Nanomedicine
Biomolecular engineering of antidehydroepiandrosterone antibodies: a new perspective in cancer diagnosis and treatment using single-chain antibody variable fragment
Rafaela L Fogaça+,1,3, Larissa M Alvarenga+,1,2,3, Thiago D Woiski4, Alessandra Becker-Finco3, Kadima N Teixeira5, Sabrina K Silva2,3, Rosana N de Moraes6, Lúcia de Noronha2,7, Magali Noiray8, Bonald C de Figueiredo4, Philippe Billiald ** ,8,9 & Juliana de Moura*, 1,2,3
1 Pós-graduação em Engenharia de Bioprocessos e Biotecnologia, Curitiba, Brazil
2 Pós graduação em Microbiologia, Parasitologia e Patologia, Curitiba, Brazil
3 Departamento de Patologia Básica - UFPR, Curitiba, Brazil
4 Instituto Pelé Pequeno Príncipe, Faculdades Pequeno Príncipe, Curitiba, Brazil
5 Faculdade de Medicina - UFPR, Toledo, Brazil
6 Departamento de Fisiologia - UFPR, Curitiba, Brazil
7 Departamento de Medicina, PUCPR, Curitiba, Brazil
8 Université Paris-Sud, Faculté de Pharmacie, INSERM UMR S1193 & UMS IPSIT, Châtenay-Malabry, France
9 Muséum National d’Histoire Naturelle, CNRS UMR 7245, Paris, France
*Author for correspondence: Tel .: +55 41 3361 1692, Fax: +55 41 3266 2042; julianademoura@ufpr.br
** Author for correspondence: philippe.billiald@u-psud.fr
*Authors contributed equally
Aim: To develop a monoclonal antibody against dehydroepiandrosterone (DHEA) and miniaturize it, gen- erating a single-chain antibody variable fragment (scFv) against DHEA as an adrenocortical carcinoma (ACC) marker. Material & methods: DHEA conjugated to keyhole limpet hemocyanin was used as an im- munogen to obtain anti-DHEA hybridomas. Variable fragments were cloned from hybridoma 5B7 total RNA, and used to detect DHEA in normal adrenal tissue and ACC cells. Results: IgM monoclonal antibody was highly specific, and the recombinant scFv preserved parental antibody characteristics, allowing tissue localization of DHEA. Conclusion: Undefined small lesions are challenges for clinicians and impact clini- cal adrenocortical tumor management. Generating an anti-DHEA scFv facilitates development of imaging tests for early diagnosis of pediatric ACC.
First draft submitted: 4 July 2018; Accepted for publication: 6 December 2018; Published online: 29 January 2019
Keywords: adrenal cortex . adrenocortical carcinoma . dehydroepiandrosterone . DHEA . monoclonal antibody . scFv . tumor marker . zona reticularis
Dehydroepiandrosterone (3B-hydroxy-5-androsten-17-one, DHEA) is a steroid hormone secreted mainly by the adrenal cortex, although the gonads, brain, adipose tissue and gastrointestinal tract can also produce it. Readily converted to its sulfated metabolite DHEA sulfate (DHEA-S), DHEA and DHEA-S are the most abundant circulating steroid hormones of the adrenocortical system [1].
In the course of human fetal development, the fetal zone represents around 85% of the adrenal cortex and is responsible for high DHEA production [2,3]. Usually, within 6 months after childbirth DHEA and DHEA-S concentrations tend to decrease drastically and begin to rise again at the age of 6-8 years when the adrenarche period initiates and the zona reticularis layer of the adrenal cortex becomes the main source of DHEA [4].
DHEA and DHEA-S are highly sensitive and reliable markers of functioning childhood adrenocortical tumors because they increase progressively when the tumor mass is still undetectable by imaging methods [5]. Adrenocortical
Future Medicine
carcinoma (ACC) is an exceedingly rare malignancy among children but is at least 20-times more common in southern Brazil because of the high frequency (0.27%) of the germline TP53 R337H mutation in the population [6,7].
Lung, liver or widespread ACC metastasis is a continuing challenge considering the uncertainty or absence of proper histopathologic evaluation, and the limited availability of tools for ACC treatment. Precise quantification and/or tissue localization of DHEA is essential to define the status of cancer patients [8].
Herein, an anti-DHEA monoclonal antibody capable of recognizing this hormone in the zona reticularis of a normal adrenal tissue and ACC cells was first obtained. Subsequently, we describe the technical approaches to clone cDNA encoding single-chain antibody variable fragments (scFv), which preserve the antigen-binding characteristics of the parental antibody. This nanostructured molecule is a promising tool that could be used in diagnostic or therapeutic strategies such as tagging or drug-delivery systems.
Materials & methods
Preparation of DHEA conjugates
DHEA-17-CMO (Steraloids, Inc., RI, USA) was coupled to keyhole limpet hemocyanin (KLH) as described previously [9]. The DHEA-17-CMO-KLH conjugate was extensively dialyzed against 50 mmol 1-1 phosphate- buffered saline (PBS, pH 7.4), and centrifuged. Protein concentration of the supernatant was determined using Micro-BCA protein assay (PI-23235, Thermo Fisher Scientific Inc., IL, USA), and aliquots were stored at -20℃ until further use. DHEA conjugated to bovine serum albumin (DHEA:BSA) was supplied by Steraloids, Inc.
