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Antibody-Drug
Conjugates
Looking Ahead to an Emerging
Class of Biotherapeutic
Amy Ritter
The cell-killing ability of a cytotoxin is joined
with the specificity of a monoclonal antibody
to produce the next generation of anticancer
therapeutics. Creating a successful antibodydrug conjugate requires careful selection of the
drug, antibody, and linker.
I
n the quest for more targeted therapies and potentially more clinically
efficacious drugs, bio/pharmaceutical
companies are increasing their research
and product development in biologics.
Although the majority of this work is focused on monoclonal antibodies (mAbs)
and recombinant proteins, progress is
being made in specialized drug types.
Antibody–drug conjugates (ADCs), which
consist of a mAb chemically linked to a
small-molecule therapeutic, are a niche
class of drugs that offer promise, particularly as oncology drugs. In August 2011,
FDA approved Adcetris (brentuximab
vedotin), codeveloped by Seattle Genetics
and Millennium Pharmaceuticals (now
part of Takeda Pharmaceutical), making it only the second ADC approved by
FDA. With the approval of Adcetris, a
drug for treating Hodgkins lymphoma
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and systemic anaplastic large-cell lymphoma and with a number of ADCs in
clinical development, the key question is
whether ADCs will be able to fill a role in
biopharmaceutical development.
ADCs at work
Adcetris consists of three parts: the chimeric IgG1 antibody cAC10, specific for
human CD30, the microtubule-disrupting
agent monomethyl auristatin E (MMAE),
and a protease-cleavable linker that covalently attaches MMAE to cAC10 (1).
Before the approval of Adcetris this
year, the only other ADC approved by
FDA was Mylotarg (gemtuzumab ozogamicin), approved more than 10 years
ago in 2000. The drug, an anti-CD33
mAb conjugated to the cytotoxin calicheamicin, was developed by Wyeth
(now part of Pfizer) and was granted
Pharmaceutical Technology January 2012 P h a r mTe c h . c o m
accelerated approval in 2000 but was
voluntarily withdrawn by Pfizer in 2010
because a required Phase III trial failed
to demonstrate a survival advantage for
Mylotarg plus chemotherapy compared
with chemotherapy alone. Despite this
setback, there are several ADCs currently in development, with more than
15 in Phase I development and several
compounds from Roche and Pfizer in
late-stage clinical trials. In the decade
that has elapsed between the first ADC
approval and the second, advances in
the understanding of cancer biology,
lessons learned from the development
of mAbs as therapeutics, and better
methods for linking small molecules to
mAbs have coalesced to advance ADCs
into the forefront of new therapies.
The most active area of development
for this class of therapeutics has been
oncology, where a mAb serves to target
the therapy to cancer cells while a potent small-molecule chemotherapeutic
provides the cell-killing efficacy. Both
mAbs and small-molecule chemotherapeutics are used individually as cancer
therapies, but an ADC is designed to
overcome the limitations of each. MAbs
are highly specific, but as therapeutics
have demonstrated only modest efficacy and often are used in combination with a conventional chemotherapy.
Chemotherapeutics are highly toxic, but
nonspecific, and so suffer from poor
side-effect profiles and dose-limiting
toxicities. In combination, the ADC
serves to keep the chemotherapuetic
bound until it reaches the cancer cell,
thereby limiting its ability to interact
with nontargeted tissues and therefore
limiting nonspecific toxicity (2).
The concept of an ADC is not a new
one, but creating a clinically successful one has been challenging. For the
therapeutic to work well, each of the
parts—the antibody, the toxin, and the
linker that holds them together—must
be carefully considered.
Choosing the right antibody
In general, mAbs as therapeutics are
selected to have high affinity for the
targeted antigen and high selectivity.
Other desirable properties in an anti-
Compositing by Dan Ward. Images: Nick Koudis/Ingram Publishing/Getty Images
Cover Story: Antibody-drug conjugates
Cover Story: Antibody-drug conjugates
body include long circulation times,
immune-effector functions, and tumorsuppressing activity (2). When choosing the antigen, it is important that it
be expressed at high levels in the tissue
of interest to maximize the amount of
ADC bound by the tumor, but at low
levels elsewhere in the body to minimize off-target toxicity. Moreover, it is
thought that internalization of the ADC
is important for its effectiveness. Many
of the chemical-linking strategies used
to construct ADCs rely on conditions
found inside a cell, either in the cytoplasm or in the lysosome, to release the
active agent (3).
