This document summarizes recent processes developed for constructing homogeneous antibody-drug conjugates (ADCs). It discusses three categories of approaches: 1) engineering amino acids in antibodies to introduce unique conjugation sites, 2) using enzymes to modify antibodies, and 3) modifying drug linkers. Several examples are provided for each approach, including payloads, drug-to-antibody ratios achieved, and advantages over conventional heterogeneous ADCs. The document concludes that while homogeneous ADCs show improved properties, recombinant engineering methods may not be applicable to existing approved antibodies, and clinical performance of homogeneous ADCs remains to be confirmed.

1. Processes for Constructing Homogeneous Antibody Drug
Conjugates
David Y. Jackson*,†
†
Igenica Biotherapeutics, 863A Mitten Road, Suite 100B, Burlingame, California 94010, United States
ABSTRACT: Antibody drug conjugates (ADCs) are synthesized by conjugating a cytotoxic drug or “payload” to a monoclonal
antibody. The payloads are conjugated using amino or sulfhydryl specific linkers that react with lysines or cysteines on the
antibody surface. A typical antibody contains over 60 lysines and up to 12 cysteines as potential conjugation sites. The desired
DAR (drugs/antibody ratio) depends on a number of different factors and ranges from two to eight drugs/antibody. The
discrepancy between the number of potential conjugation sites and the desired DAR, combined with use of conventional
conjugation methods that are not site-specific, results in heterogeneous ADCs that vary in both DAR and conjugation sites.
Heterogeneous ADCs contain significant fractions with suboptimal DARs that are known to possess undesired pharmacological
properties. As a result, new methods for synthesizing homogeneous ADCs have been developed in order to increase their
potential as therapeutic agents. This article will review recently reported processes for preparing ADCs with improved
homogeneity. The advantages and potential limitations of each process are discussed, with emphasis on efficiency, quality, and in
vivo efficacy relative to similar heterogeneous ADCs.
■ INTRODUCTION
Antibody drug conjugates (ADCs) are a rapidly growing class of
targeted therapeutic agents for treatment of cancer.1−8
Although
the number of ADCs in clinical trials has steadily increased since
2005, many have failed to reach the later stages of clinical
development; one has been withdrawn from the market
(Mylotarg in 2002), and only two (Adcetris and Kadcyla) are
currently approved by the FDA for cancer indications (Figure
1A).9−11
Thus, far, the approval rate for ADCs has not met early
expectations and is lagging behind other antibody-based
therapeutics. Based on the number of approved ADCs versus
those that have failed to progress into later stage clinical trials, the
success rate is reminiscent of that for small molecule drugs. The
reasons for the clinical failures of ADCs are often not known or
they are still under investigation. More commonly, when the
reasons for clinical failure are clear, the information is not made
available to the public domain. Emerging preclinical data suggests
that heterogeneity, a property shared by most ADCs currently in
clinical development (Table 1), may ultimately limit their
potential as therapeutic agents.12,13
ADCs are composed of a cytotoxic drug or “payload”
conjugated to a tumor selective monoclonal antibody. The
heterogeneity of conventional ADCs arises from the synthetic
processes currently used for conjugation.14
Payloads are typically
conjugated to the antibody using amino or thiol specific linkers
that react with lysines or cysteines on the antibody surface.15
A
typical antibody contains more than 50 lysines and up to 12
cysteines as potential conjugation sites (Figure 1B).16
The
optimal DAR (drugs/antibody ratio) for most ADCs, however,
ranges from 2 to 8 drugs/antibody and is dependent upon a
variety of different factors. The discrepancy between the number
of potential conjugation sites and the desired DAR, combined
with the use of conjugation methods that are not site-specific,
result in heterogeneous ADCs that vary in both DAR and
conjugation sites. Consequently, conventional heterogeneous
ADCs often contain significant amounts of unconjugated
antibody in addition to fractions with suboptimal DARs.
Unconjugated antibodies can compete for antigen binding and
inhibit ADC activity, while fractions with suboptimal DARs are
frequently prone to aggregation, poor solubility, and/or
instability that ultimately result in a poor therapeutic
window.17,18
The relative degree of ADC heterogeneity depends on the
methods used for conjugation. For example, Kadcyla, an ADC
approved in 2013 for breast cancer, is synthesized using a two-
step process in which the linker and payload are conjugated in
separate steps (Scheme 1A).19−21
The linker contains an amino-
specific NHS ester that reacts with antibody lysines in the first
step and a thiol-specific maleimide group that reacts with a
maytansinoid payload in the second step. The process affords a
highly heterogeneous mixture of ADC molecules containing
from 0 to 10 payloads/antibody with an average DAR of 3.5
drugs/antibody.22,23
Additional heterogeneity arises due to
distribution of the payloads across dozens of potential
conjugation sites. As a result, Kadcyla contains hundreds of
different ADC molecules, each with its own unique pharmaco-
logical properties.24
Conjugation of payloads to antibodies through interchain
cysteines reduces ADC heterogeneity relative to lysine
conjugation because there are fewer potential conjugation sites.
Adcetris, an ADC approved in 2011 for treatment of Hodgkin’s
lymphoma, is an example of a cysteine conjugated ADC.25−27
The process for cysteine conjugation involves partial reduction of
four antibody interchain disulfide bonds to generate up to eight
reactive thiol groups. The partially reduced antibody is
subsequently conjugated to a payload containing a thiol-specific
maleimide linker. The payload used for Adcetris is monomethyl
auristatin E (MMAE) and contains a protease cleavable
Received: March 4, 2016
Published: April 14, 2016
Review
pubs.acs.org/OPRD
© 2016 American Chemical Society 852 DOI: 10.1021/acs.oprd.6b00067
Org. Process Res. Dev. 2016, 20, 852−866
This is an open access article published under an ACS AuthorChoice License, which permits
copying and redistribution of the article or any adaptations for non-commercial purposes.

