A Single-Chain Antibody Fragment Against Human Thyroglobulin: Construction and Evaluation of Immunoreactivity

Abstract

Smaller recombinant antibody fragments are at the forefront of in vivo diagnosis and therapy. These units possess better distribution and faster clearance than larger molecules. Among these, single chain antibody fragments (scFv) are emerging as credible alternatives. These proteins are shown to have same specificities and affinities for their antigens as the parental monoclonal antibody (MAb). We have attempted to produce scFv against human thyroglobulin (H-Tg) using anti-Tg secreting hybridoma cells and PCR-based cloning approach. Hybridoma secreting anti-Tg MAb B10IV was established. cDNA was prepared from hybridoma cells. The V_H and V_L genes were amplified and cloned. The gene sequences were submitted to Genebank database (accession nos. AJ508533 and AM072962, respectively.) V_L and V_H genes were then linked together with a linker peptide and successfully cloned in pET28a and expressed as His-tag fusion protein in expression host BL21 (DE3). The scFv protein from IPTG-induced cells was purified under native conditions by immobilized metal affinity chromatography on a Ni-NTA agarose column. The yield expressed in Escherichia coli was approximately 8 mg/L. ScFv could be labeled with ¹²⁵I and its immunoreactivity evaluated in radioassays. Although scFv demonstrated specific binding to H-Tg, the immunoreactivity was low (10.3%) compared to the parental MAb B10IV, which showed immunoreactivity of 37.27%. Inhibition radioassays exhibited that scFv and MAb interact with the same epitope on the target antigen, indicating its specificity.

Introduction

Since their discovery in 1975 by somatic cell hybridization, monoclonal antibodies (MAbs) have revolutionized the field of biomedical research and proved to have tremendous potential in the diagnosis and treatment of diseases. A large number of MAbs against various tumor antigens have been evaluated for in vivo diagnosis.^(1–4) As the majority of hybridomas are of rodent origin, the foreignness and immunogenicity of the mouse monoclonals result in allergic reactions, hepatic dysfunction, and immune complex formation in the kidney, precluding their long-term use in humans.^(5–7) Another drawback of using whole MAb is the presence of high background while tumor imaging. Several attempts have been made to improve in vivo diagnosis and therapy using MAbs. One of them involves the use of the smaller formats of molecular recognition units, as these possess better distribution and faster clearance than the larger molecules.

Single-chain antibodies (scFv) are most commonly viewed as this unit. They are novel recombinant polypeptides composed of an antibody variable light chain (V_L) tethered to a variable heavy chain (V_H) by a designed peptide that links the carboxyl terminus of V_L sequence to the amino terminus of V_H sequence.⁽⁸⁾ Both chains are expressed in equimolar concentrations, and the covalent linking assists the association of V_L and V_H domains after folding. The specificities and affinities of these proteins for their antigens are usually the same as those of the MAb used for generating the recombinant antibody fragment. The scFvs, being only half the size of a Fab molecule, have lower retention time, rapid blood clearance, and better tumor penetration. They are less immunogenic, amenable to fusion with proteins and peptides, and can be readily expressed in an active form when their genes are introduced into mammalian, bacterial, or plant cells. The first in vivo application of radiolabeled scFv was demonstrated by Colcher and associates in athymic mice bearing human colon carcinoma xenografts.⁽⁹⁾ Since then, there have been many reports confirming the role of scFvs in tumor targeting.^(10–13)

Thyroglobulin (Tg) is a thyroid-specific iodinated glycoprotein with a molecular weight of 660 kDa. Various studies have resulted in establishing Tg as a biochemical tumor marker for the follow-up of patients with persistent or recurrent differentiated thyroid cancer (DTC) after total thyroidectomy.^(14–17) The treatment of well-differentiated thyroid cancer with surgery and radioiodine therapy offers a favorable prognosis. However quite a number of patients become refractory to radioiodine therapy and management of such patients during the follow-up becomes difficult. In such cases, radiolabeled MAbs may be used as an alternate treatment modality for localizing non-iodine concentrating thyroid metastasis.^(18–21)

Previously we have reported the production of anti-human Tg (H-Tg) MAbs and their clinical utility.^(22,23) Two of the anti-Tg MAbs showed promising tumor uptake in the experimental model.⁽²⁴⁾ Knowing the disadvantages of using whole murine MAb, we have attempted to produce scFv toward H-Tg using anti-Tg secreting hybridoma cells. The scFv molecule was generated by PCR-based cloning by amplification of variable heavy and variable light genes by using 5′ and 3′ oligonucleotide primers complementary to the respective ends of the genes.

Materials and Methods

Production of anti-human Tg MAb B10IV

Hybridoma-secreting anti-human Tg MAb B10IV was produced by fusing spleen cells from Balb/c mice immunized with H-Tg with mouse myeloma Sp2/0 cells, according to the method of Narkar and associates.⁽²²⁾ MAb B10IV was affinity purified using Protein A column (Bangalore genei, Bangalore, India) and was characterized in terms of specificity, affinity, and IgG subclass. The cross-reactivity of MAb B10IV with human thyroid hormones and bovine and porcine Tg was also checked.

