Bridging disulfides for stable and defined antibody drug conjugates

Article

pubs.acs.org/bc

Bridging Disulﬁdes for Stable and Deﬁned Antibody Drug

Conjugates

George Badescu, Penny Bryant, Matthew Bird, Korinna Henseleit, Julia Swierkosz, Vimal Parekh,

Rita Tommasi, Estera Pawlisz, Kosma Jurlewicz, Monika Farys, Nicolas Camper, XiaoBo Sheng,

Martin Fisher, Ruslan Grygorash, Andrew Kyle, Amrita Abhilash, Mark Frigerio, Jeﬀ Edwards,

and Antony Godwin*

PolyTherics Ltd, The London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, United Kingdom

ABSTRACT: To improve both the homogeneity and the stability of ADCs, we have developed site-speciﬁc drug-conjugating

reagents that covalently rebridge reduced disulﬁde bonds. The new reagents comprise a drug, a linker, and a bis-reactive

conjugating moiety that is capable of undergoing reaction with both sulfur atoms derived from a reduced disulﬁde bond in

antibodies and antibody fragments. A disulﬁde rebridging reagent comprising monomethyl auristatin E (MMAE) was prepared

and conjugated to trastuzumab (TRA). A 78% conversion of antibody to ADC with a drug to antibody ratio (DAR) of 4 was

achieved with no unconjugated antibody remaining. The MMAE rebridging reagent was also conjugated to the interchain

disulﬁde of a Fab derived from proteolytic digestion of TRA, to give a homogeneous single drug conjugated product. The

resulting conjugates retained antigen-binding, were stable in serum, and demonstrated potent and antigen-selective cell killing in

in vitro and in vivo cancer models. Disulﬁde rebridging conjugation is a general approach to prepare stable ADCs, which does not

require the antibody to be recombinantly re-engineered for site-speciﬁc conjugation.

Cysteine residues are less common within a mAb and

therefore provide fewer sites for attachment and with higher

reactivity. An intact mAb IgG1 has four accessible disulﬁde

bonds that can be reduced to release eight free cysteine thiols

that can serve as sites for conjugation.³⁻⁵This strategy is used

to attach MMAE to brentuximab to produce the other FDA

approved ADC known as Adcetris.⁵The reaction between the

mAb and the maleimide-MMAE reagent yields an ADC

composed of a mixture of species with between 0 and 8 drug

molecules per antibody molecule.^5,6The formation of high drug

loaded species, e.g., greater than DAR 4, is undesirable as it has

been shown to lead to lower tolerability, higher plasma

clearance rates, and decreased eﬃcacy in vivo.⁶

INTRODUCTION

■

Antibody drug conjugates (ADCs) are a growing class of

chemotherapeutic agents for the treatment of cancer,

combining the tumor targeting properties of antibodies with

the cell killing properties of potent cytotoxic drugs. Although

both monoclonal antibodies (mAbs) and cytotoxic agents can

be used independently to treat cancer, the covalent attachment

of a drug to a targeting antibody allows selective delivery and

minimizes systemic toxic side eﬀects from exposure of the drug

to healthy tissue.

The clinically established approach to conjugating cytotoxic

compounds to antibodies is to use linkers that are reactive

toward either the amino side chains of lysine residues or the

thiol side chains of cysteines obtained from reducing interchain

disulﬁde bonds. Conjugation to lysine residues can be achieved

via reaction with reactive groups such as a succinimidyl moiety

to form a stable amide bond. This approach is used for the

covalent attachment of the maytansinoid derivative, DM1, to

TRA to produce the FDA approved Kadcyla.¹However, as a

mAb may have more than 80 solvent-accessible lysine residues,

heterogeneous mixtures with variable DAR species are

produced due to the numerous sites of conjugation available.²

Maleimide based conjugates have been shown to undergo

retro-addition reactions in the presence of competing thiols.⁷⁻⁹

In serum this can result in loss of the drug from the ADC by

exchange reaction with the free thiol group of circulating

albumin.^7,9Monoalkylation reagents also leave the original

Received: April 3, 2014

Revised: May 2, 2014

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Figure 1. Conjugation of a payload to a disulﬁde bridge using a bis-alkylating conjugation approach involving a sequence of Michael addition and

elimination reactions.

Figure 2. Chemical structures of the bis-alkylating reagents 1, 2, and 3 loaded with either a cytotoxic payload (MMAE) or ﬂuorescent dyes, Alexa

Fluor 488 and FITC, respectively. The ﬂuorescent dyes are linked to the disulﬁde bridging linker via a noncleavable amide bond while MMAE is

linked by an enzyme cleavable valine citrulline dipeptide and self-immolative p-aminobenzyl moiety.

disulﬁdes unbridged, potentially introducing instability to the

antibody.

speciﬁcally into TRA.¹⁴Conjugation of a cytotoxic payload is

then achieved by using an alkoxy-amine derivatized drug to

form an oxime bond. Similar approaches are possible using

azide/alkyne “click” chemistry.¹⁵Conjugation to unnatural

amino acids is a promising alternative to produce homogeneous

ADCs but has some limitations. Re-engineering mAbs to

incorporate unnatural amino acids is complex and costly. In

addition, the current methods reported for conjugation to form

an oxime use a large excess of up to 30 equiv of cytotoxic

reagent and have long reaction times of up to 4 days.¹⁴When

an ADC is derived from mAbs that have previously undergone

clinical development, re-engineering may not be cost-eﬀective

given that GMP manufacturing processes will already be in

place.

We have developed a novel way to prepare more

homogeneous and stable ADCs that does not require protein

re-engineering (Figure 1). This approach uses bis-sulfone

reagents that are selective for the cysteine sulfur atoms from a

native disulﬁde (Figure 2). These reagents undergo bis-

alkylation to conjugate both thiols derived from the two

cysteine residues of a reduced native disulﬁde bond¹⁶such as

the interchain disulﬁde bonds of a mAb. The reaction results in

covalent rebridging of the disulﬁde bond via a three carbon

bridge leaving the protein structurally intact. This approach has

also been applied for the site-speciﬁc conjugation of poly-

(ethylene glycol) (PEG) to a wide range of therapeutic proteins

One alternative to overcome a wide DAR distribution is to

use mAbs with engineered cysteine residues. Antibodies known

as THIOMABs have been described with an engineered and

unpaired cysteine residue on each heavy or light chain designed

for conjugation of a cytotoxic payload.⁹⁻¹¹Each of the cysteine

residues provides a site for conjugation of a thiol-reactive drug

reagent. When conjugated to MMAE¹⁰or DM1¹¹using a

maleimide reagent, an ADC with a DAR close to 2 is produced.

While this approach can be used to produce more

homogeneous products it does not address issues associated

with the instability of maleimide conjugates in vivo. Engineered

cysteine thiols can also be capped during production such as by

a disulﬁde forming reaction with glutathione.¹⁰Liberating a

capped thiol to make accessible for conjugation without

reducing interchain disulﬁdes is nontrivial. In addition, the

engineered cysteine residues have been shown to form triple

light chain species during production.^12,13

Another re-engineering approach is to incorporate unnatural

amino acids in the antibody as sites for conjugation and avoid

the use of maleimide chemistry. To improve speciﬁcity non-

natural amino acids can be introduced with functionalities that

enable conjugation chemistries to be used that are orthogonal

to those typically used for native amino acids. ADCs have been

synthesized by introducing a p-acetylphenylalanine residue site

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to enhance their pharmacokinetic properties while retaining

their activity.^17,18

A cytotoxic drug molecule is attached to the bis-alkylating

linker via a PEG chain producing a reagent suitable for

generating ADCs. The reagent can undergo reaction at each of

the four interchain disulﬁdes of a mAb if they are ﬁrst reduced,

to produce DAR 4 ADCs. As the disulﬁdes are reannealed the

resulting ADCs are more homogeneous and will have greater

stability compared to maleimide conjugates. Here we describe

the use of bis-alkylating conjugation of MMAE to TRA and a

TRA derived Fab (Fab_TRA) to produce biologically active model

drug conjugates.

