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Altering antibody drug conjugate binding to the Neonatal Fc Receptor impacts efficacy and tolerability Kevin J. Hamblett, Tiep Le, Brooke M. Rock, Dan A Rock, Sophia Siu, Justin N. Huard, Kip P. Conner, Robert R. Milburn, Jason W. O`Neill, Mark E. Tometsko, and William C. Fanslow Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00153 • Publication Date (Web): 01 Jun 2016 Downloaded from http://pubs.acs.org on June 3, 2016

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Molecular Pharmaceutics

Altering antibody drug conjugate binding to the Neonatal Fc Receptor impacts efficacy and tolerability Kevin J. Hamblett†*, Tiep Le, Brooke M. Rock, Dan A. Rock, Sophia Siu, Justin N. Huard, Kip P. Conner, Robert R. Milburn, Jason W. O’Neill, Mark E. Tometsko, William C. Fanslow Amgen, Inc., Seattle, WA Keywords: antibody drug conjugate, non-cleavable linker, maytansine, DM1, FcRn, CD70, therapeutic index, H435A

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Abstract

Antibody drug conjugates (ADC) rely on the target-binding specificity of an antibody to selectively deliver potent drugs to cancer cells. IgG antibody half-life is regulated by neonatal Fc receptor (FcRn) binding. Histidine 435 of human IgG was mutated to alanine (H435A) to explore the effect of FcRn binding on the pharmacokinetics, efficacy, and tolerability of two separate maytansine-based ADC pairs with non-cleavable linkers, (c-DM1 and c-H435A-DM1) and (7v-Cys-may and 7v-H435A-Cys-may). The in vitro cell-killing potency of each pair of ADCs was similar, demonstrating that H435A showed no measurable impact on ADC bioactivity. The H435A mutant antibodies showed no detectable binding to human or mouse FcRn in vitro, whereas their counterpart wild-type IgG ADCs were found to bind to FcRn at pH=6.0. In xenograft bearing SCID mice expressing mouse FcRn, the AUC of 7v-Cys-may was 1.6-fold higher than 7v-H435A-may, yet the observed efficacy was similar.

More severe

thrombocytopenia was observed with 7v-H435A-Cys-may as compared to 7v-Cys-may at multiple dose levels. The AUC of c-DM1 was approximately three-fold higher than c-H435ADM1 in 786-0 xenograft bearing SCID mice, which led to a three-fold difference in efficacy by dose. Murine FcRn knockout, human FcRn transgenic line32 SCID animals bearing 786-0 xenografts showed an amplified exposure difference between c-DM1 and c-H435A-DM1 as compared to murine FcRn expressing SCID mice, leading to a ten-fold higher dose required for efficacy despite a six-fold higher AUC of the c-H435A-DM1.

The accelerated clearance

observed for the non-cleavable maytansine ADCs with the H435A FcRn mutation led to reduced efficacy at equivalent doses and exacerbation of clinical pathology parameters (decreased tolerability) at equivalent doses. The results show that reduced ADC clearance mediated by FcRn modulation can improve therapeutic index.


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Introduction Antibody drug conjugates (ADC) represent a relatively new modality for the treatment of cancer. Brentuximab vedotin is approved for relapsed Hodgkin lymphoma and anaplastic large cell lymphoma and ado-trastuzumab emtansine is indicated for metastatic HER2 positive breast cancer that have progressed following trastuzumab and a taxane.1, 2 ADCs are comprised of an IgG antibody conjugated to a drug via a linker. Two distinct classes of linkers are available: cleavable, and non-cleavable. Most cleavable linker ADCs release the drug from the antibody following internalization of the ADC into the endo-lysosomal pathway where protease recognition, disulfide reduction, or a change in pH leads to linker-drug cleavage.3-5 Highly potent drugs including monomethyl auristatin E, pyrrolobenzodiazepines, and the maytansinoids DM1 and DM4 often employ cleavable linkers.6-8 Release of highly potent drugs with cleavable linkers typically lead to bystander activity as the released drug can cross cell membranes of targeted as well as non-targeted cells.5 Both the ADC itself or the released drug of a cleavablelinker based ADC can contribute to toxicity.9 Cleavable linkers represent the majority of ADCs in clinical trials. ADCs with non-cleavable linkers internalize into cells followed by antibody catabolism in lysosomes to generate amino acid-linker-drug.10-12

