 75.3 4.7 SW403 0.38 61.8 1.8 LS1034 0.28 > 133 9.5 SW1463 0.15 > 133 4.8 COLO 205 0.07 > 133 3.3 COLO 320 HSR 0.05 > 133 24.9 SW620 0.04 106 4.3 COLO 320DM 0.01 > 133 17.6

IC50 = half maximal inhibitory concentration a Relative protein expression determined by Western blot analysis with an anti-EGFR antibody and normalized to Caco-2 cells. b Cell viability was determined following incubation with ABBV-221 or ABBV-321 for 144 hrs. The values represent the IC50 for ATPlite cell proliferation assays done in triplicate.

Table 4. Activity of ABBV-321 in NSCLC and Head & Neck PDX Models

c c TGI(max)(%) TGD(%) Origin Modela mRNA EGFR b ABBV-221 ABBV-321 ABBV-221 ABBV-321 NSCLC CTG-1082 3.3 40 96 50 >114 CTG-1012 -0.2 36 93 71 >343 CTG-0743 4.8 58 85 ND ND CTG-0192 4.8 86 91 >71 >71 CTG-0176 3.9 0 51 0 211 CTG-0170 4.6 31 52 0 100 CTG-0166 -1.3 85 96 280 >490 CTG-0165 2.9 85 97 150 >343 CTG-0163 4.7 93 97 >71 >71 CTG-0158 4.8 82 78 318 >445 CTG-0162 3.7 67 97 89 >126 CTG-0159 3.2 33 98 14 >181 CTG-0719 7.3 77 77 >11 >11 H&N CTG-505 4.4 96 96 >88 >88 CTG-152 5.7 81 82 >186 >186 CTG-149 4.7 72 86 75 >114 CTG-786 7.1 89 91 >82 >82 CTG-434 8.2 84 78 >560 >560 a Patient-derived xenograft mouse models were treated with 0.6 mg/kg/day ABBV-321 Q7Dx6, i.p or 6 mg/kg/day ABBV-221 Q7Dx6. b Log-transformed RNA expression levels (Log2(FPKM+0.1)-Qnorm) c TGI(max) and tumor growth delay TGD(max) for each treatment group of three mice (n=3) relative to the control antibody (huIgG)-treated group

Figures Legends

Figure 1. ABBV-321 Binding to Cell Lines Expressing EGFR. Cell binding of AM1, AM1 S238C, and ABBV-321 were assessed by FACS analysis of a murine NR6 cell line expressing human wild- type EGFR (wild-type), and an NR6 cell line expressing human EGFRC271A,C283A.

Figure 2. In vivo Efficacy of ABBV-321 in Glioblastoma. The in vivo tumor growth inhibition of ABBV-321 was compared to that of ABBV-221 as monotherapy in mice implanted with the SNO199 PDX model (A), U87MG xenograft model (B) and SNO207 PDX model (C). ABBV-321 as monotherapy and in combination with depatux-m were compared in the GBM-1 PDX model (D). The ADCs or antibodies were administered at doses indicated in the figure. Each point indicates the average tumor volume of six (A), eight (B), five (C) or six (D) xenografts and the error bars reflect the standard error of the mean.

Figure 3. In vivo Efficacy of ABBV-321 in Multiple Tumor Indications. The in vivo tumor growth inhibition of ABBV-321 is shown for mice implanted with the EBC-1 (A), FaDu (B), A253 (C) LoVo (D), SW48 (E) and HCT-116 (F) xenograft models and mesothelioma model PDX #1174 (G). In (D) and (F) growth was compared to ABBV-221. PDX #1174 also included cisplatin treatment animals (G). Each point in the graph indicates the average volume of 5 (G) or 8 (A to F) xenografts. Error bars reflect the standard error of the mean. The ADCs, antibodies and cisplatin were administered at doses indicated in the figure.

Targeting Multiple EGFR Expressing Tumors with a Highly Potent Tumor-Selective Antibody Drug Conjugate

Mark G Anderson, Hugh D Falls, Michael J. Mitten, et al.

Mol Cancer Ther Published OnlineFirst August 26, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-20-0149

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