Download Mechanism of DNA Alkylation by Duocarmycin SA

Mechanism of DNA Alkylation by Duocarmycin SA
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Structures of Second-Generation Duocarmycin ADCs
Medarex (MDX-1203)
Synthon
(SYD-985)
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Phase I
Non-Hodgkin's lymphoma;
Renal cancer
Phase I
Breast cancer;
Gastric cancer;
Solid tumours
92
MDX-1203 (Medarex) Duocarmycin-Based ADC
Drug B
Peptide Linker
Intracellular Activation of MDX-1203
Protecting Group
Lysosomal protease
Drug A

The duocarmycin payload is released
from the antibody through protease
cleavage of the dipeptide linker, and is
then converted from its pro-drug form to
the cyclopropane via carboxylesterase
cleavage of the carbamate.

The carboxylesterase CES2 is claimed
to be present in higher concentrations in
cancer cells, suggesting that the active
duocarmycin molecule should not be
activated in normal cells.
Carboxylesterase
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Duocarmycin Dimers (A-A Cross-linkers)
Patent: WO2015095212

The duocarmycin dimers are
extremely cytotoxic, and studies
have suggested potencies in the
low picomolar to femtomolar
ranges in certain cell lines.

For example, Genentech have
shown that a CBI dimer containing
a pentamethylene linker has an
average
cytotoxicity of 6pM
across a number of cell lines (e.g.,
BJAB, HCC1937)

Pfizer also
programme
dimers.

One example in their patent has
low picomolar potencies (i.e., 9pM
in N87).
have an
developing
active
CBI
Patent: WO2015110935
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The Pyrrolobenzodiazepines (PBDs)
Structures of the naturally occurring anthramycin (1), the PBD C8-Conjugates
GWL-78 (2) and KMR-28-39 (3), and examples of C7/C7’-linked (4) and
C8/C8’-linked (e.g., DSB-120, 5a, n = 3 and SJG-136, 6a, n = 3) PBD Dimers.
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Evolution of the Pyrrolobenzodiazepine (PBD) Family
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Mechanism of Action of the Pyrrolobenzodiazepines (PBDs)
A. The mechanism of covalent binding of a PBD to DNA once it has located in a low energy position within
the minor groove;
B. Molecular model of the crystal structure of anthramycin (PDB ID 274D) covalently bound to G19
(magenta) of the sequence 5’-CC(G)AACGTTGG-3’, as an example of a PBD-DNA adduct. Due to the
perfect fit of anthramycin in the DNA minor groove (as a consequence of its 3-dimensional shape created
by the C11a(S) chiral centre), normal base-pairing is maintained (cyan) with negligible distortion of the
minor groove.
97
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Modes of Interaction of Pyrrolobenzodiazepine Dimers with DNA
Diagram of the various possible adduct types that can be form ed
between SJG-136 and DNA (i.e., interstrand and intrastrand crosslinks, and mono-adducts) (X = any base).
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Modes of DNA Cross-Linking by SJG-136
A & C, Schematic diagram and molecular models, respectively, of the interstrand cross-linked adduct
formed by interaction of SJG-136 (1) with Pu-GATC-Py. B and D, Schematic diagram and molecular
models, respectively, of the intrastrand adduct formed by interaction of SJG-136 (1) with Pu-GAATG-Py.
Note: In models C and D, SJG-136 and the DNA strands to which it is bound are colored purple, whereas
the non-covalently bonded DNA strands are shown in green.
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Examples of C8/C2'- and C2/C2’-Linked PBD Dimers

Examples of C8/C2'- and C2/C2’-linked PBD dimers synthesized by the
Thurston, Kamal and Lown groups.

