Development of a novel monoclonal antibody to B7-H4: characterization and biological activity

Qian Y, Xu C, Brockmeyer Nh, Wu N, Yao H, Shen L, Wu Z, Altmeyer P, Yao

Description

Objective B7-H4, a member of the B7 family of immunoregulatory receptors, may participate in the negative regulation of cell-mediated immunity. Aberrant B7-H4 expression is detected in some tumors and it plays a role in the occurrence and development of tumors. The aim of this study was to elucidate the functional and structural properties of B7-H4. Methods We developed a monoclonal antibody (mAb) against the extracellular domain of B7-H4 through immunization of Balb/c mice with 3T3-mB7-H4 cells which expressed extrinsic B7-H4. A stable hybridoma cell line was established. Then, we analysised the characterization of the mAb through Enzyme linked immunosorbent assay (ELISA), Immunoprecipitation (IP), western blotting, Immunohistochemical (IHC), and tested the biological activity of the mAb. Results ELISA, IP, and western blotting analyses indicated that the mAb specifically recognized B7-H4. In addition, flow cytometry demonstrated that the mAb exhibits excellent reactivity when applied to leukemic cells. IHC staining revealed that the mAb stained in a predominantly diffuse plasmalemmal or cytoplasmic pattern when applied to certain tumor tissues. The preliminary results of the mAb's biological activity showed that the mAb could effectively inhibit the function of B7-H4 in the inhibition of T cell, while promotingg the growth of T cells and the secretion of Interleukin-2 (lL-2), Interleukin-4 (IL-4), Interleukin10 (IL-10) and Interferon-γ (IFN-γ). Conclusion This mAb will be a valuable tool for the further investigation of B7-H4 function.