Hybridoma generation & selection
Immunization procedure
All animal procedures were approved by the local Animal Research Ethical Committee (registered under number 643-2014). Four BALB/c mice were immunized via subcutaneous injection of DHEA:KLH (15 µg), four-times at three different intervals of 3, 2 and 1 week, respectively. Complete Freund’s adjuvant was used in the first injection whereas boosters were administered with incomplete Freund’s adjuvant. 3 days before cell fusion, DHEA:KLH (5 µg) in PBS was injected intravenously.
Serum titration
Titration and serum samples obtained from immunized mice were routinely screened using ELISA, starting from the second round of immunization. Microtitration plates (Nunc™ MaxiSorp, Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) were coated overnight at 4℃ with 100 ul of target proteins (DHEA:BSA), BSA or HSA (human serum albumin) at 10 µg ml-1 in 50 mol 1-1 carbonate buffer, pH 9.6. After washing with PBS pH 7.4 containing 0.05% Tween 20 (PBST), 150 ul of blocking solution (PBS/2% casein) was added to each well and incubated for 1 h at 37℃. Serial dilutions of control serum from preimmune mice and serum from immunized mice diluted in PBS/0.5% casein were then added to the wells. After 1 h of incubation at 37°C, bound antibodies were detected by adding antimouse immunoglobulin-peroxidase conjugate (A4416, Sigma-Aldrich, MO, USA) for 1 h at 37℃. Finally, 100 ul of the chromogenic substrate solution (50 mmol 1-1 citrate-phosphate buffer pH 5.0, 0.2 mg ml-1 of o-phenylenediamine dihydrochloride and 0.02% [v/v] of hydrogen peroxide) was added to each well and incubated for 15 min in the dark. The reaction was stopped with 20 ul/well of 1.0 mol 1-1 M sulfuric acid, and absorbance was read at 492 nm on a microplate ELISA reader (Bio-Rad model 550, Japan). At least three washes with PBST were performed between each intermediate step. Titers were measured for each individual mouse after every immunization.
Production of mouse hybridomas
3 days after the intravenous booster, the spleen from a hyperimmunized mouse was collected and the isolated splenocytes were mixed with mouse myeloma cells (P3X63Ag8.653 cell line) at a ratio of 5:1 and centrifuged. The cell pellet was washed twice with RPMI and resuspended in prewarmed (37°℃) 50% PEG 1500 (P7181, Sigma- Aldrich, MO, USA) added dropwise over a 1-min period. Cells were then centrifuged at 20℃ for 5 min at 500 x g. Hypoxanthine-aminopterin-thymidine medium was added and cells were then cloned by limiting dilutions. Hybridomas were allowed to grow for 15 days before ELISA screening using DHEA:BSA, BSA, KLH and HSA. Positive clones for DHEA:BSA but not for other antigens were recloned and maintained in culture until stabilization.
Isotyping & purification of anti-DHEA antibody
Monoclonal antibody isotyping was performed using the hybridoma cells supernatant and IsoStrip™ kit (11493027001, Roche Diagnostics, Basel, Switzerland). Antibodies from clone 5B7B3C6 were purified using a HiTrap IgM Purification HP column prepacked with Sepharose High Performance (GE Healthcare Life Sciences, Uppsala, Sweden).
Cloning of cDNAs encoding antibody V-domains & in silico analysis
Total RNA was isolated from a freshly subcloned hybridoma. Two independent batches were prepared to ensure accuracy. cDNA encoding the IGHV (immunoglobulin heavy-chain variable region) domain was amplified using degenerate universal primers VHRevU (5’-GAG GTS MAR CTG CAG SAG TCW GG) and VHForU (5’-GAC AGT GGA TAR ACM GAT GG), the latter one priming to the CH1 y domain [10]. We also used primers IGH-Rev (5’-CA GGT CAA GCT GCA GCA GTC AGG) and IGH-For (5’-AGA GTG GTG CCT TGG CCC CAG) that prime to the 5’ end of mouse IGHV FR1 and the 3’ end of IGHV FR4, respectively [11].
In order to amplify cDNA encoding the mouse IGKV domain, we used the VkForU primer that anneals to the constant region of the k chain paired with each one of the nine k chain-specific 5’ primers (VkRev1 to 9) as recommended previously [10]. PCR products were purified using an Invisorb Spin PCRapid kit (Invitek, Berlin, Germany). Bidirectional sequencing of PCR products was performed and the accuracy of the sequences was confirmed by sequencing the amplified cDNAs after cloning into pGEM-T Easy vector (Promega Corpo- ration, Madison, WI, USA). Sequences were analyzed in silico using web interface IMGT tools and databases (IMGT/DomainGapAlign) [12].
Single-chain antibody variable fragment anti-DHEA modeling & docking
An anti-DHEA scFv structure model was obtained by template-based homology modeling with the program Basic Local Alignment Search Tool (BLAST) [13] in the Protein Data Bank and was built using Swiss Model ProMod3 [14,15]. Model quality was analyzed using Global Model Quality Estimation (GMQE) and the Qualitative Model Energy Analysis (QMEAN) Z-score [16]. Compatibility between the 3D structure and its primary sequence, and its validation were assessed using Verify3D [17] and MolProbity [18], respectively.
The 3D structures of DHEA (zinc_3807917) and DHEA-S (zinc_4096458) ligands were obtained from ZINC12 (http://zinc.docking.org/) and analyzed using Avogadro software [19]. ScFv and ligands were prepared using AutoDock Tools (Scripps Research Institutes, CA, USA), which were used as the graphical user interface for AutoDock Vina docking software [20]. Gasteiger partial charges were assigned after merging the nonpolar hydro- gens. Torsions were applied to the ligand by rotating all rotatable bonds while scFv was maintained rigid. A grid of 40 × 44 × 18 points in x, y and z directions was built with a grid spacing of 1 Å. Fifteen runs were performed for both ligands and at the end of docking, the best binding modes were analyzed considering various interactions using AutoDock Tools and Pymol (https://pymol.org/2/) programs.
