抗VEGF（血管内皮生长因子）受体的功能性纳米抗体在E.coil中的表达和纯化

Molecular Immunology 50 (2012) 35– 41 Contents lists available at SciVerse ScienceDirect Molecular Immunology jo u rn al hom epa ge: www.elsev ier .com Generation and characterization of a functional N endoth nes Mahdi B , M Keyhan A ed,e Gholamr nsd a Department o b Department o c Department o d Laboratory of e Department o a r t i c l Article history: Received 5 No Received in re 25 November Accepted 29 November 2011 Available online 29 December 2011 Keywords: VEGFR2 Nanobody Angiogenesis cepto ling st int re, we named 3VGR19, from dromedaries immunized with a cell line expressing high levels of VEGFR2. We demonstrate by FACS, that 3VGR19 Nanobody specifically binds VEGFR2 on the surface of 293KDR and HUVECs cells. Furthermore, the 3VGR19 Nanobody potently inhibits formation of capillary-like struc- tures. These data show the potential of Nanobodies for the blockade of VEGFR2 signaling and provide a basis for the development of novel cancer therapeutics. 1. Introdu Angioge and metast approach fo research ha small molec (Youssoufia tant tumor- endothelial FLK1, or kin the human kinase activ Factor (VEG stream sign inhibition o et al., 2006 seems to be ∗ Correspon Research Cent Tel.: +98 216 6 E-mail add 0161-5890/$ – doi:10.1016/j. © 2011 Elsevier Ltd. All rights reserved. ction nesis plays an important role in the growth, invasion asis of cancer. Blockade of angiogenesis is an attractive r the treatment of this disease (Folkman, 2007). Recent s focused on the development of antibodies (Abs) and ules that target the tumor-associated endothelial cells n et al., 2007; Zhang et al., 2009). One of the impor- associated receptors on the endothelial cells is vascular growth factor receptor-2 (VEGFR2, fetal liver kinase-1, ase-insert domain receptor, KDR). VEGFR2 belongs to VEGF receptor 1–3 family, which has a strong tyrosine ity and transduces the Vascular Endothelial Growth F) signals in endothelial cells, producing the down- aling that leads to cell proliferation, tube formation, the f apoptosis, and eventually tumor progression (Olsson ). Because the interaction of VEGF with its receptors essential for tumor angiogenesis, blockade of the VEGF ding author at: Department of Molecular Medicine, Biotechnology er, Pasteur Institute of Iran, Postal code 1316543551, Tehran, Iran. 48 0780; fax: +98 216 648 0780. ress: sirous zeinali@yahoo.com (S. Zeinali). receptor signaling may lead to the inhibition of neovascularization and tumor metastasis. Serum of camelidae contains an important fraction of func- tional antibodies, called heavy-chain antibodies that are naturally devoid of light chains. Camelid heavy-chain antibodies, there- fore, recognize their cognate antigens by a single variable-domain, referred to as VHH or NanobodyTM (Nb) (Muyldermans et al., 2009; Rahbarizadeh et al., 2011). Unique hydrophilic amino acids within the framework-2 region of the VHH make that Nanobodies act as autonomous single-domain antigen-binding Nanobodies. In addi- tion, the hypervariable regions [i.e. complementary determining regions (CDRs)] of Nanobodies are on average longer than those of conventional antibodies, most probably to compensate for the absence of the antigen-binding regions of the light chain (Bond et al., 2003). Nanobodies have many inherent, advantageous prop- erties, such as low molecular mass (15 kDa), a strict monomeric behavior, low immunogenicity, high affinity, high solubility and stability and high yield expression of recombinant VHH in bacteria or yeasts (Buelens et al., 2010). These characteristics make VHHs useful next-generation reagents in immunoassays and for thera- peutic applications (Goldman et al., 2006; Saerens et al., 2008). As with other recombinant antibody fragments, VHH fragments iso- lated from hyper-immunized or naïve libraries are highly specific based on the recognition of unique epitopes on target