RESULTS

■

Figure 3. SDS-PAGE analysis of a conjugation reaction mixture

between MMAE and Fab_TRAusing the bis-alkylating reagent 1.

Unreduced Fab_TRAand the Fab_TRA-bisAlk-vc-MMAE conjugate each

run as a single band at 50 kDa, while the reduced protein runs as a

single band at around 25 kDa corresponding to the V_HC_H1and V_LC_L1

chains.

Preparation of Drug Conjugates. Conjugation of both

cysteine thiols derived from a reduced disulﬁde bond using the

bis-alkylating approach is carried out in two steps and

analogous to a standard maleimide conjugation process,

namely, (1) disulﬁde reduction to release the two cysteine

thiols followed by (2) conjugation under mild conditions.

Conjugation by bis-alkylation leads to the formation of a three-

carbon bridge relinking the two cysteine residues to which the

drug payload is covalently attached.¹⁸The three-carbon bridge

is small enough and suﬃciently ﬂexible not to perturb the

tertiary structure of the antibody.²⁰

The MMAE bis-alkylating reagent 1 was prepared with a

Cathepsin B cleavable valine citrulline p-aminobenzyl ether

linker designed to allow release of MMAE intracellularly from

the ADC. A small ethylene glycol spacer of 24 repeat units and

discrete mass was also incorporated into the reagent to improve

the water solubility and enable more eﬃcient conjugation. It

was not possible to perform the conjugation without PEG

present in the reagent due to low solubility in the conjugation

media. It was also hoped that the incorporation of PEG would

reduce the propensity of the resulting ADCs to aggregate and

potentially improve the pharmacokinetic proﬁle of the

conjugates in the case of the Fab. A PEG size of 24 repeat

units was chosen as this was one of the largest sizes of PEG

available with a discrete mass.

TRA was selected as a model antibody to explore the

preparation of ADCs by bis-alkylation because of well-

established in vitro and in vivo models. In addition to the

TRA ADC, a Fab drug conjugate (Fab_TRA-bisAlk-vc-MMAE)

was prepared which, when analyzed by SDS-PAGE, gave

supporting evidence that the bis-sulfone functionality of

MMAE reagent 1 was able to rebridge an interchain disulﬁde.

Puriﬁed Fab_TRAwas ﬁrst treated with DTT to reduce the

accessible single interchain disulﬁde¹⁷and conjugation was

performed after removal of residual DTT. Analysis by SDS-

PAGE showed that conjugation conversion was nearly

quantitative (95%) using only 2 equiv of reagent 1 with only

a trace amount of reduced Fab_TRAremaining in the reaction

mixture (Figure 3). When 1.5 equiv of reagent 1 was used, 88%

conversion was observed. Unreduced Fab_TRAran as a single

band at 50 kDa (Lane 2), but the reduced Fab_TRA, after

treatment with DTT, shifted in apparent molecular weight

running as a single band at 25 kDa (Lane 3). This band

corresponded to the V_HC_H1from the heavy chain and the V_LC_L

from the light chain which dissociate in the presence of SDS

after reduction of the disulﬁde and migrate together as they are

the same size. In contrast, the Fab_TRA-bisAlk-vc-MMAE

conjugate migrated as a single band at 50 kDa indicating that

the disulﬁde had been bridged by the bis-alkylating ADC

reagent (Lane 4).

The rebridging of the Fab_TRAinterchain disulﬁde bond using

reagent 1 was further conﬁrmed by running SDS-PAGE under

reducing conditions (Figure 4a) with no dissociation of the

heavy and light chain fragments observed as would be expected

for unconjugated Fab_TRA. This result also demonstrated that the

bis-thioether bonding to the Fab_TRAwas more stable than the

original disulﬁde bond as the sample survived incubation with

10 mM DTT for 30 min prior to loading on the gel, which

might be expected to cause dissociation of the reagent from the

Fab_TRAleading to heavy and light chain dissociation. Analysis of

the Fab_TRA-bisAlk-vc-MMAE conjugate by SE-HPLC (Figure

4b) indicated high purity with no indication of any product

aggregation. RP-HPLC also showed that a single product was

formed with a purity greater than 95% (Figure 4c).

Conditions similar to the preparation of Fab_TRA-bisAlk-vc-

MMAE were used to conjugate reagent 1 to TRA forming

TRA-bisAlk-vc-MMAE. Although there is a total of 16 disulﬁde

bonds in an intact IgG₁, the 12 intrachain disulﬁde bonds are

less readily reduced and are less aﬀected even by high

concentrations of reducing agents.²¹The four accessible

interchain disulﬁde bonds can therefore be used for conjugation

under moderate reducing conditions. Analytical HIC was used

to analyze the reaction mixtures, as conjugation with MMAE

gave suﬃcient separation of the diﬀerent DAR species to

determine the overall DAR distribution. Varying the concen-

tration of reductant and the stoichiometries of reactants (mAb

to reagent 1), it was possible to aﬀect the DAR distribution.

When 6 equiv of MMAE reagent 1 were added to fully reduced

TRA it was possible to achieve 78% of the DAR 4 variant and

no unconjugated TRA (Figure 5). Lower average DAR could

also be achieved if desired by adapting reaction conditions. A

sample was prepared with an average DAR of 2.8 and with 2−4

drug molecules conjugated accounting for greater than 95% of

the whole sample. A minimal amount of unconjugated mAb

remained (<1%).

Stability of Conjugation. It has been reported that

instability of maleimide conjugates in serum results in

deconjugation of the drug and subsequent cross conjugation

of the drug to other thiol containing species including serum

albumin.^7,9To determine whether similar cross conjugation

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Figure 4. Analytical characterization of puriﬁed Fab_TRA-bisAlk-vc-MMAE: (a) SDS-PAGE analysis demonstrating disulﬁde bridging. After reduction

the Fab_TRA-bisAlk-vc-MMAE conjugate remains as a single band at 50 kDa indicating that the disulﬁde was bridged by the ADC reagent as the

interchain disulﬁde is no longer reducible. (b) SE-HPLC analysis and (c) RP-HPLC analysis demonstrating the purity and homogeneity of the Fab

drug conjugate which elutes as single peak under both analytical techniques.

Figure 5. HIC analysis of TRA and bis-alkylating MMAE reagent 1 reaction mixture. (a) HIC chromatogram, (b) histogram showing DAR

distibution based on relative peak areas from HIC chromatogram.

occurred for conjugates prepared with the bis-alkylating linker,

a serum stability study was conducted using an Alexa Fluor 488

ﬂuorescently labeled bis-alkylation reagent 2 conjugated to

TRA. A maleimide functionalized Alexa Fluor 488 reagent was

conjugated to TRA as a control. Both conjugates were

incubated in either 50% rat or undiluted human serum for up

to 96 h at 37 °C. Serum samples were analyzed by SE-HPLC

with ﬂuorescence detection to monitor the liberation of free

reagent and/or formation of albumin-Alexa Fluor 488 adducts.

