SYNTHESIS AND CHARACTERIZATION OF CETUXIMAB

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SYNTHESIS AND CHARACTERIZATION OF CETUXIMABDOCETAXEL AND PANITUMUMAB-DOCETAXEL ANTIBODY-DRUG CONJUGATES FOR EGFR-OVEREXPRESSING CANCER THERAPY Dylan M Glatt, Denis R. Beckford Vera, Shamit S Prabhu, J. Christopher Luft, S. Rahima Benhabbour, and Matthew C Parrott Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00672 • Publication Date (Web): 18 Sep 2018 Downloaded from http://pubs.acs.org on September 19, 2018

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Molecular Pharmaceutics

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SYNTHESIS AND CHARACTERIZATION OF CETUXIMAB-DOCETAXEL AND PANITUMUMAB-DOCETAXEL ANTIBODY-DRUG CONJUGATES FOR EGFROVEREXPRESSING CANCER THERAPY

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Dylan M. Glatt1, Denis R. Beckford Vera2, Shamit S. Prabhu1, J. Christopher Luft1, S. Rahima Benhabbour1,3*, Matthew C. Parrott2*

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Division of Pharmacoengineering and Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, 125 Mason Farm Road, Chapel Hill, North Carolina, 27599, USA 2

Department of Radiology, Biomedical Research Imaging Center, University of North Carolina at Chapel Hill, Marsico Hall, 125 Mason Farm Road, Chapel Hill, North Carolina, 27599, USA 3

UNC-NCSU Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Marsico Hall, 125 Mason Farm Road, Chapel Hill, North Carolina, 27599, USA *Corresponding Author: S. Rahima Benhabbour, PhD 125 Mason Farm Road 4205 Marsico Hall University of North Carolina at Chapel Hill Chapel Hill, NC 27599 (919) 843-6142 [email protected] Keywords: antibody-drug conjugate, ADC, panitumumab, cetuximab, docetaxel, EGFR

1 ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract The safety and efficacy of anticancer antibody-drug conjugates (ADC) depend on the

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selection of tumor-targeting monoclonal antibody (mAb), linker, and drug, as well as their

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specific chemical arrangement and linkage chemistry. In this study, we used a heterobifunctional

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crosslinker to conjugate docetaxel (DX) to cetuximab (CET) or panitumumab (PAN). The

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resulting ADCs were investigated for their in vitro EGFR-specific cytotoxicity and in vivo

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anticancer activity. Reaction conditions, such as reducing agent, time, temperature, and

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alkylation buffer, were optimized to yield potent and stable ADCs with consistent batch-to-batch

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DARs. ADCs were synthesized with DARs from 0.4 to 3.0, and all retained their EGFR affinity

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and specificity after modification. ADCs were sensitive to cell surface wildtype EGFR

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expression, demonstrating more cytotoxicity in EGFR-expressing A431 and MDA-MB-231 cells

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lines compared to U87MG cells. A431 tumor-bearing mice treated once weekly for four weeks

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with 100 mg/kg cetuximab-docetaxel ADC (C-SC-DX, DAR 2.5) showed durable anticancer

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responses and improved overall survival compared to the same treatment regimen with 1 mg/kg

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DX, 100 mg/kg CET, or a combination 1 mg/kg DX and 100 mg/kg CET. New treatment options

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are emerging for patients with both wild-type and mutated EGFR-overexpressing cancers, and

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these studies highlight the potential role of EGFR-targeted ADC therapies as a promising new

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treatment option.


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Introduction Antibody-drug conjugates (ADCs) are an advanced class of combination therapy that

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chemically link a monoclonal antibody (mAb) with a drug, like a toxin, radionuclide, or

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immunostimulatory molecule1. The mAb functions as the vehicle to bind and accumulate at

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tumor cells, while the drug (or payload) acts as the cell killing agent. The effect of this tumor

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targeting feature of ADCs is that they can increase the therapeutic window for cytotoxic

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payloads by simultaneously lowering the therapeutic dose and increasing the maximum tolerated

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dose. In 2015, approximately 45 different anticancer ADCs were in clinical trials against

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approximately 35 unique targets2. Most of these ADCs target cell surface receptors expressed on

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malignant cells of hematological orgin and deliver payloads with cytotoxic activities at or below

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single digit nanomolar IC50s. Few, however, target solid tumors that contain more broadly

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expressed tumor-associated antigens like HER2 or EGFR3. Therefore, we synthesized an ADC

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targeting EGFR and investigated its activity in a solid tumor. To avoid toxicity to healthy tissues

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that we would expect with a traditional ADC payload in this setting, we conjugated a modestly

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potent cytotoxic drug, docetaxel, and studied the activity of the ADC compared to monotherapy

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and combination therapy.

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Docetaxel (DX) is a semisynthetic cytotoxic agent4 used to treat cancer by inhibiting

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microtubular depolymerization. It is a cornerstone of both first-line and second-line combination

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regimens. It is approved by the U.S. Food and Drug Administration (FDA) for the treatment of

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numerous solid malignancies, which include locally advanced or metastatic breast cancer, locally

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advanced or metastatic non-small cell lung cancer (NSCLC), metastatic castration-resistant

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prostate cancer, advanced gastric adenocarcinoma, and advanced squamous cell carcinoma of

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head and neck cancer5. However, DX is highly lipophilic, practically insoluble in water4, exhibits



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poor (8%) oral bioavailability6, and eliminates rapidly from circulation (triphasic, t1/2, mean = 3.4

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h7)8,9. The DX commercial product (Taxotere®) uses high concentrations of ethanol and

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polysorbate 80 to solubilize DX so it can be administered by intravenous (IV) infusion.

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Taxotere® can cause serious and sometimes fatal hypersensitivities in both animals and humans8-

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10

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docetaxel has proven effective in many regimens across pathologically diverse cancers, its use is

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limited due to dose-related toxicities and sub-therapeutic efficacy.

