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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5608_Библиотеки_им_академика_М_И_Перельмана.pdf
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origin of the parent antibody, and methods used for humanization also inuence the
performance of the ADC [15]. Even the cell culture media used for antibody production can affect the activity of an ADC.For example, different glycosylation patterns have been observed in antibodies produced in different media that resulted in
signicant changes in immunogenicity [16]. Large manufacturing batches worth
millions of dollars have been ruined because of subtle changes in the cell culture
media and stories of process scientists losing their jobs over such incidents are common. Most antigen targets are not patentable because they are not human inventions
so researchers have begun patenting specic epitopes on the targets in order to claim
ownership of specic binding sites. Antibodies that bind to different epitopes on a
target can have profoundly different activities and, in some cases, different epitopes
can provide freedom to operate (FTO) for targets being investigated by competitor
companies [17]. New epitopes are therefore often treated like new targets from a
legal and regulatory perspective.
Occasionally the antibody component used in an ADC possesses antitumor
activity without any payloads attached. Two of the currently approved ADCs,
Kadcyla and Enhertu, fall into this category and utilize a previously approved cancer drug, trastuzumab, as their antibody component. Trastuzumab, which binds to
growth-stimulating receptor (ERbB2 aka HER2) expressed on breast and other
solid tumors, blocks binding to its ligand and inhibits tumor growth. Most other
ADCs contain antibodies that lack inherent anti-tumor activity, and the antibody
only serves as a delivery vehicle for the cytotoxic payload. As a result, some
researchers believe that the antibody component of an ADC primarily serves as a
slow-release mechanism to help maintain steady-state concentrations and improve
the overall pharmacological prole of the payloads. This perspective is well supported since clinical data have shown that systemic concentration of the payloads
remains relatively constant in patients dosed with ADCs [18]. In contrast, there is
very little clinical evidence to support claims that ADCs can selectively deliver their
payloads to tumors while sparing normal tissues.
Many other factors need to be considered when selecting an appropriate antibody
to use for constructing an ADC therapeutic. For example, minor variations in antibody subtypes can signicantly impact the conjugation process, especially when it
involves the reduction of disulde bonds. Different IgG subtypes vary in the number
of interchain disuldes and afford different numbers of available cysteines upon
reduction. Usually, antibodies of the IgG1 subtype are preferred for cysteine conjugation; due to the relatively mild conditions required for disulde reduction necessary to yield an optimal number of eight cysteines for conjugation. Species
cross-reactivity is another important property to consider since the efcacy of the
ADC in tumor models is more predictive of the efcacy expected in humans if the
antibody also binds the analogous mouse antigen with equal afnity [19].
12 Development ofAntibody-Drug Conjugates
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12.5 Selection ofLinkers andPayloads
The remaining two components that signicantly impact the success of an ADC are
the linker and the drug or payload (Fig.12.3). Finding the right combination of
linker and payload has proven to be very difcult because the potency of an ADC
often does not correlate with the potency of the unconjugated payload [20]. In addition, the conjugation of a small molecule payload to an antibody, which is often
100× larger in size, limits the amount of payload that can be administered to patients.
Conventional small molecule drugs can often reach plasma concentrations of 20μM
or more, even when relatively small doses (10–100mg) are administered. In order
for an antibody to reach a plasma concentration of 20μM (1.5mg/mL), a dose of at
least 20 grams would be required. Treatments of this size would not be safe or costeffective so the payload must be extremely potent (IC50< 10nM) to enable more
practical doses of ADC in the 50–500mg range.
The payload potency alone is not sufcient to ensure ADC activity, however,
since there are numerous other factors that contribute to the activity of an ADC.These
include the payload’s mechanism of action (MoA), the location of its target within
the cell (cytoplasm or nucleus), the membrane permeability, and the linker used for
conjugation. The conjugation of a highly potent drug payload to an antibody can
result in an ADC with poor activity if the payload never reaches its target or falls off
of the ADC prematurely. Conversely, highly potent payloads with low membrane
permeability will have poor activity in their unconjugated state but can yield
extremely potent ADCs because the permeability of the payload is determined by
the antibody.