For the bis-alkyl TRA conjugate prepared using Alexa Fluor

488 labeled reagent 2, the SE-HPLC chromatogram remained

largely unchanged over 96 h incubation in both rat and human

serum indicating that the disulﬁde conjugation was extremely

stable to serum exposure (Figure 6). The main peak eluted at

11.0 min corresponding to the starting TRA-Alexa Fluor 488

conjugate. In contrast, incubation of the TRA maleimide Alexa

Fluor 488 conjugate in both rat and human serum resulted in

chromatograms with very diﬀerent proﬁles after 96 h. The peak

corresponding to the TRA maleimide conjugate at 11.0 min

decreased in peak area by 47% (rat) or 24% (human) and

several new peaks were observed. New peaks eluting at 11.9

and 16.3 min corresponded to an albumin adduct and

deconjugated Alexa Fluor 488 dye, respectively. The albumin

adduct, visible in both species’ serum, indicated that

deconjugation and cross conjugation to albumin from the

maleimide derived TRA conjugate had occurred.

incubations indicated the formation of aggregates. A second

new peak observed eluting at 13.3 min was consistent with a

light chain-Alexa Fluor 488 conjugate based on comparison

with molecular weight standards. This suggests that there was

some dissociation of TRA-Alexa Fluor 488 conjugate over time

as the heavy and light chains were not covalently linked

through the interchain disulﬁde bonds. These signs of antibody

instability were not observed for the conjugate prepared using

the disulﬁde rebridging reagent 2.

Experiments were also conducted to monitor the change in

DAR over time for MMAE TRA conjugates incubated in an

artiﬁcial serum composed of 20 mg/mL HSA in PBS. Artiﬁcial

serum was used as endogenous IgGs in whole serum would

overlap with the conjugate peaks in the analytical HIC method

used for DAR analysis.

The drug was conjugated to TRA either using MMAE

reagent 1 or maleimide-val-cit-PAB-MMAE. The reactions

yielded conjugates with an average DAR of 2.3 for the bis-

alkylating reagent and 3.2 for the maleimide reagent. Each

conjugate at a ﬁnal concentration of 1.0 mg/mL was incubated

at 37 °C for up to 120 h and loss of MMAE was monitored by

measuring the change over time of HIC peak areas

corresponding to each DAR species. The conjugate prepared

using reagent 1 showed no decrease in DAR after 120 h. In

contrast, analysis of the incubation mixtures of the maleimide

conjugate showed that there was a decrease in the average DAR

indicating loss of MMAE with time (Figure 7). It is likely that

the deconjugated MMAE cross-conjugated to the HSA present

In addition to linker deconjugation, some evidence of

antibody instability was also seen with the maleimide

conjugates. A new early peak eluting at 8.5 min for both sera

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Figure 6. Serum stability of TRA-Alexa Fluor 488 conjugates prepared using either bis-alkylating or maleimide conjugation; maleimide conjugate

incubated for (a) 0 h, (b) 96 h in rat serum, (c) 96 h in human serum; bis-alkylation conjugate incubated for (d) 0 h, (e) 96 h in rat serum, (f) 96 h

in human serum. The peaks were identiﬁed as follows: peak 1, Trastuzumab-Alexa Fluor 488 conjugate; peak 2, albumin-Alexa Fluor 488 conjugate;

peak 3, released Alexa Fluor 488; peak 4, aggregated conjugate; peak 5, light chain-Alexa Fluor 488 conjugate.

in the mixture, although this was not detected using this

method.

Both mAb and Fab conjugates were found to have retained

binding activity which was comparable to the unconjugated

parent molecule (Figure 8) demonstrating that the bis-

alkylating linker has a minimal impact on receptor binding.

Internalization of Conjugates. Confocal microscopy was

used to monitor internalization of both TRA and Fab_TRA

conjugates prepared using the FITC labeled bis-alkylating

reagent 3. Following incubation with SK-BR-3 cells at 37 °C for

4 h, visualization of the cells showed that both Fab and mAb

conjugates had been internalized as eﬃciently as the

unconjugated antibody (Figure 9). No internalization was

observed in the A549 cell line (data not shown).

Retained Binding Activity of Conjugates Prepared

Using Bis-Alkylating Reagents. The binding of TRA and

TRA MMAE conjugates to the extracellular domain of HER2

was measured by ELISA to determine whether antigen speciﬁc

binding was retained after conjugation.

In Vitro Cytotoxicity of Conjugates Prepared Using

the Bis-Alkylating Linker 1. The in vitro potency of TRA-

bisAlk-vc-MMAE (DAR 2.8) and Fab_TRA-bisAlk-vc-MMAE

(DAR 1) conjugates was determined using the HER2-positive

human breast cancer cell lines, SK-BR-3 and BT-474, and the

antigen-negative cell lines, A549 and MCF-7.

Fab_TRA-bisAlk-vc-MMAE was found to reduce viability of SK-

BR-3 cells and its activity was found to match that of free

MMAE (Figure 10). No antiproliferative activity was observed

for unconjugated Fab_TRAin the concentration range tested

(data not shown). When tested in an antigen-negative cell line,

Figure 7. Change in DAR over time for ADCs incubated with HSA as

measured by analytical HIC. The DAR of the maleimide based

■

conjugate (red - -) is seen to decrease over time while little change is

●

observed in the TRA-bisAlk-vc-MMAE DAR (blue - -).

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Figure 8. Antibody−drug conjugates obtained using bis-alkylating MMAE reagent 1 retained the speciﬁc antigen-binding of the naked antibody

(ELISA) in both Fab and mAb formats.

Figure 9. Antibody conjugates obtained using the disulﬁde bis-alkylating approach internalize into HER2-positive cells (SK-BR-3). (a) Trastuzumab

detection with an anti-human mAb conjugated to Alexa Fluor 488. (b) TRA-bisAlk-FITC conjugate. (c) Fab_TRA-bisAlk-FITC conjugate.

A549, the potency of Fab_TRA-bisAlk-vc-MMAE was about 3

orders of magnitude lower than that of free MMAE.

activity. Fab_TRA-bisAlk-vc-MMAE was well tolerated and

ﬂuctuations in body weight were similar between groups.

In the TRA-bisAlk-vc-MMAE study, mice were treated with

10 mg/kg, 20 mg/kg, or 30 mg/kg based on TRA with dosing

every fourth day for up to 21 days. The treatment regimen was

chosen based on published BT-474 xenograft studies with

trastuzumab based ADCs and because the speciﬁc BT-474

model used was particularly fast-growing in comparison.

Dosing was stopped when tumor volume reached less than

10% of the day 1 mean volume so the number of doses received

were 4, 5, and 6 for the 30 mg/kg, 20 mg/kg, and 10 mg/kg

groups, respectively. Control groups were treated with either 20

mg/kg TRA, 0.4 mg/kg free MMAE, or vehicle on the same

dosing schedule.