Dimer C differs from the others in that it possesses only one electrophilic N10C11 imine moiety, and so cannot cross-link DNA.
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Structure-Activity Relationships (SARs) for the
Pyrrolobenzodiazepines (PBDs) and Indolinobenzodiazepines (IGNs)
Summary of the Structure Activity Relationships (SARs) for the PBD monomers (A), the PBD dimers (B),
and the indolinobenzodiazepine analogues (C). Modifications which enhance activity are shown in blue,
and those which reduce activity are in red.
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DNA Cross-Linking Ability of SJG-136
(a) Autoradiograph of a neutral agarose gel showing DNA interstrand cross-linking of linear 32P-end-labeled pBR322 DNA (0.2 nM) by SJG-136
following a 2 h incubation at 37 °C. The lanes are: C, double-stranded DNA control; 0, single-stranded DNA control; and 0.001, 0.003, 0.01,
0.03, 0.1, 0.3, 1.0, 3.0, 10.0 μM SJG-136. DS and SS are double- and single-stranded DNA, respectively;
(b) Quantification of the gel in (a) to showing the concentration dependence of DNA cross-linking for SJG-136 in linear
DNA.
32P-end-labeled
pBR322
"Reprinted with permission from Gregson, S. J., et al, (2001) Design, synthesis, and evaluation of a novel pyrrolobenzodiazepine DNA-interactive agent with highly efficient crosslinking ability and potent cytotoxicity, Journal of Medicinal Chemistry, 44, 737-748 (2001). Copyright © 2001 American Chemical Society."
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Footprinting and In Vitro Stop Assay Results for SJG-136
A, Footprinting gels showing the interaction of SJG-136 with the MS2 DNA sequence (10 nM) at concentrations of 0.1, 1, 3, 10, 30, and 100 μM (left panel
= top strand [MS2-F]; right panel = bottom strand [MS2-R]). The labels to the right of each gel correspond to the potential cross-linking sites;
B, T-Stop assay showing the effect of incubation time on the ability of SJG-136 (at 1.0 μM) to inhibit transcription. The right-hand side of the panel shows
that most stop sites are already visible after only 15 mins incubation of SJG-136 and the DNA duplex together prior to the addition of transcription buffer,
and that there is little significant change up to 60 mins incubation. The left-hand side of the panel shows that, once transcription had started (t = 0), there
was little effect upon adding SJG-136 at either 30 or 60 min time points (labeled as −30 and −60 min, respectively).
Martin, C., Ellis, T., McGurk, C.J., Jenkins, T.C., Hartley, J.A., Waring, M.J. and Thurston, D.E., “Sequence-Selective Interaction of the Minor-Groove
Interstrand Cross-Linking Agent SJG-136 with Naked and Cellular DNA: Footprinting and Enzyme Inhibition Studies”, Biochemistry, 44, 4135-4147 (2005).
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HPLC/MS Studies of the Interaction of SJG-136 With Oligonucleotides
A. Structures of the double-stranded oligonucleotides sequences used to study the interaction with of SJG-136 in initial using HPLC/MS studies
methodology;
B. Graph of % cross-linking versus time for duplexes Seq-1 to Seq-4 for a molar ratio of 4:1 (SJG-136/DS-DNA);
C. Same data as in Panel B but plotted against log time to provide rate data from the gradients (Units = % cross-linking, log h−1).
Note: For both Panels (B and C), 0 h corresponds to approximately 5 mins after initial mixing of the duplex DNA and SJG-136. All data points are
the means of triplicate measurements from independent experiments, with error bars showing ± standard errors.
Narayanaswamy M., Griffiths, W.J., Howard, P.W., Thurston D.E., “An assay combining high-performance liquid chromatography and mass spectrometry to measure DNA
interstrand cross-linking efficiency in oligonucleotides of varying sequences”, Analytical Biochemistry, 374, 173-181 (2008).
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Comparison of the reaction rates of SJG-136 with 12-mer duplex oligonucleotides
containing Pu-GATC-Py, Pu-GATG-Py, Pu-GAATC-Py and Pu-GAATG-Py sequences.
Reactions were monitored by HPLC in separate experiments with a 4:1 molar ratio of SJG136/DNA, and with adduct molecular weights and stoichiometries confirmed by MS.
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New Mechanism of Action: PBDs Bonding to Terminal Guanine Residues
Mantaj J, Jackson PJM, Karu K, Rahman KM, Thurston DE. Covalent Bonding of Pyrrolobenzodiazepines (PBDs) to Terminal Guanine Residues within Duplex and
Hairpin DNA Fragments. PLoS One. 2016;11(4):e0152303.
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Examples of Methods of Attachment of PBD Dimers
A. N10-attachment (e.g., Stemcentrx and Genentech):
B. N10-attachment with second imine deactivated (ADC Therapeutics):
C. C2-attachment (e.g., Seattle Genetics, ADC Therapeutics) with or without PEG:
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Examples of Linker Technologies
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PBD Dimers Connected to C-ring via a PEG Unit
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Intra-tumour Catabolites Used for Determining Efficacy of
Antibody Drug Conjugates
ZHANG, D., YU, S.-F., MA, Y., XU, K., DRAGOVICH, P. S., PILLOW, T. H., LIU, L., DEL ROSARIO, G., HE, J., PEI, Z., SADOWSKY, J. D., ERICKSON, H. K.,