A classic example of the lack of correlation between the potency of an ADC with
its unconjugated payload is shown in Fig.12.4. Two structurally and mechanistically similar tubulin inhibitor payloads (MMAE and MMAF) differ in potency by
roughly 200-fold in their unconjugated state (avg. IC50=0.4 and 100nM, respectively), but when conjugated to trastuzumab, the relative potencies of the resulting
ADCs are reversed and the MMAF ADC is actually more potent than the analogous
MMAE ADC [21]. The potency of the conjugated MMAF increased by orders of
Fig. 12.4 (a) Cytotoxicity of unconjugated MMAE & MMAF payloads and trastuzumab ADCs
against HER2 positive SkBr3 cells. (b) Tubulin binding afnity of MMAE vs MMAF. (c) Tubulin
inhibition assay showing the superior potency of MMAF over MMAE
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magnitude relative to the unconjugated drug (Fig.12.4a), while the MMAE ADC
had roughly equal potency with the unconjugated payload. Both of the MMAE
ADCs are less potent than analogous MMAF ADCs regardless of the linkers used
for conjugation. ADC potency does correlate with the tubulin binding afnity of the
payloads however, and with their potency in tubulin polymerization assays where
MMAF is the clear winner (Fig.12.4b, c).
The dramatic increase in the potency of the MMAF ADCs relative to unconjugated MMAF (Fig.12.4a) is consistent with the fact that unconjugated MMAF has
extremely poor membrane permeability. Conjugation to trastuzumab gives both
payloads equal access to their tubulin target because the payloads are internalized
with the antibody at similar rates. MMAF is actually a more potent tubulin inhibitor
than MMAE (Fig. 12.4b), which is consistent with the superior potency of the
MMAF ADCs. Moreover, the poor permeability of MMAF causes it to accumulate
to lethal concentrations inside the cell, while MMAE is free to diffuse out into
nearby cells. The superior permeability of MMAE is believed to contribute to the
enhanced invivo activity of MMAE ADCs against tumors with low antigen expression. This property has been named “Bystander effect” and some researchers believe
that the increased permeability of MMAE makes it more effective against certain
tumors because it is able to diffuse into nearby cancer cells.
In contrast, MMAF remains in the intercellular space and is cleared more rapidly
than MMAE.Some ADC researchers believe the “bystander effect” is also responsible for the systemic off-target toxicity observed in patients treated with ADCs
[22]. This hypothesis is well founded since the free payloads are no longer selective
for antigen-expressing cells after they are released from the ADC.
12.6 ADC Technology
The benets of combining small molecule potency with the specicity of antibodies
are often accompanied by new challenges during the development process. ADCs
can be considered either as turbo-charged antibody therapeutics with improved
potency or as tumor targeting chemotherapeutics enhanced by antibodies. The dual
complexicity of ADCs raises numerous questions regarding CMC and regulatory
requirements such as whether to le an NDA or BLA [23]. Some researchers believe
that the antibody component of an ADC simply serves as a slow-release delivery
system for the payload and therefore ADCs should be classied as small molecule
drugs. Others believe that the payloads just enhance the activity of an existing antibody therapeutic; like Kadcyla & Enhertu do for trastuzumab. Regardless of which
side of the fence you stand on, ADCs are currently considered to be biologics by the
FDA and therefore follow a similar development path as antibody therapeutics [24].
In reality, the ADC development process is more rigorous than that for antibodies
(or small molecules) because additional safety considerations are necessary due to
the cytotoxic payloads that are released. Some researchers say that “developing an
12 Development ofAntibody-Drug Conjugates
353
ADC is similar to developing a new drug and a new biologic simultaneously and
therefore requires twice the amount of work.”
Another major factor that further complicates ADC development is heterogeneity. Eight of the 11 approved ADCs are heterogeneous mixtures of distinct molecules that differ in the number of payloads perantibody and their location [10].