In some instances, developers have
been able to leverage experience gained
through the development of mAb therapies to create their ADC. Trastuzumab
emtansine (T-DM1) is an ADC in Phase
III, which combines trastuzumab, (Herceptin), which targets human epidermal
growth factor receptor 2 (HER2) receptors
in breast and stomach cancer, with a maytansine derivative DM1, a small-molecule
cytotoxin that binds to tubulin to prevent
microtubule formation, through a nonreducible bis-maleimido-trixyethylene glycol linker (4). Trastuzumab was developed
by Genentech (now part of Roche) and
was approved by FDA in 1998 for use in
women with metastatic breast cancer who
have tumors that overexpress the HER2
protein. The maytansine derivative DM1
and linking technology were developed
by ImmunoGen. In the case of the ADC
trastuzumab emtansine, developers were
able to use a target that had already been
validated and a well-characterized antibody with a known safety and efficacy
profile as the starting point for an ADC.
Choosing the right
cytotoxic small molecule
The earliest versions of ADCs used standalone chemotherapeutics such as doxorubicin, methotrexate, or vinca alkyloids
as the cytotoxic arm of the conjugate.
Clinical-trial results using these ADCs
were disappointing, and it is thought that
part of the problem was the relatively low
potency of the toxins used (2). The newer
classes of cytotoxins are at least 100-fold
more potent than the older molecules,
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with in vitro potency against tumor cell
lines of 10−9 to 10−11 M (5).
There are only a few major chemical
classes of toxins being explored. They
can be divided into two types, those that
cause damage to DNA and those that interfere with tubulin polymerization. Calicheamicin, used in Mylotarg and in Pfizer’s inotuzumab ozogamicin, an ADC in
Phase III trials, binds to the minor groove
of DNA and induces double-strand DNA
breaks that result in cell death. Duocarmycins, isolated originally from Streptomyces bacteria, are DNA minor-groove
binding alkylating agents (2). Fully synthetic duocarmycin derivatives are being
used by the biopharmaceutical company
Syntarga (acquired by the pharmaceutical
company Synthon in June 2011) for ADC
constructs (see sidebar).
Microtubule disruptors are represented by two major classes: maytansinoids and auristatins. Maytansinoids
are deriviatives of maytansine, a natural product originally isolated from the
shrub Maytenus serrata. ImmunoGen
has focused on development of this class
of cytotoxic small molecules and associated linker technologies and has been devloping maytansinoid ADC compounds
singularly and in partnership with other
companies. In addition to trastuzumab
emtansine, which is being codeveloped
by Roche and ImmunoGen, another example of a maytansinoid ADC being developed by ImmunoGen is the company’s
IMGN901, which uses the maytansinoid
DM4. Auristatins are synthetic analogs of
dolostatin 10, a natural product derived
from a marine mollusk, Dolabela auricularia. Like the maytansinoids, auristatins
are microtubule disruptors. Millennium
and Seattle Genetics’ ADC Adcetris is a
conjugate of an anti-CD30 mAb to monomethyl auristatin E (MMAE). Seattle
Genetics focuses on the development of
auristatin-conjugated ADCs, using the
auristatins MMAE and monomethyl auristatin F (MMAF) and proprietary linkers.
Choosing the right linker
Developing the right linker and method
of attachment is a crucial part ADC
development. “Many areas around the
process have improved, however, the
Pharmaceutical Technology January 2012 P h a r mTe c h . c o m
linker strategy for ADC manufacturing and their application has certainly
contributed perhaps the most in moving the field forward,” says Grant Boldt,
director of business development at the
CMO SAFC. The creation of linkers that
are stable in circulation but labile upon
binding of the ADC to its target has resulted in the current generation of ADCs
having better stability and lower systemic
toxicity than earlier ADCs, according to
Boldt. Early versions of ADCs, including
Mylotarg, suffered from instability while
in circulation. The linkage between the
mAb and the cytotoxic small molecule
were destroyed by endogenous proteases
in the blood, and the premature release of
the cytotoxin resulted in side-effect profiles similar to that of an unconjugated
chemotherapeutic. The current generation of linkers is more resistant to degradation in the blood while still allowing
release of the payload at the target. Choice
of a linker is influenced by which toxin is
used, as each toxin has different chemical
constraints (6).