antigens. see front matter © 2011 Elsevier Ltd. All rights reserved. molimm.2011.11.013 elial growth factor receptor-2; angioge ehdania , Sirous Zeinali a,∗ , Hossein Khanahmada zadmaneshc, Alireza Khabiri a, Steve Schoonoogh eza Hassanzadeh-Ghassabehd,e, Serge Muylderma f Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran f Animal Breeding and Genetics, Animal Science Research Institute of IRAN (ASRI), Iran f Virology, Pasteur Institute of Iran, Tehran, Iran Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium f Structural Biology, NSF, VIB, Brussels, Belgium e i n f o vember 2011 vised form 2011 a b s t r a c t Vascular endothelial growth factor re blockade of the VEGF receptor signa metastasis. Nanobodies are the smalle antibodies occurring in camelids. He / locate /mol imm anobody against the vascular is cell receptor orteza Karimipoura , Nader Asadzadehb , , Mahdi Habibi Anbouhia, ,e r-2 (VEGFR2) is an important tumor-associated receptor and can lead to the inhibition of neovascularization and tumor act antigen binding fragments derived from heavy chain-only describe the identification of a VEGFR2-specific Nanobody, 36 M. Behdani et al. / Molecular Immunology 50 (2012) 35– 41 Antigen-specific Nanobodies have been reported for a wide-range of targets ranging in size from immunogenic proteins as part of cells, parasites or viruses, to individual enzymes or toxins and to low-molecular weight haptens (Doyle et al., 2008; Hmila et al., 2010; Lafay In this specific Na Nanobodies Furthermor tube forma 2. Materia 2.1. Cell lin HEK293 is a stably t per cell (Ba grown in D were grown Serum Grow on plastic fl Recomb from R&D s 2.2. Cell ba Two you six times su cell line. Ab resuspende adjuvant fo incomplete Pre-imm tion. The im samples by binant VEG polyclonal bits with ca followed by 2.3. Library Four day ple was col prepared u supplier. Th ously (Thys peripheral b transcriptio were ampli CTG GCT GC (5′-GGT AC (VHH-CH2 bodies) we nested prim GG-3′) and GTG ACC TG respectively NotI, was li with PstI an was transfo repertoire w helper pha four consec coated with 100 �g/ml (100 �L per well) recombinant extracellu- lar domain of VEGFR2. After each selection, bound virions were eluted with 100 mM triethylamine (pH 10.0) and after trans- fer to a fresh tube, immediately neutralized with 1 M Tris–HCl, . Pha g E. ndiv ble p iogal s re hase arac odies VHH perip , nu d by sequ in th ag, u rme ed as smic is-Se s we aded ted o mole ted i ti-H orpt finity of N nity VEG naly e VE odies 1000 ach e bi nsta ine heth rst N g of R19 d. If of th first N s wh her N two due at t CS a VEG 2 ne arac ase N e et al., 2009; Roovers et al., 2007; Thys et al., 2010). paper, we present the first examples of VEGFR2- nobodies and demonstrate the ability of one of these , named 3VGR19, to bind VEGFR2 on the cell surface. e, we show that Nanobody 3VGR19 inhibits capillary tion in vitro. ls and methods es and proteins , 293KDR and HUVECs were used in this study. 293KDR ransfected cell line expressing about 2.5 ×106 VEGFR2 cker and Backer, 2001). HEK293 and 293KDR were MEM medium supplemented with 10% FBS. HUVECs in M199 medium supplemented with 10% FBS and Low th Supplement (Invitrogen). Cultures were maintained ask and incubated at 37 ◦C in 5% CO2. inant extracellular domain of VEGFR2 was purchased ystem. sed-immunization and serum response ng male camels (Camelus dromedarius) were injected bcutaneously at monthly intervals with human 293KDR out 5 × 107 cells were washed three times with PBS, d in 2 ml PBS and mixed with 2 ml Freund’s complete r the first immunization, and with 2 ml of Freund’s adjuvant for the following immunizations. une and immune sera were collected before each injec- mune response was monitored by titration of serum ELISA on methanol fixed 293KDR cell line and recom- FR2. The bound camel antibodies were detected with rabbit anti-camel IgGs (obtained by immunizing