2. maleimide linker (Scheme 1B). Although Adcetris is less
heterogeneous than Kadcyla, it is composed of dozens of
different ADC molecules containing 0 to 8 payloads with an
average DAR of 3.6 drugs/antibody.28
Like most cysteine
conjugated ADCs, Adcetris has a reduced half-life in vivo
compared to the parent antibody, cAC10. The diminished half-
life has been attributed to rapid clearance of high DAR species
(>4 drugs/antibody) and to partial loss of interchain disulfide
bonds during the conjugation process.29,30
Although different processes for lysine and cysteine
conjugation are used to synthesize Adcetris and Kadcyla, both
ADCs contain thio-succinimide bonds between the payload and
the antibody, which originate from the use of maleimide linkers
in the conjugation processes. Kadcyla contains a thio-
succinimide between the linker and the payload (Scheme 1A),
while Adcetris contains a thio-succinimide bond between the
linker and the antibody (Scheme 1B). Thio-succinimide groups
are known to undergo undesired side reactions such as
elimination or thiol exchange that can result in premature
release of the payloads from the ADC and lead to reduced
potency and/or increased systemic toxicity.31,32
Despite the known limitations of conventional heterogeneous
ADCs, most ADCs currently in clinical development utilize
similar conjugation methods to those described in Scheme 1. As a
result, they are likely to possess similar pharmacological
properties to Adcetris and Kadcyla, in addition to other less
successful ADCs that may have performed poorly in clinical
trials. In order to improve the pharmacological properties of
current and future ADCs, new site-specific conjugation processes
for synthesizing homogeneous ADCs are now being devel-
oped.33−36
Site-specific conjugation processes for constructing homoge-
neous ADCs can be divided into three different categories. Two
are focused on antibody modification (engineered amino acids
and enzyme mediated), while the third category is focused on
linker modification. The categories can be subdivided further
based on the specific processes that are used (Table 2). Examples
from each process were selected based on availability of sufficient
preclinical data to enable comparison with similar conventional
Figure 1. (A) Number of ADCs in different stages of clinical development from 2006 to 2014. (B) Structure of a typical IgG antibody showing lysines
(red), cysteines (yellow), and glycans (green) as potential conjugation sites.16
Table 1. Examples of Heterogeneous ADCs Currently in Clinical Trials for Cancer Indicationsa
ADC Sponsor Indications Status Payload Linked to Target
Adcetris Seattle Genetics HL and ALCL approved MMAE cysteine CD30
Kadcyla Genentech/Roche breast cancer approved DM1 lysine Her2
inotuzumab ozogamicin Pfizer NHL and ALL Phase III calicheamicin lysine CD22
lorvotuzumab mertansine Immunogen SCLC Phase II DM1 lysine CD56
glembatumumab vedotin Celldex BC, melanoma Phase II MMAE cysteine GPNMB
PSMA-ADC Progenics prostate Phase II MMAE cysteine FOLH1
SAR-3419 Sanofi DLBCL, ALL Phase II DM4 lysine CD19
ABT-414 Abbvie glioblastoma Phase II MMAE cysteine EGFR
BT-062 Biotest mult. myeloma Phase II DM4 lysine CD138
HLL1-Dox Immunomedics CLL, MM, NHL Phase II doxorubicin cysteine CD74
Immu-130 Immunomedics CRC Phase II SN-38 cysteine CEACAM5
Immu-132 Immunomedics solid tumors Phase II SN-38 cysteine EGP1
SYD985 Synthon breast cancer Phase II duocarmycin cysteine Her2
SAR-3419 Sanofi DLBCL, ALL Phase II DM4 lysine CD19
IMGN853 ImmunoGen solid tumors Phase I DM4 lysine FOLR1
IMGN529 ImmunoGen BCL,CLL, NHL Phase I DM1 lysine CD37
ASG-22M6E Astellas solid tumors Phase I MMAE cysteine nectin-4
AGS-16M8F Astellas RCC Phase I MMAF cysteine AGS16
AMG 172 Amgen RCC Phase I DM1 lysine CD27L
AMG 595 Amgen glioblastoma Phase I DM1 lysine EGFR8
BAY94-9343 Bayer solid tumors Phase I DM4 lysine mesothelin
a
Source: www.clinicaltrials.gov.
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3. heterogeneous ADCs. Homogeneous ADCs derived from these
processes have only just begun to enter clinical trials. Whether
they will outperform their heterogeneous counterparts in clinical
trials remains uncertain, but preclinical data suggest that
homogeneous ADCs are likely to dominate future clinical trials
and will lead to improved clinical results.
■ ENGINEERED AMINO ACID APPROACHES
Early attempts to construct homogeneous ADCs were
performed by reduction of interchain disulfide bonds followed
by conjugation of payloads to all eight interchain cysteines.37,38
The process resulted in a loss of four interchain disulfide bonds
and frequently resulted in ADC aggregation, instability, and/or
poor solubility due to hydrophobic properties of the payloads
that were available at that time. These fully loaded ADCs
containing eight drugs/antibody also demonstrated poor
pharmacokinetic properties and offered no significant advantages
over analogous heterogeneous ADCs with lower DARs.17,30,39
The conjugation methods currently used for preparing conven-
tional heterogeneous ADCs such as Adcetris or Kadcyla were
Scheme 1. (A) General Process for Synthesizing ADCs such as Kadcyla via Lysine Conjugation; (B) General Process for
Synthesizing ADCs, such as Adcetris, via Cysteine Conjugation
Table 2. Summary of Different Processes for Constructing Homogeneous ADCs
Institution Approach
Conjugation
Site Linker Type Payload(s) DAR Reference
Engineered
A.A.s
Seattle Genetics engineered cysteines HC (S239) maleimide MMAE, PBD 2 42, 50, 51
Genentech engineered cysteines HC(A114),
LC(V205)
maleimide MMAE,
DM1
2 or 4 43−46, 48
ETH engineered cysteines N or C terminus aldehyde cemadotin 2 47
AmBrx/WuXi eng. p-acetyl Phe HC (S115) alkoxyamine MMAD 2 55
Sutro/Cellgene eng. p-azido-Phe HC (S136) alkyne MMAF 2 56, 57
Allozyne/Medimmune eng. azido-Lys HC (K274) alkyne AF or PBD 2 58
Enzyme
mediated
Pfizer/Rinat transglutaminase (mTG) LL QGA tag 1° amine MMAD 2 59−61
Innate Pharma transglutaminase (BTG) HC
(Q295,Q297)
1° amine MMAE 4 62−64
Catalent/Redwood
Biosciences
formylglycine generating
enzyme (FGE)
C XPXR tag hydrazone DM1 2 65−67
NBE Therapeutics sortase A LPETG tag 1° amine DM1 2 or 4 68
Sanofi/Genzyme glycosyl transferase HC(N297)−
CHO
alkoxyamine MMAE 1 or 2 69
SynAffix endoglycosidase HC(N297)-
AzidoLys
alkyne DM1 2 70
Linker-based Seattle Genetics hydrophilic linkers interchain
cysteines
maleimide Auristatin T 8 72
Polytherics disulfide bridging interchain
cysteines
bis-sulphone MMAE 4 74−76
University College of
London (UCL)
next generation maleimides
(NGMs)
interchain
cysteines
dithiophenylmaleimide
dibromopyridazinedione
Doxirubicin 4 77−89
Igenica interchain cross-linking interchain
cysteines
dibromomaleimide MMAF 4 90, 91
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4. developed in order to overcome the potential liabilities
associated with fully loaded ADCs. Heterogeneity was
considered an acceptable penalty for the benefits gained by
lowering DARs. It has now become apparent that new methods
for synthesizing homogeneous ADCs are necessary for ADCs to
reach their full therapeutic potential.