Construction, expression & purification of scFv
Synthetic genes encoding 5B7 IGHV A or B fused to the IGKV (immunoglobulin k variable region) counterpart via a (Gly4Ser)3 peptide linker and a C-terminal His6 tag were designed with the codon sequence optimized at Genscript USA Inc. (Piscataway, NJ, USA) for prokaryotic expression and the terminal restriction sites (Pst1/XhoI) chosen for in-frame cloning into pSW1 expression vector [21]. A step-by-step protocol has been reported previously [10].
ScFv were expressed in the periplasm of HB2151 bacteria cultured in 2XTY medium with ampicillin (0.05 g 1-1) and induced at A600 = 0.6-0.8 with isopropyl ß-D-1-thiogalactopyranoside (IPTG) (0.8 mmol 1-1) for 16 h at 16℃ under agitation (75 r.p.m.). Cells were pelleted (5000 x g, 20 min at 4°C) and periplasmic proteins were recovered after mild osmotic shock and buffer exchange via dialysis against PBS (pH 7.4).
Purification of (His)6-tagged scFvs was carried out using immobilized-metal affinity chromatography (IMAC) chromatography [22]. Periplasmic extracts (20 ml) were incubated with 0.5 ml of Ni2+-NTA-Agarose beads (Qiagen, Les Ulis, France) for 2 h and then loaded onto a microcolumn. After washing with PBS, pH 7.4, containing 20 mmol l -1 imidazole, bound proteins were eluted with PBS pH 7.4 containing 150 mmol l-1 imidazole in 0.5 ml fractions. Fractions containing proteins as evaluated using the Micro-BCA protein assay were pooled and extensively dialyzed against PBS, pH 7.4. Preparations were analyzed by SDS-PAGE on a homogeneous 12.5% gel under nonreducing conditions and western blotting using the 6x-His Tag monoclonal antibody (antibody MA1-21315, Thermo Fisher Scientific Inc., IL, USA). This step was followed by incubation with an HRP-conjugated goat antimouse
antibody (A4416, Sigma-Aldrich, MO, USA). Immunocomplexes were then detected using the chemiluminescence substrate (GE Healthcare Life Sciences, Uppsala, Sweden) or 4-chloro-1-naphthol (CN) and 3,3’-diaminobenzidine tetrahydrochloride (DAB) solution. Samples were also analyzed after size-exclusion HPLC on a calibrated Superdex 75 10/300 GL column (GE Healthcare Life Sciences, Uppsala, Sweden).
Immunoassays
Western blot
Free BSA or DHEA-BSA conjugate (5 µg) was assayed by SDS-PAGE (12.5 %) under nonreducing conditions followed by transfer to polyvinylidene difluoride (PVDF) membrane and Red Ponceau staining. For Western blotting, the membrane was blocked with 5% (w/v) nonfat dry milk and 0.3% (v/v) Tween 20 in PBS. The strips were then incubated with hybridoma supernatants. Immunocomplexes were detected after incubation with HRP-conjugated goat anti-mouse antibody (SAB3701047, Sigma, MO, USA) and staining using CN/DAB as a substrate. Three washing steps with PBS/0.05% Tween 20 were performed between each intermediate step.
Direct ELISA
An immunotiter plate (Nunc™ MaxiSorp, Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) was coated overnight at 4℃ with 100 ul of DHEA:BSA (0.312-5 µg ml-1) in 0.1 mol 1-1 carbonate buffer, pH 9.6. Alternatively, BSA, KLH and HSA were used for coating. An amount of 150 ul blocking solution (PBS/2% BSA) was added and the microplate was incubated for 1 h at 37℃. Next, 5B7 antibody solution (2.5, 5, 10 and 20 µg 1-1 in PBS/0.5% BSA) was added to the wells and the microplate was incubated for 1 h at 37℃. Bound antibodies were detected by adding HRP-conjugated goat antimouse antibody (A4416, Sigma-Aldrich, MO, USA) for 1 h at 37℃. Next, the developing solution described above was added to each well. The reaction was stopped with 20 ul of 1.0 mol l -1 sulfuric acid, and the A490 nm was read on a microplate ELISA reader. Each sample was analyzed in triplicate. Specific binding was defined by subtracting the binding observed in wells coated with BSA (unspecific binding) from that observed in wells coated with DHEA:BSA (total binding).
ELISA with scFv as the primary antibody was carried out under similar conditions of coating, blocking and primary antibody (successive dilutions of periplasmic extracts) incubation. Immunocomplexes were revealed after incubation with 6x-His Tag monoclonal antibody for 90 min at 37℃ followed by 1 h at 37℃ incubation with HRP-conjugated goat antimouse antibody, and colorimetric development was carried out as described above. In all ELISA procedures, at least, three washing steps with PBST were performed between each intermediate step.
Surface plasmon resonance (SPR) analysis
The BIAcore T100 instrument and all reagents were obtained from GE Healthcare Life Sciences (Uppsala, Sweden). BSA and DHEA:BSA were covalently attached to a CM5-sensor chip (approximately 2545 and 1306 resonance unit (RU), respectively).
In order to screen the antibody-producing hybridoma, culture supernatants diluted 1:2 in PBS, pH 7.4, were passed over the immobilized targets at a flow rate of 30 ul min-1, at 25℃, during 1 min. Regeneration of the chip was carried out after injection of guanidine 6 M followed by injection of NaOH 10 mmol 1-1 (40 ul min-1, 30 s) between the successive samples.