Amino acid-linker-drug catabolites

containing maytansine, such as Lysine-MCC-DM1, are transported from the lysosome to the cytoplasm by the lysosomal transporter SLC46A3.13 Once the catabolite reaches the cytoplasm the catabolite inhibits tubulin polymerization which leads to cell death. Lysine-MCC-DM1, the catabolite of non-cleavable linker ADC Ab-MCC-DM1, is significantly less potent in cell-based


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assays than DM1 itself.14 Non-cleavable linker ADC potency is derived solely from internalization and production of the catabolite as non-cleavable linker-ADC catabolites do not readily enter cells and thus do not exhibit bystander activity.5 One hundred percent of LysineMCC-DM1 injected into rats was recovered within 24 hours and no metabolites were observed (unpublished data). The poor cell membrane permeability and lack of metabolism suggest that non-cleavable linker ADC toxicity is primarily due to the ADC in contrast to cleavable linker ADCs where both the released drug and the ADC itself can mediate the toxicity. The majority of ADCs currently in clinical trials are conjugated to antibody native lysines or cysteines, which generate heterogeneous drug antibody ratio (DAR) profiles.15 Purified fractions of cleavable monomethyl auristatin E (MMAE) conjugates with different DARs demonstrated that ADC half-life and therapeutic index inversely correlated with DAR.16 Much of the current focus of ADC optimization is directed toward the generation of homogeneous site-specific ADCs using different techniques such as engineered cysteines, non-natural amino acids, enzymes, or reagents to bridge native disulfides in an attempt to improve therapeutic index.17-23 Junutula et al. demonstrated that the pharmacokinetic profile and therapeutic index of a site-specific cysteine antibody drug conjugate was superior to a heterogeneous ADC.20 The neonatal Fc receptor (FcRn) specifically binds to the IgG class of antibodies and protects IgG from catabolism, leading to the longest half-life of the five Ig classes in humans, despite the smallest molecular weight of the five Ig classes.24, 25 Amongst the four IgG subclasses IgG1, IgG2, and IgG4 half-lives are approximately 21 days in humans, whereas IgG3 is only about 7 days.26

IgG3 antibodies contain different residues in the Fc domain than the other IgG

subclasses leading to decreased pH-dependent FcRn binding and reduced half-life.27 To mimic this observation, Firan et al. mutated a histidine to alanine of a humanized IgG1 antibody at


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position 435 which significantly reduced antibody half-life in mice.28 To create mouse models that more closely mimic human biology, murine FcRn knockout mice were crossed with mice expressing the human FcRn transgene.29 These mice, designated as as Tg32 were back-crossed with immunodeficient SCID mice to generate Tg32-SCID mice to explore the role of human FcRn interactions in disease models that require immunodeficient animals.30 The contributions of multiple factors on ADC pharmacokinetics make it challenging to investigate the key attributes that impact the efficacy and toxicity of ADCs. Here we explored the impact of pharmacokinetic changes on the efficacy and tolerability of non-cleavable linker ADCs via modulation of binding to FcRn. To create ADCs with modified pharmacokinetics, antibody pairs of IgG1 wild-type and IgG1 with a mutated Histidine 435 were generated. The Histidine 435 site was mutated to alanine to attenuate binding to FcRn at pH=6.0 and to reduce antibody drug conjugate exposure compared to the corresponding wild-type IgG conjugate.24 To explore the role of FcRn on non-cleavable linker ADCs we used anti-CD70 antibodies as a model system. CD70, a type II transmembrane glycoprotein, is expressed in renal cell cancer, non-Hodgkin lymphoma and other solid tumor malignancies.31,

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Restricted normal tissue

expression of CD70 on activated T cells, B cells and dendritic cells coupled with expression on cancer cells makes it an attractive target for cancer treatment.

Anti-CD70 antibody drug

conjugates robustly internalize into CD70 expressing cells leading to efficacy in preclinical xenograft models.32

H435A mutations were introduced into two anti-CD70 antibodies, a

chimeric antibody (cBU69) conjugated in a heterogeneous fashion using lysine conjugation and fully human cysteine mutant antibody (7v-Cys) conjugated to a site-specific cysteine. cBU69 and 7v bind human CD70 and do not cross react with murine CD70. Use of non-cleavable linker


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ADCs, which lack bystander efficacy and toxicity of released drug were used to facilitate interpretation of the impact of pharmacokinetic modulation.