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. In addition, serious DX-related toxicities can limit dose intensity5,11-13. Therefore, while

On one hand, reformulating toxic and sub-therapeutic chemotherapeutic drugs as an ADC

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can rescue the drug by altering the drug’s pharmacokinetics and uptake by tumor tissue. For

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example, maytansine failed phase I clinical trials as a single agent due to dose-limiting toxicity

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and subtherapeutic efficacy14-18 but has demonstrated markedly improved safety and efficacy

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when conjugated to the anti-human epidermal growth factor receptor 2 (HER2) mAb

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trastuzumab as the ADC ado-trastuzumab emtansine19. Compared to single agent maytansine, the

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double-drug ADC ado-trastuzumab emtansine exhibits a long circulating half-life (~ 4.6 days)20

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and robust tolerability21, including at least a two-fold higher safety margin compared to

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maytansine alone in non-clinical safety studies22. These pharmaceutical improvements are likely

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due to improved intracellular delivery23,24 and preferential uptake of ado-trastuzumab emtansine

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by HER2-positive cancer cells25,26.

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On the other hand, improving mAb potency to more broadly expressed antigens, without

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sacrificing safety, may improve responses to mAb-based monotherapy and combination

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therapies. Like HER2, the epidermal growth factor receptor (EGFR) is overexpressed in a large

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percentage of patients across pathologically-diverse cancers27. FDA-approved anti-EGFR mAbs

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panitumumab (PAN) and cetuximab (CET) inhibit EGFR-activation to starve tumors of critical


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pro-growth signals. While neither PAN nor CET is indicated as monotherapy in the first-line

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setting, both can sensitize tumors to additional chemotherapy or radiation28,29. In particular,

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combination DX plus PAN30,31 or CET32,33 has shown improved anticancer activity compared to

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single agents, and may also decrease EGFR-related drug resistance observed with some patients

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treated on single agent EGFR targeted therapies (e.g., erlotinib and gefitinib) that are at risk for

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resistance34,35.

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We hypothesized that an anti-EGFR-DX ADC could overcome the biopharmaceutical

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limitations of DX and simultaneously eliminate the necessary combinational regimen of anti-

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EGFR mAbs plus chemotherapy required to elicit the desired anticancer response. Studies herein

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describe the synthesis and antitumor activity of a series ADCs linking DX to PAN or CET. We

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synthesized the ADCs with consistent batch-to-batch drug-to-antibody ratios (DARs) without

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reducing affinity or specificity to EGFR. ADCs improved selectivity of DX to EGFR-expressing

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cancer cells, enhanced cytotoxicity of anti-EGFR mAbs to EGFR-expressing cancer cells, and

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exhibited potent anticancer activity in a human EGFR-overexpressing tumor model.



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Materials and Methods

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Cell lines and cell culture

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A431 human epidermoid carcinoma, U87MG human malignant glioblastoma, and MDA-

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MB-231 human mammary gland/breast triple negative cancer cells were purchased from the

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American Type Culture Collection (ATCC) and cultured as described. A431 and U87MG cells

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were cultured in high glucose DMEM (Gibco 11995-065) supplemented with 10% (v/v) fetal

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bovine serum (FBS, Sigma F2442) and 100 U/mL penicillin-streptomycin (PS, Gibco 15140-

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122). MD-MB-231 cells were cultured in RPMI 1640 (Gibco 11875-093) also complete with

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10% (v/v) FBS and 100 U/mL PS. Cells were maintained in a humidified incubator of 5% CO2 at

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37 °C and passaged at 80-85% confluency. All experiments were performed with cells between

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passage 5 and 20.

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Antibodies, chemotherapeutic drugs, and chemicals

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Cetuximab (Erbitux®, Eli Lilly), panitumumab (Vectibix®, Amgen), and docetaxel

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(Taxotere®, Sanofi NDC 0955-1020-01) were purchased from the University of North Carolina

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Pharmacy, and used as received. Docetaxel (TSZ CHEM RS019) was prepared as a 10 mM stock

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in DMSO fresh for each assay. All other chemicals were purchased, as the highest grade, from

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Fischer Scientific.

128 129

Long chain-docetaxel prodrug (LC-DX) synthesis

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A flame-dried round-bottom flask was charged with DX (80 mg, 9.9 × 10-5 mol, 1.0

131

equiv) and a catalytic amount of 4-dimethylaminopyridine (DMAP) (12 mg, 9.9 × 10-5 mol, 1

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equiv) in dry CH2Cl2 (20 mL) under argon, and the solution was stirred for 10 min at room

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temperature. LC-SPDP (succinimidyl 6-[3(2-pyridyldithio)propionaamido]hexanoate, Thermo


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21651, 42 mg, 9.9 × 10-5 mol, 1 equiv) was added, and the reaction mixture stirred at room

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temperature overnight. The reaction was monitored by TLC (CH2Cl2: MeOH 95:5 v/v) for

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completion. The solution was evaporated under vaccuo, and the crude product was purified by

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preparative TLC in CHCl3:MeOH (95:5) (Rf = 0.39). The silica gel was removed by filtration

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through a fine fritted funnel, and the filtrate was evaporated under vaccuo to give the desired

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product as a white powder (LC-DX) (72 mg, 65%). LC-DX prepared at 250 µg/mL in

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acetonitrile (ACN) was assessed for purity by reverse-phase high-performance liquid

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chromatography (RP-HPLC). Samples of LC-DX were spiked with DX or LC-SPDP, and run

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under the same RP-HPLC conditions. LC-DX elutes at 15.9 min, whereas DX elutes at 14.4 min

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and LC-SPDP elutes at 10.2 min. 1H NMR (400 MHz, CDCl3): δ (ppm) = 0.06 (s, 3H, –C15CH3),

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1.1 (s, 3H, –CH19), 1.2 (s, 9H, –H7’-9’), 1.27 (s, 3H, –H19), 1.74 (s, 3H, –H18), 1.93 (m, 2H, –