DHEA:BSA binding kinetics of the purified 5B7 antibody and the purified scFvs were evaluated in similar conditions but using the single-cycle kinetic method. Kinetic constants (kon, koff) were deduced from the analysis of association and dissociation rates of at least four different antibody concentrations. The dissociation constant Kp was calculated as Kp = koff/kon. Sensorgrams were analyzed using the BIAevaluation version 2.0.2 software. All experiments were carried out in duplicate. The interaction of scFv with immobilized DHEA:BSA was also analyzed in similar conditions after preincubation of scFv (1 µmol 1-1) with either DHEA or cortisol (0-500 µg ml-1).
Immunohistochemistry
The specificity of 5B7 IgM and scFv antibodies was evaluated by their precise staining pattern in immunohisto- chemistry using a kit (Spring Bioscience Corporation, CA, USA). Different paraffin-embedded tissues were used: normal adrenal of a 41-year-old man submitted to adrenalectomy due to clear cell kidney cancer, a virilizing child- hood ACC and an adult liver (as negative control for DHEA expression). The tissues samples were obtained and approved by the Pequeno Príncipe Hospital Ethics Committee (ACC and liver, CAAE: 50622315.0.0000.0097) and the Health Sciences Center Ethics Committee at Universidade Federal do Paraná (normal adrenal, Certificate of
Presentation for Ethical Consideration [CAAE]: 78692617.4.0000.0102). After deparaffinization and rehydration of the sectioned tissues (4 um thick), the endogenous peroxidase was inactivated with 5% hydrogen peroxide solution. Antigenic recovery was carried out by dipping the samples in Immuno Retriever solution in a water bath at 99℃ for 40 min. After cooling and washing, the samples were incubated overnight with hybridoma supernatant or scFv at 4℃ in a moist chamber. All slides were washed and incubated with Enzyme Advance HRP Link for 30 min at room temperature, washed and stained with 3,3-diamino-benzidine and hematoxylin. Except for the assays using scFv, tissues samples were also incubated with monoclonal antihistidine antibody (1:800, Thermo Fisher Scientific Inc., Rockford, IL, USA) for 15 min at room temperature.
Quantification of DHEA immunostaining in the normal adrenal
The micrograph images used to quantify the anti-DHEA IgM and scFv markup were obtained with Axio Scan Z1 (ZEISS®, Jena, Germany) using 10% of an optical microscope at 40-fold magnification and ZEN Software v 2.3 (ZEISS®, Jena, Germany). The anti-DHEA was quantified in 20 random 64 x 64 um2-sized fields per adrenal area applying a mask into adequate levels of the positive tissue using the Image Pro Plus software. The Shapiro-Wilk test was used to verify the normality of results. The Kruskal-Wallis and Dunn’s test were used to check the difference in markup between the adrenal zones. p < 0.05 was considered significant. Statistical analysis was carried out using GraphPad Prism v 6.05.
Results
Measurement of anti-DHEA antibody titers in mouse sera
Following mouse immunization with DHEA conjugated to KLH, the titer of anti-DHEA antibodies was measured in mouse sera by ELISA using DHEA-BSA as an antigen. Mouse sera were diluted from 1:100 to 1:12,800. A titer >1:12,800 indicated that the immunization process in mice was good enough to allow cell fusion. No cross reactivity was detected with BSA and HSA (data not shown).
Screening & selection of antibody-producing hybridoma
Culture supernatants from growing hybridomas at the 15th day post cell fusion were screened by ELISA against immobilized DHEA:BSA. Among 22 identified clones, three (5B7B3, 5B7C2 and 2F5B7) were selected for their immunoreactivity and were characterized by ELISA against different immobilized proteins (BSA, DHEA:BSA, KLH, and HSA), along with Western blot and SPR analyses (Figure 1A, B & C). Antibodies from cell culture supernatants of these three clones reacted specifically with DHEA:BSA in ELISA and Western blot, whereas no signal was observed with clone 2H12 derived from the same cell fusion, and was identified as a nonbinder or nonsecreting antibody clone. We performed SPR analysis of cell culture supernatants for ranking and found that the 5B7B3 clone presented the best interaction with DHEA, slightly stronger than 5B7C2 (Figure 1C & D). A signal with intermediate intensity was observed for the clone 2F5B7. The second reason to select the 5B7B3 hybridoma was because cells were growing very well, even after subcloning, and gave rise to the 5B7B3C6 stable clone, which was designated as 5B7 hereafter and used in all subsequent analyses.
Structural characterization of the 5B7 antibody
In order to confirm the monoclonality and perform detailed functional analysis on clone 5B7, we followed the recommendations of Bradbury et al. [23] comprising isotyping, cloning, sequencing the antibody V-domains and finally expressing them as a functional recombinant antibody fragment.