Most new methods for synthesis of homogeneous ADCs
require recombinant engineering in order to introduce unique
functional groups into the antibody for site-specific conjugation.
In early examples of site-specific conjugation, Rader and co-
workers incorporated selenocysteine into antibodies to obtain
ADCs with one or two drugs per antibody.40,41
More recently,
several different engineered amino acid approaches have been
used successfully to generate homogeneous ADCs with two,
four, or eight drugs per antibody. In most cases, the engineered
ADCs have outperformed similar heterogeneous ADCs in vitro
and in vivo, yet there are potential limitations that should be
considered prior to clinical development. For instance,
recombinant methods for antibody re-engineering are not
applicable to existing “off-the-shelf” antibodies, which might be
desirable in some cases. Other potential challenges for
recombinant approaches include identification of optimal
conjugation sites, possible immunogenicity and use of antibody
expression systems which have not yet been clinically validated.
Whether the benefits of ADC homogeneity will outweigh the
additional time and cost associated with developing these
methods is still unclear, but significant progress has been made
toward producing homogeneous ADCs with improved pharma-
cological properties.
Engineered Cysteines. The first examples in which
recombinant antibody engineering was used to improve ADC
homogeneity involved two opposite strategies, removal or
addition of cysteine residues. Carter and co-workers systemati-
cally removed interchain cysteines by replacement with serine in
cAC10, the anti-CD30 antibody used in Adcetris.42
The
remaining cysteines were then conjugated to the well-known
auristatin payload (MC-vc-Pab-MMAE) to yield homogeneous
ADCs containing two or four drugs/antibody (Scheme 2A). The
resulting ADCs were found to have comparable pharmacological
properties to analogous heterogeneous ADCs. This led the
authors to conclude that improved homogeneity had a minimal
effect on the therapeutic index of ADCs; however, the loss of
interchain disulfides in the engineered ADCs may have masked
potential gains derived from improved homogeneity.
An alternative approach by Junutula and co-workers led to a
different conclusion. Cysteine mutations were introduced into
position 114 on the heavy chain of an anti-MUC16 antibody,
3A5.43
The mutations provided two unique unpaired thiol
groups suitable for conjugation to payloads containing conven-
tional maleimide linkers. The process afforded ADCs that
Scheme 2. Engineered Cysteine Approaches toward Homogeneous ADCsa
a
(A) Replacement of four hinge interchain cysteines with serine followed by reduction and conjugation with conventional maleimide linkers affords
homogeneous ADCs with 4 drugs/antibody; (B) Cysteine point mutations are introduced into the heavy and/or light chains of an antibody. The
engineered cysteines are expressed in an oxidized state. Mild reduction followed by re-oxidation of interchain disulfides yields unpaired cysteines in
reduced form suitable for conjugation with conventional maleimide linkers to afford homogeneous ADCs with 2 or 4 drugs/antibody. (C) Cysteines
are introduced at the N-terminus of H or L chains and conjugated to aldehyde linkers to form a stable thiazolidine linkage with the antibody.
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5. contained predominantly two drugs/antibody, however, addi-
tional reduction and oxidation steps were required to obtain
mutant antibodies in a form suitable for conjugation (Scheme
2B). The thio-mAb ADC (aka TDC) demonstrated comparable
efficacy to a conventional heterogeneous ADC, yet the relative
toxicity of the TDC was significantly reduced for an improved
therapeutic index. In a subsequent study, Boswell and co-workers
used a similar approach to construct anti-STEAP1 ADCs and
obtained comparable results.44
The engineered cysteine approach was later applied to
alternative payloads via site-specific conjugation of engineered
trastuzumab to a maytansine payload (DM1).45
The DM1
payload is analogous to that used in Kadcyla, a heterogeneous
trastuzumab ADC approved in 2013 for treatment of Her2
positive breast cancer.19
The pharmacological properties of the
resulting homogeneous TDCs were compared with Kadcyla and
the TDCs demonstrated improved safety in both rat and cyno
toxicity studies. Interestingly, the pharmacokinetic profile of the
TDC was comparable to the Kadcyla benchmark. This led the
researchers to conclude that the improved safety profile was
likely due to removal of the higher DAR species present in
Kadcyla rather than improved linker stability.
A follow-up study by Pillow and co-workers utilized an oxime
linker for conjugation of DM1 to trastuzumab through
engineered cysteines on both heavy and light chains to generate
homogeneous TDCs with four drugs/antibody.46
Further
improvements in efficacy and safety were observed for the
TDC versus Kadcyla. The improved properties were attributed
to enhanced stability of the oxime linker, however, other factors
such as different linkers (SMCC vs MPEO or MPA), conjugated
through different side chains (Lys vs Cys), at different locations
on the antibody, likely contributed to the observed differences in
pharmacological properties. As a result, the relative contributions
of homogeneity or linker stability to the observed improvements
in therapeutic index could not be determined from these studies.
In summary, the engineered cysteine approaches afforded
homogeneous TDCs with superior therapeutic windows over
conventional ADCs. The improvements were attributed
primarily to improved linker stability and elimination of high
DAR species that are present in heterogeneous ADCs.