Linkers can be divided into two broad
categories: cleavable and noncleavable.
Cleavable linkers rely on processes inside the cell to liberate the toxin, such
as reduction in the cytoplasm, exposure
to acidic conditions in the lysosome, or
cleavage by specific proteases within the
cell. Noncleavable linkages require catabolic degradation of the conjugate for
release of the cytotoxic small molecule.
The released cytotoxic small molecule
will retain the linker and the amino acid
by which it attached to the mAb. Importantly, both classes are designed to release
the cytotoxic small molecule only after
the ADC has reached the interior of the
cancer cell (2).
There are a limited number of
chemical moities on proteins, including mAbs, that are available for chemical modification. Linkers can attach to
the mAb through the amino groups of
lysine residues, or by the thiol groups
on cysteine residues. Attachment is a
pseudorandom process: in theory, any
of the targeted amino acids within the
mAb, either cysteine or lysine, can be
modified (3). According to Boldt, the
conjugation reaction results in a het-
Cover Story: antibody-drug conjugates
erogeneous mixture of conjugated species, but the proportion of each species
in the mixture is reproducible from
batch-to-batch and quantifiable.
Putting it all together
Producing the ADC requires both
biologic-based and small-molecule
manufacturing. “One of the biggest
challenges in manufacturing ADCs is
controlling all the components that go
into the final conjugation step,” says
Boldt. “Namely, the three main components that make up an ADC (e.g.,
antibody, linker, and payload) are all
manufactured in very different ways.
For example, it is not uncommon for
these components to be manufactured
by synthetic chemistry and mammalian cell culture. Thus, there presents a
challenge in ensuring all these components have been manufactured under
cGMP, and subsequently bringing
them all together to generate the final
ADC under cGMP, as well.”
The biologics portion of the ADC
and the high-potency API require very
different handling methods, and manufacturers must make sure that handling requirements for both are met.
“It is imperative that manufacturers
emphasize the protection of the product from workers as well as the protection of workers from the product,” says
Jason Brady, head of business development, conjugates and cytotoxics at the
CMO Lonza. Clinical ADC manufacturing is executed in an aseptic biological manufacturing environment to
protect the product from contamination, explains Brady. Once conjugated
with the high-potency API (which is
manufactured in a high-containment
environment), the resulting ADC also
is handled under high-containment
conditions. The level of containment is
determined by occupational exposure
limits for the high-potency API and
resulting ADC. The environment must
provide manufacturing personnel with
isolation from cytotoxic chemicals in the
occupational exposure range of 5 ng/m3
of air. Also important is that facility design includes design of equipment and
process contact surfaces that permit
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The importance of linker technology Although antibody-drug conjugates (ADCs)
offer promise for delivering a drug payload—
often a cytotoxic small molecule—with
greater specificity through its attachment to a
monoclonal antibody, one challenge is to create
the link between the antibody and the drug
molecule that remains stable until reaching
the target cell but that does not affect the
mechanism of action of the cytotoxic agent.
Meeting both needs has presented a stumbling
block for several ADCs in development. In these
cases, inappropriate drug choice or unstable
linking technologies have resulted in clinical-trial
failures (1).
One company active in ADC linker technology
is Synthon through its acquisition in June 2011
of Syntarga, a company specializing in antibody
payload chemistries. “We have a new family
of duocarmycin derivatives—our warhead
molecules—that we link to antibodies,” says
Vincent de Groot, former CEO of Syntarga and
vice-president of ADCs at Synthon. Duocarmycins
are small-molecule DNA minor groove
binding alkylating agents with potency in the
subnanomolar to picomolar range, according to
the company.
While this class of drug has been around for
some time, says de Groot, Phase II clinical trials
using the unconjugated compound proved to be
too potent to offer a therapeutic window with
adequate safety. However, given its mechanism
of action—interacting with DNA—duocarmycin
can kill tumor cells in all phases of the cell cycle,
not just in the mitosis phase. Therefore, it offers
great potential in treating solid tumors, where
cells are dividing slowly or not at all, providing
its cytotoxicity en route to the target cell can be
limited, according to de Groot.