TRA-bisAlk-vc-MMAE was well tolerated with no dose

related eﬀect on body weight observed. Tumor growth was

delayed at all three doses and the conjugate demonstrated a

dose-related eﬃcacy in the model (Figure 13). Apparent

curative activity was attained at the two higher doses which

each elicited a TGD of 30.2 days with 10 out of 10 tumor-free

survival. The 10 mg/kg dose also resulted in the maximum

TGD of 30.2 days with tumor regression but no tumor-free

survival. As expected, only a marginal tumor growth delay was

obtained for groups treated with TRA or free drug.

The TRA-bisAlk-vc-MMAE conjugate was found to have

potent antiproliferative activity in both antigen positive cell

lines (Figure 11). In the antigen negative cell lines, the

conjugate was found to decrease viability but the potency was

about 3 orders of magnitude lower than that of free drug

supporting antigen selective delivery of the MMAE.

In Vivo Eﬃcacy of TRA and Fab_TRA- MMAE

Conjugates. The ability of the TRA-bisAlk-vc-MMAE (DAR

2.8) and Fab_TRA-bisAlk-vc-MMAE (DAR 1) conjugates to

inhibit tumor growth in vivo was evaluated in a BT-474 breast

cancer model. The conjugates were administered to female

CB.17 SCID mice bearing subcutaneous BT-474 tumor

xenografts.

In the Fab_TRA-bisAlk-vc-MMAE conjugate study, mice were

treated with 20 mg/kg of the Fab conjugate with dosing on

alternate days over 25 days. Control groups were treated with

either 20 mg/kg TRA (every fourth day), 0.3 mg/kg free

MMAE (alternate days), or vehicle (alternate days). For

Fab_TRA-bisAlk-vc-MMAE conjugate and free MMAE dosed

groups each dose was equivalent with respect to the amount of

MMAE administered.

Fab_TRA-bisAlk-vc-MMAE was found to be highly active

against the BT-474 xenografts resulting in a marked delay in

tumor growth (Figure 12). Treatment with Fab_TRA-bisAlk-vc-

MMAE resulted in a median time to end point (TTE) of 66.3

days, corresponding to a tumor growth delay (TGD) of 48.7

days. In contrast, TRA (TTE 24.6 days) and free drug (TTE

21.8 days) each elicited only marginal TGDs of 7.0 days and 4.2

days, respectively, which did not translate to meaningful

DISCUSSION

■

ADCs are a proven strategy for improving the therapeutic

window of cytotoxic drugs. The high levels of toxicity

associated with the administration of highly potent dolasta-

tin^22,23and maytansinoids²⁴⁻²⁶has prevented their use as

therapies. Despite the limitation of these compounds as small

molecule cytotoxic agents, their derivatives MMAE and DM1

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Figure 10. In vitro potency of Fab_TRA-bisAlk-vc-MMAE. (a,b) Potent antiproliferative eﬀect is seen for both the conjugate and free drug in HER2

positive cell lines SK-BR-3 and BT-474. (c,d) In antigen negative cell lines the conjugate was found to decrease viability but the potency was two to

three orders of magnitude lower than that of free drug depending on the cell line. (e) IC₅₀values for free drug and conjugate in both antigen positive

and negative cell lines.

have demonstrated clinical eﬃcacy when incorporated into the

approved ADCs, Adcetris^27,28and Kadcyla,²⁹respectively.

Despite the clinical success of many ADCs in both

hematological and solid tumors, they are far from the promise

of a “magic bullet”. Most clinical-stage ADCs have very similar

maximum tolerated doses with dose limiting toxicities due to

oﬀ-target toxicity. Strategies to produce ADCs with a wider

therapeutic window need to focus on improving eﬃcacy and

tolerability, which is linked to having better deﬁned ADC

structures. ADCs with more than four drug molecules are more

toxic, clear faster, and are less eﬀective.⁶Although higher drug

loading has been found to increase potency in vitro^5,30,31this

does not translate to a marked improvement of eﬃcacy in vivo.⁶

In experiments investigating the in vitro cytotoxicity of MMAE

conjugates of cAC10 (brentuximab), an anti-CD30 mAb, a

trend in potency was observed of DAR8 > DAR4 > DAR2.⁶

However, in vivo the higher loaded ADC was found to have

lower activity than the DAR 4 and DAR 2 species at an

equivalent drug dose. This unexpected diﬀerence in potency is

attributed to the diﬀerent pharmacokinetic properties of the

conjugates. The ADC with eight drug molecules loaded was

found to clear faster than the lower DAR conjugates resulting in

reduced exposure.

drug molecules attached.^5,6,31A lower average DAR can be

achieved after partial reduction, but this still yields a

distribution of conjugates with up to 30% of the ADC bearing

over four drug molecules. Each of the individual DAR species

within this mixture is also likely to be a mixture of diﬀerent

isomers with diﬀerent conjugation sites and potentially diﬀerent

pharmacological properties. Although problems with broad

DAR distribution can be overcome by engineering new sites for

conjugation (e.g., cysteine residues or non-native amino acids)

the re-engineering involved may lead to increased development

timelines and problems with manufacturability/scalability and

ultimately increased costs.

We have shown that disulﬁde bridging reagents can be used

as an alternative thiol conjugation method to mono-alkylation

that results in a narrower DAR distribution and can produce an

ADC with DAR 4 as the major product. The TRA-bisAlk-vc-

MMAE ADC prepared contained only low amounts (<1%) of

unconjugated antibody. Optimal ADCs will contain little or no

unconjugated parent antibody since this can act as a

competitive inhibitor, preventing binding of the ADC to the

target cell.

The conjugates formed using the bis-alkylating linker were

stable in serum and in the presence of high concentrations of

albumin over 5 days. Maleimide conjugates by comparison were

shown to be unstable with cross conjugation reactions to

albumin occurring. In a study comparing conjugates with

diﬀerent sites of conjugation and associated diﬀerences in

stability, the more stable ADCs elicited better eﬃcacy and

Current strategies for preparing ADCs via conjugation to

naturally occurring amino acids produce heterogeneous

mixtures with varying amounts of drug conjugated.^2,6

Maleimide conjugation of mAbs after full reduction of the

interchain disulﬁde bonds generates ADCs with up to eight

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Figure 11. In vitro potency of TRA-bisAlk-vc-MMAE ADC (DAR 2.8). (a,b) Potent antiproliferative eﬀect is seen for both the conjugate and free

drug in HER2 positive cell lines SK-BR-3 and BT-474. (c,d) In antigen negative cell lines the conjugate was found to decrease viability but the

potency was 2 to 3 orders of magnitude lower than that of free drug depending on the cell line. (e) IC₅₀values for free drug and conjugate in both

antigen positive and negative cell lines.

Figure 12. In vivo eﬃcacy of Fab_TRA-bisAlk-vc-MMAE (DAR = 1).

survival in in vivo cancer models.⁹Instability leading to

deconjugation disarms the ADC resulting in increasing

amounts of naked antibody which reduces eﬃcacy, acts as a

competitive inhibitor, and is also likely to have a longer half-life

and thus may compromise subsequent treatment cycles.

Figure 13. In vivo eﬃcacy of TRA-bisAlk-vc-MMAE ADC (DAR =

2.8).