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C7HCH2), 2.4 (s, 3H, –C21OCH3), 2.59 (t, 2H, –CH2C1”), 3.06 (t, 2H, –CH2NHCO), 4.19 (d, 1H,

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–H5), 4.3 (d, 1H, –H7), 4.85 (d, 1H, –H2), 5.25 (s, 1H, –OH), 5.4 (d, 1H, –H10), 5.55 (t, 2H, –

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H13), 5.64 (d, 2H, –H20), 6.2 (d, 1H, –H2’), 6.6 (d, 1H, –H3’), 7.22-7.53 (m, 8H, –Ar-H26-28 and

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Ar-H30-35), 8.05 (d, 2H, –Ar-H25,29), 8.4 (d, 1H, –Ar-NCH). 13C NMR (100 MHz, CD3OD): δ

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(ppm) = 9.9 (–C19), 14.3 (–C18), 20.8 (–C22), 22.6 (–C16,17), 25.9 (–C1”OCH2CH2), 26.4

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(C1”OCH2CH2CH2), 28.1 (–C7’-9’), 29.6 (–(CH2)14C1”), 31.9 (–C6,14), 43.1 (–C15), 44.5 (–C3), 45

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(–CHBr), 46.4 (–C3’), 57.5 (–C8), 71.8 (–C13), 72.1 (–C7), 74.4 (–C2), 75 (–C10), 75.3 (–C20),

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78.9 (–C6’), 79.9 (–C1), 80.9 (–C4), 84.2 (–C5), 126.3 (–C31,33,35), 128.9 (–C32,34), 129.2 (–C26,28),

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130.2 (–C24,25,29), 133.6 (–C27), 135.5 (–C11), 138.9 (–C12), 154.2 (–C5’), 167 (–C23), 168.3 (–

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C21), 169.6 (–C1), 170.8 (–C1”), 172.6 (–CONH), 211.5 (–C9).

155



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Short chain-docetaxel prodrug (SC-DX) synthesis A flame-dried round-bottom flask was charged with DX (129.3 mg, 1.6 × 10-4 mol, 1.0

157 158

equiv) and a catalytic amount of DMAP (20 mg, 1.6 × 10-4 mol, 1 equiv) in dry CH2Cl2 (30 mL)

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under argon. The solution was stirred for 10 min at room temperature. SPDP (succinimidyl 3-(2-

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pyridyldithio)propionate, Thermo 21857, 50 mg, 1.6 × 10-4 mol, 1 equiv) was added, and the

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reaction mixture was stirred at room temperature overnight. The reaction was monitored by TLC

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(CH2Cl2: MeOH 95:5 v/v) for completion. The white precipitate of dicyclohexyl urea byproduct

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was filtered through a fritted funnel, and the filtrate was evaporated under vaccuo. The crude

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product was purified by preparative TLC in CHCl3:MeOH (95:5) (Rf = 0.26). The silica gel was

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removed by filtration through a fine fritted funnel, and the filtrate was evaporated under vaccuo

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to give the desired product as a white powder (118 mg, 73%). SC-DX prepared at 250 µg/mL in

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ACN was assessed for purity by RP-HPLC. SC-DX was spiked with DX or SPDP and run under

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the same conditions. SC-DX elutes at 16.8 min, whereas DX elutes at 14.4 min and the SPDP

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linker elutes at 10.8 min. 1H NMR (400 MHz, CDCl3): δ (ppm) = 0.07 (s, 3H, –C15CH3), 1.1 (s,

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3H, –CH19), 1.2 (s, 9H, –H7’-9’), 1.25 (s, 3H, –H19), 1.33 (m, 28H, –(CH2)14CH3), 1.75 (s, 3H, –

171

H18), 1.83 (m, 2H, –C7HCH2), 2.4 (s, 3H, –C21OCH3), 2.84 (t, 2H, –CH2C1”), 2.97 (t, 2H, –

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CH2NHCO), 4.19 (d, 1H, –H5) 4.3 (d, 1H, –H7), 4.85 (d, 1H, –H2), 5.16 (s, 1H, –OH), 5.4 (d, 1H,

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–H10), 5.47 (t, 2H, –H13), 5.62 (d, 2H, –H20), 6.2 (d, 1H, –H2’), 7.22-7.53 (m, 8H, –Ar-H26-28 and

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Ar-H30-35), 8.05 (d, 2H, –Ar-H25,29), 8.4 (d, 1H, –Ar-NCH). 13C NMR (100 MHz, CD3OD): δ

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(ppm) = 9.9 (–C19), 14.2 (–C18), 20.8 (–C22), 22.6 (–C16,17), 26.4 (C1”OCH2CH2CH2), 28.2 (–C7’-

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9’),

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71.8 (–C13), 74.5 (–C2), 75 (–C10), 76.5 (–C20), 78.8 (–C6’), 79.9 (–C1), 81 (–C4), 84.3 (–C5),

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119.9 (Ar-SPDP), 121.5 (Ar-SPDP), 126.3 (–C31,33,35), 128.9 (–C32,34), 129.2 (–C26,28), 130.2 (–

32.9 (–(CH2)C1”), 33.5 ((–(CH2CH2)C1”), 35.5 (–C6,14), 43.1 (–C15), 46.5 (–C3), 57.6 (–C3’,8),


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C24,25,29), 133.6 (–C27), 135.5 (–C11), 138.9 (–C12), 149.3 (Ar-SPDP), 155.1 (–C5’), 159.6 (Ar-

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SPDP), 167 (–C23), 167.9 (–C21), 169.7 (–C1), 170.6 (–C1”), 211.5 (–C9).

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Characterization of LC-DX and SC-DX RP-HPLC was used to determine concentration and monitor stability of DX, LC-DX, and

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SC-DX. All samples were prepared in 50/50 water/ACN, injected on the ThermoFisher HPLC

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equipped with a C-18 column, and monitored at 265 nm. Briefly, 20 μL of sample was injected

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and run at 1 mL/min on a gradient starting with 0.1% (v/v) TFA in ddH2O (solvent A) and 0.1%

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(v/v) TFA in ACN (solvent B) at a 95/5 A/B ratio and ending at 0/100 A/B ratio after 20 min.