There are two important requirements for ADC
linkers: stability in the blood and ADC lability
inside the target cell for release of the active
species (2). Synthon has three linker chemistries;
its SpaceLink technology reversibly links the
drug by means of a linear releasable linker,
and the MultiLink technology allows linkage of
multiple drugs to the antibody. The company’s
AbLoad technology offers a synthetic approach
to introduce the chemical group that reacts with
the antibody (i.e., Ab-reactive group) in the final
step of the linker-drug construct synthesis. The
key to Synthon’s SpaceLink technology is that
the linker molecule can reversibly bind to a drug
molecule’s hydroxyl group, which is particularly
Pharmaceutical Technology January 2012 P h a r mTe c h . c o m
by Rich Whitworth
complementary to the duocarmycin class of
drugs because the hydroxyl group is an essential
part of a precursor molecule that rearranges into
the active species spontaneously.
Synthon’s chief scientific officer Ian Anderson
provides the following example. “The antibody
directs the ADC to the cell of interest, for
example an anti-HER2 would go to HER2 on
the breast-cancer cell, where it would then
be internalized as the ADC. There is then a
lysosomal protease cleavage within the cell to
liberate the duocarmycin moiety. The drug, once
cleaved, spontaneously turns into the active
species.” Essentially, the linker–drug undergoes
spontaneous electronic cascade self-elimination
followed by spontaneous cyclization elimination
and finally spontaneous rearrangement into the
active species, at which point duocarmycin is free
to bind to and alkylate DNA and thus exerting its
cytotoxic effect.
The next part of the conjugation involves
attaching the linker–drug molecule to the
antibody, and there are two main methods,
according to de Groot. The first method is
to attach the linker–drug molecule to free
cysteine residues after antibody reduction,
and the second method is to attach the linker–
drug molecule to lysine residues. Lysines are
positioned throughout the antibody molecule,
so there is little control over where the linker–
drug positions itself, but if cysteine attachment
distribution can be narrowed, the number of
species variants produced can be controlled.
Both approaches are being pursued by the
industry according to de Groot.
Looking ahead, the next generation of ADCs
will likely use site-directed coupling of the
linker–drug to the antibody, which is an area of
active ADC research, says Anderson. Having more
control over the conjugation process, through
the use of nonnatural amino acids, for example,
will enable pharmaceutical companies to
develop better-characterized, more homogenous
ADCs, and the potential for increased efficacy
and safety.
References
1. S. Webb, “Pharma Interest Surges in Antibody Drug Conjugates,” Nat. Biotechnol. online,
DOI:10.1038/nbt0411-297, Apr. 8, 2011.
2. V. de Groot, “Novel ADC Linker–Drug Technology for Next Generation ADC Products,” presented at Peptalk–Protein Science Week (Cambridge Health Institute, San Diego, Jan. 10–14,
2011).
clean-in-place and steam-in-place to
remove minute traces of residual drug
contamination during both interbatch
and product changeover cleaning, according to Brady.
Room for improvement
As ADCs advance in the clinical
pipeline so does the technology to
manufacture ADCs to control certain
product and process conditions. “New
technology that can limit the heterogeneity of ADC products is something
that will be important in the future,”
says Brady. “ADCs made via current
technologies are heterogenous mixtures. Heterogeneity can be controlled
and measured by robust and reproducible manufacturing processes and
proper analytics, but new technologies will likely emerge to inf luence
and improve ADC manufacturing,” he
explains. Some fraction of the finished
drug product consists of unconjugated
antibody. The remaining portion of the
finished drug product contains conjugated antibody with a variable number
of the cytotoxic small molecules conjugated at different sites on the antibody.
Controlling the number and location of
cytotoxic molecules conjugated to the
antibody is being pursued as a means to
create a more uniform product and as a
way of being able to explore structure–
function relationships by varying the
site of attachment of the cyotoxin.