Engineered cysteine conjugated through nonmaleimide link-
ers have also been reported. For example, Casi and co-workers
introduced cysteine residues at the N-termini of antibody heavy
and light chains to enable site-specific conjugation with
aldehydes. The engineered antibody was conjugated with a
cemadotin aldehyde derivative. The conjugation process resulted
in efficient formation of a cleavable thiazolidine linkage between
the payload and the antibody intended to slowly release the
payload in vitro (Scheme 2C). The ADCs contained four
payloads per antibody and demonstrated moderate potency
against antigen expressing cells in vitro, but their in vivo efficacy
was not reported.47
An important lesson learned from engineered cysteine
approaches for synthesizing homogeneous ADCs was the
discovery that the location of the conjugation site can have a
dramatic impact on ADC activity. Shen and co-workers
introduced cysteine mutations into three different sites on
trastuzumab heavy and light chains.48
The resulting thiomabs
were used to construct homogeneous ADCs via conjugation with
an auristatin payload (MC-VC-MMAE) that contained a
cleavable, self-emolative dipeptide maleimide linker analogous
to that used in Adcetris.27
The three conjugation sites (LC-
V205C, HC-A114C, and Fc-S396C) were selected based on
differences in solvent accessibility and local charge. All three thio-
trastuzumab-MC-VC-MMAE ADCs demonstrated comparable
homogeneity to each other with DARs ranging from 1.7−1.9
drugs/antibody and varied only in their conjugation sites.
Remarkably, the ADCs demonstrated substantially different
pharmacological properties in vivo, attributed primarily to
differences in linker stability.
Native LC/MS analysis of the ADCs revealed that linker
stability correlated with the rate of maleimide hydrolysis to a
ring-opened form that was less prone to premature release of the
payload. The observed differences in the rates of hydrolysis were
postulated to result from subtle variations in the microenviron-
ments at different conjugation sites. This hypothesis was later
confirmed by Tumey and co-workers who synthesized
heterogeneous trastuzumab ADCs using maleimide linkers.49
The ADCs were subsequently hydrolyzed in vitro to the ring-
opened isoform, and their pharmacological properties were
compared with analogous ADCs containing conventional (ring-
closed) maleimide linkers. As expected, the ADCs containing the
ring-opened form demonstrated improved stability and superior
efficacy over the conventional (ring-closed) ADCs. Overall, the
results demonstrated that the conjugation site can significantly
effect ADC activity, suggesting that optimal conjugation sites
might be different for each ADC.
Several important breakthroughs in ADC technology have
been made through use of engineered cysteines. For example,
researchers at Seattle Genetics recently reported SGN-CD33A,
an ADC that contains a highly potent pyrrolobenzodiazepine
dimer (PBD) payload that is 10−100 times more potent than
current tubulin inhibitor payloads such as MMAE or DM1.50
Earlier attempts to construct conventional ADCs using PBD
payloads often resulted in aggregate formation due to poor water
solubility of PBDs. Conjugation via engineered cysteines enables
the synthesis of homogeneous ADCs containing only two PBDs
per antibody and reduces aggregation to an acceptable level.
Moreover, the resulting ADC had superior potency over a well-
known anti-CD33 ADC (Mylotarg) in AML tumor models with
a multidrug-resistant phenotype. Positive results were also
reported for an anti-CD70 ADC containing similar PBD
payloads conjugated through engineered cysteines at position
239 on the heavy chains.51
The engineered cysteine approach
propelled both of these homogeneous ADCs into early clinical
development and preliminary results have been very encourag-
ing.
Engineered Non-Natural Amino Acids. Non-natural
amino acids (nnAAs) have been used as alternatives to cysteine
for providing site-specific conjugation sites.52−54
The nnAA
approach offers several advantages over engineered cysteines due
to the diversity of different side chains that can be introduced as
potential conjugation sites. For instance, liabilities associated
with engineered cysteines and maleimide linkers can be
eliminated and new payloads can be tested that might be
incompatible with conventional methods for cysteine con-
jugation. In addition, the incorporation of nnAAs with different
side chains on antibody heavy and light chains would enable
payloads with different mechanisms of action to be conjugated to
the same antibody.
A number of different processes in which non-natural amino
acids were used to afford ADCs with improved properties have
recently been reported. For example, stop codon suppression
technology (EuCODE) was used to express antibodies
containing p-acetyl phenylalanine (pAF) at position 115 on the
heavy chains. Site-specific conjugation of the pAF side chains to
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6. an auristatin payload (MMAD) containing an alkoxyamine linker
forms a stable oxime linkage with the antibody. The conjugation
process affords ADCs with 2 drugs/antibody (Scheme 3A).55
The resulting non-natural amino acid drug conjugate (NDC)
was compared to an analogous thiomab drug conjugate (TDC)
containing engineered cysteines instead of pAF at identical
locations on the heavy chains. The TDC payload contained an
analogous oxime linker to the NDC, but a maleimide group was
added to enable conjugation with cysteine. Differences in activity
between the NDC and TDC could therefore be attributed to the
presence (or absence) of a thio-succinimide link to the antibody.
The results demonstrated that the homogeneous NDC out-
performed the TDC in vivo, attributed in part, to the absence of a
thio-succinimide group in the NDC.
Engineered non-natural amino acids provide new options for
linker chemistry that are not possible with conventional
conjugation methods or engineered cysteines. For example,
Zimmerman and co-workers used non-natural amino acids in
combination with a cell free expression system to incorporate p-
azidomethyl L-phenylalanine (pAMF) into dozens of different
sites on Trastuzumab.56,57
The pAMF amino acid was designed
for conjugation to alkyne linkers via strain-promoted azide−
alkyne cycloaddition copper free click chemistry (Scheme 3B).
The results were consistent with previous approaches in that
ADC activity was highly dependent on the conjugation site.
Antibody expression and conjugation efficiency were also
affected by the location of the pAMF, suggesting that optimal
conjugation sites may be different for each antibody. The data
supported this suggestion, because the HC-Ala114 conjugation
site previously used for making NDCs and TDCs was found to be
inferior to Ser136 based on antibody expression and conjugation
efficiencies. Further studies are needed to determine whether
conjugation sites will remain optimal when applied to different
antibodies.