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Solvent B is held at 100% for 2 min at which time the column re-equilibrates at the stating 95/5

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A/B for 3 min before the next injection (total run time 25 min). Stability of LC-DX and SC-DX

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were measured as function of pH (pH 5 and 7.4, 37 °C) and in the presence DTT (10 mM DTT,

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pH 7.4, 37 °C) as a function of time. Yield of LC-DX and SC-DX was determined by subtracting

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the moles generated in reaction from the starting moles of DX, and then dividing by the starting

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moles of docetaxel. Cell viability data were collected by Promega’s CellTiterGlo ATP assay

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after 72 h continuous exposure to A431 human epidermoid carcinoma cells. IC50s were

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determined by non-linear regression of log(drug) v. response in GraphPad PRISM 6.

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Supplementary Figures 1-3).

197 198 199

PAN-DX and CET-DX antibody-drug conjugate (ADC) synthesis ADCs were prepared using a modified reduction-alkylation procedure previously

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described36. Briefly, panitumumab (from Vectibix®) was buffer exchanged to 10 mg/mL in 25

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mM sodium borate pH 8.1, 25 mM NaCl, 5 mM EDTA (borate buffer), and subsequently treated



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with 10 mM TCEP also prepared in borate buffer at 8 mole TCEP/mole PAN (low DAR) or 24

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mole TCEP/mole PAN (high DAR) for 1-2 h at 40 °C under gentle agitation. Reduced PAN was

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purified from the reducing agent and buffer exchanged into ice cold 40/60 (%v/v) DMSO/0.1 M

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Tris-HCl pH 8.1 with 1 mM EDTA (Tris/DMSO buffer) by PD-10 column filtration. Antibody

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fractions were pooled and concentrated by ultrafiltration over a 30 kDa MWCO Amicon filter.

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SC-DX or LC-DX prepared at 10 mM in DMSO was diluted and added dropwise (20 mole DX-

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prodrug/mole PAN for low DAR or 40 mole DX prodrug/mole PAN for high DAR) to reduced

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PAN at room temperature under gentle agitation. This reaction proceeded for 30 min at 40 °C

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and continued overnight at 4°C. Excess N-acetyl cysteine (NAC) prepared in Tris/DMSO buffer

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was added to quench unreacted DX-prodrug. ADCs were filtered by centrifugation through a 0.1

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µm filter, treated with excess N-ethyl maleimide (NEM) to quench unreacted free thiols, and

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purified by PD-10 column filtration pre-equilibrated with 10 mM PBS pH 7.4 150 mM NaCl.

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Purified ADC was concentrated to at least 5 mg/mL by ultrafiltration over a 30 kDa MWCO

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Amicon filter and stored at 4 °C in 10 mM PBS pH 7.4 150 mM NaCl until further use.

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Cetuximab ADCs (from Erbitux®) were prepared as described except the reducing conditions

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were 1.5 mole TCEP/mole CET for low DAR and 2.75 mole TCEP/mole CET for high DAR.

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Three independent lots of ADCs were synthesized and characterized for reaction yield, DAR,

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binding affinity, and cytotoxicity (Table 2, Figures 3 and 4) before progressing to in vivo

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studies.

221 222

Bioanalytical characterization of ADCs

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Protein concentration was determined using a Nanodrop 2000/2000c. Free thiol

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concentration was determined using by Ellman assay using L-cysteine-HCl-H2O as a standard.


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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in an

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XCell SureLockTM Mini-Cell electrophoresis system using 4-12% bis-tris gel (NuPAGE Novex

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NP0322BOX) with MOPS running buffer (NuPAGE NP0001). Matrix Assisted Laser

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Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) was performed in a

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TOF/TOFTM 5800 system using a matrix of sinapinic acid. Size-exclusion high-performance

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liquid chromatography (SE-HPLC) was performed at room temperature on an Agilent 1200

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series chromatographic system. Samples were injected onto Agilent Bio SEC-5 size-exclusion

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column (5 µm, 300 Å; 7.8 x 300 mm) and Agilent Bio SEC-5 size-exclusion column (5 µm, 100

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Å; 7.8 x 300 mm) connected in series using EDTA 0.01 M in PBS as the isocratic mobile phase.

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The flow rate was maintained at 1 mL/min and the elution was monitored by UV

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spectrophotometer at 254 and 280 nm. (Supplementary Figures 4-9).

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Data in Table 2 are generated from N=3 batches of ADCs synthesized under the exact

237

same conditions. Pooled mixtures were used for all subsequent in vitro and in vivo studies. Drug-

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to-antibody ratio (DAR) was determined by applying to following calculation: DAR = (Mass of

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ADC – Mass of mAb)/(Mass of DX), where mass of ADC and mAb were determined by

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MALDI-TOF MS and Mass of DX was set equal to 807.879 Da. Thiol concentration was also

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monitored throughout the ADC synthesis, in particular before and after mAb was reduced and

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after DX prodrug was alkylated to form the ADC. Thiol concentration was converted from

243

concentration to number of free thiols by Ellman assay using a freshly prepared cysteine

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standard. Ellman served as an orthogonal method to monitor the reduction-alkylation chemistry

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and quantify DAR. ADC yield was calculated by measuring the difference between the moles of

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mAb in the starting 10 mg/mL stock and the moles of ADC following gel filtration.



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Concentration of mAb or ADC was determined by BCA protein assay using a standard of human

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pooled IgG.