There can be thousands of different molecular entities in a single ADC drug
(Fig.12.5). Kadcyla (aka T-DM1), an ADC approved for breast cancer in 2013, is a
prime example of a heterogeneous ADC [25]. Kadcyla is composed of a payload
conjugated to an antibody via lysine side chains to afford a complex mixture of different molecules that contain between 0 and 10 payloads perantibody and an average drugs/antibody ratio (DAR) of 3.7. Since the payloads are dispersed over
multiple conjugation sites, the ADC is composed of a cocktail of thousands of different molecules, each with its own unique properties. Like Kadcyla, most currently
approved ADCs are heterogeneous mixtures that range from dozens to thousands of
different molecules. ADC heterogeneity arises from conventional conjugation
methods that use lysine or cysteine reactive linkers that are not site-specic. Since a
typical antibody has up to 12 cysteines and more than 70 lysines available for conjugation and a typical ADC contains only three to four drugs perantibody, most
conventional ADCs differ in both DAR and conjugation sites (Fig.12.5).
Over the past decade, substantial effort has been focused on reducing ADC heterogeneity resulting in a plethora of new site-specic conjugation methods [26].
Signicant progress has been made toward producing homogeneous ADCs, but
only two of the currently approved ADCs (Enhertu & Polivy) can be considered
homogeneous [27, 28]. The homogeneity of Enhertu was not achieved through
recombinant antibody engineering or other site-specic methods, but through new
linker technology that enabled the conjugation of eight payloads perantibody without precipitation or aggregation of the ADC.
Fig. 12.5 Heterogeneity of ADCs with payloads conjugated to lysine or cysteine
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D. Y. Jackson
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Fig. 12.6 ADCs containing dibromomaleimide linkers maintain covalent bonds between the
heavy and light chains and yield homogeneous ADCs with 4 drugs/mAb
Other linker-based approaches for synthesizing homogeneous ADCs include the
use of dibromomaleimide linkers that crosslink interchain cysteines and yield
homogeneous ADCs with 4 drugs perantibody (Fig. 12.6) [29]. This innovative
approach does not require recombinant engineering and maintains covalent bonds
between the antibody heavy and light chains for improved stability invivo. Similar
linker-based approaches for generating homogeneous ADCs have been reported,
but have not yet progressed to clinical development [30].
12.7 ADC Clinical Development
Despite the difculties that many companies have faced during ADC development,
11 ADCs have now been approved for cancer (Table12.1), and at least 20 more are
in late-stage clinical trials (Table12.2). A vast majority of these ADCs carry tubulin
inhibitor payloads, which suggests that tubulin may be an ideal target for ADC payloads. Alternatively, the frequent use of tubulin inhibitor payloads might be a reection of the risk-averse nature of pharmaceutical companies that chose to follow a
similar path as their predecessors. Indeed, the popularity of MMAE as an ADC
payload probably arises from the fact that it was used in Adcetris, the third approved
ADC and arguably the most efcacious ADC to date.
When considering the unprecedented clinical activity involving ADCs, it is
important to remember that more ADCs fail in the clinic than those that succeed.
Dozens of ADC clinical trials have been terminated, discontinued, or withdrawn.
The large number of ADC failures illuminates the signicant challenges that the
ADC eld has experienced over the last 20years [31]. A partial list of ADC failures
is shown in Table12.3 which suggests that there is ample room for improving the
therapeutic properties of ADCs.
Eight of the 11 ADCs currently approved for cancer share one or more of the four
major ADC components discussed previously (see Fig.12.3). For example, Mylotarg
and Besponsa have identical linkers and payloads but target different antigens
(CD33 & CD22) [32, 33]. Kadcyla and Enhertu share the same target (HER2) and
the same antibody (trastuzumab) but have different linkers and payloads; while four
12 Development ofAntibody-Drug Conjugates
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Drug ADC Company Year Indications Target Payload MOA
Mylotarg Gemtuzumab ozogamicin Pzer/Wyeth 2000 AML CD33 Calicheamicin DNA alkylator
Adcetris Brentuximab vedotin SEAGEN 2011 Hodgkin’s lymphoma CD30 MMAE Tubulin inh.