One strategy for controlling the site
of attachment has been developed by
researchers from Genentech, a member
of the Roche Group. They describe precise site-specific conjugation of human
IgG1 to MMAE by replacing Ala114 at
the junction of the CH1 and the variable heavy-chain domain with cysteine
to create an engineered antibody called
a THIOMAB. This site was chosen because it does not participate in antigen
binding or effector functions. According to Jagath Reddy Junutula, senior
scientist at Genentech, the process
for creating a THIOMAB differs only
slightly from that of a conventional
mAb. The THIOMAB is subjected to
partial reduction to remove cysteine
and glutathione adducts. The partial
reduction also breaks interchain disulfide bonds, which must be reformed by
a reoxidation step. After reoxidation,
the engineered cysteine residues are
available for conjugation.
Genentech researchers used this
process to conjugate MMAE to a THIOMAB version of an antibody against
MU16, a cell-surface protein expressed
in ovarian cancer cells. The THIOMAB
conjugate was shown to be homogenous
and to contain a single drug molecule
attached to each heavy chain, for a total
of two MMAE molecules per ADC. The
THIOMAB–MUC16 was found to have
comparable efficacy to a conventionally
produced ADC and to be better tolerated
in two preclinical species (7). In a subsequent study, a different cytotoxin, DM1,
was conjugated to a THIOMAB version
of trastuzumab. Results were similar,
with the THIOMAB T–DM1 displaying
comparable efficacy and better tolerability in preclinical species than its conventionally produced counterpart (8).
According to Junutula, the reoxidation
step is the only thing that distinguishes
manufacture of a THIOMAB drug conjugate from that of a conventional ADC.
“We can make up to grams scale without
any difficulty. And the results are huge—
you have a homogenously conjugated cytotoxic drug to the antibody,” he says.
While the THIOMAB uses the
substitution of one amino acid for
another to control the site of conjugation, several groups are working toward incorporating nonnatural amino
acids into the mAb for to control the
site of conjugation and also to provide
an expanded repertoire of functional
groups that could be used for linker
chemistry. The biopharmaceutical
company Ambrx has developed expression systems in E. coli, yeast, and
Chinese hamster ovary (CHO) cells
that can be used for such substitutions and which can be scaled up to
volumes required for commercial
manufacturing. Ambrx’s expression
systems contain engineered transfer
RNAs that will read through a stop
codon called amber, as well as engineered tRNA synthetases that will
aminoacylate the orthoganal tRNA
with an Ambrx nonnatural amino
acid. The expression system will insert
a nonnatural amino acid whenever the
amber stop codon is encountered (9).
Sutro Biopharma, a provider of
protein-synthesis technology, also is
developing a platform for introducing
nonnatural amino acids, but in a cellfree translation system that is reported
to be scalable to commercial production
volumes (10). The system is based on an
extract of E. coli, and because it is an
open system, the tRNA charged with
a nonnatural amino acid can be added
directly to the reaction mix as a reagent.
Looking ahead
The future of ADCs in the biopharmaceutical market will ultimately depend on their clinical success. Companies and researchers are seeking
to meet that challenge by optimizing
the selection of all the components in
the ADC—the antibody, linker, and
cytotoxin—and successfully combining manufacturing techniques for
both high- potency APIs and biologics. ADCs are sometimes described as
armed antibodies, and their cytotoxic
components as warheads. Whether
ADCs will prove to be an effective
weapon against cancer or other diseases has yet to be seen as more are
tested in the clinic.
References
1. FDA, “Label for Adcetris, BLA 125338,”
FDA Approved Drug Products: Drugs@
FDA, accessed Dec. 20, 2011.
2. V.S. Goldmacher and Y.V. Kovtun, Ther.
Deliv. 2 (3), 397–416 (2011).
3. F. Dosio, P. Brusa and L. Cattel, Toxins 3,
848–883 (2011).
4. H.A. Burris, Expert Opin. Biol. Ther. 11
(6), 807–819 (2011).
5. A. Beck et al., Discov. Med. 10 (53), 329–
359 (2010).
6. S.V. Govindan and DM Goldenberg,
Scientific World Journal 10, 2070–2089
(2010).
7. Junutula et al., Nat. Biotechnol. 26 (8),
925-932 (2008).
8. Junutula et al., Clin. Canc. Res. 16, 4769–
4778 (2010).
9. A. Ritter Pharm. Tech. 35 (6), 36–39
(2011).
10. Zawada et al., Biotech. Bioeng. 108 (7),
1570–1578 (2011). PT
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