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EGFR affinity was assayed by saturation binding to EGFR-overexpressing A431 cancer

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cells in the whole-cell enzyme linked immunosorbent assay (ELISA). Briefly, freshly harvested

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cells were prepared at 2 x 105 cells/mL in complete media, seeded at 2 x 104 cells per well in

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Costar 3903 96-well plates, and cultured for 24 h at 37 °C and 5% CO2. The following morning,

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cells were blocked (200 µL/well) with 5% (w/v) non-fat dry milk (LabScientific M0841) in PBS

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(Gibco D-PBS 1X 14190-144) at 4 °C for 1 h. After aspirating the blocking buffer, serially

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diluted treatments (50 μL/well) in 1% (w/v) milk in PBS (ELISA buffer) were added and

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incubated with cells at 4 °C for 1 h. Cells were washed with 3 sequential flushes (200 µL/well)

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of with 0.05% (w/w) Tween-20 (Fischer 9005-64-5) in PBS buffer (PBS-T). To each well, 50 μL

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of HRP-conjugated anti-human Fab-specific secondary antibody (Sigma A0293) were prepared

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at 1:10000 in ELISA buffer and incubated at 4 °C for 30 min. Cells were washed, incubated with

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Ultra-TMB blotting solution (Thermo Scientific 37574, 100 μL/well) for 10 min at RT while

261

shielded from light, quenched with 2 N H2SO4 (100 μL/well), and absorbance read at 450 nm.

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Equilibrium dissociation constant (Kd) was determined by non-linear fit using the one site

263

specific binding model in GraphPad PRISM 6. All data are reported as blank corrected and

264

normalized by cell number.

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EGFR-dependent cytotoxicity of CET-DX and PAN-DX ADCs in human cancer cell lines In vitro cytotoxicity of ADCs was performed on A431, MDA-MB-231, and U87MG

268

cells. Briefly, cells were seeded at 5000 cells per well in 100 μL complete DMEM and incubated

269

for 48 h at 37 °C and 5% CO2. Media was aspirated, and cells were treated with 50 μL of serially


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270

diluted ADC prepared in complete DMEM and incubated at 37 °C. Cells were exposed to

271

treatment for either 4 h or 72 h, aspirated and thoroughly washed, and cell viability was

272

measured at 72 h using the Promega CellTiter-Glo Luminescent Cell Viability Assay (G7571)

273

following the supplied protocol. Data were analyzed and IC50s determined using the

274

log(inhibitor) vs. response model in GraphPad PRISM 6.

275 276

Pharmacokinetics in healthy and tumor-bearing mice

277

Athymic (nu/nu) 6-8 week-old mice were randomized into 8 groups of 3 (24 mice total)

278

and mAb or ADC at 10 mg/kg (prepared at 2 mg/mL in PBS, approximately 200 μg/mouse) were

279

administered IV by tail vein injection. Separately, athymic (nu/nu) 6-8 week-old mice were

280

implanted with 1 x 106 A431 cells (100 μL/mouse prepared at 1 x 107 cells/mL in D-PBS) in the

281

rear flank by subcutaneous xenograft. Tumors grew to approximately 150 mm3, at which time

282

mice were randomized into 4 groups of 3-4 mice, and administered 10 mg/kg PAN, CET, P-SC-

283

DX (DAR 2.5), or C-SC-DX (DAR 2.5) by tail vein IV injection. In both studies, 10-20 µL of

284

blood was collected by tail nick in a 500 μL Eppendorf pre-treated with 2 μL 0.5 M EDTA.

285

Concentration-time profiles were generated by determining plasma concentrations of ADC or

286

mAb by ELISA (previously described), using a calibration curve with fresh PAN, CET, P-SC-

287

DX, and C-SC-DX of known concentration (Figure 5, Supplementary Figure 10,

288

Supplementary Table 1). Area under the curve of mAb and ADC was calculated from the

289

plasma concentration versus time curve. Half-life was determined in GraphPad PRISM 6 by

290

nonlinear fit with a one-phase exponential decay.

291



292 293

Singe-dose efficacy in tumor-bearing mice Athymic (nu/nu) 6-8 week-old mice were implanted with 1 x 106 A431 cells (100

294

μL/mouse prepared at 1 x 107 cells/mL in D-PBS) in the rear flank by subcutaneous xenograft.

295

Tumors grew to approximately 150 mm3, at which time mice were randomized into four groups

296

of 3-4 mice, and administered 10 mg/kg ADC or 10 mg/kg mAb by tail vein IV injection.

297

Tumors were monitored for size (mm3) and mice were monitored for body weight (g). Mice were

298

sacrificed when tumors exceeded 1 cm in any direction (Supplementary Figure 11).

299 300

Multi-dose efficacy study in tumor-bearing mice

301

Athymic (nu/nu) 6-8 week-old mice were implanted with 1 x 106 A431 cells (100

302

uL/mouse prepared at 1 x 107 cells/mL in D-PBS) in the rear flank by subcutaneous xenograft.

303

Tumors grew for approximately to approximately 150 mm3, at which time mice were

304

randomized into 9 groups of 6 mice and administered ADC, mAb, DX, or a combination of mAb

305

and DX by tail vein IV injection weekly for four weeks. Doses of ADC were selected as 10

306

mg/kg (approximately 200 μg/mouse) and 100 mg/kg (approximately 2000 μg/mouse) based on

307

the concentration-time pharmacokinetic data, the A431 IC50, and the single dose 10 mg/kg ADC

308

efficacy data. PAN and CET were dosed at 100 mg/kg to be equimolar to the 100 mg/kg ADC

309

dose. Likewise, DX was dosed at 1 mg/kg to be equimolar to the 100 mg/kg ADC dose. Tumors

310

were monitored for size (mm3) (Figure 6 and 7), survival plotted by group (Supplementary

311

Figure 12), and mice were monitored for body weight (g) (Figure 8). Mice were sacrificed when

312

tumors exceeded 1 cm in any direction. No animals were sacrificed due to body weight decrease.