Kadcyla Trastuzumab emtansine Genentech 2013 Breast cancer HER2 DMI Tubulin inh.
Besponsa Inotuzumab ozogamicin Pzer/Wyeth 2017 ALL CD22 Calicheamicin DNA alkylator
Table 12.1 ADCs currently approved for cancer indications
Enhertu Trastuzumab deruxtecan Daiichi/AZ 2019 Breast cancer HER2 Dxd Topo 1 inh.
Padcev Enfortumab vedotin AsteIIaslSEAGEN 2019 Urothelial cancer Nectin-4 MMAE Tubulin inh.
Polivy Polatuzumab vedotin Genentech/Roche 2019 Large Bcl CD79b MMAE Tubulin inh.
Trodelvy Sacituzumab govitecan Immunomedlcs 2020 TNBC TROP-2 SN38 Topo 1 inh.
Blenrep Belantamab mafodotin GSK 2020 Multiple myeloma BCMA MMAF Tubulin inh.
Tivdak Tisotumab vedotin SEAGEN 2021 Cervical cancer TF MMAE Tubulin inh.
Zynlonta Loncastuximab tesirine ADC Therapeutics 2021 B-cell lymphoma CD19 PBD DNA crosslinker
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ADC name Sponsor Indications Status Target Linker (type) Payload MOA
Depatuxizumab Mafodotin Abbvle Glioblastoma Phase Ill EGFR MC MMAF Tubulin inh.
Mirvetuximab Soravtansine ImmunoGen Ovarian Phase Ill FolR alpha SPDP (disulde) DM4 Tubulin inh.
Table 12.2 ADCs in late stage development in 2021
Vadastuximab Talirine Seattle Genetics AML Phase Ill CD33A VCP (cleavable) PBD DNA alkylator
Trastuzumab duocarmazine Synthon MBC (breast) Phase Ill Her2 VCP (cleavable) Duocarmycin DNA alkylator
Rovalpituzumab tesirine AbbVie SCLC Phase Ill DLL3 VCP (cleavable) PBD DNA crosslinker
Telisotuzumab vedotin Abbvie Solid tumors Phase Ill c-Met VCP (cleavable) MMAE Tubulin inh.
Camidanlumab tesirine ADC Therapeutics AML Phase Il CD25 VCP (cleavable) PBD DNA crosslinker
Loncastuximab tesirine ADC Therapeutics DLBCL Phase Il CD19 VCP (cleavable) PBD DNA crosslinker
Indatuximab ravtansine Biotest MM Phase Il CD138 SPDP (disulde) DM4 Tubulin inh.
BMs-986148 BMS Solid tumors Phase Il Mesothelin VCP (cleavable) Tubulysin Tubulin Inh.
Glembatumumab vedotin Celldex Melanoma Phase Il gpNMB VCP (cleavable) MMAE Tubulin inh.
Patritumab Deruxtecan Daiichi Sankyo Breast cancer Phase Il Her3 GGFG (cleavable) DXd Topo I inh.
Pinatuzumab vedotn Genentech/Roche DLBCL, NHL Phase Il CD22 VCP (cleavable) MMAE Tubulin inh.
Enapotamab vedotin GenMab Solid tumors Phase Il AxI VCP (cleavable) MMAE Tubulin inh.
Tetosumab vedotin GenMab Ovarian/solid Phase Il TF VCP (cleavable) MMAE Tubulin inh.
Labetuzumab govitecan Immunomedics CRC Phase Il CEACAM5 PEG (cleavable) Camptothecin Topo 1 inh.
OBI-999 OBI Pharma Solid tumors Phase Il Globo H MCVC (thiobridge) MMAE Tubulin inh.
PSMA-ADC Progenics Prostate Phase Il PSMA VCP (cleavable) MMAE Tubulin inh.
Disitamab vedotin Remegen/Mabplex Solid tumors Phase Il Her2 VCP (cleavable) MMAE Tubulin
STRO-002 Sutro Ovarian Phase Il FoI-Ra p-AMF/D8CO Hemiasterlin Tubulin inh.