Whole molecule IgGs may be limited in their tumor

penetration due to their large size restricting diﬀusion and

extravasion from the vasculature.³²One approach to improve

penetrability is to use smaller antibody fragments, such as

domains, scFvs, minibodies, and other engineered formats.^33,34

A limitation of these smaller formats is that their circulation

half-lives are too short for suﬃcient tumor exposure and

accumulation. By contrast, whole IgGs have very long

circulation half-lives due to FcRn mediated recycling. However,

the longer an ADC is in circulation, the greater the chance it

will bind oﬀ-target sites and degrade, which may lead to

increased systemic toxicity. An optimal half-life must balance

the requirements for suﬃcient tumor exposure and accumu-

H

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lation while ensuring ultimate elimination pathways limit

toxicity. Many studies have shown the utility of PEG in

improving the biodistribution of fragment antibodies for drug

conjugate and tumor radioimaging applications, often with

relatively small PEG sizes of less than 5 kDa that increase the

apparent size of the fragment to above the renal ﬁltration cutoﬀ

threshold.³⁵Avoiding kidney exposure and directing clearance

toward the liver, which has greater capacity for detoxiﬁcation,

may be beneﬁcial in improving tolerability. With the disulﬁde

conjugation approach described here, modifying the length of

the PEG chain incorporated between the reactive bis-sulfone

and the drug would confer increased half-life to a Fab conjugate

as can be achieved by protein PEGylation,^38,39allowing the

optimization of the pharmacokinetic proﬁle of the Fab for drug

conjugate use.

Monovalent antibody formats may also avoid the antigen

barrier which has been proposed as a factor contributing to the

limited penetration of mAbs in many solid tumors due to their

bivalent and high aﬃnity binding.³⁶Also, the bivalent binding

of antibodies may cross-link antigens and lead to unwanted

activation of signaling pathways. One example is the agonistic

eﬀect seen for DN-30 anti-Met bivalent antibody but not as a

Fab or single Fab arm antibody.^37,40

mented with 10% fetal bovine serum (FBS), 100 U/mL

penicillin, and 100 μg/mL streptomycin. All media were

obtained from Life Technologies. Cell lines were grown at 37

°C and 5% CO₂in a CO₂air-jacket incubator (Binder).

The extracellular domain of the HER2 receptor (ECD/

HER2) was purchased from Stratech Scientiﬁc Ltd., UK. Nunc

Maxisorp microtiter plates were purchased from Thermo

Scientiﬁc. Anti 6xHis antibody conjugated to HRP was

purchased from Clontech Laboratories Inc.

Preparation of Bis-Sulfone-PEG(24)-COOH. Bis-sulfone-

PEG(24)-COOtBu was prepared from H₂N-PEG(24)-COOt-

Bu according to the method described previously for methoxy

PEG bis-sulfone synthesis.^18,19Removal of the t-butyl

protecting group was achieved by stirring the bis-sulfone-

PEG(24)-COOtBu (0.98 g, 0.58 mmol) in 50% triﬂuoroacetic

acid in DCM (8 mL) at room temperature for 2 h. The solvents

were removed under reduced pressure, then the residue was

dissolved in acetone (30 mL). The solution was ﬁltered

through a sintered glass ﬁlter to remove insoluble material, then

cooled in a dry ice bath to give a white precipitate. The

precipitate was collected by centrifugation (4566 g, 30 min).

The resulting oﬀ-white solid was dried under vacuum (0.82 g,

1

85%). H NMR (400 MHz, CDCl₃) δ 2.49 (s, 6H, Ar−CH₃),

The conjugation of a cytotoxic payload via a three carbon

disulﬁde bridge to produce a biologically active Fab drug

conjugate was exempliﬁed using a Fab derived from TRA. A

Fab fragment has a single interchain disulﬁde that is readily

reduced. After reduction of Fab_TRA, MMAE was successfully

conjugated using the bis-alkylating reagent 1. The resulting

drug conjugate was homogeneous with respect to DAR with a

single drug molecule conjugated, and was structurally intact

with the disulﬁde bridged by the linker.

Conjugation of MMAE to a mAb or Fab using the bis-

alkylating reagent approach was shown to produce eﬀective

ADCs. The conjugates retained binding and demonstrated

antigen-selective in vitro cytotoxicity. Both mAb and Fab

conjugates were also found to be well tolerated and eﬃcacious

in vivo. We believe bis-alkylation is a general approach to

producing stable and more homogeneous ADCs that is eﬃcient

and predictable and does not rely on reengineering of the

antibody for site-speciﬁc conjugation.

2.59 (t, 2H, PEG−CH₂−COOH), 3.47−3.79 (m, PEG), 4.33

(m, 1H, SO₂−CH₂−CH−CH₂−SO₂-), 7.03 (s, 1H, PEG-NH-

carbonyl), 7.36 (d, 4H, SO₂Ar), 7.66 (d, 2H, COAr), 7.69 (d,

4H, SO₂Ar), 7.81 (d, 2H, COAr).

Synthesis of Bis-Sulfone-PEG(24)-MMAE (Reagent 1).

HATU (14 mg, 0.036 mmol) was added to a stirred suspension

containing Na₂CO₃(4 mg, 0.036 mmol), H₂N-val-cit-PAB-

MMAE (53 mg, 0.048 mmol), and bis-sulfone-PEG(24)-

COOH (40 mg, 0.024 mmol) in anhydrous DCM/DMF

(3:1, 1 mL) and stirred for 48 h under an argon atmosphere.

The crude mixture was puriﬁed using an anion exchange

column eluting with MeOH followed by column chromatog-

raphy eluting with MeOH/DCM (5:95 v/v). Compound 1 was

isolated as a colorless solid (46 mg, 71%). ¹H NMR (400 MHz,

CDCl₃) δ 0.60−0.99 (m, aliphatic side chains), 2.43 (s, Me-Ts),

3.36−3.66 (m, PEG), 7.15−7.28 (m, Ar), 7.31 (d, J = 8.3 Hz,

Ar), 7.54−7.62 (m, Ar), 7.79 (d, J = 8.3 Hz, Ar).

Preparation of Boc-NH-PEG(2 kDa)-FITC. Boc-NH-PEG(2

kDa)-NH₂(107 mg, 0.056 mmol) was dissolved in DCM (1

mL). Triethylamine (8.6 μL, 0.062 mmol) was then added

followed by FITC (100 mg, 0.25 mmol) in DMF (1 mL). The

resulting mixture was stirred at room temperature under an

argon atmosphere for 24 h. Volatile solvents were removed by

evaporation, then the product was precipitated by addition of

acetone (20 mL). The precipitate was collected by

centrifugation (4566 g, 30 min), then dried under vacuum to

EXPERIMENTAL PROCEDURES

■

Materials. TRA (Herceptin) was produced by Roche.

Maleimide-val-cit-PAB-MMAE and NH₂-val-cit-PAB-MMAE

were purchased from Concortis Biosystems. Boc-NH-PEG(2

kDa)-NH₂was purchased from Creative PEGWorks. Mal-

eimide-C5 and all other PEG precursors were purchased from

IRIS Biotech Gmbh. Alexa Fluor 488 C₅-maleimide and Alexa

Fluor 488 NHS ester were purchased from Invitrogen. All other

reagents and solvents were purchased from Acros Organics or

Sigma-Aldrich and used as received. ¹H NMR data were

collected using a Bruker 400 or 600 MHz spectrometer in the

NMR solvent stated. Chemical shifts δ are given in ppm

downﬁeld from tetramethylsilane.