313


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314

Results

315

Synthesis of docetaxel prodrugs A short-chain docetaxel prodrug (SC-DX) and long-chain docetaxel prodrug (LC-DX)

316 317

were prepared to a yield greater than 65% and purity greater than 98% by esterification of the 2’-

318

hydroxyl of docetaxel (DX) using succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or

319

succinimidy 6-[3(2-pyridyldithio)propionaamido]hexanoate (LC-SPDP) , respectively

320

(Supplementary Figure 1). SC-DX and LC-DX were shown to be more than 50% stable at pH

321

7.4 but only 20% stable at pH 5 when incubated in PBS at 37 °C for 24 h (Supplementary

322

Figure 2). DX rapidly released from both LC-DX and SC-DX in the presence of DTT at 37 °C

323

in a time- and concentration-dependent manner (Table 1, Supplementary Figure 1 and 3). LC-

324

DX was found to be approximately 2.5-fold less potent (1083 nM) than SC-DX (472 nM) and

325

10-fold less potent than DX (107.9 nM) to EGFR-expressing A431 cancer cells (Table 1,

326

Supplementary Figure 1).

327 328 329

Table 1. Synthesis and characterization of short chain- (SC-DX) and long chain- (LC-DX) docetaxel prodrugs DX, Linker, or DX-prodrug1

330 331 332 333

Yield (%)

IC50 (nM)

Chemical Stability in presence of 10 mM DTT (% remaining) 1h

16 h

38 h

DX

--

107.9

--

--

--

LC-SPDP

--

>40000

97

88

80

LC-DX

65

1083

96

61

49

SPDP

--

>40000

97

82

58

SC-DX

73

472.4

95

43

25

1

See Supplementary Figure 1. “DX” = docetaxel; “LC” = succinimidy 6-[3(2pyridyldithio)propionaamido]hexanoate; “SC” = succinimidyl 3-(2-pyridyldithio)propionate; “LC-DX” = long chain DX prodrug “SC -DX” = short chain DX prodrug



334 335

Synthesis of panitumumab-docetaxel and cetuximab-docetaxel ADCs Synthesis of panitumumab-docetaxel (PAN-DX) and cetuximab-docetaxel (CET-DX)

336

ADCs was performed using a reduction-alkylation scheme previously reported with several

337

minor modifications36 (Figure 1).

338


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339 340 341 342 343 344 345

Figure 1. Reduction-alkylation scheme to generate anti-EGFR antibody-drug conjugates (ADCs) composed of the anti-EGFR mAb cetuximab (CET) or panitumumab (PAN) with the short-chain docetaxel prodrug (SC-DX) or long-chain docetaxel prodrug (LC-DX). Resulting short-chain conjugates were labeled “C-SC-DX” and “P-SC-DX” for CET-SC-DX and PAN-SC-DX conjugates, respectively; long-chain conjugates were labeled “C-LC-DX” and “P-LC-DX” for CET-LC-DX and PAN-LC-DX, respectively. Drugto-antibody ratio (DAR), or the moles DX per mole of mAb (n), was quantified by measuring mass of ADCs using MALDI-TOF MS.



346

ADCs were synthesized by a sequential two-step reduction-alkylation process in which

347

mAb disulfides of CET or PAN were reduced to generate free thiols (step 1) and DX was

348

alkylated to free thiols on reduced mAbs to form covalent disulfide bonds (step 2). The number

349

of DX molecules per mAb, or drug-to-antibody ratio (DAR), was targeted by controlling the

350

number of free thiols generated on the mAb during reduction. Alkylation was performed in

351

excess DX. Reduction conditions were screened by assessing reduced CET and PAN mixtures

352

for the number of free thiols generated, structural purity, and EGFR binding affinity. Of the four

353

reducing agents screened (Supplementary Figure 4), tris(2-carboxyethyl)phosphine (TCEP)

354

was selected as the reducing agent for both PAN and CET reactions, and subsequently TCEP

355

reduction conditions were optimized.

356

Reduced, full-length CET and PAN were isolated, and confirmed to be intact and with

357

available free thiols for DX alkylation. Co-solvent and percent co-solvent, time, and temperature

358

used in the DX alkylation were screened, and the final conditions were selected based on

359

improvements to DX solubility, relative reaction rates, yield, and retained antigen interactions

360

(Supplementary Figures 5 and 6). Based on these studies, alkylation to form ADCs was

361

performed at 40 °C for 1-2 h, followed by overnight incubation at 4 °C in 100 mM Tris-HCl pH

362

8.1 with 40% (v/v) DMSO (Figure 1).

363


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364 365 366 367 368

Figure 2. A) Native (non-reducing) SDS-PAGE, B) ELISA to EGFR-overexpressing A431 cells, and C) MALDI-TOF mass spectrometry (MS) demonstrate that PAN-DX and CET-DX ADCs retain structure and EGFR binding affinity, yet have increased mass compared to parent PAN and CET mAbs. Note: MS data are representative and only shown for C-SC-DX and P-SC-DX conjugates; representative mass spectra of C-LC-DX and P-LC-DX are also included as Supplementary Figures 10 and 11. 19 ACS Paragon Plus Environment


369

Eight ADCs with DAR 1.4 to 2.7 were synthesized in N=3 batches. Batches were

370

prepared under the same conditions and pooled for biophysical characterization (Figure 2, Table

371

2, Supplementary Figures 7-9). ADCs showed similar structural purity and migration through a

372

sodium dodecyl sulfate polyacrylamide gel when compared to native mAb (Figure 2A). PAN-

373

containing ADCs showed a high molecular weight band (> 160 kDa), consistent with native

374

PAN. CET-containing ADCs contained a small population of fragment band around 22 kDa not

375

observed in the native CET. Moreover, C-SC-DXhigh and C-LC-DXhigh showed multiple bands

376

between 110 kDa and 160 kDa not observed in the “low” DAR ADCs or native CET. Taken

377

together, CET appeared more susceptible to fragmentation during reduction-alkylation than

378

PAN, particularly at higher concentrations of reducing agent. Saturation binding data was

379

collected using EGFR-expressing A431 cells to determine a dissociation constant (Kd) between

380

320 pM and 390 pM for CET-DX ADCs, equivalent to 330 pM for native CET. Similary, a

381

dissociation constant (Kd) between 380 and 410 pM was determined for PAN-DX ADCs,

382

equivalent to 370 pM for native PAN (Figure 2B). ADC chemical yields were greater than 40%

383

(range 30-50%) (Table 2) and ADCs were produced to similar monomeric purity

384

(Supplementary Figure 9). Importantly, the molecular weight of ADCs was increased

385

compared to native mAb, confirming successful alkylation of DX to reduced CET and PAN

386

(Figure 2C). Increases in molecular weight were observed in both parent (m/z = 1) and daughter

387

(m/z = 2 and m/z = 6) fragment species by MALDI-TOF MS (Supplementary Figures 7 and 8).