Sujets

• Monoclonal antibodies
• Serology
• Biological activity

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

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Eur J Med Res (2011) 16: 295-302 © I. Holzapfel Publishers 2011
DEv El o PMEn t o f a n o v El Mo n o c l o n a l a n t Ib o Dy t o b 7-H4:
c Ha Ra c t ERIz a t Io n a n D b Io l o g Ic a l a c t Iv It y
1,2 1 1 1 3 3 1 1y . Qian , l . Shen , c . Xu , z . Wu , n . H. b rockmeyer , P. a ltmeyer , n . Wu , H. y ao
1State Key l aboratory for Diagnosis and t reatment of Infectious Diseases, Institute of Infectious Diseases, f irst a ffiliated Hospital,
z hejiang University School of Medicine, Hangzhou, c hina
2Institute of Immunology, z hejiang University, Hangzhou, c hina
3c linic for Dermatology, v enerology and a llergology, Ruhr-University, b ochum, g ermany
Abstract In t Ro DUc t Io n
Objective: b7-H4, a member of the b7 family of
immunoregulatory receptors, may participate in the nega- t he b7 family transmits both costimulatory and
cointive regulation of cell-mediated immunity. a berrant hibitory signals to t cells, thus controlling t
cell-medib7-H4 expression is detected in some tumors and it ated immune responses and tolerance. b7-H4 (also
plays a role in the occurrence and development of tu- known as b7S1 and b7x) is a recently discovered
mors. t he aim of this study was to elucidate the func- member of the b7 family [1-2], and delivers a
co-intional and structural properties of b7-H4. hibitory signal that down-regulates t cell activation,
Methods: We developed a monoclonal antibody (ma b) thereby preventing t cell proliferation, cytokine
secreagainst the extracellular domain of b7-H4 through im- tion, and the development of cytotoxicity [1,3-4]. In
munization of balb/c mice with 3t 3-mb7-H4 cells vitro experiments have shown that b7-H4 inhibits t
which expressed extrinsic b7-H4. a stable hybridoma cell activation by down-regulating Il -2 production and
cell line was established. t hen, we analysised the char- arresting the cell cycles of both c D4+ and c D8+ t
acterization of the ma b through Enzyme linked im- cells. In vivo experiments also support the assamption
munosorbent assay (El ISa ), Immunoprecipitation that b7-H4 functions as an inhibitor to t
cell-mediat(IP), western blotting, Immunohistochemical (IHc ), ed immunity [1, 3-4].
and tested the biological activity of the ma b. b7-H4 plays an important role in the immune
reResults: El ISa , IP, and western blotting analyses indi- sponse mediated by tumors. b7-H4 mRn a transcripts
cated that the ma b specifically recognized b7-H4. In are detected extensively in the spleen, lung, thymus,
addition, flow cytometry demonstrated that the ma b and other normal tissues; however, the protein is not
exhibits excellent reactivity when applied to leukemic detectable in these tissues [1]. In contrast, an
increascells. IHc staining revealed that the ma b stained in a ing number of studies using human tumor samples
predominantly diffuse plasmalemmal or cytoplasmic have revealed that b7-H4 is overexpressed in various
pattern when applied to certain tumor tissues. t he tumors, including breast [5-6], ovarian [5, 7], renal [8],
preliminary results of the ma b’s biological activity prostate [9], and non-small cell lung cancers [10], and
showed that the ma b could effectively inhibit the that b7-H4 expression, as assessed using Rt -Pc R and
function of b7-H4 in the inhibition of t cell, while IHc , is associated with disease progression. In
addipromoting the growth of t cells and the secretion of tion, b7-H4 is expressed in tumor-associated
suppresInterleukin-2 (Il -2), Interleukin-4 (Il -4), Interleukin- sive macrophages [11] and the serum level of soluble
10 (Il -10) and Interferon-γ (If n -γ). b7-H4 is elevated in patients with renal cell carcinoma
Conclusion: t his ma b will be a valuable tool for the and ovarian cancer [12-13]. Previous studies [5-6, 14]
further investigation of b7-H4 function. have shown that the high expression of b7-H4 protein
in breast cancer decreases the number of
tumor-infilKey words: b7-H4; monoclonal antibody; immunologic trating lymphocytes and prevents tumor cell apoptosis.
techniques; biological activity t herefore, the b7-H4 protein is a negative regulator
of the antitumor immune response and may play an
Abbreviations: ma b: monoclonal antibody; El ISa : important role in promoting tumor growth. t o
eluciEnzyme linked immunosorbent assay; IP: Immuno- date the functional and structural properties of
b7precipitation; IHc : Immunohistochemical; Il -2: Inter- H4, several different epitope-specific antisera against
leukin-2; Il -4: Interleukin-4; Il -10: Interleukin-10; b7-H4 have been raised in rabbits or goats. However,