Alternative DNA targeting payloads such as pyrrolobenzodia-
zepine dimers (PBDs) have been successfully conjugated to
antibodies through non-natural amino acids to yield homoge-
neous NDCs with two drugs/antibody.58
For example, VanBrunt
and co-workers used a cell-based mammalian expression system
to produce variants of a Her2 specific antibody (4D5) in high
yield (1.7 g/L). The variants contained a non-natural lysine
analog (N6−2-azidoethoxycarbonyl-L-lysine) modified with a
terminal azide group. The azido-lysine derivative was engineered
into positions on either chain of the antibody (HC-274 or LC-
70) to enable site-specific conjugation via copper assisted alkyne
cycloaddition (CuAAC) click chemistry (Scheme 3C). Auristatin
F or PBD payloads containing alkyne linkers were conjugated
with high efficiency (>95% conversion) after 4 h at room
temperature to afford NDCs with 1.9 drugs/antibody. The
conjugation process forms a stable triazole linkage with the
antibody. NDC stability was determined in vivo via single
intravenous injections in rats and found to be comparable to
unconjugated trastuzumab. In addition, the PBD NDC
demonstrated superior efficacy over an analogous auristatin
NDC in Her2 positive BT474 tumor bearing mice after three
weekly doses at 1 mg/kg. This result further validated the use of
PBDs as alternatives to conventional tubulin inhibitors.
Scheme 3. Synthesis of Homogeneous ADCs via Engineered Non-Natural Amino Acidsa
a
(A) p-acetyl phenylalanine (pAF) is substituted for Ser 115 on the H-chain. Conjugation of pAF with alkoxyamino linkers forms a stable oxime
linkage and yields NDCs with 2 drugs/antibody. (B) p-azidomethyl phenylalanine (pAMF) is substituted for Ser 136 on the heavy chain.
Conjugation of pAMF to alkyne linkers via strain-promoted azide−alkyne cycloaddition (SPAAC) affords NDCs with 2 drugs/antibody; (C)
Incorporation of an azido-lysine analog into antibody heavy or light chains, followed by site-specific conjugation to alkyne linkers forms a stable
triazole linkage with the antibody.
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7. ■ ENZYME-MEDIATED APPROACHES
Transglutaminase. Alternative methods for site-specific
conjugation have been reported in which enzymes are used to
site-specifically modify antibodies with unique functional groups
for conjugation. For example, Strop and co-workers introduced a
microbial transglutaminase (mTG) recognition sequence tag
(LLQGA) into 90 different positions on an anti-EGFR
antibody.59,60
The glutamine tag served as an acyl donor for
enzymatic ligation to primary amines catalyzed by mTG
(Scheme 4A). The tagged antibodies were enzymatically
conjugated with MMAD payloads containing cleavable (Ac-
Lys-vc-MMAD) or noncleavable (amino-PEG6-MMAD) pri-
mary amine linkers. Twelve sites were considered adequate for
ADC synthesis based on conjugation efficiencies, and two sites
(one each on the heavy and light chains) were selected for further
evaluation. The glutamine tags were engineered into different
antibodies and consistently afforded ADCs with DARs > 1.8
drugs/antibody determined by native MS analysis.61
ADCs were
prepared from the cleavable Ac-Lys-VCP-MMAD payload using
Scheme 4. Enzyme Mediated Approaches for Synthesizing Homogeneous ADCsa
a
(A) A glutamine tag (LLQGA) is inserted into the antibody for transglutaminase mediated conjugation with payloads containing primary amino
groups. The mTG transfers the amino group to the glutamine tag forming a stable amide linkage with the antibody. (B) A recognition sequence
(CXPXR) is inserted into antibody heavy chains. Treatment with formylglycine generating enzyme (FGE) converts the cysteine tag into
formylglycine to enable site-specific conjugation with hydrazine linkers. (C) Sortase A mediated synthesis; a recognition sequence (LPETG) is
introduced at the C-termini of the heavy and/or light chains. Conjugation to payloads containing a pentaglycine linker using Sortase A forms a stable
amide linkage with the antibody. (D) Site-specific conjugation via glyco-engineering. Sialic acid groups are introduced on Asn297 of the heavy chains
and oxidized to aldehydes with periodate. Conjugation of the aldehyde with alkoxyamino linkers forms a stable oxime linkage with the antibody. (E)
Endoglycosidase trimming of native glycans exposes two GlcNAc groups on Asn 297 which are then attached to to a GalNAc derivative modified
with an azide group. The azide is then conjugated to payloads containing alkyne groups (BCN) via click chemistry.
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8. transglutaminase, and their pharmacological properties were
evaluated in vivo.
In general, the mTG modified ADCs had comparable efficacy
to control ADCs synthesized via conventional methods. In
addition, they demonstrated improved stability and reduced
toxicity in rodents, attributed in part to the formation of stable
amide linkages with the antibody. Payloads containing non-
cleavable linkers were also reported to conjugate with high
efficiency, but their potency was not reported. The noncleavable
ADCs could have been informative controls since drug release
would likely be restricted to antibody degradation. Instead, the
control ADCs used in this study were heterogeneous, had higher
DARs, and contained payloads conjugated to different sites. As a
result, the relative impact of the amide linkage resulting from
mTG mediated conjugation remains uncertain.
Alternatively, transglutaminase can be used without introduc-
ing a sequence tag.62
Early studies showed that deglycosylation of
antibodies at position N297 enables site-specific bacterial
transglutaminase (BTG) mediated conjugation to the native
glutamine at position Q295.63
Lhospice and co-workers later
produced aglycosylated variants of cAC10 (the anti-CD30
antibody in Adcetris) with an N297Q mutation.64
The mutation
enabled site-specific conjugation of MMAE payloads to Q295
and Q297 on the heavy chains using BTG. The conjugation
process afforded ADCs containing four payloads/antibody with
70% efficiency, but significant amounts of lower DARs were
present. Nonetheless, the pharmacological properties of the
transglutaminase modified ADCs were comparable to Adcetris.
Overall, the study results expanded the transglutaminase
approach to include ADCs with 4 drugs/antibody and
demonstrated that aglycosylated antibodies could be used
without adverse effects.
Formylglycine Generating Enzyme (FGE). Alternative
enzyme-mediated approaches have been used to construct
homogeneous ADCs. Drake and co-workers introduced the
recognition sequence (CXPXR) for formylglycine generating
enzyme (FGE) into eight different sites on a generic IgG1
antibody (1 light and 7 heavy chain sites).65−67
The approach is
reminiscent of engineered cysteine approaches except that
cysteine is introduced as a pentapeptide insertion rather than a
single point mutation. Treatment of the mutant antibodies with
FGE results in site-specific conversion of cysteine to
formylglycine. The inserted peptide reduces the number of
potential conjugation sites relative to other recombinant
approaches due to structural constraints, but the aldehyde
functionality enables new conjugation chemistries to be
explored. Two of the heavy chain labeled sites resulted in highly
aggregated antibodies, and one was predicted to be immuno-
genic. Three of the five remaining sites were selected for
evaluation using trastuzumab as a benchmark antibody. The
aldehyde-tagged antibodies were site-specifically conjugated via
hydrazino-iso-Pictet-Spengler (HIPS) chemistry to a maytansine
payload containing an appropriately modified hydrazine linker
(Scheme 4B).