388

DAR was calculated by dividing the difference in mass between the ADC and the native mAb,

389

and then dividing by the molecular weight of DX. DAR of CET and PAN ADCs increased with

390

increases in the concentration of reducing agent and concentration of DX prodrug (Table 2).

391

Ellman assay was used as an orthogonal technique to MALDI-TOF to monitor reduction-


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392

alkylation and final drug load. For the remainder of the manuscript, rather than “low” or “high,”

393

ADCs under discussion will include DAR.

394



395

Table 2. Bioanalytical characterization of CET-DX and PAN-DX ADCs mAb

Moles of TCEP

DX prodrug

Moles of SCDX or LC-DX

ADC

DAR

Kd (nM)1

Yield (%)

IC50 (p-value)2

0

--

0

CET

--

0.33 ± 0.03

--

3670 ± 1960

C-SC-DXlow

1.4 ± 0.9

0.35 ± 0.03

48.4 ± 7.9

233 ± 96.9 (0.0057)

LC-DX

C-LC-DXlow

1.5 ± 0.8

0.32 ± 0.02

52.8 ± 17

782 ± 335 (0.0177)

SC-DX

C-SC-DXhigh

2.5 ± 0.4

0.39 ± 0.03

40.1 ± 9.2

129 ± 57.8 (0.0047)

C-LC-DXhigh

2.5 ± 0.2

0.34 ± 0.03

39.2 ± 14

376 ± 148 (0.0076)

PAN

--

0.37 ± 0.02

--

4680 ± 343

P-SC-DXlow

2.5 ± 0.9

0.38 ± 0.04

54.5 ± 13

204 ± 107 (0.0351)

LC-DX

P-LC-DXlow

2.6 ± 0.6

0.38 ± 0.02

49.5 ± 13

1050 ± 444 (0.0986)

SC-DX

P-SC-DXhigh

2.7 ± 0.7

0.41 ± 0.04

46.3 ± 9.7

266 ± 126 (0.0379)

P-LC-DXhigh

2.7 ± 0.5

41.8 ± 12

1120 ± 424 (0.1065)

Cetuximab

SC-DX 1.5

20

2.75

40 LC-DX

0

--

0

SC-DX Panitumumab

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

8

20

24

40 LC-DX

396 397 398

Page 22 of 50

1

0.41 ± 0.03

Kd (nM) values determined by saturation binding in whole-cell ELISA using EGFR-expressing A431 cells IC50s were analyzed to determine differences in cytotoxicity between CET ADCs and native CET, and separately with PAN ADCs and native PAN. Differences were determined by one-way ANOVA using Tukey’s post-test to determine statistical significance with pairwise comparisons. 2


Page 23 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

399 400


Characterization of anti-EGFR ADCs ADC potency was first studied as function of DX-prodrug and DAR (Figure 3) using

401

EGFR-expressing A431 cells. PAN and CET ADCs were more potent when synthesized with the

402

short-chain-linked docetaxel prodrug (SC-DX) compared to the long-chain-linked docetaxel

403

prodrug (LC-DX). For example, C-SC-DX (DAR 2.5, 129 nM) was found to be more potent than

404

C-LC-DX (DAR 2.5, 376 nM) (p=0.0452), and P-SC-DX (DAR 2.7, 266 nM) was found to be

405

more potent than P-LC-DX (DAR 2.7, 1120 nM) (p=0.0437). Increased potency was observed

406

for CET ADCs with higher DAR. For example, C-SC-DX (DAR 2.5, 129 nM) was more potent

407

than C-SC-DX (DAR 1.4, 233 nM) (p=0.0437). Because P-SC-DX (DAR 2.7) and P-SC-DX

408

(DAR 2.5) statistically exhibited the same DAR, binding affinity, and cytotoxicity to EGFR-

409

expressing A431 cells, they were combined together and treated as “P-SC-DX (DAR 2.5)”. P-

410

LC-DX (DAR 2.7) and P-LC-DX (DAR 2.6) also exhibited the same DAR, binding affinity, and

411

cytotoxicity, so we took the same approach and treated the pooled lot as “P-LC-DX (DAR 2.6)”.

412

All CET ADCs were found to be more cytotoxic to EGFR-positive A431 cells than CET alone;

413

however, P-SC-DX but not P-LC-DX was found to be more cytotoxic to A431 cells than PAN

414

alone (Figure 3, Table 2).