We first identified the 5B7 isotype as IgM k and used reverse transcriptase polymerase chain reaction (RT-PCR) to analyze the cDNA encoding the antibody V-domains (Figure 2A & B). No cDNA amplification was observed through the antisense primer VHForU, which hybridizes to the mouse constant y chain region, near the variable- constant region junction, which confirmed that 5B7 is probably not an IgG. Nevertheless, a 400 base pair cDNA was amplified using primers (IGH-Rev and IGH-For), which anneal to the 3’- and 5’-extremities of the IGHV domain. Sequencing of the purified PCR product led to a scrambled sequence. We therefore cloned the PCR product into a PGEMT vector and conducted sequencing of ten plasmids after cloning in bacteria. Surprisingly, two cDNAs encoding two distinct functional IGHV domains were identified and registered in GenBank as IGHV 5B7 scFv A (accession number MH194343) and 5B7 ScFv B (MH194344) (Figure 2C). For the IGKV domain, only one set of primers of the nine used led to significant amplification of a 350 base pair PCR product. Sequencing using the forward and reverse primers allowed us to demonstrate that IGKV encodes a functional V-domain. The
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| DHEA-BSA | 593 | 654 | 400 | 69 |
| Corrected signal | 459 | 455 | 181 | 18 |
| Specificity (%) | 77.4 | 69.6 | 45.2 | 26.1 |
Figure 1. Screening and selection of hybridoma secreting dehydroepiandrosterone-binding antibodies. (A) ELISA screening of hybridoma supernatants using various immobilized antigens (BSA, DHEA:BSA, keyhole limpet hemocyanin, human serum albumin). Bars from the left to the right: hybridoma 2H12, 2F5B7, 5B7C2 and 5B7B3. (B) Western blot analysis of hybridoma supernatants after SDS-PAGE of BSA (1) or DHEA:BSA (2). M: Molecular weight marker. (C and D) SPR analysis of hybridoma supernatants using immobilized DHEA:BSA in which sensorgrams of 5B7B3 (blue), 5B7C2 (red), 2F5B7 (green), 2H12 (purple) supernatants diluted 1:2, and purified IgM 5B7B3C6 (40 nmol l-1) in green are shown. Antibody samples were injected at t = 0 s. (D) Expression of the corrected signal and arbitrary ranking. BSA: Bovine serum albumin; DHEA: Dehydroepiandrosterone; KLH: Keyhole limpet hemocyanin; HSA: Human serum albumin.
closest germline mouse genes and their corresponding identity percentage, as well as complementary determining regions, framework and canonical structures were identified (Figure 2C & D). The aberrant MOPC21-derived transcript was not amplified [24].
The scFv model employing docking
The structure model of scFv 5B7 consists of two B-sheets domains corresponding to IGHV and IGKV connected by a long loop (recombinant (G4S)3) linker. The model was built using 5JYM (1B chain) Protein Data Bank as template, which is a recombinant scFv solved by x-ray diffraction (2.45 Å resolution). The target and template presented 79.75% identity and the entire sequence of the target was covered. Values for GMQE = 0.82 and QMEAN = 0.49 show that the residue positioning inside the model is satisfactory. In addition, the Z-score value (<1.0) indicates a high quality model in agreement with experimental structures of similar size. Concerning the environmental assessment of each amino-acid residue, Verify3D pointed a probable correct folding (94.85% of residue with a 3D-1 d average score ≥0.2). The model was validated using MolProbity without residues with steric impediment (98.7% of all residues were in allowed regions in the Ramachandran plot).
Nine scFv 5B7A binding modes were found for both ligands, DHEA and DHEA-S, and the best one presented the lowest binding energy. Considering the binding energy values, the interaction between scFv5B7A and steroid
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G47
A13
47
R14
13
K14
| CDR1 | CDR2 | CDR3 | |
|---|---|---|---|
| IGHVa | GYTFTSYW | INPTTGYT | RYGNYY |
| Canonical class | 1/10A | Similar to 2/10A | – |
| IGHVb | GYFTTDYE | IDPETGGT | RYGNYY |
| Canonical class | Similar to 1/10A | Similar to 1/10A | – |
| IGKV | SASSSVSYMH | YDTSKLA | QQWSSNPFT |
| Canonical class | 1/10A | 1/7A | Similar to 1/9A |
CDR: Complementary determining region; IGHV: Immunoglobulin heavy-chain variable region.
A
PR
B
176
LYS
9171
TR
170
GL
72
G.
88
ARG 177
107
4
ALA 100
86
73
TYR
06
SE
47
TH
43
ARG 101
218
SER 174
4!
GL
42
VA 96
AL
48
C
1
2
D
2
1
hormones was highly similar to both DHEA and DHEA-S indicating that scFv 5B7A is able to recognize both hormones.
Regarding the electrostatic and hydrophobic interactions that may be established with the residues of scFv5B7A, the oxygen atom of the fourth ring of the steroid nucleus in both DHEA establishes a hydrogen bond with Ser187 (Ser L69 in IGKV) present in the variable light chain. Polar interactions with Tyr105 and Tyr106 (Tyr H116 and H117 in IGHV) side chains from scFv 5B7A seem to be important to stabilize the interaction with DHEA (Figure 3A).
DHEA was also docked to the scFvB model and the analysis indicated superficial interaction involving a few residues. Polar interactions are possible between -NH groups of the peptide bond of Thr173 and Gly172, and Gly172 and Ser171 and the hormone. The Thr173 lateral chain allows the most stable interaction between DHEA and scFvB (Figure 3B). Mutated residues are likely responsible for the different folding of scFvA (Figure 3C) and scFvB (Figure 3D).
Functional characterization of the 5B7 antibody
The 5B7 antibody was obtained by hybridoma cell culture and purified from culture supernatant before being further characterized.
Western blot analysis confirmed the ability of 5B7 to react with DHEA:BSA but not to free BSA (Figure 4A). ELISA allowed us to demonstrate the dose-dependent reactivity of the antibody with DHEA:BSA while no reactivity with BSA was observed under the same experimental conditions (Figure 4B). Binding of 5B7 to DHEA-BSA was also analyzed in real time (Figure 4C). We observed a relatively quick and specific association with BSA-DHEA, and a very slow dissociation. Using BIAevaluation software, the kinetic constants were kon = 3639 M-1 s-1 and koff = 6.74 10-4 s-1, respectively, resulting in an apparent dissociation constant Kp = 185 nmol 1-1.