The pharmacological properties of the resulting homogeneous
ADCs were compared to Kadcyla, a heterogeneous trastuzumab
ADC with an analogous maytansine payload. Consistent with
previous approaches, the homogeneous trastuzumab ADCs
demonstrated comparable potency, improved stability, and
reduced toxicity in vivo compared to Kadcyla. Contrary to
results from previous approaches, the conjugation site did not
significantly impact ADC efficacy, and minimal differences in
tumor growth inhibition were observed in xenograft tumor
models. The differences between Kadcyla and the FGE tagged
ADCs were attributed to the presence of high DAR species
present in Kadcyla. Although Kadcyla contains an analogous
maytansine payload, it is a heterogeneous ADC and is prepared
using different linker chemistry conjugated to lysines at different
sites, properties which likely contributed to the observed
differences in activity of the ADCs.
To simplify the overall aldehyde tagging approach, antibody
expression was carried out in cells overexpressing FGE.
Efficiencies for the cysteine to formylglycine conversion ranged
from 86% to 98% depending on the conjugation site.
Conjugation efficiencies between the tagged antibody and the
hydrazine payload were typically 75% or higher, although 8−10
equiv of the payload were required to obtain ADCs with two
drugs/antibody. Moreover, preparative hydrophobic interaction
chromatography (HIC) was required to remove unconjugated
antibody in the final purification step, which raises questions
regarding scalability of the process to a level required for clinical
development.
Sortase A. Alternative enzyme-mediated approaches have
been used to generate homogeneous ADCs. For example, Beerli
and co-workers engineered a recognition sequence for a
transpeptidase (sortase A) to the C-termini of the heavy and/
or light chains of antibodies.68
Sortase A catalyzes transfer of
polyglycine substrates to the C-terminus of the pentapeptide
sequence motif, LPXTG, resulting in a stable amide linkage with
the antibody (Scheme 4C). ADCs were constructed via
conjugation of engineered trastuzumab and cAC10 variants
containing an LPETG recognition motif with MMAE or DM1
payloads containing pentaglycine linkers. The resulting ADCs
had improved homogeneity vs conventional Kadcyla and
Adcetris controls and contained approximately 1.8 drugs/
antibody after purification by affinity chromatography. Notably,
the homogeneous trastuzumab ADC demonstrated comparable
efficacy to Kadcyla in Her2 expressing xenograft tumors despite
have a lower DAR. Additional studies with ADCs containing
auristatin payloads are ongoing to demonstrate the versatility of
the methods for use with alternative payloads.
Glycosyltransferases. Additional enzyme-mediated ap-
proaches to site-specific conjugation have been reported which
do not require recombinant engineering. For example, Zhou and
co-workers used glycosyltrasferases to incorporate terminal sialic
acid moieties into glycans linked to the native Asn297
glycosylation site in trastuzumab and two other antibodies.69
Mild oxidation of the engineered sialic acid groups with sodium
periodate yields two aldehyde groups for site-specific conjugation
to appropriately modified payloads. The glyco-engineered
antibodies were conjugated to auristatin payloads (MMAE and
MMAD) containing amino-oxi linkers resulting in formation of a
stable oxime linkage with the antibody (Scheme 4D). Unlike
other approaches discussed so far, the method does not require
recombinant antibody engineering, but relatively large quantities
of the linker-payload (24 equiv) were required to produce ADCs
containing 1 or 2 drugs/antibody. The in vivo stability and
pharmacokinetic properties of the ADCs were not reported, but
they demonstrated comparable activity to conventional controls
in xenograft tumor models.
Endoglycosidase. Another nonrecombinant approach to
site-specific conjugation that involves glycan remodeling at
Asn297 was reported recently by van Geel and co-workers.70
The
multistage process begins by treatment of the antibody with
endoglycosidase to remove core GlcNAc moieties. This enables
site-specific attachment of azide-modified GalNAc analogues
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9. using glycosyltransferases. The resulting azide-labeled antibody
is then conjugated to payloads with bicyclononyne (BCN)
linkers via copper-free click chemistry (Scheme 4E). A variety of
different linker-payload constructs were synthesized using five
different payloads combined with cleavable or noncleavable
linkers. Conjugation efficiencies were typically >95%, and the
ADC (trastuzumab-BCN-PEG-DM1) outperformed Kadcyla in
efficacy studies.
■ LINKER-BASED APPROACHES
The engineered amino acid and enzyme mediated approaches for
site-specific conjugation have been successful in producing
ADCs with improved homogeneity and led to significant
improvements in other ADC properties such as stability,
potency, and safety. Moreover, most of the methods discussed
thus far yielded ADCs with comparable or superior therapeutic
windows when compared to conventional heterogeneous
benchmarks. The relative impact of homogeneity on ADC
activity remains uncertain, however, because the homogeneous
ADCs often contained different payloads and conjugation sites
than those used in the benchmark ADCs. Conjugation methods
that leverage the same conjugation sites as conventional ADCs
should enable the impact of homogeneity on ADC activity to be
determined with greater confidence because other variables can
be eliminated. The majority of the linker-based processes for
constructing homogeneous ADCs utilize interchain cysteines for
conjugation to afford homogeneous ADCs with four or eight
drugs/antibody. The processes are chemically driven and differ
from previously discussed processes in that they are focused on
linker modifications. As a result, they can be applied to existing
“off the shelf” antibodies and do not require recombinant
antibody re-engineering or unconventional expression systems.