415


Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

416 417 418 419

Figure 3. Cytotoxicity of anti-EGFR ADCs to EGFR-overexpressing A431 cancer cells. IC50s (n=3) of mAbs and ADCs was determined by non-linear regression using GraphPad PRISM following 4h incubation with three separate ADC preps (N=3). Data were analyzed by one-way ANOVA and differences were determined using Tukey’s post-test. * = p < 0.05, ** = p < 0.01 24 ACS Paragon Plus Environment

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420


C-SC-DX (DAR 2.5) and P-SC-DX (DAR 2.5) exhibited the greatest cytotoxicity in the

421

initial in vitro cytotoxicity screen against high-EGFR expressing A431 cells. Therefore, we

422

selected C-SC-DX (DAR 2.5) and P-SC-DX (DAR 2.5) for additional experiments designed to

423

study the relationship between cytotoxicity and EGFR expression (Figure 4). Immortalized

424

human cancer cell lines A431, MDA-MB-231 and U87MG were used for the study. A431 cells

425

express between 5 x 105 and 5 x 106 wildtype EGF receptors per cell56, whereas U87MG cells

426

express no wildtype EGFR57. Conflicting reports claim very low58 and very high59 wildtype

427

EGFR expression on MDA-MB-231 cells. Saturation binding with cetuximab or panitumumab

428

suggests MDA-MB-231 cells express similar levels of EGFR as A431 cells (Figure 4A). P-SC-

429

DX (DAR 2.5) was found to be more cytotoxic to A431 cells compared to U87MG cells

430

(p=0.012) and at least 10-fold more potent in A431 and MDA-MB-231 cells (100–150 nM)

431

compared to U87MG cells (1909 nM). Similarly, C-SC-DX was found to be more cytotoxic to

432

A431 cells compared to U87MG cells (p=0.0068) and at least 9-fold more potent in A431 and

433

MDA-MB-231 cells (80–120 nM) compared to U87MG cells (1092 nM). DX alone was found to

434

be equipotent in A431, MDA-MD-231, and U87MG cells (p=0.5035). DX (101.7 nM) was more

435

potent than P-SC-DX (p=0.0001) and C-SC-DX (p=0.0390) in U87MG cells; however, in A431

436

cells, C-SC-DX (63.7 nM, p=0.7079) and P-SC-DX (156 nM, p=0.7079) were equipotent and

437

statistically non-inferior to DX (109 nM). No change in cytotoxicity was observed when P-SC-

438

DX (DAR 2.5) was incubated with A431 cells for either 4 h (186 nM) or 72 h (156 nM). C-SC-

439

DX (DAR 2.5) incubated with A431 cells for either 4 h (120 nM) or 72 h (63.7 nM) was

440

similarly equipotent. Furthermore, 4 h incubation of P-SC-DX (186 nM, p=0.6296) or C-SC-DX

441

(120 nM, p=0.9555) was equally cytotoxic as 72 h incubation of DX with A431 cells (109 nM).

442

Taken together, P-SC-DX and C-SC-DX demonstrated rapid and specific EGFR-dependent



443

cytotoxicity, and DX is at least, if not more, cytotoxic than ADCs against non-target, low- and

444

non-EGFR expressing tissues.


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445 446 447 448 449 450

Figure 4. Saturation binding (A) and cytotoxicity (B) of P-SC-DX and C-SC-DX were studied on U87MG and A431 cells to explore the relationship between of EGFR expression level and cytotoxicity. A431 cells were incubated with ADCs for either 4 h or 72 h to investigate the role of incubation time on cell viability (C).



451 452

ADC pharmacokinetics and single dose efficacy in A431-tumor bearing mice A single intravenous dose of 10 mg/kg ADC was studied in both healthy and A431

453

tumor-bearing mice in order to determine the area under the curve (AUC), a surrogate measure

454

of exposure of the tumor to the ADC. Neither the linker nor DAR impacted the AUC of ADCs in

455

healthy mice. Interestingly, however, while PAN-DX ADCs showed nearly superimposable

456

elimination as native PAN in healthy mice, CET-DX ADCs showed modestly reduced

457

elimination from healthy mice compared to native CET (Supplementary Figure 10). In A431

458

tumor-bearing mice, the AUC of P-SC-DX (12.1 μM*h) was 5-fold lower than in healthy mice

459

(64.4 μM*h ). Similarly, the AUC of C-SC-DX in A431 tumor-bearing mice (15.9 μM*h) was 3-

460

fold lower than in healthy mice (50.1 μM*h). While C-SC-DX exhibited a similar half-life of

461

75.4 h and 81.8 h in healthy and A431 tumor-bearing mice, P-SC-DX half-life was only 21.6 h in

462

A431 tumor-bearing mice compared to 67.3 h in healthy mice (Figure 5, Supplementary Table

463

1). Both mAbs and ADCs saw a 2-fold to 10-fold reduction in circulating plasma concentrations

464

in the A431-tumor bearing mice 1 h post-dose, which significantly decreased the AUC in A431-

465

tumor bearing mice compared to healthy mice.


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466 467 468 469 470


Figure 5. Pharmacokinetic profile (n=3 or 4) of P-SC-DX (DAR 2.5) (A) and C-SC-DX (DAR 2.5) (B) were studied in both healthy (solid lines) and A431 tumor-bearing mice (dashed). In each panel, circles represent the native PAN and CET controls and squares represent the ADC.



471

A single dose efficacy study was performed in A431 tumor-bearing mice

472

(Supplementary Figure 11). C-SC-DX (DAR 2.5) (10 mg/kg, equivalent of 0.14 mg/kg DX)

473

exhibited modest in vivo anticancer activity. Mice had a median OS of 37 days compared to 23

474

days for mice dosed treated with CET alone (10 mg/kg). Conversely, P-SC-DX (2.5) (10 mg/kg,

475

equivalent of 0.15 mg/kg DX) did not exhibit in vivo anticancer activity. Mice had a median OS

476

of 27.5 days compared to 25 days for mice treated with PAN alone (10 mg/kg).

477 478 479

In vivo efficacy and OS of A431 tumor-bearing mice treated with C-SC-DX and P-SC-DX Based on the single dose efficacy and pharmacokinetic data, A431 tumor-bearing mice

480

were treated weekly for four weeks with either 10 mg/kg or 100 mg/kg ADC. Mice were

481

sacrificed when tumors exceeded 1 cm in any direction. Treatment with 100 mg/kg C-SC-DX

482

(DAR 2.5, equivalent of 1.4 mg/kg DX) reduced tumor volumes compared to mice treated with

483

10 mg/kg C-SC-DX (p

SYNTHESIS AND CHARACTERIZATION OF CETUXIMAB

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