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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5886_Библиотеки_им_академика_М_И_Перельмана.pdf
360
A)
D. Y. Jackson
NH
O
S S
S S
S S
S S
1) partialreduction
TCEP,pH7
S S
SH
HS
SH
SH
S S
2) payload conjugation
MC-VCP-MMAE
S S
SH
HS
HS
S S
Adcetris
O
S
X 4
H
N
N
O
O
O
O
O
N
H
HO
2
NH
O
H
N
N
H
O
O
O
O
O
N
N
N
N
H
O
S S
SH
HS
HS
S S
B)
Adcetris
anti-CD30-MC-VC-pAB-MMAE
+ CO2
+
MMAE
+ mAb
HN
Fig. 12.8 (a) Synthesis of Adcetris, Polivy, Padcev and Tivdak via partial reduction of disuldes
followed by conjugation with MMAE. (b) Cathepsin-mediated cleavage of the val-cit linker followed by elimination of a self-immolative p-aminobenzyl group to release unmodied MMAE
Fig. 12.9 Kaplan Meier Curve of progression-free survival in patients treated with Adcetris versus
standard of care, Methotrexate. (Source: Adcetris prescribing information)
properties of Adcetris are combined, it demonstrates a real benet to patients without the severe toxicity observed with Mylotarg. Adcetris now serves as a benchmark
ADC for many others in the clinic and three additional approved ADCs share the
same MMAE payload. Adcetris continues to be one of the most efcacious ADCs
approved thus far (Fig.12.9).
S-S
S-S
S-S
S
-S
S-S
S-
S
12 Development ofAntibody-Drug Conjugates
361
12.8.3 Kadcyla
Trastuzumab emtansine (aka Kadcyla) was the third ADC to reach the market and
was approved in 2013 [44]. Kadcyla was the rst ADC to be directed against solid
tumor types and it differs from earlier ADCs in a number of other ways. Like
Adcetris, Kadcyla carries a tubulin inhibitor payload (DM1), but the payload is
conjugated to lysines (instead of cysteines) to afford a highly heterogeneous ADC
with an average DAR of 3.7 drugs/antibody (Fig.12.10a) [45]. Kadcyla’s linker also
differs from most other ADCs in that it is noncleavable and does not release its payload until the entire ADC is degraded to peptide fragments [46]. Kadcyla’s target
antigen, human epidermal growth factor receptor 2 (HER2), is expressed in breast
tumors but is also found in normal breast tissue and numerous other solid tissues
and organs. Consequently, the broad expression prole of its target results in a rather
narrow therapeutic window for most patients [47].
Kadcyla’s developer Roche (formerly Genentech) had a clever solution to this
problem that eventually spawned a whole new approach to personalized medicine.
In order to increase the likelihood of clinical success, cancer patients were prescreened for HER2 expression and only patients with favorable expression proles
were admitted into the trial. Roughly a third of breast cancer patients expressed
Her2 at levels considered to be adequate for effective treatment, which left the
majority of patients looking for other treatment options. This prescreening approach
was rst used during the development of Kadcyla’s parent antibody component
known as Herceptin (approved for breast cancer in 1998) and has contributed signicantly to the commercial success of both products [48].
To avoid competition between two of their own products, Roche marketed
Kadcyla to extend the product lifetime of Herceptin and timed its release to coincide
with the expiration of Herceptin’s patent. Herceptin is now a generic drug known as
trastuzumab and Kadcyla has replaced it as one of Roche’s top selling drugs for
O
O
N
S
O
N
O
O
N
O
O
O
O
Cl
N
O
OH
O
N
H
O
O
S
N
O
O
OH
N
H
O
O
O
O
O
N Cl
O
-S-S-
-S-S-
NH
2
1) linker attachment
O
O
NHS
N
O
N
H
O
O
N
H
-S-S-
-S-S-
2) payloadconjugation
O
lysosome
O
DM1
N
O
N
DM1
Peptide
Fragments
-S-S-
-S-S-
O
H2N
OH
+
(activemetabolite)
O
O
N
H
O
N
H
O
Fig. 12.10 (a) Synthesis of Kadcyla via a two-step conjugation process. (b) Lysosomal degradation of Kadcyla releases the DM1 payload still connected to lysine
362
D. Y. Jackson
cancer. Nonetheless, trastuzumab remains one of the most successful antibody therapeutics to date. Based on the clinical results for both Herceptin and Kadcyla however, it is widely accepted that neither drug would have been approved without
prescreening patients for high HER2 expression. Ironically, the success of HER2 as
a cancer target can be attributed primarily to patient selection rather than target
selection. This “personalized medicine” is unpopular with some doctors but has
become a common practice in oncology and will likely continue to play a part in the
future success of many other ADC therapeutics [49].