HOP, C. E. C. A. & KHOJASTEH, S. C. 2016. Chemical Structure and Concentration of Intratumor Catabolites Determine Efficacy of Antibody Drug Conjugates.
Drug Metabolism and Disposition, 44, 1517-1523.
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Other New Analytical Method: DM1 Evaluation in Human Serum
 On-line solid phase extraction (SPE)—liquid chromatography–tandem
mass spectrometry (LC–MS/MS) used for the quantitation of maytansinoid
(DM1) in human serum
 DM1 contains a free thiol moiety, likely to readily dimerize or react with
other thiol-containing molecules in serum
 Samples pre-treated with a reducing agent [tris (2-carboxyethyl)
phosphine] (TCEP) and further blocked with N-ethylmaleimide (NEM).
 DM1-NEM exhibited sufficiently stability under all relevant analytical
conditions and no DM1 losses from the ADC were observed.
 The assay was used for DM1 determination in human serum
concentration after the intravenous administration of an investigational
antibody drug conjugate (ADC) containing DM1 as payload.
HEUDI, O., BARTEAU, S., PICARD, F. & KRETZ, O. 2016. Quantitative analysis of maytansinoid (DM1) in
human serum by on-line solid phase extraction coupled with liquid chromatography tandem mass spectrometry Method validation and its application to clinical samples. J Pharm Biomed Anal, 120, 322-32.
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PBD-Based ADCs at the Clinical Stage
Company
Product Name
Seattle Genetics
Indication
Target Payload Linkage
Phase
SGN-CD33A (vadastuximab talirine) AML
CD33
SG3211
C2 Val-Ala
I->III
Seattle Genetics
SGN-CD33A (vadastuximab talirine) AML (+SoC)
CD33
SG3211
C2 Val-Ala
I
Seattle Genetics
SGN-CD33A (vadastuximab talirine) AML (ASCT)
CD33
SG3211
C2 Val-Ala
I/II
Seattle Genetics
SGN-CD33A (vadastuximab talirine) AML (+HMA)
CD33
SG3211
C2 Val-Ala
III
Seattle Genetics
SGN-CD33A (vadastuximab talirine) MDS (+HMA)
CD33
SG3211
C2 Val-Ala
I->II
Seattle Genetics
Seattle Genetics
Seattle Genetics
SGN-CD70A
SGN-CD123A
SGN-CD19B
NHL/RCC
AML
NHL/RCC
CD70
CD123
CD19
SG3211
SG3211
SG3211
C2 Val-Ala
C2 Val-Ala
C2 Val-Ala
I
I
I
Stemcentrx (recently
acquired by AbbVie)
Rovalpituzumab teserine (Rova-T;
SC16LD6.5)
SCLC
DLL3
SG3249
N10 Val-Ala
I->III
Stemcentrx (recently
acquired by AbbVie)
Rovalpituzumab teserine (Rova-T;
SC16LD6.5)
CisR Ovarian
DLL3
SG3249
N10 Val-Ala
I
ADC Therapeutics
ADC Therapeutics
ADC Therapeutics
ADCT-301
ADCT-301
ADCT-402
CD25
CD25
CD19
SG3249
SG3249
SG3249
N10 Val-Ala
N10 Val-Ala
N10 Val-Ala
I
I
I
ADC Therapeutics
ADCT-402
Lymphoma
AML
CD19-positive
hematological tumors
CD19-positive
hematological tumors
CD19
SG3249
N10 Val-Ala
I
*Source: World ADC Berlin 2016 (ADC Therapeutics Presentation)
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Examples PBD-Based ADCs at Pre-Clinical Stage
•
At least 5 pre-clinical studies involving PBD-based ADCs are currently ongoing:
Company
Product Name
Indication
Target Payload Linkage
Phase
Seattle Genetics
SGN-CD352A
Multiple myeloma
CD352
SG3211
C2 Val-Ala
Pre-clinical
Genentech
Trastuzumab linked ADC
Breast cancer
HER2
Undisclosed Cleavable
Pre-clinical
Genentech
hu7C2-linked ADC
Breast cancer
HER2
Undisclosed Cleavable
Pre-clinical
Pierre Fabre
TBA
Axl-expressing cancers Axl
Cleavable
Pre-clinical
•
All PBD-based ADCs listed above contain maleimide Ab-attachment moieties.
*Source: World ADC Berlin 2016 (ADC Therapeutics Presentation)
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Interest in PBD-Based Payloads Enhanced Due
to AbbVie Acquiring StemCentrx in 2016
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BMS Works with AbbVie to Explore PBD-Based Payloads
 Bristol-Myers Squibb will team up with AbbVie to run an early-stage lung
cancer study combining BMS’ marketed checkpoint inhibitors with AbbVie’s
experimental antibody drug conjugate Rova-T (rovalpituuzumab tesirine).
 The Phase I/II study, scheduled to start later this year, will involve
combination therapy of BMS’ YervoyTM (ipilimumab) and OpdivoTM
(nivolumab) (which have licenses for melanoma and non-small lung cancer
among others) with Rova-T for relapsed extensive-stage small cell lung
cancer (SCLC).
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PBD-CBI (G-A Cross-Linking) Dimers as ADC Payloads
Adenine
Guanine
CBI-PBD heterodimer with dual mode of alkylation
and the University of Auckland
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PBD-CPI Dimers: G-A Cross Linking Mechanism
Example of a PBD-CPI
(Bizelesin) dimer:
Snapshot of an MD simulation of the
Hurley hybrid PBD-CPI dimer (blue)
with its PBD and CPI units covalently
bound to the G18 (magenta) and A8
(yellow) bases of the sequence 5’GCC(G)AATTAGC-3’, with excellent
accommodation in the minor groove
and little distortion of the central AT
base-pairing (cyan).
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Publications on PBD-CBI Dimers
The use of molecular dynamics simulations to evaluate
the DNA sequence-selectivity of G-A cross-linking PBDduocarmycin dimers
Jackson Paul, Rahman Khondaker Miraz and Thurston David E.
Bioorganic Medicinal Chemistry Letters (In Press)
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PBD-CBI Dimers as ADC Payloads: Poster at AACR 2016 (New Orleans)
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Indolinobenzodiazepines (IGNs)