If n -γ: Interferon-γ; Rt -Pc R: reverse transcription there is little ma b available that can be used for IHc
polymerase chain reaction; HRP: horseradish peroxi- or other analyses. In the present study, we developed a
dase; f It c : fluorescein isothiocyanate; o D: optical new ma b against the extracellular domain of b7-H4.
density. t his development had great utility for immunoblot-2. Yao_Umbruchvorlage 12.07.11 11:30 Seite 296
296 EURo PEa n Jo URn a l o f MEDIc a l RESEa Rc H July 25, 2011
ting, indirect immunofluorescence staining, IP, flow (KPl c ompany) for 10~20 min at room temperature.
cytometry, and IHc staining. a nd it also had inhibitive c olor development was stopped with 2 M H So and2 4
biological function to b7-H4. a ccordingly, this mono- the absorbance was measured at 450 nm using a Model
clonal antibody will provide a powerful tool for the 680 microplate reader (bio-Rad, c a , USa ).
further investigation of b7-H4 function.
WESt ERn b l o t t In g a n a l y SIS
Ma t ERIa l S a n D MEt Ho DS
c ell lysate preparation and western blotting analysisPRo DUc t Io n of a n t I-b 7-H4 Mo n o c l o n a l a n t Ib o Dy
were performed according to previously described
6l iving 3t 3-mb7-H4 cells (5×10 ), which were pre- procedures. In brief, cell lysates were denatured for 10
pared as described previously [15], were used as im- min at 95°c with SDS-Pa g E sample loading buffer,
munogens to immunize 6 balb/c mice (Shanghai l ab- electrophoresed on 10% SDS-Pa g E gels, and
transoratory a nimal c enter, c hinese a cademy of Sciences) ferred to polyvinylidene difluoride membranes. t he
one injection biweekly (every 2 weeks) repeated 4 membranes were blocked with 5% nonfat milk in
times. t he 2 mice with the highest antibody titer as de- t bSt [20 mM t ris-Hc l (pH 7.5), 150 mM n ac l, and
termined by El ISa were boosted intraperitoneally 0.05% (v/v) t ween 20 and incubated with 3E8 ma bs
with 3t 3-mb7-H4 cells 3 days before cell fusion. Peri- or goat anti-b7-H4 polyclonal antibody (R&D
Systoneal macrophages from normal balb/c mice used as tems, Mn , USa ) for 2 h at room temperature. a fter
feeder layer cells were prepared 1 day prior to fusion. thorough washing, the blots were incubated with HRP
Spleen cells from immunized animals were fused with conjugated goat anti-mouse IgM antibodies (Santa
Sp2/0 myeloma cells (a t c c , va , USa ) [16]. More c ruz) or rabbit anti-goat Igg antibodies (Santa
than 100 independent hybridomas were obtained from c ruz).t he reaction was developed using Ec l reagents
2 fusions. Pooled culture fluids from individual hy- (a mersham, n J, USa ) and analyzed using a v ersaDoc
bridoma cultures were screened for their reactivity with MP5000 imaging system (bio-Rad).
the lysates of 3t 3-mb7-H4 cells using direct El ISa
and by IP analysis. l ysates of 3t 3 cells (a t c c ) not IP a n a l y SIS
transfected with b7-H4 were used as a control [17]. a
positive hybridoma line of 3E8 was established by lim- b7-H4 was immunoprecipitated from the 3t
3-mb7iting dilution. Isotypes of the ma bs produced were H4 or 3t 3 cells lysates. Individual samples (350 µg
identified using a mouse monoclonal isotyping kit protein/sample) were immunoprecipitated using 3E8
(a bD Serotec, n c , USa ). f urther, the hybridoma cells ma b (2 µg/sample) coupled to protein l -Sepharose
were injected intraperitoneally into a balb/c mouse to beads. t he purified recombinant b7-H4 protein [18]
obtain ascites containing high concentrations of the immunoprecipitated with normal mouse IgM was used
ma b. t he ma b was purified from mouse ascites using as an antibody control [16]. Western blotting was
cara protein l Ultral ink c olumn (Pierce, Il , USa ), and ried out using goat anti-b7-H4 polyclonal antibody as
then stored at a concentration of 1.5 mg/ml . the primary antibody, followed by rabbit anti-goat Igg
antibody coupled with HRP. t he reaction was
develEn z y ME l In KED IMMUn o So Rb En t a SSa y oped as described above.
63t 3-mb7-H4 or 3t 3 cells (1 × 10 ) were lysed in 200 f l o W c y t o MEt Ry a n a l y SIS
µl c ell l ysis buffer (c ell Signaling t echnology, Ma ,
USa ) for 30 min at 4 °c . t he insoluble material was In order to analyze the specificity of antibody, leu -
then removed by centrifugation at 8000 × g for 10 kemic cells such as U937, t HP-1, Hl 60, and MM1R
min. t he concentration of protein in the lysate was cells (a t c c ) were used in this study. Each of cells (2
6determined using a bc a protein assay kit (Pierce) with × 10 ) was incubated with 3E8 ma b or goat
anti-b7bSa as the standard. H4 polyclonal antibody for 30 min at 4 °c . t he
nort he lysate of 3t 3-mb7-H4 cells, diluted to 10