1

aﬀord a yellow solid (76 mg, 59%). H NMR (400 MHz,

CDCl₃) δ 1.40 (s, 9H, t-Bu), 3.20−3.28 (m, 2H, PEG), 3.39−

3.44 (m, 1H), 3.47−3.51 (m, 2H), 3.52−3.72 (m, 183H, PEG),

3.73−3.87 (m, 2H), 5.20 (br s, 1H), 6.50 (dd, 2H, J = 8.6, 2.3

Hz), 6.58 (d, 2H, J = 8.6 Hz), 6.67 (d, 2H, J = 2.3 Hz), 7.03 (d,

1H, J = 8.3 Hz), 7.53−7.58 (m, 1H), 7.94 (dd, 1H, J = 8.3,1.7

Hz), 8.09 (d, 1H, J = 1.7 Hz), 9.22 (br s, 2H), 9.44 (s, 1H,

NH).

Preparation of H₂N-PEG(2 kDa)-FITC. Boc-NH-PEG(2

kDa)-FITC (73 mg, 0.031 mmol) was dissolved in DCM

(3.6 mL), then triﬂuoroacetic acid (0.4 mL) was added and the

resulting mixture stirred at room temperature for 2 h. Volatiles

were removed by evaporation under reduced pressure. The

crude product was dissolved in acetone (20 mL). The solution

All cell lines were purchased from the American Type

Culture Collection (ATCC), except for A549 which was

purchased from the Health Protection Agency Culture

Collection (HPACC). Cells were maintained in McCoy’s 5a

(SK-BR-3), DMEM:F12 (BT-474), Dulbecco’s modiﬁed Eagle

medium (DMEM) supplemented with 200 mM glutamine

(A549), or Minimum Essential Medium (MEM) supplemented

with 10 μg/mL insulin (MCF-7). All media were comple-

I

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was ﬁltered through a sintered ﬁlter to remove insoluble

material, then cooled in a dry ice bath to give a yellow

precipitate. The precipitate was collected by centrifugation

(4566 g, 30 min), then dried under vacuum to aﬀord a yellow

3.58−3.68 (m, 46H, CH₂OPEG + PhSO₂CH₂), 4.28−4.35 (m,

1H, CH), 7.54 (br, 1H, NH), 7.34, 7.64 (AB dd, SO₂Ar, 8H, J =

8.4 Hz), and 7.61, 7.82 (AB dd, COAr, 4H, J = 8.5 Hz).

Preparation of Bis-Sulfone-PEG(12) Amine TFA Salt. The

bis-sulfone-PEG(12)-NH-Boc (0.147 g, 125 μmol) was

dissolved in formic acid (2 mL) and the resulting mixture

was stirred at room temperature. After 16 h, the reaction was

concentrated under vacuum and the residue was resuspended in

water (1.5 mL), and puriﬁed by reverse-phase (C-18)

chromatography (gradient elution H₂O/MeCN buﬀers con-

tended 0.1% TFA, 0% to 100%) to aﬀord the desired bis-

sulfone-PEG(12) amine as a TFA salt (0.097 g, 68%). ¹H NMR

(600 MHz CDCl₃,) δ 2.46 (s, 6H, PhCH₃), 3.16 (m, 2H,

CH₂O), 3.48−3.52 (m, 2H, CH₂O), 3.59−3.73 (m, 46H,

CH₂OPEG + PhSO₂CH₂), 3.80 (m, 2H, CH₂O), 4.28−4.34

(m, 1H, CH), 7.53 (br, 1H, NH), 7.36, 7.66 (AB dd, SO₂Ar,

8H, J = 8.4 Hz), and 7.64, 7.86 (AB dd, COAr, 4H, J = 8.5 Hz).

Preparation of Bis-Sulfone-PEG(12)-Alexa Fluor 488

(Reagent 2). To a mixture of bis-sulfone-PEG(12) amine

(0.005 g, 4.80 μmol), Alexa Fluor 488 NHS ester (0.0025 g,

3.88 μmol), and anhydrous DMF (0.5 mL) under an argon

atmosphere was added sodium bicarbonate (0.0010 g, 11.9

μmol), and the reaction mixture was stirred at room

temperature. After 56 h, the reaction mixture was neutralized

by adding acetic acid (50 μL), puriﬁed by reverse-phase (C-18)

chromatography (gradient elution H₂O/MeCN buﬀers con-

tended 0.1% TFA, 0% to 100%) to aﬀord the desired bis-

sulfone-PEG(12)-Alexa Fluor 488 (0.002 g, 33%) as a red solid.

Purity by HPLC at 488 nm 91.2%. ESI-MS, m/z: 1543.1 (10%)

[M + 1H]⁺; 772.1 (100%) [M + 2H]²⁺.

1

solid (47 mg, 76%). H NMR (400 MHz, CDCl₃) δ 3.15 (s,

2H, PEG), 3.45−3.94 (m, 167H, PEG), 6.59 (dd, 2H, J = 8.6,

2.3 Hz), 6.66 (d, 2H, J = 8.6 Hz), 6.78 (d, 2H, J = 2.3 Hz), 7.07

(d, 1H, J = 8.3 Hz), 7.76 (br, 2H, NH), 7.94 (d, 1H, J = 8.3

Hz), 8.29 (s, 2H), 9.31 (br, 2H, PhOH).

Preparation of Bis-Sulfone-PEG(2 kDa)-FITC (Reagent 3).

4-[2,2-Bis[(p-tolylsulfonyl)methyl]acetyl) benzoic acid-NHS

ester (bis-sulfone NHS) was prepared according to a previously

described method.¹⁸H₂N-PEG(2 kDa)-FITC (23 mg, 0.010

mmol) was dissolved in DCM (6 mL) with the bis-sulfone

NHS ester (24.9 mg, 0.042 mmol) and pyridine (38 μL). The

resulting mixture was stirred at room temperature under argon

for 48 h. Volatiles were removed by evaporation under reduced

pressure. The crude product was dissolved in acetone (20 mL).

The solution was ﬁltered through a sintered ﬁlter to remove

insoluble material, then cooled in a dry ice bath to give a yellow

precipitate. The precipitate was collected by centrifugation

(4566 g, 30 min), then dried under vacuum to aﬀord a yellow

solid (15 mg, 60%). ¹H NMR (400 Hz, CDCl₃) δ 2.49 (s, 6H,

PhCH₃), 3.11 (s, 1H, PEG), 3.45−3.94 (m, 167H, PEG), 4.35

(m, 1H, CH), 6.59 (dd, 2H, J = 8.7, 2.3 Hz), 6.67 (d, 2H, J =

8.6 Hz), 6.77 (d, 2H, J = 2.2 Hz), 7.07 (d, 1H, J = 8.3 Hz), 7.37

(d, 4H, J = 8.0 Hz), 7.66 (d, 2H, J = 8.4 Hz), 7.71 (d, 4H, J =

8.0 Hz), 7.75−7.81 (m, 2H), 7.84 (dd, 2H, J = 8.4 Hz), 7.87−

7.92 (m, 1H), 8.00−8.03 (m, 1H), 8.14−8.28 (m, 2H), 9.0 (br

s, 1H).