Methods Reagents 786-0 cells were obtained through the American type culture collection and held in a repository at Amgen.

786-0 cells were cultured in supplemented Roswell Park Memorial

Institute media with 10% fetal bovine serum. The chimeric anti-CD70 antibody cBU69 and the fully human anti-CD70 antibody 7v were conjugated to the maytansinoid DM1 (ImmunoGen, Inc.) using a non-cleavable linker.33

Briefly, wild-type cBU69 and cBU69-H435A were

modified with the hetero-bifunctional linker succinimidyl-4-(N-maleimidomethyl)cyclohexane1-carboxylate (SMCC). Following removal of the excess SMCC, maleimide labeled antibody was incubated with DM1 to generate cBU69-MCC-DM1 (c-DM1) and cBU69-H435A-MCCDM1 (c-H435A-DM1). Conjugation of the fully human anti-CD70 antibodies with engineered site-specific cysteines 7v-Cys and 7v-H435A-Cys to 4'-bromomaytansine and 7v to SMCC-DM1 was performed as described previously.13 Antibody and DM1 concentrations were calculated by solving a pair of Beer's Law equations at 252 and 280 nm.34

FcRn Equilibrium Binding Assessment by Biacore FcRn binding affinity (KD) was measured by surface plasmon resonance (SPR) using an ‘IgG down’ approach, immobilizing the antibody on the chip to eliminate concerns of binding avidity from the estimated parameters which occurs when FcRn is immobilized on the chip. All samples of recombinant human and murine FcRn protein with associated β-2 microglobulin (β2-M) were


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prepared similarly. Briefly, the protein stocks were first diluted in binding buffer (0.1M Hepes buffered saline, pH = 6.0, 350mM NaCl, 0.1% Tween-20) prior to passage through a 50 kDa molecular weight cut-off spin concentration device (Amicon-Ultra, Millipore) to separate high MW aggregate. The resultant filtrate was analyzed by SEC-RALS and by SDS-PAGE (data not shown) to verify the absence of high MW aggregate and the presence of β2-M (bands at ~30 and ~12 kDa in reduced SDS-PAGE) prior to binding analysis. For SPR, CM4 sensor surfaces were prepared by coupling (in 10 mM Sodium Acetate, pH = 5.0) up to 1100 RU of goat anti-human H + L chain-specific F(ab’)2

(Jackson ImmunoResearch) to all flow cells using standard

EDC/NHS-based protocols provided by the manufacturer (GE Healthcare). For binding analysis, 500 RU of anti-CD70 mAb ligands were captured on flow cells 2-4 in binding buffer maintained at 10 µL/min flow rate, and the baseline allowed to stabilize prior to injection of FcRn analyte. For murine FcRn binding, concentrations between 27 – 2000 nM were injected at 20 µL /min for 750 s to achieve steady-state; for human FcRn binding, concentrations between 61-15000 nM were injected at 50 µL /min for 200 s. Surfaces were regenerated between injections using 2 x 30 s injections of pH = 8.0 buffer followed by 2 x 30 s injections of 2 M NaCl. All sensorgrams were double-referenced by first subtracting the response of the reference cell (Fc1), followed by subtraction of the average response of a series of buffer injections for each respective flow cell. Data analysis was performed in BiaEval v3.0 (GE Healthcare) using a standard 1:1 binding model to fit the steady state RU responses from each injection series.

Cell Growth Inhibition 786-0 cells were cultured in a 96-well tissue culture plate and incubated at 37°C, 5% CO2 for approximately 4-6 hours. After the initial incubation, serial dilutions of ADC were added to cell


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cultures and continuously incubated at 37°C, 5% CO2 for 4 days prior to measurement of cellular ATP levels using the CellTiter-Glo reagent (Promega). The ATP levels directly correlated with viable cell number under the conditions employed. Luminescence was measured using a Wallac EnVision 2103 multilabel reader from Perkin Elmer with a reading time of 0.1 second per well.

Animal Care and Use Female SCID and Tg32-SCID mice were cared for in accordance to the Guide for the Care and Use of Laboratory Animals, 8th Edition. Mice were group-housed at an AAALAC internationalaccredited facility in sterile ventilated micro-isolator (or static) housing on corn cob bedding. All research protocols were approved by the Institutional Animal Care and Use Committee. Animals were fed ad libitum with pelleted feed and water via automatic watering system or water bottle. Animals were maintained on a 12:12 hr light: dark cycle in rooms and had access to enrichment opportunities.