Another signicant difference between Kadcyla and earlier ADCs involves the
linker. Kadcyla contains a relatively stable noncleavable (SMCC) linker that minimizes the release of the DM1 payload. Interestingly, Kadcyla’s SMCC linker was
originally intended to serve as a negative control for comparison with less stable
disulde linkers so researchers were surprised when Kadcyla demonstrated efcacy
in xenograft tumor models. Since Kadcyla’s linker does not actually release its payload, researchers identied a metabolite that contains the DM1 payload and the
SMCC linker still connected to the lysine to which it was originally conjugated
(Fig.12.10b) [50]. This led to the conclusion that activity was dependent upon the
lysosomal degradation of Kadcyla into peptide fragments. Whether this metabolite
is solely responsible for Kadcyla’s antitumor activity is unknown and other potential explanations such as the potential contribution of the intact ADC to Kadcyla’s
activity were not investigated [51].
The presence of lysine in the active metabolite increases its hydrophilicity relative to the unmodied payload and reduces its permeability. The reduced permeability diminishes the potency of the metabolite and enables rapid clearance after its
released from the cell. It also reduces potential bystander effects on surrounding
cancer cells that are thought to be benecial in some cases. Studies conducted after
Kadcyla’s approval have shown that Kadcyla’s linker is actually less stable than
originally designed [52]. Researchers found that the maleimide group linking DM1
to the MCC linker can undergo thiol exchange with serum albumin, which results in
premature release of the DM1 payload and contributes to systemic off-target toxicity. Since most ADCs currently in clinical development contain maleimide groups
in the linkers, they are likely to possess similar linker instability. The fact that most
ADC researchers continue to use maleimide linkers, suggests that they are not aware
of these stability issues, or they believe that maleimide instability is necessary for
optimal performance.
Overall Kadcyla’s efcacy was determined to be statistically signicant and
patients with breast cancer gained about 3months survival over the standard of care.
Whether or not Kadcyla offers improved efcacy over Herceptin (trastuzumab) is
debatable since the two have never been compared head-to-head in a clinical trial.
Based on Kaplan-Meyer plots for both drugs, Kadcyla may afford a slightly
improved response over trastuzumab (Fig.12.11).
12 Development ofAntibody-Drug Conjugates
Fig. 12.11 Kaplan Meyer plot showing survival of patients treated with Kadcyla or Herceptin
[53]. (Source: KADCYLA Prescribing Information. Genentech, Inc. 2022)
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12.8.4 Besponsa
Four years after the approval of Kadcyla, Inotuzumab ozogamicin (Besponsa)
gained FDA approval in 2017 and was the fourth ADC approved for cancer indications. Besponsa is developed by Pzer for the treatment of adults with relapsed or
refractory B-cell precursor acute lymphoblastic leukemia. The ADC is composed of
an anti-CD22 monoclonal antibody conjugated to the payload N-acetyl-gammacalicheamicin via a bi-functional 4-(4-acetylphenoxy) butanoic acid linker. The
linker and payload components of Besponsa are identical to those used in Mylotarg
and are conjugated via lysine residues on the antibody (Fig.12.7). Besponsa however demonstrates better homogeneity and stability compared to Mylotarg due to
improvements in the conjugation process that enabled higher DARs and reduced the
amount of unconjugated antibody. Nonclinical data suggest that the anticancer
activity of inotuzumab ozogamicin is due to the binding of the ADC to CD22expressing tumor cells, internalization of the ADC, and intracellular release of the
calicheamicin payload after hydrolytic cleavage of the linker in the lysosome.
Release and activation of calicheamicin induce double-strand DNA breaks and
eventually lead to cell cycle arrest and apoptosis [54].
.According to the prescribing information provided by the drug’s manufacturer, Pzer, Besponsa increases overall survival from 6.2 to 7.7 months
(Fig.12.12). However, the product insert states, “The analysis of overall sur-
vival (OS) did not meet a prespecied boundary for statistical signicance of
P=0.0104.” In Laymen’s terms, they are not sure if Besponsa actually works,
because the treatment group was too small to support their claims statistically.
