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Abstracts of Oak Ridge Posters |
1
Department of Biotechnological Science, Kinki University, 930 Nishi-mitsuya, Uchita, Naga-gun, Wakayama 649-6493, Japan
2
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
a author for correspondence: fax 81-736-77-4754, e-mail morimoto{at}bio.waka.kindai.ac.jp
Bispecific monoclonal antibodies (BsMAbs) have two different binding regions that simultaneously recognize two different antigens (1)(2). BsMAbs have been studied for use in immunodiagnosis (3)(4) and immunotherapy (5)(6).
Most BsMAbs are derived from the IgG class, whereas most hybridomas raised against carbohydrate antigens, including the tumor-associated antigens, are IgM MAb producers. IgM antibody-enzyme conjugates are of limited use in enzyme immunoassays because of steric effects, reduction of their activities, and high nonspecific binding. For these reasons, BsMAbs prepared from IgM MAbs have been especially used for cancer immunodiagnosis. Their production by hybrid-hybridoma fusion is one method (7), but it is not an effective one. F(ab')2µ fragments have been successfully prepared from their IgMs, and their performance has been evaluated (8)(9)(10)(11). In this report, homogeneous bispecific F(ab')2µ fragments (BsFµ fragments) were efficiently prepared from IgMs.
Mouse IgM monoclonal antibodies 9CA10 and APM05 recognize
carcinoembryonic antigen (CEA) and calf intestine alkaline phosphatase
(AP), respectively. Their F(ab')2µ fragments
were prepared by pepsin digestion and purified by hydrophobic
interaction HPLC (8)(10)(11). The
purities of the fragments from the IgMs were >96% by size exclusion
HPLC using a TSKgel G3000SWXL column (7.8 mm
i.d x 30 cm; Tosoh), and the yields were 54% (APM05) and
53% (9CA10), respectively. The F(ab')2µ of
9CA10 was reduced to Fab'µ-SH with 50 mmol/L
2-mercaptoethylamine (Sigma) in phosphate-buffered saline (PBS)
containing 10 mmol/L EDTA at 37 °C for 1 h, and was separated
from 2-mercaptoethylamine by size exclusion HPLC on a TSKgel
G3000SWXL column. Immediately after HPLC, the
Fab'µ-SH was converted to a thionitrobenzonic
acid (TNB) derivative with dithionitrobenzene, and
Fab'µ-TNB was applied to a TSKgel
G3000SWXL column to remove any excess TNB and
dithionitrobenzene. Fab'µ-TNB (9CA10) was
reacted with Fab'-SH (APM05) to form S
S bonds between
Fab'µ (9CA10) and Fab'µ
(APM05) at a molar ratio of 2.5:1, followed by incubation at 4 °C
for 16 h under N2 gas. Ammonium sulfate
(70% saturation) was added to the reaction mixture, and the mixture
was centrifuged at 10 000g for 20 min. The precipitate was
dissolved with 50 mmol/L phosphate buffer (pH 7.4) containing 2.0 mol/L
ammonium sulfate. The solution was applied to a TSKgel Ether-5PW column
(7.5 mm i.d. x 7.5 cm; Tosoh) equilibrated with the same buffer and
eluted with a linear gradient of ammonium sulfate from 2.0 to 0
mol/L at a flow rate of 0.5 mL/min. Three peaks were eluted at 32 min
(peak 1), 40 min (peak 2), and 46 min (peak 3). The proteins of peaks 1
and 3 were Fab'µ-TNB and
Fab'µ-SH, respectively. The protein eluted in
peak 2 showed immunoreactivities against both antigens (data not shown)
and was identified as BsFµ. The elution time of
peak 2 suggests that the hydrophobicity of the
BsFµ fragment is the average of those of the
parental F(ab')2µ fragments. The
BsFµ fragments were purified to
homogeneity in a one-step procedure using hydrophobic interaction HPLC
(Fig. 1A
), and the final yields of the
BsFµ fragments were 35–40% of the theoretical
values.