Tumor volume and animal weights were measured two or three

times a week. Tumor length and width were measured with electronic digital calipers. Tumor volume (mm3) was calculated as (W2 X L)/2 where width (W) is defined as the smaller of the two measurements and length (L) is defined as the larger of the two measurements. Tumor volumes and weights were measured twice weekly. Subcutaneous tumor efficacy experiments were performed in a masked fashion with one individual measuring tumor volume and animal weights while a separate individual prepared test articles and dosed the animals. Serum samples were collected from mice and analyzed by immunoassay.

SCID mice were purchased from

Charles River (Cat No. 236) and B6.Cg-Fcgrt PrkdcTg(FCGRT)32Dcr/DcrJ, referred to as Tg32-SCID, were licensed from Jackson Laboratory (Cat No. 018441).


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Doses are shown as nmol/kg for drug dose equivalents of maytansine and DM1 for 7v and cBU69, respectively, or as mg/kg for the equivalent antibody dose levels.

Efficacy of 7v-Cys-may and 7v-H435A-Cys-may in subcutaneous 786-0 xenografts Five million 786-0 cells mixed with growth factor reduced matrigel from BD Biosciences were implanted subcutaneously into SCID mice.

Once tumor volumes grew to approximately

180 mm3, animals were randomized into treatment groups of ten animals each. Animals were administered a single intravenous dose in the tail vein of either vehicle, 7v-Cys-may or 7vH435A-Cys-may at 30 nmol/kg and 100 nmol/kg, corresponding to antibody doses of 2.2 mg/kg and 7.3 mg/kg for 7v-Cys-may and 2.3 mg/kg and 7.7 mg/kg for 7v-H435A-Cys-may, respectively. Efficacy of c-DM1 and c-H435A-DM1 in subcutaneous 786-0 xenografts SCID mice.

Five million 786-0 cells mixed with growth factor reduced matrigel were

implanted subcutaneously into SCID mice. Once tumor volumes grew to approximately 130 mm3, animals were randomized into treatment groups of ten animals each.

Animals were

administered a single intravenous dose in the tail vein of either vehicle, c-DM1 or c-H435ADM1 at 34 nmol/kg (1.1 mg antibody/kg) and 102 nmol/kg (3.3 mg antibody/kg). Tg32-SCID mice. Five million 786-0 cells mixed with growth factor reduced matrigel were implanted subcutaneously into Tg32-SCID mice. Once tumor volumes grew to approximately 210 mm3, animals were randomized into treatment groups of seven animals each. Animals were administered a single intravenous dose in the tail vein of either vehicle, c-DM1 at 31 nmol/kg (1.0 mg antibody/kg), c-DM1 at 94 nmol/kg (3.0 mg antibody/kg) or c-H435A-DM1 at 310 nmol/kg (10.0 mg antibody/kg).


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Tolerability of 7v-Cys-may, 7v-H435A-Cys-may, and 7v-MCC-DM1 in SCID mice SCID mice were separated into groups of mice and administered a single intravenous dose (via tail vein) of vehicle, 7v-Cys-may at 300, 1000, or 2000 nmol/kg, (22, 73, and 147 mg antibody/kg, respectively), 7v-Cys-H435A-may at 300 or 1000 nmol/kg, (23 and 77 mg antibody/kg, respectively), or 7v-MCC-DM1 at 940 or 1880 nmol/kg (27 and 53 mg antibody/kg, respectively). Four days after injection animals were euthanized and blood was collected via cardiac puncture for clinical pathology. Serum total antibody concentrations Multi-Array® 96-well standard microplate wells (Meso Scale Discovery [MSD]) were coated with 30 µL of murine anti-human Fc mAb (Amgen, Inc.) at 1-3 µg/mL in BLOTTO/Tween-20 (0.01%) and incubated overnight. After washing the plate, standards, QCs, and matrix blank plus study samples were loaded into plate wells (25 µL) subsequent to a 50-fold dilution of serum in BLOTTO/Tween-20 buffer. Next, 25 µL of murine anti-human Fc mAb (Ruthenium) SULFOTAG-conjugate (1-2 µg/mL) was added to the microplate wells prior to an additional 3-4 hr incubation with light shaking. Following additional wash steps, standard MSD read buffer containing tripropylamine electrochemiluminescent additive was added (150-200 µL). Data were collected using a Meso Scale Discovery Sector Imager 6000 plate reader equipped with Discovery Workbench™ software version 3.0. For pharmacokinetic analysis of the resultant data non-compartmental analysis was performed on the individual serum concentration-nominal time data using Phoenix software (Enterprise version 6.3.0.395 Pharsight Corp). Statistics Group comparisons for pharmacokinetic data were performed using the Mann-Whitney test and comparison of hematology or clinical chemistry parameters following ADC exposure was