Hydrophilic Linkers. As discussed previously, ADCs with
eight payloads/antibody conjugated through interchain cysteines
were shown to possess suboptimal pharmacological properties,
attributed in part to the hydrophobicity of the conventional
payloads that were used for their preparation. With this in mind,
researchers at Seattle Genetics hypothesized that hydrophilic
Scheme 5. Linker Based Approaches for Synthesizing Homogeneous ADCsa
a
(A) Hydrophylic linkers; reduction of interchain cysteines followed by conjugation with payloads containing a hydrophylic linker yields ADCs with
8 drugs/antibody. (B) Reduction of interchain disulfides followed by conjugation with bis-sulfone linkers forms a three-atom disulfide bridge and
yields ADCs with 4 drugs/antibody. (C) NGMs (next generation maleimides). Interchain disulfide reduction followed by addition of a
dithiophenylmaleimide linker containing an alkyne group enables conjugation with payloads containing an azide group. The conjugation reaction
forms a triazole linkage with the antibody and affords ADCs with 4 drugs/antibody. (D) Dibromopyridazinedione reacts with interchain cysteines to
afford ADCs with 4 linkers/antibody. Each linker contain two different azide groups to enable conjugation of two different payloads.
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860

10. linkers and payloads would enable construction of fully loaded
ADCs (DARs = 8 drugs/antibody) without compromising other
desired properties. Earlier work by Doronina et al. had shown
that dipeptide linkers could be used for conjugating auristatins
through their C-terminus to yield heterogeneous ADCs with
improved potency over conventional auristatin ADCs.71
Lyon and co-workers synthesized similar auristatin derivatives
with reduced hydrophobicity by replacing the C-terminal
phenylalanine in auristatin F with a more hydrophilic amino
acid, threonine. The resulting derivative (auristatin T) was then
linked through the C-terminus to a hydrophilic dipeptide
maleimide linker (Scheme 5A).72
The hydrophilic payload was
then conjugated to an anti-CD70 antibody (h1F6) to afford
homogeneous ADCs containing eight drugs/antibody. The fully
loaded ADCs were compared to ADCs containing conventional
linkers (MC or MC-VC-PAB) and payloads (MMAF) and
demonstrated slower clearance and improved efficacy over the
conventional ADCs.
The results led the authors to conclude that “reducing
hydrophobicity of homogeneous ADCs improves pharmacoki-
netics and therapeutic index”, as reflected in the title of the article.
ADC hydrophylicity correlated with improved pharmacological
properties, however, with the ADCs compared in the study, and
they all contained different payloads, connected to different
linkers in different orientations (N or C terminus). Since these
factors would also have a significant effect on the overall
properties of the ADCs, the relative contribution of reduced
hydrophobicity to the improved therapeutic index could not be
determined with confidence.73
Bis-alkylating Linkers. Most linker based approaches for
synthesizing homogeneous ADCs utilize bifunctional linkers
designed to cross-link antibody interchain cysteines and afford
homogeneous ADCs containing four drugs/antibody. One
example of this approach was reported by Badescu and co-
workers who synthesized bis-sulfone linkers designed to cross-
link two cysteines and form a 3-carbon bridge (Scheme 5B).74,75
The bis-sulfone cross-linking group was attached to MMAE
through a cleavable PEG spacer and conjugated to trastuzumab
to afford ADCs that were 78% DAR4. The resulting ADCs were
more stable than conventional maleimide ADCs under various
conditions and were moderately potent against Her2 positive
cells in vitro. Efficacy studies were performed, and the bis-
alkylated ADCs demonstrated superior potency to unconjugated
trastuzumab, although multiple high doses (>10 mg/kg) were
required for tumor growth inhibition.
In a follow-up study, Godwin and co-workers used a similar
payload to construct ADCs containing predominately 1, 2, 3, or 4
drugs per antibody by changing the stoichiometry of the
conjugation reaction.76
The ADCs were purified by preparative
hydrophobic interaction chromatography (HIC) to remove
undesired DAR fractions and tested in a BT474 xenograft tumor
model. ADC efficacy correlated with increased DAR and again,
multiple high doses (>10 mg/kg) were required for tumor
growth inhibition. A second study in JIMT-1 xenografts was
performed, and the bis-alkylated ADCs demonstrated improved
potency compared to T-DM1. Since neither unconjugated
trastuzumab nor analogous heterogeneous ADC controls were
included in these studies, the relative impact of the bis-alkylating
linkers on the overall therapeutic index of the ADCs could not be
determined.
Next-Generation Maleimides (NGMs). Alternative re-
agents have been used for cross-linking interchain cysteines
with improved efficiency over the previously discussed bis-
sulfone linkers. Researchers at UCL (University College of
London) published a series of papers in which substituted
maleimides were shown to be highly efficient cysteine cross-
linking reagents.77−79
Early applications for substituted
maleimides included disulfide protection, protein pegylation,
and fluorescent labeling.80−82
Moody and co-workers later
reported that bromomaleimide-linked bioconjugates are cleav-
able in mammalian cells,83
and numerous antibody-based
applications with substituted maleimides have recently been
reported.84−87
For example, Caddick and co-workers at University College of
London (UCL) reported the synthesis of homogeneous
trastuzumab ADCs using a three-step process. Interchain
disulfides were reduced with TCEP followed by addition of an
N-propargyl-3,4-dithiophenylmaleimide linker to form a dithio-
maleimide linkage with the antibody. Addition of an azido-
doxirubicin derivative resulted in formation of a triazole linkage
with the payload (Scheme 5C). The process was applied to
trastuzumab and afforded homogeneous ADCs with four drugs/
Scheme 6. (A) Synthesis of Homogeneous ADCs via Cysteine Cross-Linkinga
and (B) Hydrophobic Interaction Chromatography
(HIC) Analysis Comparing a DBM Cross-Linked ADC (Blue) with an Analogous Conventional ADC (Red)
a
Interchain disulfides are reduced with TCEP and then conjugated to payloads containing dibromomaleimide DBM linkers to afford homogeneous
ADCs with 4 drugs/antibody.
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11. antibody. The ADCs demonstrated comparable antigen binding
affinity compared to the parent antibody, but in vivo
pharmacological properties were not reported. Unlike conven-
tional methods for cysteine conjugation, covalent bonds between
antibody H and L chains are maintained which is expected to
improve the stability of the cross-linked ADC.88
Dibromopyridazinediones. Maruaini and co-workers later
published a similar cross-linking approach using a dibromopyr-
idazinedione linker that contained dual orthogonal alkyne
functional groups. The linker was designed to enable chemo-
selective conjugation with two different azide derivatives using
click chemistry (Scheme 5D). Doxirubicin and a fluorophore,
both modified with azide groups, were conjugated sequentially to
Herceptin resulting in an ADC with four payloads and four
fluorophores. The dual labeled ADC was stable in plasma and
selectively killed Her2 positive BT474 cells at μM concen-
trations. Although the pharmacological properties of the ADCs
in vivo were not reported, the results suggested that a similar
strategy could be used for conjugating two different payloads to
an antibody with a single linker.89
Dibromomaleimides. In order to determine the effect of
interchain cysteine cross-linking on the in vivo properties of
ADCs, Behrens and co-workers synthesized a derivative of
monomethyl auristatin F that contained a dibromomaleimide
(DBM) linker instead of a conventional maleimide.90
The DBM-
MMAF payload was designed to cross-link interchain cysteines
to form a dithiomaleimide linkage with the antibody (Scheme 6).