rab- mel IgGs isolated on protein A and protein G columns), HRP-conjugated anti-rabbit-IgG. construction and specific VHH selection s after the last antigen injection, a 150 ml blood sam- lected and peripheral blood lymphocytes (PBLs) were sing Lymphoprep (Greiner Bio-one), as instructed by e VHH library was constructed as described previ- et al., 2010). Basically, total RNA was extracted from lood lymphocytes, and cDNA was prepared by reverse n (RT) with an oligo (dT) primer. VH and VHH genes fied with the leader-specific primer CALL001 (5′-GTC T CTT CTA CAA GG-3′) and CH2-specific primer CALL002 G TGC TGT TGA ACT GTT CC-3′). The 600-bp fragments without CH1 exon corresponding to heavy-chain anti- re purified from agarose gel and re-amplified using er A6E (5′-GAT GTG CAG CTG CAG GAG TCT GGR GGA primer 38 (5′-GGA CTA GTG CGG CCG CTG GAG ACG G GT-3′) containing the restriction sites PstI and NotI, . The amplified PCR product, digested with PstI and gated into the pHEN4 vector which was also digested d NotI (Arbabi Ghahroudi et al., 1997). Ligated material rmed into electro-competent E. coli TG1 cells. The VHH as displayed on phage after infection with M13K07 ges. VEGFR2-Specific phage virions were enriched by utive rounds of in vitro selection on microtiter plates pH 8.0 growin ning, i of solu d-1-th contain solid p 2.4. Ch Nanob The tive in aligned groupe CDR3 cloned His6 t transfo obtain peripla on a H protein was lo centra with a evalua with an UV abs 2.5. Af binding Affi ies and (SPR) a AB). Th Nanob 5 and After e HCl. Th rate co determ mine w of a fi bindin of 3VG injecte signal of the in case of anot by the can be change 2.6. FA The VEGFR well ch lactam ge particles were finally used to infect exponentially coli TG1 cells. After the third and fourth rounds of pan- idual colonies were randomly picked and expression eriplasmic VHHs was induced with 1 mM isopropyl- actopyranoside (IPTG). The periplasmic extract, which combinant VHH, was tested for antigen recognition in a ELISA. terization, expression and purification of anti-VEGFR2 nucleotide sequence of each clone that scored posi- lasmic extract-ELISA was determined. Sequences were mbered according to IMGT (Lefranc et al., 2005) and MEGA-5 software (Tamura et al., 2011) according to ences. The VHH genes of the selected clones were re- e pHEN6C expression vector, in fusion with a C-terminal sing BstEII and PstI. The recombinant pHEN6C was d into E. coli WK6 cells and Nanobody expression was described previously (Conrath et al., 2001). Briefly, the proteins were extracted by osmotic shock and loaded lect column (Sigma). After washing with PBS, the bound re eluted with 500 mM imidazole. The eluted fraction on Superdex 75 column (Pharmacia Biotech) and con- n Vivaspin concentrators (Sartorius Stedim Biotech) cular mass cutoff of 5 kDa. The purity of the protein was n a Coomassie stained SDS–PAGE and Western blotting is tag antibody. The final yield was determined from the ion at 280 nm. measurements and analysis of the simultaneous anobody pairs constants for the binding between selected nanobod- FR2 were determined by surface plasmon resonance sis using the Biacore T200 analytical system (Biacore GFR2 was immobilized on the CM-5 sensor chip. The were diluted in HBS buffer to concentrations between nM and injected at a flow rate of 20 �L per minute. cycle, the chip was regenerated with 20 �L of 100 mM nding sensorgrams were used to calculate the kinetic nts kon and koff by the BIA evaluation software and to the equilibrium dissociation constant (KD). To deter- er Nanobodies bind VEGFR2 simultaneously, an excess anobody (3VGR19 from group 1) was injected. After 3VGR19 Nanobody reached an equilibrium, a mixture and a second Nanobody (3VGR17 from group 2) was the two Nanobodies bind antigen simultaneously the e Nanobody mixture increases compared to the signal anobody alone. No difference in the signal is