A
1
2
1
2
B
1.6
T
1.2
T
492 nm
A492
0.8
T
T
0.4
T
T
T
0.0
20
10
5
2.5
1.25
0.62
0.31
0.16
[IgM] µg ml-1
C
120
100
80
Signal (RU)
60
40
20
0
1
Ť
1
t
-20
5
10
20
40
nmoI.l-1
-200
0
200
400
600
800
Time (s), t = 0 injection of first concentration
Design, production & characterization of the 5B7 scFv
IgM are very large and multivalent structures (970 kD) with high avidity, which may impact their adaptability to in vitro immunoassays. Thus, we decided to design the smallest monovalent functional unit (scFv, 25 kD) with a C-terminal (His)6 tag for extensive characterization of 5B7. Because of two distinct natural IGHV sequences, we synthesized two distinct genes designated as scFv 5B7A and scFv 5B7B based on the IGHV identity. These genes were cloned into vector pSW1 (Pst1/Xho1) (Figure 5A) before being expressed in the periplasm of bacteria. ScFv 5B7A was produced in the periplasm of bacteria and reacted with DHEA:BSA in a dose-dependent manner after ELISA (Figure 5B). Conversely, no signal was observed when the periplasmic extract of bacteria expressing scFv 5B7B was used, even if this scFv was present in the periplasmic extract, as demonstrated after western blotting (not shown). The recombinant scFv 5B7A was isolated using IMAC chromatography. A major band of 25 kDa was observed after SDS-PAGE and Western blot analysis (Figure 5C). We did not purify scFv 5B7B because no significant binding-signal was observed in ELISA. Therefore, we discontinued the study with scFv 5B7B.
Interaction between scFv 5B7A and DHEA:BSA was also analyzed by SPR (Figure 6A). The best fit was observed with the heterogeneous ligand kinetic model (Figure 6B), which accounts for the presence of ligand species that bind the analyte independently. Kinetic constants are indicated in Table 1. The Kp deduced from this kinetic model was close to the one deduced from the Langmuir model used for the first evaluation attempt (Kp = 77 nM),
rsg
A
B
VI
(GAS)3
VL
His6 6
2.5
H
*
BamHI
2.0
Pstl
Nhel Xhol
A492 nm
1.5
1.0
Cloning in pSW1
1
0.5
1:2
1:4
1:8
Periplasm dilution
0.0
1:16
5.0
2.5
1:32
PelB
VI H
1.25
(GAS)3
VL
His6
0.625
DHEA:BSA (µg ml-1)
0.3125
*
C
p/o
rbs
Pstl
BamHI
M
1
Nhel Xhol
2
3
M
1
2
3
120 kDa→
Expression
85 kDa
50 kDa→
66 kDa-
35 kDa →
scFv 5B7
29 kDa-
25 kDa→
20 kDa
| KD1 (nmol l-1) | ka1 (M-1.s-1) | kd1 (s-1) | KD2 (nmol l-1) | ka2 (M-1.s-1) | kd2 (s-1) | ×2 | Rmax1 (RU) | Rmax2 (RU) | |
|---|---|---|---|---|---|---|---|---|---|
| Values | 51 | 1.73 × 104 | 8.8 × 10-4 | 555 | 9.1 × 108 | 503.2 | 8.5 | 56 | 76 |
| Error | 1 | 1.9 × 102 | 1 × 10-5 | 10 | 7.7 × 106 | 45 | |||
| RU: Resonance unit. |
which was slightly better than the one observed for the parental monoclonal antibody. In addition, the specificity of the signal was confirmed by the ability of free DHEA to inhibit scFv binding to immobilized DHEA:BSA, whereas preincubation of scFv with cortisol did not interfere with scFv/DHEA:BSA interaction (Figure 6C).
Immunohistochemistry & staining quantification
The IgM 5B7 and scFv recognized DHEA, which was well compartmentalized in a normal adrenal gland from a 41-year-old man (Figure 7A-D). A staining gradient was observed, beginning with a weak immunolabeling on the zona glomerulosa and the zona fasciculata (ZF) and abruptly very intense at the zona reticularis (ZR) (Figure 7B & C) where the quantified staining was remarkably higher than that in any other adrenal compartment regardless
A
600
Signal (RU)
400
200
0
0
400
800
1200
Time (s)
1
1
1
1
4
150
300
600
1200
2400
nmol.I-1
B
C
120
60
Signal (RU)
80
Signal (RU)
40
40
20
0
1
1
1
1
1
0
0
400
800
1200
0
1
10
100
250
500
100
250
500
Time (s)
scFv (1 µmol 1-1)
+ cortisol at 0 to 500 µg ml-1
Cortisol at 100 to 500 µg ml-1
of anti DHEA-IgM or scFv (Figure 7E; p < 0.0001). Stronger staining was detected in the ACC removed from a boy presenting with a virilizing syndrome and high levels of DHEA and DHEA-S, using the intact IgM from hybridoma media (Figure 8A) or the miniaturized 25 kDa scFv (Figure 8B) (3% of the total IgM size) as primary antibodies. There was no DHEA staining in control liver cells (Figure 8C & E) where DHEA is not produced. In addition, nonspecific reactions were not observed when IgM or scFv was omitted (Figure 8D).