CX-2029 Cytomx/Abbvie Solid tumor Phase Il CD71 VCP (cleavable) MMAE Tubulin
Source: Clinical trials.gov
12 Development ofAntibody-Drug Conjugates
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ADC Name Sponsor Indications Status Target Linker Payload MOA
Coltuximab ravtansine Immunogen ALL, NHL Phase Il CD19 SPDP DM4 Tubulin inh.
MLN2704 Takeda Prostate Phase Il PSMA SPP DM1 Tubulin inh.
MDX-1203, BMS936561 BMS RCC Phase I CD70 MC-VC Duocarmycin DNA binder
Cantuzumab ravtansine Immunogen Gastric Phase Il CANAG/MUC1 SPDP DM4 Tubulin inh.
Anetumab ravtansine Bayer/NCI Solid tumors Phase Il Mesothelin SPDP (cleavable) DM4 Tubulin inh.
Denintuzumab mafodotin SEAGEN DLBCL Phase Il CD19A MC (stable) MMAF Tubulin inh.
Glembatumumab vedotin Celldex BC, melanoma Phase Il gpNMB VCP (cleavable) MMAE Tubulin inh.
Pinatuzumab vedotin Genentech DLBCL, NHL Phase Il CD22 VCP (cleavable) MMAE Tubulin inh.
Rovalpituzumab tesirine Abbvie Small cell lung Phase Il DLL-3 VCP (cleavable) PBD DNA crosslinker
Vadastuximab talirine SEAGEN AML Phase Il CD33A VCP (cleavable) PBD DNA alkylator
ABBV-838 Abbvie MM Phase I CD319 VCP (cleavable) MMAE Tubulin inh.
AGS67E Astellas AML Phase I CD37 VCP (cleavable) MMAE Tubulin inh.
BAY 1129980 Bayer Solid tumors Phase I LYPD3 VC (cleavable) MMAE Tubulin inh._
Table 12.3 Examples of ADCs discontinued or terminated due to inadequate safety
BAY 1187982 Bayer Gastric Phase I FGFR2 NHS-caproyl MMAW Tubulin inh.
CDX-014 Celldex RCC Phase I TIM-1 VCP (cleavable) MMAE Tubulin inh._
HKT288 Novartis Ovarian Phase I Cadherin-6 SPDP (disulde) DM4 Tubulin inh.
IMGN289 ImmunoGen NSCLC Phase I EGFR MCC (stable) DMI Tubulin inh._
LOP628 Novartis Solid tumors Phase I CD117 MCC (stable) DMI Tubulin inh._
SGN-CD123A SEAGEN AML Phase I CD123A VCP (cleavable) PBD DNA alkylator
SGN-CD19B SEAGEN ALL, NHL Phase I CD19B VCP (cleavable) MMAE Tubulin inh.
SGN-CD48A SEAGEN Mult. Myeloma Phase I CD48A glucuronidase MMAE Tubulin inh._
SGN-CD70A (SGN-75) SEAGEN NHL, RCC Phase I CD70 MC (stable) MMAF Tubulin inh._
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Mylotarg or Besponsa
D. Y. Jackson
ADCs: Adcetris, Polivy, Padcev, and Tivdak share identical linkers and payloads but
have different antibodies directed against different targets [34–36].
Only one ADC property is shared by all 11 approved ADCs; they are all designed
to release toxic payloads in tumors. The following section will discuss the pros and
cons of the currently approved ADCs, the synthetic processes that were used to
make them, and the technology that contributed to their approval. The remaining
sections of this chapter will focus on the evolution of ADCs starting with a review
of those currently approved. New ADCs currently in late-stage development and
predictions for future ADC therapeutics will also be discussed. The objective of this
chapter is to provide information about the risks and benets that are associated
with ADCs for the treatment of cancer in addition to summarizing their development history.