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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed in a 6% slab gel under nonreducing conditions (12), and the proteins were stained with Coomassie Brilliant Blue R-250. The BsFµ fragment and its parental F(ab')2µ fragments showed a similar migration pattern (144–146 kDa) during SDS-PAGE, suggesting that the BsFµ is composed of four chains [two pairs of a truncated heavy chain and an intact light chain derived from the parental F(ab')2µ].
The dissociation constants (KD) of the BsFµ and its parental F(ab')2µ fragments with their specific antigens were measured by surface plasmon resonance using a BIAcore biosensor (Pharmacia Biosensor). The BsFµ and its parental F(ab')2µ fragments were injected separately and immobilized on the chip coated with goat anti-mouse (IgG + IgM) antibodies (Jackson ImmunoResearch Laboratories). When CEA (50 mg/L; Scripps Laboratories) was injected on the chips coated with BsFµ or its parental F(ab')2µ fragments, the intensities increased 29 and 30 relative response units, respectively. The intensities of BsFµ (50 mg/L) and its parental F(ab')2µ fragments (50 mg/L) against AP increased 23 and 24 relative response units, respectively. For CEA, the KDs were 249 nmol/L (BsFµ) and 212 nmol/L [F(ab')2µ (9CA10)], respectively. The KDs against AP were 13 nmol/L (BsFµ) and 10 nmol/L [F(ab')2µ (APM05)], respectively.
The immunoreactivity of BsFµ bound to AP was
evaluated by sandwich enzyme immunoassay and compared with that of the
F(ab')2µ covalently conjugated to AP. A 96-well
microtiter plate (Nunc-intermed; MaxiSorp) was coated with goat
anti-CEA polyclonal antibody (prepared in our laboratory) and blocked
by incubation with 2 g/L bovine serum albumin in PBS
(pH 7.4). After washing, the plate was incubated at 25 °C for 60 min
with 100 µL of CEA at various concentrations
(0, 25, 50, 100, 200, 400 µg/L) and 100 µL of the
BsFµ bound to AP (absorbance at 280 nm in
PBS = 0.004), IgM covalently labeled with AP (absorbance at 280 nm
in PBS = 0.002), or F(ab')2µ covalently
labeled with AP (A280 nm in PBS =
0.004). After another washing, 100 µL of the substrate solution (2
g/L p-nitrophenylphosphate in 50 mmol/L carbonate
buffer, pH 9.5, containing 10 mmol/L MgCl2) was
added to the plate for 20 min at 25 °C, and the reaction was
terminated by adding 100 µL of 0.5 mol/L NaOH. The AP
activity was measured by the absorbance at 405 nm (Fig. 1B
).
The detection limits of the CEA assay (defined as 2 SD above the zero
concentration calibrator) were 0.90 µg/L for
BsFµ and 3.4 µg/L for
F(ab')2µ, respectively. The immunoreactivity of
BsFµ against CEA was almost the same as that of
the F(ab')2µ covalently labeled with AP in the
range 0.90–400 µg/L. The nonspecific binding of
BsFµ was 27% (0.90 x 100/3.4 = 27)
lower than that of F(ab')2µ.
Because the covalently labeled conjugate that was prepared from F(ab')2µ and AP is a complex polymer molecule and has multiple antigen binding sites in one conjugate molecule, the conjugate cannot bind to the antigen at a molar ratio of 1:1. The BsFµ bound to the AP conjugate can bind more efficiently to the antigen at a 1:1 molar ratio. To detect lower concentrations of the antigen, BsFµ may be more useful than the covalently labeled conjugate. The BsFµ bound to the AP conjugate was stable for at least 3 months at 4 °C.
In conclusion, we showed that homogeneous BsFµ fragments prepared from IgMs recognized both antigens without any loss in immunoreactivity. The BsFµ bound to AP allowed sensitive and reliable measurement of the antigen and may be generally applicable for immunodiagnosis.
References
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), anti-CEA F(ab')2µ
fragment covalently labeled with AP (
), or BsFµ bound
to AP (
) was added to the well, followed by the addition of 100 µL
of CEA solution at the concentration indicated on the x
axis. p-Nitrophenylphosphate was added, and the
absorbance at 405 nm was measured.