Distinguished from PBD dimer ADC constructs by four unique structural features:
 Two additional D-rings attached to the C-rings of the PBD skeletons.
 Conversion of one N10-C11 imine to a non-electrophilic secondary amine to create a payload
that mono-alkylates rather than cross-links DNA.
 The central aniline ring within the C8/C8’-linker to which the antibody is attached, a concept
originally developed by Sanofi.
 Joining of the DGN462 payload to the antibody via a cleavable disulfide linker.
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Tetrahydroisoquinolinebenzodiazepine (THIQBD) Dimers
Patent: WO2016115191

Contains a 6-7-6-6 (A-B-C-D) scaffold with G-G cross-linking ability.

A variety of analogues have been produced, and when conjugated to an antibody, cytoxicities in the
low picomolar to femtomolar ranges have been obtained.

For example, when conjugated to the anti-fucosyl GM1 antibody, the ADC outlined below was
shown to have a cytotoxicity of 30pM against N87 cells.
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Pro-PBDs and Related Payload Technologies






pro-PBDs (latent DNA cross-linkers)
pro-DRH-417- containing SMDC
pro-IBD (indolinobenzodiazepines)
pro-PBD-Hoechst dye
pro-PBD-seco-CBI hybrids
Distamycin hybrids
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Endocyte’s Concept of Pro-PBD Conjugates
Slide provided by: Iontcho R. Vlahov, VP Discovery Chemistry, Endocyte, Inc.
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Calicheamicins (DNA-Cleaving Agent)
Structure of calicheamicin
Caliche clay found in central Texas (USA) of the type that
the calicheamicin-producing microorganism
(Micromonospora echinospora ssp calichensis) was first
isolated from.
 Calicheamycin is a natural
product isolated from the
bacterium
Micromonospora
echinospora sp. Calichensis.
 It was discovered in caliche
clay (a type of soil found in
Texas)
by
Wyeth-Ayerst
researchers (now Pfizer).
 It works by cleaving DNA
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Calicheamicins (DNA-Cleaving Agent)
Structure of Calicheamicin γ1
Molecular model of Calicheamicin γ1
interacting with DNA
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Mechanism of Biological Action of Dynamicin A
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Synthesis of ADCs Containing Calicheamicin
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Example: MylotargTM (Gentuzumab ozogamicin)
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129
MylotargTM Loses Licence in 2010
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Novel DNA-Interactive Agents
• 1,5-Diaryl-3-oxo-1,4-pentadienyl agents (University of Saskatchewan)
• Major-groove binding agents (Femtogenix Ltd)
• PNU-159682 (bioactive metabolite of nemorubicin; 3,000-fold more
active than doxorubicin)
• Rebeccamycin (inhibitor of topoisomerase, intercalates into DNA).
• Sandramycin (DNA topoisomerase I inhibitor)
• Sanguinarine (induces DNA damage)
• Sinefungin (blocks methylation of bases in DNA and RNA)
• Telomestatin (telomerase inhibitor)
Rebeccamycin
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Sandramycin
131
Examples of Mechanistic Studies
Gel-Based Studies
DNA Footprinting