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determined using one-way ANOVA with Tukey's multiple comparisons correction using GraphPad Prism version 6.03. Group tumor volumes are shown as means plus or minus standard error of the mean (SEM) and plotted as time post cell implantation.

Statistical significance of observed differences

between the rate-based T/C of group log-transformed tumor volumes was evaluated by one-way ANOVA with Tukey's multiple comparisons correction using GraphPad Prism version 6.03.35 Results Generation of antibody drug conjugates. The anti-CD70 chimeric antibody BU69 with a point mutation at position H435 and wild-type antibody were conjugated to DM1 using the noncleavable linker SMCC to generate cBU69-MCC-DM1 (c-DM1) and cBU69-H435A-MCC-DM1 (c-H435A-DM1) with DARs of 4.5. The fully human anti-CD70 antibody 7v containing a sitespecific cysteine mutation (7v-Cys) was genetically engineered at position H435 to generate 7vH435A-Cys. 7v-Cys and 7v-H435A-Cys were conjugated to 4'-bromomaytansine to generate 7v-Cys-maytansine (7v-Cys-may) and 7v-H435A-Cys-maytansine (7v-H435A-Cys-may) with DARs of 2.0 and 1.9, respectively. The matched DARs of each ADC pair eliminates the potential for differential pharmacokinetics due to drug loading differences of the ADCs. Figure 1.


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In vitro characterization of ADCs. The in vitro potency of each pair of anti-CD70 ADCs was evaluated using the CD70 expressing renal cell carcinoma line 786-0 growth inhibition assay. The results show that the potency of c-H435A-DM1 and c-DM1 were indistinguishable in this assay (Figure 1A). Growth inhibition potency results for the 7v pair of ADCs, 7v-Cys-may and 7v-H435A-Cys-may, showed similar potency between the two (Figure 1B). For both pairs of antibodies introduction of the H435A mutation did not impact the ADC target mediated cell kill. To determine the in vitro binding affinity of each antibody to FcRn, the antibody was captured on the chip surface followed by injection of FcRn to measure binding affinity. Both cDM1 and 7v-Cys-may bound to murine FcRn at pH=6.0 with affinities of 720 and 960 nM, respectively (Table 1). In contrast, neither c-H435A-DM1 nor 7v-H435A-Cys-may were observed to bind to murine FcRn, as expected. Similarly, c-DM1 and 7v-Cys-may bound to human FcRn with affinities of 2.08 µM and 2.56 µM, respectively at pH=6.0 (Table 2), yet neither 7v-H435A-Cys-may nor c-H435A-DM1 showed any measurable binding to human FcRn by Biacore. Figure 2.


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Human site-specific ADC pharmacokinetics and efficacy in SCID mice.

In order to

compare the efficacy and pharmacokinetics of 7v-Cys-may and 7v-H435A-Cys-may, SCID mice (murine FcRn) bearing 786-0 subcutaneous xenografts were treated with two dose levels of 7v-Cys-may or 7v-H435A-Cys-may, 30 and 100 nmol/kg of maytansine. Serum concentration of the total antibody for each ADC is shown in Figure 2A. 7v-H435A-Cys-may was eliminated from the animals more rapidly than the wild-type 7v-Cys-may at both dose levels. The AUC over 14 days, AUC(0-14d), was 782,100 ng/mL*day for 7v-Cys-may and 484,950 ng/mL for 7v-H435A-Cys-may at the 100 nmol/kg dose (Table 3). At the 30 nmol/kg dose, AUC(0-14d) was determined to be 166,800 ng/mL*day for 7v-Cys-may and 104,950 ng/mL*day for 7vH435A-Cys-may. Relative AUC(0-14d) observed for 7v-H435A-Cys-may as compared to 7vCys-may was approximately 62% at both dose levels.

Efficacy was observed with both

conjugates at 30 and 100 nmol/kg dose levels (Figure 2B) yielding significant delay in tumor


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growth compared to vehicle p

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