364
Fig. 12.12 Kaplan-Meyer plot for survival of Besponsa treated patients. (Source: BESPONSA
Prescribing Information. NewYork, NY: Pzer Inc.)
D. Y. Jackson
Since a typical treatment course can cost tens of thousands of dollars or more,
patients are again faced with a difcult decision.
12.8.5 Polivy
Polatuzumab vedotin (aka Polivy) is an ADC developed by Roche and SEAGEN for
diffuse large B-cell lymphoma. It was the fth ADC approved in the US and it contains an identical linker and payload to Adcetris, Padcev, and Tivdak (see Fig.12.8)
[55]. The FDA granted accelerated approval for Polivy in 2019 based on a single
clinical trial. Because the results were based on only 40 patients, additional clinical
trials are required to conrm any clinical benet. Polivy’s target is CD79b, a component of the B-cell antigen receptor that is highly expressed in B-cell lymphoma.
The ADC contains a CD79b-specic antibody with an average of 3.5 MMAE molecules per antibody linked via a cathepsin cleavable valine-citrulline inker. Like
Adcetris, Polivy’s linker is designed to release the antimitotic tubulin inhibitor, MMAE.
The conjugation process for Polivy (and most other ADCs conjugated through
cysteine residues) utilizes a thiol-reactive maleimide linker. The reaction of the
maleimide linker with cysteine proceeds rapidly with high yields. Consequently,
maleimide linkers are used in a majority of ADCs currently in clinical development
[56]. Ironically, maleimide-based linkers are known to be inherently unstable,
which has likely contributed to the inadequate safety of many ADCs [57]. In fact,
most ADCs currently in development (including seven of the approved ADCs) contain unstable maleimide-derived linkers, which are prone to thiol exchange or elimination reactions in plasma [58]. Thiol exchange often results in transfer of the
12 Development ofAntibody-Drug Conjugates
Fig. 12.13 Overall survival of patients with DLBCL treated with Polivy vs chemotherapy
365
payloads to an unpaired cysteine in serum albumin, the most abundant protein in the
blood. Once transferred, the payload is no longer selective for tumor antigens resulting in systemic exposure and off-target toxicity. Although stable maleimide alternatives are available for cysteine conjugation, they haven’t been utilized thus far [59].
.Unlike most other ADCs, the recommended number of treatments for Polivy is
ve cycles and off-target toxicity is a major issue. The extreme toxicity of Polivy is
reected by a 7% mortality rate, but the overall survival probability was considered
sufcient to justify its approval in 2019 (Fig.12.13). It is currently unclear whether
Polivy will demonstrate meaningful efcacy in future clinical trials, yet annual
gross sales are expected to reach one billion by 2023 [35].
12.8.6 Padcev
Enfortumab vedotin (PADCEV) is a nectin-4-directed antibody and microtubule
inhibitor conjugate approved in 2019 for the treatment of adult patients with locally
advanced or metastatic urothelial cancer and bladder cancer [36]. Like Adcetris,
Polivy, and Tivdak, Padcev carries a tubulin inhibitor payload (MMAE) conjugated
to an antibody via a protease cleavable dipeptide linker (see Fig.12.8). Padcev was
developed by Astellas in a partnership with SeaGen [60].
Padcev’s target is nectin-4, a cell adhesion molecule found in a wide variety of
tissues. Nectin-4, also known as polio virus receptor-like 4 (PVRL4), is thought to
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D. Y. Jackson
contribute to tumor proliferation and metastasis. The broad expression of the
Nectin-4 on epithelial tissues is consistent with the toxicity prole, which includes
peripheral neuropathy, ocular disorders, and skin reactions. In fact, there were serious adverse reactions in 46% of patients treated with Padcev. The most common
reactions were urinary tract infection (6%), cellulitis (5%), febrile neutropenia
(4%), diarrhea (4%), sepsis (3%), acute kidney injury (3%), dyspnea (3%), and rash
(3%). Fatal adverse reactions occurred in 3.2% of patients, including acute respiratory failure, aspiration pneumonia, cardiac disorder, and sepsis (each 0.8%) [61].
Padcev was the sixth ADC approved by the FDA and the third ADC to use
MMAE as a payload. Consequently, two thirds of the approved ADCs carried tubulin inhibitor payloads. The success of these ADCs either reects the risk-averse
business practices of most pharmaceutical companies, or it suggests that tubulin
inhibitors may have unique properties that are optimal for use as ADC payloads.