N7 Guanine Major Groove: Hot aqueous piperidine → Cleavage.

N3 Adenine Minor Groove: Thermal cleavage of adducts.

Competition experiments with minor and major groove binding
agents.
A.
1000
1000
Seq-1, RT
26.0 mins
500
500
0
mAU

mAU

0
0
10
20
30
40
Minutes
1000
B.
2/Seq-1 adduct,
RT 27.0 mins
Seq-1, RT
26.0 mins
500
Free 2,
RT 38.5 mins
0

Determine whether a molecule intercalates DNA
0
0
10
20
40
2/Seq-1 adduct,
RT 27.0 mins
C.
mAU
HPLC-MS Assays and Biophysical Studies
30
Minutes
1000

500
Seq-1, RT
26.0 mins
500
Free 2,
RT 38.5 mins
0
1000
500
0
0
10
20
30
40
Minutes


Designed duplex and hairpin oligonucleotides

Inosine replacement studies

FRET-based DNA melting studies
High-field NMR and/or X-Ray Studies
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mAU
1000
132
132
mAU
Viscosity studies
mAU

6. Small-Molecule Payloads Currently at the Discovery or Development Stages



RNA Polymerase II and III Inhibitors
•
α-Amanitin, β-Amanitin, Epsilon-Amanitin, gamma
•
Amatoxin – Heidelberg Pharma

•
Tubulysins (e.,g. A)
•
Pironetin
•
Podophyllotoxin
Tubulin Polymerisation Inducer
•
Epothilone A and B
•
Ixabepilone
•


Protein toxins – PE18, SarcinDI, Diptheria toxin, Bouganin toxin


Cytokines, chemokines (Philogen)

Novel Tubulin Inhibitors
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•
Bafilomycin A (V-ATPase Inhibitor)
Rapamycin Kinase Inhibitors
AZD8055
Chaetosin (Specific inhibitor of lysine-specific methyltransferase SU)

Ansamitocin P-3 (Oskar)
Apoptolidin (F0F1-ATPase Inhibitor)
Methyltransferase Inhibitors

•
•

Spliceostatins
Cryptophycins (Genentech)
Major-groove binding agents (Femtogenix Ltd)
ATPase Inhibitors
•

•
1,5-Diaryl-3-oxo-1,4-pentadienyl agents
(University of Saskatchewan)
Tubulin Inhibitors
•
Novel DNA-Interacting Agents
Actin Cytoskeleton Polymerisation Inhibitors
•

Cucurbitacin E
HDAC Inhibitor
•
Tubastatin A

eIF4A Inhibitors (e.g., Flavaglines)

Oxysterol Binding Protein (OSBP) Interactors (e.g., OSW-1)

Leptomycin
133
Spliceostatin-Based RNA Splicing Inhibitors

Under development by Pfizer

Pre-mRNA splicing is catalyzed by the large ribonucleoprotein spliceosome.

Spliceosome assembly is a highly dynamic process in which the complex transitions through a number of
intermediates.

The potent antitumour compound Spliceostatin A (SSA) has been shown to inhibit splicing and to interact with an
essential component of the spliceosome, SF3b.

Potent anti-proliferative agents capable of targeting both actively dividing and quiescent cells.
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Thailanstatin A:
A Novel Spliceostatin Analog
 Antibody-thailanstatin conjugates have been
shown to disrupt RNA splicing in tumour cells.
 They have potent cytotoxicity in a variety of cell
lines including multi-drug resistant lines, and are
inactive in non-target expressing cells.
Spliceostatin
 The patent literature suggests conjugates
prepared at Pfizer have a superior safety profile
to the approved ADC T-DM1.
Thailanstatin A
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