Preparation of 4-(2,2-Bis[(p-tolylsulfonyl)methyl]acetyl)

Benzoic Acid HOBt Ester. Under an argon atmosphere, a

stirred mixture of 4-(2,2-bis[(p-tolylsulfonyl)methyl]acetyl)

benzoic acid (1.00 g, 2.0 mmol), prepared as described

previously,^16,181-hydroxybenzotriazole (0.27 g, 2.0 mmol),

and anhydrous tetrahydrofuran (20 mL) were cooled using an

ice bath. Neat 1,3-diisopropylcarbodiimide (313 μL, 2.0 mmol)

was then added dropwise at ∼0 °C. The reaction mixture was

stirred for 20 min, the ice-bath was then removed, and the

reaction mixture stirred at room temperature. After 16 h, the

resulting precipitate was isolated using a no. 3 sintered glass

funnel and washed with tetrahydrofuran (2 × 5 mL) and dried

in vacuo. The crude solid was suspended in methanol (15 mL)

and collected by ﬁltration after stirring for 1 h to aﬀord the

Preparation of Fab_TRA. Digestion buﬀer (20 mM NaH₂PO₄

pH 7.0, 10 mM EDTA, 50 mM cysteine, 50 mL) was warmed

to 37 °C. TRA (150 mg) was resuspended in ultrapure water

(7.5 mL). An aliquot (5 mL, 100 mg) of the resulting solution

was then diluted with the prewarmed digestion buﬀer (40 mL).

Papain (5 mL, 1 mg/mL) was then added to the TRA solution

to give a TRA:papain ratio of 20:1 (w/w). The mixture was

mixed gently then incubated at 37 °C for 30 min with gentle

mixing. Puriﬁcation was carried out by aﬃnity chromatography

on a protein L column (Pierce) followed by hydrophobic

interaction chromatography using a XK 16/10 column (GE

Healthcare) packed in-house with Toyopearl, Phenyl-650S

resin (Tosoh Bioscience LLC).

Conjugation to Fab_TRAUsing Bis-Alkylating MMAE

Reagent 1. Conjugation of reagent 1 to Fab_TRAwas carried

out using methods described previously for conjugation of PEG

to disulﬁde bonds using the bis-alkylating linker.^17,18

Conjugation to TRA Using Bis-Alkylating Reagents.

Conjugation of bis-alkylating MMAE reagent 1, bis-alkylating

Alexa Fluor 488 reagent 2, or bis-alkylating FITC-reagent 3 to

TRA was carried out using methods described previously for

conjugation of PEG to disulﬁde bonds.^17,18

Conjugation to TRA Using Maleimide-val-cit-PAB-MMAE.

TRA (10 mg/mL) in 20 mM sodium phosphate pH 8.0, 150

mM NaCl, 20 mM EDTA was treated with tris(2-

carboxyethyl)phosphine (TCEP, 2 mol equiv) at 40 °C for

60 min. The reductant was then removed by buﬀer exchange

into fresh pH 8.0 phosphate buﬀer by gel ﬁltration (PD-10

column, GE Healthcare). Determination of free thiols using

5,5′-dithiobis(2-nitrobenzoic acid) showed that there were

approximately four SH groups per mAb. Maleimide-val-cit-

PAB-MMAE (1.5 equiv per thiol) in DMSO (ﬁnal concen-

tration 5% v/v) was added and the resulting mixture incubated

1

desired active HOBt ester (0.84 g, 68%) as a white solid. H

NMR (600 MHz CDCl₃,) δ 2.49 (s, 6H, PhCH₃), 3.52 (m, 2H,

PhSO₂CH₂), 3.65 (m, 2H, PhSO₂CH₂), 4.49 (q, 1H, CH, J =

6.5 Hz), 7.39, 7.74 (AB dd, SO₂Ar, 8H, J = 8.6 Hz), 7.49 (m,

2H, PhH, HOBt), 7.60 (m, 1H, PhH, HOBt), 7.93, 8.31 (AB

dd, COAr, 4H, J = 8.4 Hz), 8.14 (d, 1H, PhH, HOBt, J = 9.2

Hz).

Preparation of Bis-Sulfone-PEG(12)-NH-Boc. Under an

argon atmosphere, a mixture of 4-(2,2-bis[(p-tolylsulfonyl)-

methyl]acetyl) benzoic acid HOBt ester (0.12 g, 194 μmol), α-

amino-ω-(t-butyloxycarbonyl)-dodecae(ethylene glycol) (0.10

g, 155 μmol) and anhydrous chloroform (4 mL) was stirred at

room temperature. After 16 h, the reaction mixture was

concentrated down to 1 mL of total volume, and puriﬁed by

ﬂash silica chromatography (gradient elution 100% DCM to

DCM:MeOH 10:90 v/v) to aﬀord the desired bis-sulfone-

1

PEG(12)-NH-Boc (0.15 g, 87%) as a white solid. H NMR

(600 MHz CDCl₃,) δ 1.42 (s, 9H, (CH₃)₃), 2.46 (s, 6H,

PhCH₃), 3.28 (m, 2H, CH₂O), 3.45−3.56 (m, 4H, CH₂O),

J

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at 40 °C overnight. Unreacted reagent was removed by gel

ﬁltration (PD-10, PBS).

buﬀer B (50 mM sodium phosphate pH 7.0, 20% isopropanol)

in 50 min. The ﬂow rate was 1 mL/min and the temperature

was set at 30 °C. Detection was carried out by following UV

absorption at 214, 248, and 280 nm.

Conjugation to TRA Using Alexa Fluor 488 C₅-Maleimide.

TRA (10 mg/mL) in 20 mM sodium phosphate pH 8.0, 150

mM NaCl, 20 mM EDTA was treated with dithiothreitol

(DTT, 3 mol equiv) at 40 °C for 60 min. The reductant was

then removed by buﬀer exchange into fresh pH 8.0 phosphate

buﬀer by gel ﬁltration (PD-10 column, GE Healthcare).

Determination of the number of free thiols present using

5,5′-dithiobis(2-nitrobenzoic acid) showed that there were

approximately 3.5 SH groups per mAb. Alexa Fluor 488 C₅-

maleimide (1.2 equiv per thiol) in MeCN (ﬁnal concentration

5% v/v) was added and the resulting mixture incubated at 4 °C

overnight. Unreacted reagent was removed by gel ﬁltration

(PD-10, PBS).

Analytical Hydrophobic Interaction Chromatography

(HIC). Analytical HIC of conjugates was carried out using a

TOSOH, TSKgel Butyl-NPR column (35 × 4.6 mm)

connected to a Dionex Ultimate 3000RS HPLC system. The

method consisted of a linear gradient from 80% buﬀer A (1.5 M

ammonium sulfate in 50 mM sodium phosphate, pH 7.0) to

86% buﬀer B (20% isopropanol (v/v) in 50 mM sodium

phosphate) over 20 min at a ﬂow rate of 0.8 mL/min. The

temperature was maintained at 30 °C throughout the analysis

and UV detection was carried out at 214 nm and 280 nm. For

each analysis an injection of 10 μg of mAb or ADC was carried

out.

Analytical Size Exclusion Chromatography (SEC). An

Acquity UPLC BEH200 column (Waters) connected to a

Dionex HPLC system was used for SEC. The mobile phase was

PBS, pH 7.4, containing 10% (v/v) isopropanol or 15% (v/v)

acetonitrile and the ﬂow rate was kept constant at 0.25 mL/

min. The column was maintained at 30 °C throughout the

analysis. The analysis was carried out in a 24 min isocratic

elution with UV detection at 214, 248, and 280 nm.