Discussion
Our present tools and findings provide new insights for future preclinical studies on diagnostic and therapeutic options for childhood ACC, which are presently insufficient, as discussed widely [8,25]. Our approach presents a two- step strategy from the hybridoma to recombinant technology, by reshaping the IgM macromolecule to its essential binding portion fragment, the scFv anti-DHEA. Although both antibodies (IgM and scFv) were found to be useful in immunoassays, our main goal was to demonstrate their capabilities of recognizing specific DHEA-producing tissue targets. Our strategy for exploitation of the antibody specificity was possible based on the well-known DHEA gradient at different normal adrenal compartments to the childhood virilizing ACC.
Antibody 5B7 was selected because the hybridoma cell line appeared to be stable after subcloning, grew well and could secrete sufficient amounts of antibody under conventional cell culture conditions. The screening was efficient even if carried out without any prior quantification of the antibody in the supernatant or isotyping. The entire process enabled us to identify a hapten-specific probe even though we expected an IgG after the long-term immunization protocols used. Altogether, this demonstrates that the developed process is effective to provide valuable immunological tools. Despite the need for well-characterized probes in immunodiagnosis, very
IgM
scFv
A
1
500um
500um
ZG
B
ZG
ZF
ZF
ZR
ZR
M
M
P
ZĞ
C
ZG
ZF
ZF
ZR
ZR
M
M
D
E
2500
2500
Stained area (um2)
2000
Stained area (um2)
2000
1500
1500
1000
1000
1-
500
500
0
0
M
ZR
ZF
ZG
M
ZR
ZF
ZG
Adrenal zone
Adrenal zone
A
B
50pm
50um
C
D
E
0
0
0.
6
O
8
0
®
50pm
few monoclonal anti-DHEA antibodies have been reported and among the few marketed, none is characterized at the molecular level.
One major point with monoclonal antibodies derived from hybridomas is the need for validation at the outset otherwise major flaws in their reliability may arise, especially when they are directed against nonimmunogenic haptens. An extensive study carried out with 6120 commercial antibodies indicates that less than half of these antibodies recognized only their specific target [26]. These antibodies may bind to more than one target either because they are actually a mixture of antibodies with multiple specificities or simply because of crossreactivities and lack of specific targeting. This underlines the need for better molecular characterization of monoclonal antibodies prior to their use and possibly fine tuning their specificity after in vitro maturation, as was clearly pointed out by Bradbury and 111 co-signatories [23,27,28]. Here, we characterized the antibody 5B7 according to conventional criteria such as the process for subcloning, the isotype and the sequence that encodes the functional unit. cDNA cloning allowed identification of the unique functional IGKV sequence. Reagents are rarely validated to the same degree.
No aberrant MOPC21-derived transcript was detected, and this may be related to the myeloma fusion partner type we used (P3X63Ag8.653 and not Sp2/0-Ag14). However, two productive IGHV were identified. This point was of concern but consistent with a recent study showing that 32% of 126 hybridomas analyzed were able to express additional productive V genes [29]. Here the presence of two functional IGHV is possibly not a consequence of insufficient cloning of the hybridoma. It could be related to simultaneous fusion of the myeloma with more than one spleen cell or to a single spleen cell but with several rearrangements, or possibly postfusion rearrangements after prolonged in vitro cultivation. All these points underline the need to express the natural antibody as a recombinant antibody fragment and to assume that it is a defined monospecific molecular entity. Despite being made of two closely related IGHV (87% sequence identity), both recombinant scFvs exhibited significant differences. ScFv 5B7B did not bind DHEA:BSA in ELISA even if SPR analysis revealed weak antigen-binding activity (data not shown). Corroborating docking analysis indicates that the mutated residues may be particularly important for proper folding, disfavoring more stable interactions and, consequently, influencing the functionality of the scFvB fragment.
The real-time SPR analysis indicated a better affinity of the monovalent scFv 5B7A for the target compared with the parental pentameric IgM molecule. There are several potential explanations for this. First, the IgM with a potential mixture of heavy chain could have impaired functionality with a decreased number of functional binding sites per IgM unit, resulting in a lower apparent binding activity or the presence of two closely related but different paratopes can lead to cross reactivity thus compromising specificity [29]. Another element to be considered is that BSA is decorated at the surface of many DHEA molecules in broad to close vicinity and is not accessible at the same time to the ten antigen-binding sites of IgM, which lack flexibility. In contrast, the binding of each scFv molecule is independent and does not suffer from such steric hindrance because scFv remains as a minimal size monovalent antigen-binding unit. Using label-free DHEA as an analyte and the antibody or scFv as an immobilized ligand could help confirm these data. However, the response of SPR is directly related to the mass changes on the sensor surface, and because the molecular mass of DHEA is much lower than 1 kDa, specialized enhancement technologies are needed to acquire a detectable signal [30,31].
Here, scFv 5B7 was shown to bind DHEA without cross reacting with cortisol. However, in case of unexpected off-target activity or the need to alter the antigen-binding function of scFv, several strategies including error-prone PCR, in vitro scanning saturation mutagenesis, and maturation can be successful [27,32,33]. For all these reasons, scFv offers several advantages over conventional monoclonal antibodies, including the possibility for engineering.
The scFv 5B7A is able to recognize DHEA by immunohistochemistry and is probably suitable for recognizing it in living tissues, such as primary ACC and metastasis. This is a scFv-histidine minibody with a nanostructure size (<100 nm), which can further be self assembled as a sensor for living conditions because it can be reformatted and conjugated to radionuclides [34], cytotoxic drugs [35], enzymes [36], fluorophores [37], nanoparticles [38-40] and other molecules to generate bifunctional anti-DHEA molecules for diagnostic and therapeutic preclinical protocols.