observed ere binding of one Nanobody interferes with the binding anobody. This happens when the epitopes, recognized Nanobodies, are identical or overlap. Alternatively, this to steric hindrance or induction of a conformational he epitope of the second Nanobody. nalysis FR2 expressing cells HUVECs and 293KDR and the gative cell HEK293 were used for FACS analysis. A terized Nanobody of unrelated specificity (an anti-�- anobody (Conrath et al., 2001)) was used as negative M. Behdani et al. / Molecular Immunology 50 (2012) 35– 41 37 Fig. 1. Camel ELISA sixth (�) injec . control wh Nanobody. PBS-1% BSA microgram for 1 h on i were incub ice. Bound anti-mouse labeled anti BSA and cel 2.7. Endoth Geltrix aliqouts we bated at 37 HUVECs ce out 1 �g of seeded onto at 37 ◦C ove tube format microscope 3. Results 3.1. Immun To raise immunized high levels and the imm tions of the the second (Fig. 1, pane data demon response to 3.2. Selectio Nanobodies From th cDNA was p coding for t PCR. The PC tor (Arbabi TG1 cells. A obtained, a the library id ). Th fect ning sing of p es w s pa om a s fro e exp PTG. ntige r VE lones e bin ces. imila pitop Nano ms a R1. A hallm ces o bclon the e e pH binan His-t chr rom purit immune response monitoring. Sera from two immunized camels were examined by tion using methanol fixed 293/KDR (A) and recombinant VEGFR-2 (B) coated plates ich purified in the same way as VEGFR2-specific About 3 × 105 cells were washed three times with and resuspended in a total volume of 100 �l. One of Nanobody was added, and cells were incubated ce. After three times washing with PBS-1% BSA, cells ated with 1 �g mouse anti-His-tag antibody for 1 h on Nanobodies were detected by staining with 0.2 �g rat -IgG PE conjugate (BD Biosciences). Excess fluorescein- body was removed by two times washing with PBS-1% ls were analyzed on a BD FACSCanto II (BD Biosciences). elial tube formation assay (in vitro angiogenesis) solution (Invitrogen) was thawed on ice and 50 �l re transferred to a 96-well tissue culture plate and incu- ◦C for 1 h to solidify. For the assays, about 4.5 × 103 lls suspended in 200 �l Medium 200, with or with- anti-VEGFR2 Nanobody or control Nanobody, were the surface of the polymerized Geltrix and incubated rnight in a CO2 incubator. The following day endothelial ion was digitally photographed under an inverted light . ization and serum respond an immune response against VEGF receptor type 2, we camels with a stably transfected cell line expressing of VEGFR2. Blood was collected before each injection, une response was monitored by ELISA using serial dilu- camel sera. Specific antibody titers raised rapidly after injection as evaluated on methanol fixed 293KDR cells l A) and on recombinant VEGFR2 (Fig. 1, panel B). These phagem (Fig. 2A after in to pan expres rounds particl of viru that fr colonie and th with I their a cific fo VHH c of thes sequen have s same e other bly for the CD region sequen 3.3. Su For into th recom ries a affinity tion ch no im strated the successful induction of a humoral immune wards VEGFR2. n and sequence analysis of VEGFR2 specific e blood lymphocytes of two immunized dromedaries, repared, mixed and used as template to amplify genes he variable domains of the heavy-chain antibodies by R fragments were ligated into pHEN4 phagemid vec- Ghahroudi et al., 1997) and transformed into E. coli library of about 4 × 107 individual transformants was nd PCR analysis of 40 randomly picked colonies from indicated that about 85% of the colonies contained a SDS–PAGE. tagged Nan single band were detect tein was ob shake flask 3.4. Affinity Evaluati Nanobodies yielding kon koff values o (KD values b for pre-immune sera (�), after second (�), after fourth (�) and after with an insert of the expected size for a VHH gene e VHH repertoire of the library was displayed on phages ion with helper phages. The library was then subjected on recombinant