The DBM-MMAF payload was conjugated to trastuzumab and a
novel anti-CD98 antibody to afford homogeneous ADCs with
four drugs/antibody.91
The ADCs selectively bound to antigen
expressing cells in vitro with comparable affinity to the parent
antibodies and inhibited growth at sub nanomolar concen-
trations. The pharmacological properties of the cross-linked
ADCs in vivo were compared to analogous heterogeneous ADCs
synthesized using conventional maleimide (MC) linkers. The
results demonstrated that the DBM linkers yield homogeneous
ADCs directly from a variety of different antibodies without
recombinant engineering. Importantly, the DBM cross-linked
ADCs demonstrated improved pharmacokinetics, safety, and
efficacy over analogous conventional heterogeneous ADCs.
The protocol for DBM conjugation requires fewer steps than
most previously discussed methods, and consistently affords
ADCs with >90% DAR 4. The reduction and conjugation
processes can be performed sequentially in one pot at room
temperature in less than 3 h. Unlike other methods for generating
homogeneous ADCs, excess reagents are unnecessary and the
process is easily scalable to gram quantities. Buffer exchange or
membrane filtration of the crude conjugation mixture affords
highly pure ADCs. The DBM cross-linking approach was the first
study in which homogeneous ADCs were directly compared to
analogous heterogeneous ADCs containing identical linkers and
payloads conjugated to identical sites. Since other variables
known to affect ADC properties were effectively removed from
the study, the relative contributions of homogeneity and
interchain cysteine cross-linking could be accurately determined.
The results demonstrated that interchain cross-linking with
DBM does not adversely affect ADC activity in vivo relative to
conventional methods. In addition, the results provided
convincing evidence that homogeneous ADCs are superior to
their heterogeneous counterparts and validated previous efforts
to construct homogeneous ADCs with defined DARs.
■ DISCUSSION
All of the processes reviewed here were successfully used to
construct ADCs with improved homogeneity over ADCs
synthesized using conventional methods. A majority of
approaches utilize recombinant antibody engineering to
introduce unique functional groups for site-specific conjugation.
The unique functional groups were introduced either as point
mutations for cysteine and non-natural amino acids or as enzyme
recognition tags. These recombinant engineering approaches
offer several potential advantages over nonrecombinant
approaches. For example, engineered cysteines can be
incorporated into dozens of different sites with minimal impact
on the functional properties of the antibody. This enables ADCs
to be optimized for conjugation efficiency, linker stability, and
potency. Engineered non-natural amino acids offer additional
advantages due to the diverse array of different functional groups
that can be introduced. Furthermore, non-natural amino acids
enable a variety of new linker chemistries to be investigated that
are not possible with conventional conjugation processes.
The flexibility offered by recombinant processes may also
represent their greatest challenge. The importance of the
conjugation site for ADC activity is well-established, but
additional factors should be considered before selecting a
development candidate. Potential effects on antibody expression,
conjugation efficiency, linker stability, aggregation, and other
factors need to be considered before selecting a specific
conjugation site. These factors can ultimately determine the
success or failure of an ADC development program. Since
antibodies share many of the same properties, it seems likely that
optimal conjugation sites will be identified that are broadly
effective when used with different antibodies. Other potential
challenges for processes involving antibody engineering include
increased development time and costs, immunogenicity of
engineered sequence tags, scalability, and use of novel linkers and
payloads that are not yet clinically validated.
In addition to homogeneity, improvements in other ADC
properties such as potency, stability and half-life were observed.
In fact, many of the homogeneous ADCs derived from these
processes out-performed conventional heterogeneous ADCs in
efficacy and safety studies. Much of their success has been
attributed to elimination of high DAR species present in
conventional ADCs. In general, experimental results are
consistent with this conclusion, and many would agree that
substantial progress has resulted from these efforts to improve
ADC homogeneity. Ironically, the relative contribution of
homogeneity to the improved properties of the engineered
ADCs could not be determined from most studies because other
factors known to effect ADC activity could not be ruled out.
For instance, recombinant approaches for making homoge-
neous ADCs were designed to introduce conjugation sites in
different locations from those used in conventional methods.
Since it is now well-established that “location matters”, the
observed differences in activity between TDCs (or NDCs) and
the conventional ADC controls could result from different
conjugation sites, rather than from elimination of high DAR
species. Enzyme mediated approaches face similar challenges
when comparing homogeneous and heterogeneous ADCs
because the conjugation sites are different. Other variables
such as linker type (cleavable or noncleavable) and payload
(maytansine or PBD) need to be carefully controlled before
reaching conclusions about the benefits of homogeneity.
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862

12. Linker based processes are more suitable for comparing
homogeneous ADCs with conventional heterogeneous ADCs
because they utilize the same conjugation sites. Once other
variables that might impact ADC activity were carefully
controlled, the relative benefits of homogeneity were revealed
for the first time and the results confirmed that efforts to improve
ADC homogeneity have been a worthwhile endeavor.
Most of the processes reviewed here are still in early phases of
clinical development. All of the methods have advantages and
limitations that will ultimately decide which approach will
become the preferred process for manufacturing homogeneous
ADCs. It is not yet clear which process will rise above the others
as a preferred method, but all of these approaches have
contributed valuable information to our knowledge base and
resulted in ADCs with improved pharmacological properties
over conventional heterogeneous ADCs. Our future challenge
will be to apply this knowledge to develop ADCs that will be
more effective as therapeutic agents. Our ability to synthesize
homogeneous ADCs provides another reason to be optimistic
about the future of ADCs.
■ AUTHOR INFORMATION
Corresponding Author
*Igenica Biotherapeutics 863A Mitten Road, Suite 100B
Burlingame, CA 94010, USA. E-mail: dyjackson@comcast.net.
Cell: 650-339-3948.
Notes
The authors declare no competing financial interest.
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