Synthetic methods and characterization of all the compounds
are listed in the supplemental materials.
2.5 In vivo Efficacy
The MUC16-55 ADC was studied for efficacy using an in
vitro cell line for viability assays that could also be grown in vivo
(Ovcar3 endogeneously expresses the target antigen MUC16).
Shown in Figure 2A is the single dose of 5 mg/kg for two ADCs
and vehicle, MUC16-55 and isotype control-55, in Ovcar3 tumors.
The isotype control lacked efficacy along with vehicle (PBS w/5%
glycerol). The targeting MUC16-55 ADC suppressed tumor
growth out to 50 days (Figure 5A). For our comparison, we
conjugated the 3A5 mAb, used in the clinical ADC, with mc-VC-
PAB-MMAE
4.2 In Vitro Cytotoxicity
Ovcar3 (MUC16+) and HEK293 (MUC16-) cells were seeded
in 96 well plates at 3000 cells per well in complete growth media
and grown overnight. For cell viability curves, serially diluted
conjugates or payloads were added to the cells at final
concentrations ranging from 300 nM to 5 pM and incubated for 8
days. To measure viability, cells were incubated with CCK8
(Dojindo) for the final 1-3 hours and the absorbance at 450nm
(OD450) was determined on a Victor (Perkin Elmer). Background
OD450 levels determined from digitonin (40 nM) treated cells were
subtracted from all wells and viability is expressed as a percentage
of the untreated controls. IC50 values were determined from a
four-parameter logistic equation over a 10-point response curve
(GraphPad Prism) in quadruplicate. All conjugate curves and IC50
values are corrected for payload equivalents.
Figure 2. In vivo efficacy of MUC16 targeting ADCs in the Ovcar3 tumor
model. Graph A shows the MUC16-55 ADC and graph B shows the 3A5-mc-
VC-PAB-MMAE ADC dosed at 5 mg/kg (in each study with the exact dosing
for control) IV in SCID mice.
randomly to the interchain disulfides (similar to the DMUC5754A
ADC) and dosed in the same Ovcar3 in vivo model.13 The 3A5-
mc-VC-PAB-MMAE ADC, when dosed once at 5 mg/kg IV,
suppressed tumor growth for a similar 50 days, while the vehicle
was not effective (Figure 2B). There was efficacy with the isotype
control ADC initially, but the tumors recovered by day 50.
4.3 Cell Surface Expression
The 3A5 antibody literature measurements were averaged and
reported in Table 2.13
4.4 Conjugation and Characterization
The Ovcar3 in vivo model was used previously for the 3A5-
mc-VC-PAB-MMAE ADC and provides an excellent comparison
between the linker payload developed in this study (vid supra).
The previous preclinical report of 3A5-mc-VC-PAB-MMAE
when dosed at 3x2 mg/kg IV displayed similar tumor suppression
at 28 days by bioluminescence. It could be speculated that if
MUC16-55 ADC were dosed in a fractionated 3x2 mg/kg IV
manor, comparable tumor control would be realized.
Three antibodies were conjugated to various linker payload
compounds using the procedure below. The targeting antibodies
used in these experiments were: (1) an internally generated
antibody and (2) an anti-MUC16 antibody from the literature, 3A5.
All the monoclonal antibodies were expressed in CHO cells and
purified by Protein A. A non-binding isotype control antibody
derived from an immunological antigen having no relation to
oncology was also used.
The antibody (10 mg/ml) in 50 mM HEPES, 150 mM NaCl,
pH 7.5, was treated with 1 mM dithiothreitol at 37 °C for 30 min.
After gel filtration (G-25, pH 4.5 sodium acetate), the maleimido
linker payload derivatives 55, 56, and 57 (1.2 equivalents/SH
group of the cysteine residue) in DMSO (10 mg/ml) was added to
the reduced antibody and the mixture adjusted to pH 7.0 with 1 M
HEPES (pH 7.4). After 1 h the reaction was quenched with excess
N-ethyl maleimide. The conjugates were purified by size
exclusion chromatography using Dulbecco’s PBS with 5%
3. Conclusion
To continue our quest to further the research on the anti-cancer
natural product maytansine, we have developed several novel
payloads and linker-payload combinations. Our second goal of
developing an ADC with a cell-penetrating payload was realized.
These payloads were potent in the in vitro Ovcar3 cell line
endogenously expressing antigen with 3 payloads possessing 3-4
fold more potency than MMAE. The Muc16 targeting ADC
(MUC16-55) was able to suppress tumors in the Ovcar3 tumor
model with similar efficacy as the clinical positive control (3A5-
mc-VC-MMAE). It’s interesting to point out that the mc-VC-
PAB-MMAE linker payload is now used in 3 FDA approved
ADCs, Adcetris, Polivy™, and Padcev™. This work completes
our dual goal of producing ADCs with cell impermeable (vid
supra) and cell permeable maytansinoid payloads (“bystander”
capable payloads).
glycerol and sterile filtered.
Protein and linker payload
concentrations were determined by UV spectral analysis. Size-
exclusion HPLC established that all conjugates used were >95%
monomeric, and RP-HPLC established that there was <0.5%
unconjugated linker payload. All conjugated antibodies were
analyzed by UV for linker payload loading values according to
Hamblett15.
Although we have presented convincing data on an alternative
linker payload and antibody delivery vehicle for an ovarian cancer
therapeutic, Regeneron had a promising parallel discovery
program for a CD3 bispecific. The MUC16xCD3 bispecific
antibody (REGN4018) has transitioned into the clinic and we
eagerly await results.14
4.5 Enzyme cleavage of compound 55
The linker payload 55 was set at 100 μg/mL final in 25 mM
sodium acetate buffer, 1 mM EDTA, pH 5.0 and pre-incubated at
37˚C. Cathepsin B (Sigma # C8571) was activated at room
temperature for 15 minutes with 1 equivalent of 30 mM DTT, 15
mM EDTA to 2 equivalents of cathepsin B stock. The activated
cathepsin B solution was added to the substrate solutions at a 1:20
molar ratio (purified H2O, instead of activated cathepsin B was
added for the control sample.) Samples were incubated at 37˚C
4. Experimental Methods
4.1 Synthesis