The fact that almost 75% of the ADCs currently in the clinic also contain tubulin
inhibitor payloads supports the notion that tubulin is a superior target for ADC
payloads.
Padcev was granted accelerated approval in 2019 based on an overall response
rate of 44% and a median duration of 7.6months (Table12.4). This might seem
impressive to most patients but closer inspection reveals that the results are based on
the data from 55 patients. Like most ADC clinical trials, the lack of a placebo control group makes it very difcult to determine the true benet of treatment. A Kaplan
Meier plot indicates a median survival benet of almost four months compared to
chemotherapy, but after 20months both treatment groups had an equal number of
survivors (Fig.12.14) [36].
12.8.7 Enhertu
Trastuzumab Deruxtecan (aka Enhertu) is an ADC that was approved for metastatic
Her2-positive breast cancer in 2020. Enhertu targets the same antigen (Her2) as
Kadcyla and even uses the same antibody trastuzumab. Trastuzumab (aka Herceptin)
was approved in 1998 for Her2-positive metastatic breast cancer and later for gastric
cancer. It was one of the rst antibodies to be approved for solid tumors and is
Table 12.4 Efcacy results for Padcev from EV201 trial
PADCEV
Endpoint
Conrmed ORR (95% CI) 44% (35.1, 53.2)
Complete response rate (CR) 12%
Partial response rate (PR) 32%
Mediana duration of response, months (95% CI) 7.6 (6.3, NE)
Source: Prescribing information
NE not estimable
a
Based on patients (N=55) with a response by BICR
n=125
F
12 Development ofAntibody-Drug Conjugates
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Fig. 12.14 Kaplan Meier plot showing % overall survival of patients treated with Padcev. (Source:
N Engl J Med. 2021 March 25; 384(12): 1125–1135)
O
H
N
N
H
O
NH
O
O
O
HN
O
N
N
OH
S S
S S
S S
S S
1) full reduction
TCEP
SH
SH
SH
SH
HS
SH
2) conjugation
SH
SH
MC-GGFG-Dxd
S
N
O
O
O
x 8
O
H
N
N
O
H
O
Fig. 12.15 Synthesis of Enhertu via reduction of interchain disuldes followed by conjugation
with a tetrapeptide linker to yield a homogeneous ADC with 8 drugs/mAb
thought to inhibit the dimerization and signaling of Her2 (ERBB2) and its other
ERBB family receptors. When Roche (formerly Genentech) realized that the commercial lifetime of Herceptin could be prolonged by attaching a cytotoxic payload
to yield a more potent drug, they developed Kadcyla, an ADC that was discussed
previously (Fig.12.10). Daiichi-Sankyo timed its release of Enhertu perfectly to
coincide with the expiration of Herceptin’s patent in the US [62]. This gave them
freedom to operate with trastuzumab but since Kadcyla, the rst trastuzumab ADC,
is patent protected until 2028 Daiichi needed to prove the superiority of Enhertu
over Kadcyla. They also needed to nd an alternative payload that could outperform
Kadcyla’s tubulin inhibitor payload, DM1.
The synthesis of Enhertu is shown in Fig.12.15. One of the properties that distinguishes Enhertu from Kadcyla (and other ADCs) is the linker. Unlike Kadcyla,
Enhertu utilizes a protease-cleavable tetrapeptide linker that releases its payload in
the lysosome. The use of the GGFG linker enabled Daiichi to circumvent SEAGEN’s
patents on cathepsin-cleavable dipeptides. More importantly, Enhertu’s peptide
368
O
D. Y. Jackson
O
OH
O
S
O
O
N
O
O
H
N
N
H
O
O
H
N
N
H
N
H
O
N
N
lysozome
F
H
N
O
O
HO
NH
O
O
O
O
N
OH
N
+ CH3-NH
2
F
Fig. 12.16 Cleavage of Enhertu GGFG peptide linker to release the DXd payload
linker improves the overall hydrophilicity of the ADC and enables the conjugation
of eight payloads perantibody. Since all eight cysteines are conjugated to a payload,
Enhertu was the rst approved homogeneous ADC [63].
