Sartorius_May21_NeutAntiMakingTheir - 17

NEUTRALIZING ANTIBODIES MAKING THEIR MARK IN NEXT WAVE OF BIOLOGICS

for comparing the internalization of multiple

monoclonal antibody, Herceptin (Trastuzumab).

antibodies at a single target, or one antibody

We constructed a concentration-response curve

in various cell types. The assay precision and

by labeling Herceptin with the pH-sensitive

workflow is such that it would be possible to

reagent, then serially diluting (1:2) before adding

compare 100s of different antibodies at once, and

to BT-474 cells.

further throughput could be achieved through

In BT-474 Her2-positive breast carcinoma

miniaturization to a 384-well format.

cells, we saw definite time and concentration-

Showing a Simple Pharmacological and Kinetic

dependent internalization of Herceptin over 48

Quantification of Antibody Internalization Using

hours. From an area under the curve (AUC) time-

Herceptin

course analysis, we calculated the EC50 value for

In addition, we experimented to determine EC50

internalization at 323 ng/mL = 2.1 nM (Figure 6).

values for the internalization of a clinically used

Our calculated EC50 value is similar to the known

Figure 6: Quantitative pharmacological analysis of pH-sensitive dye-labeled Herceptin shows time and concentration-dependent
internalization and an EC50 value of 2.1 nM. Quantitative pharmacological analysis of pH-sensitive dye-labeled Herceptin. BT-474 Her2positive cells were treated with increasing concentrations of pH-sensitive dye-labeled Herceptin. The time course graph displays an
increasing normalized red area over time with increasing Herceptin concentrations (A). Area under the curve analysis of this response
displays a clear concentration dependent response with an EC50 of 323 ng/mL (B). All data shown as a mean of 3 wells ± SEM, time course
data shown as normalized red area.
B.

A.
80

80

80008000
ng/mLng/mL

3

3

2

2

40004000
ng/mLng/mL
20002000
ng/mLng/mL

40

20

20

250 ng/mL
250 ng/mL

AUC (0-48 h) x 103

500 ng/mL
500 ng/mL

AUC (0-48 h) x 103

40

1000 1000
ng/mLng/mL

60

Normalized red area (%)

Normalized red area (%)

60

1

1

0

0

EC50 323
EC50ng/mL
323 ng/mL
(21 nM)
(21 nM)

125 ng/mL
125 ng/mL

62.5 ng/mL
62.5 ng/mL
0

0

0

0

12

12

24

24

TimeTime
(h) (h)

36

36

48

48

-8

-8

-7

-7

-6

-6

-5

-5

Log [Herceptin]
Log [Herceptin]
(g/mL)
(g/mL)

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| 17


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