VEGFR2 to enrich for phage particles antigen-specific Nanobodies at their tips. After four anning, a clear enrichment for VEGFR2 specific phage as observed when monitored by comparing the titer rticles eluted from a well coated with antigen with well without antigen (Fig. 2B). One hundred and fifty m the third and fourth rounds were randomly chosen ression of their VHHs as soluble proteins was induced The soluble VHHs were then screened by ELISA for n specificity and several of them proved to be spe- GFR2. The nucleotide sequences of 45 ELISA reactive were determined. The amino acid sequence analysis ders revealed two distinct groups based on their CDR3 The 3VGR10, 3VGR19, 4VGR38, 4VGR17 Nanobodies r CDR3s and therefore are predicted to recognize the e. The sequence of Nanobody 3VGR17 is different from bodies and contains a Cys in its CDR3, which proba- n interloop disulfide bond with a second Cys located in ll VREGFR2 specific Nanobodies contain the expected ark amino acids of a VHH. The predicted amino acid f these VHHs are shown in Fig. 2C. ing and Nanobody expression xpression and purification, all binders were subcloned EN6c expression vector (Conrath et al., 2001). The t protein is directed to the periplasm of E. coli and car- ag to facilitate purification by immobilized metal-ion omatography (IMAC). After an additional gel filtra- atography, the recombinant VHH was obtained and y could be detected by coomassie stained gels after The purified proteins were confirmed to be the His obody by Western blot (Fig. 3). VHHs were present as a of ∼14 kDa. No contaminants or degradation products ed (Fig. 3). An average yield of about 4 mg purified pro- tained per liter of overnight culture grown in baffled s. measurement on of the binding between VEGFR2 and selected was performed by surface plasmon resonance analysis values in the range of 3.4 × 104 to 1.2 × 105 M−1 s−1 and f 7.7 × 10−4 to 1.0 × 10−3 s−1. A high affinity for VEGFR2 etween 5.4 and 6.8 nM) was observed for 3VGR19 and 38 M. Behdani et al. / Molecular Immunology 50 (2012) 35– 41 Fig. 2. (A) Col rker l products are th ere ex sequence of th metho Nanobody. Sen 19 Na data to a 1:1 in Fig. 3. Cooma indicated and 4VR38 Nan lower affini (Table 1). T None of ously. Sequ 4VGR17 sh tope. The la Nanobody Nanobodies to steric hin upon bindin experiment cognate ant Table 1 Kinetic rate an sured by SPR. Nanobody 3VGR19 3VGR10 4VGR17 4VGR38 3VGR17 ony-PCR analysis of randomly picked colonies from the library. The MW of the ma e right size. (B) Phage-ELISA monitoring. Phage from each rounds of panning (�) w e anti-VEGFR-2 nanobodies. Numbering and CDR designations are according to the sorgram overlays showing the binding of 20, 100, 200, 300, 400 and 600 nM 3VGR teraction model. ssie stained (A) and Western-blotting (B) SDS–PAGE analysis of purified anti-VEGFR2 N revealed by Coomassie staining or by Western blot with anti-His antibodies. The MW of th obodies. Three other Nanobodies revealed a slightly ty towards VEGFR2 (KD values between 12 and 58 nM) he sensogram curve of 3VGR19 are shown in Fig. 2D. the Nanobody pairs tested bind the antigen simultane- ences of the Nanobodies 3VGR19, 4VGR38, 3VGR10 and ow that all these Nanobodies recognize the same epi- ck of simultaneous binding of 3VGR17 with any other may be because the epitopes, recognized by the two , are identical or overlap. Alternatively, this can be due drance or induction of conformational changes induced g of a Nanobody. Therefore we continued our further s with Nanobody 3VGR19 which has the best KD for its igen. d equilibrium binding constants of the anti-VEGFR2 Nanobodies mea- kon (M−1 s−1) koff (s−1) KD (nM) 1.4 × 105 7.7 × 10−4 5.4 3.4 × 104 2.0 × 10−3 58 7.2 × 104 1.0 × 10−3 15 1.2 × 105 8.4 × 10−4 6.8 6.7 × 104 7.9 × 10−4 12 3.5. FACS a A FACS ity of the se on the cell and HUVEC as compare line HEK29 3.6. Endoth We nex to inhibit V endothe