Enhertu was also the rst ADC to carry a topoisomerase 1 inhibitor as its payload. The payload (known as DXd) is a close derivative of a well-known topoisomerase 1 inhibitor, Exatecan, that had been investigated previously in clinical trials
for a variety of solid tumor indications. Daiichi discovered the DXd payload while
testing ADCs designed to release exatecan-like molecules. The challenge faced by
Daiichi’s chemists was how to release an unmodied payload from the GGFG tetrapeptide without leaving behind remnants of the linker. Attachment of the exatecan
payload directly to the C-terminus of GGFG afforded a highly stable amide linkage
but the payload was a poor substrate for lysosomal proteases [64]. To facilitate proteolytic cleavage of the GGFG linker, researchers added a 4-aminobutyric acid
spacer that resulted in high levels of aggregate when more than 3 drugs perantibody
were conjugated. Substitution of the middle methylene group in 4-aminobutyrate
with a single oxygen atom resulted in efcient cleavage of the tetrapeptide, followed by spontaneous loss of methylamine, and release of the highly potent topoisomerase 1 inhibitor payload, known as DXd (Fig. 12.16) [65]. Enhertu has
outperformed Kadcyla in breast cancer patients with low Her2 expression and has
shown promise in treating gastric cancers. It also has a superior safety prole to
most other ADCs [66].
12.8.8 Trodelvy
Sacituzumab govitecan or Trodelvy is an ADC developed for the treatment of metastatic triple-negative breast cancer. It was originally developed by Immunomedics
and is now marketed by Gilead who acquired them in 2020. Trodelvy has the structure shown in Fig.12.17 and targets Trop-2 (tumor-associated calcium signal transducer 2), a protein expressed in a variety of different tissues and on numerous solid
tumors. Sacituzumab is the humanized antibody component of Trodelvy that is specic for Trop-2. The payload, SN-38, is a derivative of camptothecin, a potent topoisomerase 1 inhibitor [67] and it is the active metabolite of Irinotecan, a
chemo-therapeutic drug that was approved in 1996 for the treatment of colon cancer
and small cell lung cancer [28].
HO
12 Development ofAntibody-Drug Conjugates
N
N
N
O
S
N
O
x 8
NH
O
O
NH
O
O
N
7
H
2
cleavage
O
N
O
H
site
H
N
O
O O
O
N
O
369
N
O
O
Fig. 12.17 Structure of Trodelvy showing cleavage of the carbonate ester and release of the SN-38
payload, a potent topoisomerase1 inhibitor (shown in blue)
Like Enhertu, Trodelvy contains eight payloads perantibody and is nearly homogeneous. The most distinguishing feature of Trodelvy compared to other approved
ADCs is its unusually complex linker. It is unclear why Trodelvy’s linker contains
so many different functional groups when the cleavage mechanism for releasing the
payload is the hydrolysis of the carbonate ester directly attached to the payload
(Fig.12.17). One can only speculate about the linker selection criteria that were
used, but a possible explanation for the complex linker design is that it provided
Immunomedics with the freedom to operate (FTO). Indeed, the chances of nding
a similar design in competitor patent claims would be very small. Linker stability
was probably not considered to be important since carbonate esters of this type are
susceptible to serum esterases that cause premature release of the SN38 payload.
This instability is reected in the pharmacological properties of Trodelvy, which has
a relatively short half-life in plasma of about 15hours. Some researchers believe
that Trodelvy’s activity arises primarily from a bystander effect rather than conventional internalization and lysosomal degradation [68, 69].
The efcacy of Trodelvy was determined in the ASCENT trial of 529 patients
with metastatic triple-negative breast cancer. Based on the results in which patients
treated with Trodelvy survived an average of 5months longer than those on standard chemotherapy, Trodelvy received accelerated approval in 2020 (Fig.12.18).
Later in 2021, Trodelvy was approved for metastatic urothelial cancer based on a
single-arm trial of 112 patients. In both trials, the median overall survival was nearly
double that of patients who received chemotherapy.
12.8.9 Blenrep
Belantamab mafodotin (aka BLENREP) is a recently approved ADC [70] and the
rst that uses MMAF (monomethyl-auristatin Phe) as a payload. MMAF is a closely
related analog to MMAE, the payload found in Adcetris, Padcev, Polivy, and Tivdak.
Both MMAF and MMAE are anti-mitotic tubulin polymerization inhibitors that
disrupt microtubule formation during mitosis. The two payloads share a common
tetrapeptide core structure and differ only at the C-terminus where MMAF contains
a phenylalanine moiety and MMAE contains a pseudoephedrine group (Fig.12.19a).