Alexa Fluor 488 Conjugate Serum Stability. Alexa Fluor

488-TRA conjugates were diluted with 50% rat or human

serum at a concentration of 0.1 mg/mL in sterile tubes. Sodium

azide was added (ﬁnal concentration 1 mM), and then the

mixtures were split into 4 equal aliquots. One aliquot was

immediately frozen at −80 °C. The remaining samples were

transferred to a CO₂incubator and kept at 37 °C. Aliquots were

removed from the incubator after 24, 48, and 96 h and

transferred to a −80 °C freezer. After the ﬁnal time point,

mixtures were analyzed by SE-HPLC using the method

described above including ﬂuorescence detection with an

excitation wavelength of 495 nm and emission wavelength of

525 nm.

Anti-HER2 ELISA. Maxisorp microtiter plates (96-well) were

coated overnight at 4 °C with TRA, Fab_TRA, TRA-bisAlk-vc-

MMAE, and Fab_TRA-bisAlk-vc-MMAE (5 μg/mL in 0.05 M

carbonate/bicarbonate buﬀer, pH 9.6). The plates were washed

three times with PBS/0.05% Tween 20 (PBS-T) and blocked

with 3% BSA/PBS-T for 1 h at room temperature. Serial

dilutions of ECD/HER2 were subsequently added and the

plates incubated for 3 h at room temperature. After 3 washes in

PBS/T, HRP-conjugated anti-6xHis antibody was added for 1 h

at room temperature to detect bound ECD/HER2. Tetra-

methyl benzidine (TMB) substrate and stop reagents were then

added in sequence, and the absorbance read at 630 nm using a

Spectramax M3 microplate reader. GraphPad Prism 5 software

using one site-speciﬁc binding with hill slope ﬁt was used for

analysis.

Cell Viability Assays (Cell Titer Glo). The eﬀect of TRA-

bisAlk-vc-MMAE and Fab_TRA-bisAlk-vc-MMAE MMAE con-

jugates on the viability of HER2-positive breast cancer cell lines

was assessed by Cell Titer Glo luminescence assay (Promega).

HER2-negative cell lines were included in the assays to conﬁrm

antigen selectivity. Cells were seeded in 96-well white opaque

plates (Corning) at a density of 5 × 10³to 1 × 10⁴/well

dependent on the cell line. Cells were allowed to attach for 24 h

at 37 °C and 5% CO₂in a humidiﬁed atmosphere. Following

incubation, cell culture medium was removed and serial

dilutions of TRA MMAE conjugates, TRA, and free drug

were added. Cells were incubated at 37 °C and 5% CO₂for 96

h and viability in response to treatment was quantiﬁed

according to the manufacturer’s instructions. Brieﬂy, Cell

Titer Glo reagent was added to the wells and plates were

incubated on a plate shaker for 3 min and 300 rpm. Cells were

incubated for 20 min at room temperature to allow stabilization

of the signal and luminescence was subsequently measured on a

SpectraMax3 micro plate reader (Molecular Devices). A four-

parameter curve ﬁtting model was applied (GraphPad) to

generate a dose−response curve. Response was expressed as %

viability of untreated controls.

Internalization Assay. SK-BR-3 and A549 cells were seeded

on poly(^D-lysine) coated coverslips in 24-well plates at a

density of 2 × 10⁵/well or 3 × 10⁴/well, respectively. Cells were

allowed to attach for 24 h at 37 °C and 5% CO₂in a humidiﬁed

atmosphere. Cells were subsequently surface labeled for 30 min

on ice by replacing the cell culture medium with 4 μg/mL TRA,

TRA-bisAlk-FITC, or Fab_TRA-bisAlk-FITC in serum-free

medium. Following incubation, control plates were washed

with PBS and coverslips were carefully mounted on microscope

slides in ProLong Gold antifade reagent (Life Technologies).

The remaining plates were washed three times with serum free

medium, transferred to a CO₂air-jacket incubator, and

incubated for 4 h at 37 °C and 5% CO₂to allow internalization.

Cells were subsequently washed three times with PBS and

acetate buﬀer (pH 2.5) to remove membrane bound TRA or

TRA-FITC conjugates, respectively. Samples were ﬁxed with

4% paraformaldehyde in PBS, washed three times with PBS,

and either mounted on microscope slides as described above

(FITC conjugates) or permeabilized with 0.5% Triton-X100 in

PBS (TRA). Binding and internalization of unconjugated TRA

was detected by means of Alexa Fluor 488 conjugated goat anti-

ADC Stability in the Presence of Free Thiols. TRA-bisAlk-

vc-MMAE was diluted to a ﬁnal concentration of 1 mg/mL

with PBS pH 7.4 containing human serum albumin (HSA) at a

concentration of 20 mg/mL. Sodium azide was added (ﬁnal

concentration 1 mM), and then the mixtures were split into 4

equal aliquots. One aliquot was immediately frozen at −80 °C.

The remaining samples were transferred to a CO₂incubator

and kept at 37 °C. Aliquots were removed from the freezer after

24, 48, and 120 h and transferred to a −80 °C freezer. After the

ﬁnal time point, mixtures were analyzed by analytical

hydrophobic interaction chromatography using a method

modiﬁed from the standard protocol with a ProPac 2.1 mm

× 100 mm HIC-10 column (Fisher Scientiﬁc). The method

consisted of a linear gradient from 100% buﬀer A (50 mM

sodium phosphate pH 7.0, 1.5 M ammonium sulfate) to 100%

K

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human IgG secondary antibody (Life Technologies). Cells were

imaged with a Leica SP5 laser scanning confocal microscope.

In Vivo Eﬃcacy Studies. Xenografts were initiated in female

severe combined immunodeﬁcient mice (Fox Chase SCID,

C.B-17/Icr-Prkdcscid, Charles River Laboratories) with BT-474

human breast carcinomas maintained by serial subcutaneous

transplantation in SCID mice. On the day of tumor implant,

each test mouse received a 1 mm³BT-474 tumor fragment

implanted subcutaneously in the right ﬂank, and tumor growth

was monitored as the average size approached the target range.

Tumors were measured in two dimensions using calipers, and

volume was calculated using the formula:

w²× l

tumor volume(mm³) =

2

where w = width and l = length, in mm, of the tumor.

The animals were fed ad libitum water (reverse osmosis, 1

ppm of Cl), and NIH 31 Modiﬁed and Irradiated Lab Diet. The

mice were housed on irradiated Enrich-o’cobs Laboratory

Animal Bedding in static microisolators on a 12 h light cycle at

20−22 °C and 40−60% humidity. On day 1 of the study, mice

were randomized into treatment groups (n = 10), and dosing

was initiated. All agents were administered intravenously (i.v.)

into the tail vein in a dosing volume of 0.2 mL per 20 g of body

weight (10 mL/kg), scaled to the body weight of the individual

animal.

In Vivo Eﬃcacy of Fab Conjugate. Mice were treated with

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Fab conjugate) or 20 mg/kg TRA (every fourth day for four

doses).

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MMAE (based on TRA) with dosing every 5 days for up to 7

occasions. Dosing was stopped when the tumor volume

reached less than 10% of the day 1 mean volume. Control

groups were treated with either 20 mg/kg TRA, 0.4 mg/kg free

MMAE, or vehicle on the same dosing schedule.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: antony.godwin@polytherics.com.

Notes

■

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank our PolyTherics colleagues for their help, Dr.

Maurizio Muroni for providing analytical support, and Prof.

Steve Brocchini and Dr. Matthew Baker for critical review of

the manuscript.

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Article Doi

Bridging disulfides for stable and defined antibody drug conjugates

DOI: 10.1021/bc500148x

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