MODULAR ANTIBODY ENGINEERING: ANTIGEN BINDING IMMUNOGLOBULIN Fc CH3 DOMAINS AS BUILDING BLOCKS FOR BISPECIFIC ANTIBODIES (mAb) - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 561

CD16a FcRn

400 1800

1600

350

pH 6.0 pH 7.5

1400

300

1200

250

1000

RX

200

800

150

RX-TNF353-2

600

100

400

50

200

0

0

50 200

Time (s) Time (s)

FIGURE 39.4 Binding of mAb 2 RX-TNF353-2 and rituximab (RX) to human CD16a and FcRn.
Antibodies were captured with Biacore chips loaded with Protein A followed by addition of human
CD16a. Biacore chips loadedwith human FcRnwere used tomeasure its interactionwith the antibodies.

with antibodies in 100% autologous serum before killed B
cells were quantified as above. Again, mAb 2 RX-TNF353-2
displayed the same CDC potency compared to unmodified
rituximab (Figure 39.5B).

To assess if these data were specific for this particular
mAb 2 or could be extended to other model types of mAb 2 ,
the CH3 domain of Fcab TNF461-1 was used to replace the
CH3 domain of the monoclonal antibody trastuzumab (HC).
Trastuzumab is a monoclonal antibody recognizing the
receptor HER2/neu and is approved for the treatment of
HER2/neu overexpressing breast cancer patients [8]. Fcab
TNF461-1 was selected for TNF- a binding by yeast display
similarly as Fcab TNF353-2, but the composition of amino
acids forming the binding site for the cytokine is quite
different. The resulting mAb 2 HC-TNF461-1 and its parent
trastuzumab were expressed in HEK 293 cells and purified
to homogeneity as described earlier.
ADCC experiments were performed to assess the relative
potencies of mAb 2 HC-TNF461-1 versus its parental anti-
body. The human lung cancer cell line Calu-3, which
expresses HER2/neu on its surface, was used as target cells.
Antibody opsonized Calu-3 cells were incubated with pri-
mary NK cells as effector cells for 4 h before dead cells were
quantified. The data revealed that the mAb 2 was equipotent
to trastuzumab to elicit ADCC killing of Calu-3 cells.
Addition of excess of TNF- a did not alter the potency of
HC-TNF461-1 to kill Calu-3 cells (Figure 39.6) demonstrat-
ing that TNF- a occupation of the binding site in the CH3
domain did not negatively influence NK-cell recruitment
mediated by the CD16a binding site located in the CH2
domain. In the absence of NK cells, no dead Calu-3 cells
were observed indicating that the mechanism of killing was
due to ADCC (not shown). Collectively, these results dem-
onstrated that the immuno-effector functionalities of two
different mAb 2 proteins are unchanged compared to their
parental monoclonal antibodies.
To determine if binding of mAb 2 RX-TNF353-2 to TNF- a
was different compared to cytokine binding of the Fcab,
ELISA experiments were performed. For both proteins, com-
parable EC 50 values were calculated (EC 50 TNF353-2:

(A) Primary B cells, NK/B = 5:1

120

100

80

60 ADCC

40

20

RX
RX-TNF353-2
hu lgG1

0

Primary B cells, 100% serum

(B)

70

60

50

40

CDC

30

20

10

0

mAb ( μ g/mL)

FIGURE 39.5 Immuno-effector functions of mAb 2 RX-TNF353-
2 are unchanged compared to rituximab. (A) ADCC of opsonized
primary human B cells incubated with autologous NK cells at a 5:1
ratio for 4 h. Dead cells were stained with 7-amino-actinomycin D
(7-AAD) and the percentage of 7-AAD positive cells was measured
by flow cytometry. (B) Opsonized human B cells were incubated in
100% autologous serum for 4 h. Dead cells were quantified by
7-AAD staining as stated earlier.