Anti-DHEA IgM and scFv have presented specificity in detecting DHEA with higher immunostaining in the adrenal reticular zone. The stronger staining found in a 41-year-old male ZR in the present study indicates the total amount of DHEA recognized by IgM 5B7, and the related scFv is almost three-times higher in the ZR than in ZF. Recently, using high-resolution MALDI-Fourier transform-ion cyclotron resonance and mass spectrometry imaging (MSI), Sun et al. [41] compared male and female ZR and ZF subzones based on the DHEA-S, where DHEA-S was highly abundant in the ZR, while in the males, DHEA-S presented with very low intensity and localized mainly in the ZF. It also suggests that the DHEA production rate in the ZR is faster than the sulfate acquisition occurring between ZR and ZF. Taken together, future studies combining both MSI and sensors using different anti-DHEA(s) approaches could detect the co-localization of DHEA and DHEA-S at higher resolution.
Detection of tissues producing high levels of DHEA may aid the localization of DHEA sources in virilizing conditions such as ACC, which are very common in children from southern Brazil. Very little DHEA is produced by the gonads, and that would be easily detectable, but could still be a concern in targeted therapy. The most common organs requiring differential diagnosis along with other types of lesions are the lungs and liver, and therefore CT scans of the chest and MRI of abdomen are usually requested for all ACC patients [8,25]. Technetium bone scans are also used in the initial ACC evaluation for possible metastatic sites. However, it is common to find a lack of definition using these protocols.
The antibody 5B7 produced here was re-engineered as a minimal size antigen-binding molecule (scFv) because functional antibody V-domains were identified correctly. This molecule can now be reformatted and optimized for testing in an animal model of childhood ACC [42]. Strategically, the model should be designed only to reproduce a very specific common situation in clinical practice, that is, when the primary ACC is surgically dissected, and the oncologist is facing a problem to characterize a new and small lesion detected by imaging in the absence of supraphysiologic DHEA/DHEA-S blood levels. Use of a recombinant antibody fragment conjugated with a sensor for identification should be tested in this animal model to detect small ACC lesions.
Small ACC lesions occur more frequently within the first 2 years of follow-up after dissection of advanced staging ACC, usually when these tumors are too small to produce high DHEA/DHEA-S blood levels [25]. The same animal model would be suitable to evaluate engineered antibody fragments conjugated to a killing molecule, associated or/not with the modified standard Children Oncology Group treatment protocol [43], or with one of the emerging target therapies [44].
Remarkably, hemorrhage and necrosis are common features of malignant ACC [25,45], which contribute to humoral antigen spread and reduction of the physical barrier to the nanocomposite. Basically, scFv should not be tested in an animal model reproducing large pediatric ACC with elevated systemic levels of DHEA/DHEA-S, as scFv would promptly react with the circulating soluble DHEA/DHEA-S. Previous studies faced similar situations using
antiprostate-specific antigen or antiprostatic acid phosphatase monoclonal antibodies for radioimmunoscintigraphy to image prostate cancer, as reviewed by Neal et al. [46]. These authors expressed concerns about the safety issues and standardization of methods using monoclonal antibodies and radioimmunoscintigraphy. Currently, such large pediatric lesions detected by magnetic resonance imaging (MRI) or positron emission tomography-computed tomography (PET-CT) associated with elevated DHEA/DHEA-S levels would be more appropriately treated by surgical dissection [45].
Future perspective
Special imaging using scFv or other antibody fragments coupled with radionuclide or metallic nanoparticles with appropriate pharmacokinetic and pharmacodynamic relationships would contribute to the definition of ACC lesions.
Our present findings may impact medical and biological research, but further studies are necessary to prepare optimized antibody fragments for diagnosis and humanized antibodies, naked or conjugated with drugs, toxins or radioactive molecules [47], for ACC therapy.
Conclusion
A detailed molecular protocol was conducted to select and characterize an anti-DHEA IgM monoclonal antibody. Its recombinant fragment was engineered to the scFv size (~ 25 kDa) capable of specifically recognizing normal and pathological tissue targets with minimal cross reactions to other noble structures. This study paves the way for a new generation of theranostic nanomedicine for adrenocortical tumor detection and treatment.
Summary points
· Efficient hybridoma protocols were conducted to obtain polyclonal antibodies and select a very specific monoclonal anti-dehydroepiandrosterone (DHEA) IgM.
· The cDNA cloning strategy to generate a single-chain antibody variable fragment (scFv) allowed characterization of anti-DHEA IgM at the molecular level.
· Soluble scFv (25 kDa) preserved the parental IgM (970 kDa) features.
· The generated scFv was able to trace DHEA, an adrenocortical carcinoma (ACC) marker.
· Anti-DHEA IgM and scFv were able to identify stronger DHEA staining in the zona reticularis of the normal adrenal gland and ACC cells.
. Further preclinical studies are necessary to test anti-DHEA scFv in vivo aiming for diagnosis and treatment of ACC metastasis.
Financial & competing interest disclosure
This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq, Brazil (Ciência sem Fronteiras - PVE, grant No.401355/2014-4). The authors are grateful to the following institutions for their support to the respective authors: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES (RL Fogaça, TD Woiski and SK Silva) and CNPq (A Becker- Finco, BC de Figueiredo, P Billiald and J de Moura). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research
The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. For investigations involving human subjects, informed consent has been obtained from the participants involved. In addition, animal experiments were carried out in compliance with the ‘Guide for use of Animals in Research or Education’ (Conselho Nacional de Controle de Experimentação Animal - CONCEA, Brazil).
Tsg
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