12.8 ADCs Approved forCancer
12.8.1 Mylotarg
Gemtuzumab ozogamicin (Mylotarg) was the rst immunoconjugate to receive
FDA approval to treat patients with acute myeloid leukemia (AML) [32]. It was
originally developed by Wyeth in the 1990s as an immunoconjugate, before the
term “ADC” was coined, and was later approved in the year 2000. Mylotarg is an
ADC in which a humanized anti-CD33 mAb is covalently linked to N-acetyl gamma
calicheamicin through an acid-labile linker (Fig.12.7). The presence of the hydrazone function in the linker allows the rapid release of calicheamicin from its conjugated state under the acidic conditions found in a lysosome [8].
The target of gemtuzumab ozogamicin, CD33, is a 67Kd adhesion molecule
expressed on both normal and malignant cells that bind 2,3-linked sialic acid residues. Upon binding to its CD33 target, Mylotarg is rapidly internalized, and the
payload is released in the lysosome. Once released the calicheamicin payload
induces double-strand DNA breaks, subsequently inducing cell cycle arrest and
apoptotic cell death. The safety of Mylotarg was questioned very early in development due to 8 fatalities out of 131 patients in the dosing arm of the pivotal clinical
trial. Although there is a 6% chance that a patient will not survive the treatment,
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Fig. 12.7 Synthesis of gemtuzumab ozogamicin (Mylotarg) and inotuzumab ozogamicin
(Besponsa), which contain identical calicheamicin payloads
12 Development ofAntibody-Drug Conjugates
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Mylotarg was granted accelerated approval from the FDA based on phase 1 data.
Considering the high fatality rate and cost of treatment, combined with marginal
efcacy (there was no statistically signicant difference between the treatment arm
and the control group in overall survival), it is not surprising that Mylotarg was
removed from the market in 2010.
Numerous other problems contributed to the temporary demise of Mylotarg. For
instance, the original ADC contained an average of only two to three payloads
perantibody conjugated stochastically to over 70 lysines on the antibody surface.
The low drug/antibody ratio (DAR) resulted in a highly heterogeneous mixture of
ADC molecules with up to 30% unconjugated antibody [37]. The presence of an
unconjugated antibody is problematic because it can function as a competitive
inhibitor of the ADC by occupying target binding sites. These factors together with
the inherent linker instability of Mylotarg likely contributed to its discontinuation.
Mylotarg was eventually reapproved in 2017 after several more clinical trials demonstrated that the benets outweigh the risk [38]. Most ADCs that have followed the
approval of Mylotarg have shown modest, though somewhat gradual improvement,
and the benets they offer to cancer patients are becoming more apparent.
12.8.2 Adcetris
Nearly a decade after the initial approval of Mylotarg, a second ADC, brentuximab
vedotin (aka Adcetris), was approved for Hodgkin’s lymphoma in 2011 [39].
Adcetris is composed of an anti-CD30 (TNFRSF8) antibody conjugated to a potent
anti-mitotic agent, monomethyl auristatin E (MMAE) via a protease cleavable
dipeptide linker [40]. Adcetris differs from Mylotarg in many ways that have led to
its continued commercial success. Perhaps the most important difference is that
Adcetris contains an average of four drugs/antibody conjugated to interchain cysteines after partial reduction of the antibody (Fig.12.8a) [41]. Since there are only
eight interchain cysteines available for conjugation compared to 70 or more lysines
used for conjugation on Mylotarg, the heterogeneity of Adcetris is signicantly
reduced and the fraction of unconjugated antibody is less than 5%.
The use of a tubulin inhibitor payload in Adcetris may provide other less tangible
benets such as bystander killing of nearby tumor cells [42]. Another component
that was improved in Adcetris is the linker, which is signicantly more stable than
Mylotarg’s linker. As a result, Adcetris is less prone to premature release of the
payloads. In addition, the linker used in Adcetris contains a p-aminobenzyl functional group that self-destructs upon cleavage of the aryl amide bond to release the
unmodied payload (Fig. 12.8b) [43]. In contrast to Adcetris, the payload on
Mylotarg is released by acid hydrolysis of the hydrazone bond, and later in the
nucleus the payload is activated by attack of the glutathione on the disulde.
Lastly, the expression prole for the Adcetris target, CD30, is more restricted to
specic cell types while CD33 is more broadly expressed. When the improved