RNA isolation, cDNA cloning, and nucleotide sequencing

Total RNA was isolated from approximately 3 × 10⁶ hybridoma cells using TRIzol kit (Gibco BRL, Life Technologies, Carlsbad, CA) and the first strand cDNA was synthesized using Revert-Aid H-minus first strand cDNA synthesis kit (MBI Fermentas, St. Leon-Rot, Germany) as per the manufacturer's instructions. The antibody variable domains (V_H and V_L) were amplified in a total reaction volume of 50 μL containing cDNA as template, 200 μM dNTP mixture, 10 pM of forward and reverse primers, 2 mM MgCl₂, and 1 U of Taq DNA polymerase using the following protocol: 4 min at 94°C, 35 cycles of 1 min at 94°C, 90 s at 55°C, 1 min at 72°C, and a final extension of 10 min at 72°C. The coding region of variable heavy chain (V_H) and variable light chain (V_L) was amplified using primers adapted from Chaudhary and associates.⁽²⁵⁾ V_H was amplified with the forward primer V_HFor (5′-ATTTT AAAAG GTGTC GTCGAC GATGT GCAGC TGGTG GAGTC TGG-3′) and V_HRev (5′-AGACTG ATGGGG GTGTTG GGATCC TTATT GAGGA GACTG TGAGA GTGGT TCC-3′). The primers were designed to take advantage of the conservation of sequences at the 5′ and 3′ ends within the variable regions. However, direct amplification of the V_L gene resulted in non-specific gene amplification. Hence V_L gene was singled out using specific primers after amplifying the light chain with a degenerate set of primers, which were obtained from Shantha Biotechnics (Hyderabad, India) as a kind gift. The V_L gene was then pulled out from the amplified product and the linker peptide added sequentially using primers V_LFor (5′-CATATG GAAAAT GTGCTC ACCCAG TCTCC-3′) and V_LRev1 (5′-TTTGAG CTCCAG CTTGGT CCC-3′); V_LRev2 (5′-GATTTA CCCTCT TTGAGC TCCAGCT-3′); V_LRev3 (5′-CCAGAT CCTGAG GATTTA CCCTCT-3′); V_LRev4 (5′-GGATTC GGAGCC AGATCC TGAGGA-3′); V_LRev5 (5′-GTCGAC TTTGGA GGATTC GGAG-3′). The primers provided amplified V_H with SacI/BamHI and amplified V_L with NdeI/SacI restriction sites, respectively (underscored). The amplified gene segment was subcloned into TA cloning vector using InsT/A clone PCR product cloning kit (MBI Fermentas). The constructs (VHTA and VLTA) were used to transform DH5-α bacterial cells, and the clones were analyzed by colony PCR using the protocol mentioned above. Plasmid (VHTA and VLTA) was extracted from positive transformants and further checked for the presence of correct insert and orientation by digesting with SacI/BamHI and NdeI/SacI, respectively. Selected plasmids were sequenced on both chains using the M13 primers (Bangalore genei).

Expression vector construction and screening of small expression cultures

Plasmid from clones VLTA and VHTA was extracted and double digested with NdeI/SalI and SalI/BamHI, respectively. The fragments were purified using Gel-extraction kit (Qiagen, Hilden, Germany) and equimolar concentrations of both fragments were ligated together in the presence of 1 μL of 1 U of enzyme T4 DNA ligase. The assembled scFv was selected out by PCR with V_LFor and V_HRev primers using the protocol described above. For expression and easy detection, scFv was cloned in frame into N-terminal His-tag of pET 28a after digestion with NdeI and BamHI. BL21 (DE3) competent cells were transformed with the construct and recombinant clones verified by restriction analysis. The selected recombinant clones were screened by growing the clones in medium supplemented with 50 μg/mL of kanamycin. The cells were grown for 3 h at 37°C and induced with Isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM followed by incubation for 3 h at 37°C. The cells were harvested (both before and after induction) and analyzed by 15% SDS-PAGE.

Overexpression and purification

The recombinant bacteria harboring scFv were harvested from 1 L culture after induction with 1 mM IPTG at 37°C for 18 h. Cells were suspended in lysis buffer containing 50 mM NaH₂PO₄ (pH 8.0), 300 mM NaCl, 10 mM imidazole, and 1 mg/mL lysozyme, then sonicated and centrifuged at 10,000 rpm for 30 min. The scFv protein was purified by immobilized metal affinity chromatography (IMAC) using Ni-NTA agarose (Qiagen) according to the manufacturer's protocol. The recombinant scFv was detected by Western blot analysis using anti-His-tag antibody.

Specificity against H-Tg

Western blot analysis was carried out to check the specificity of scFv against H-Tg. Affinity purified scFv was separated by PAGE and transferred electrophoretically onto PVDF membrane (Millipore, Billerica, MA). After blocking the non-specific sites with 5% BSA and 5% non-fat dry milk in TBS, membrane was incubated overnight in cold with H-Tg (1 μg/mL), followed by anti-Tg MAb B10IV overnight in cold. After washing off the unbound antibody, the membrane was exposed to HRP-conjugated sheep anti-mouse IgG (Amersham Life Sciences, Buckinghamshire, United Kingdom). Immune complex was detected by using the substrate tetramethylbenzidine (TMB).

Immunoreactivity against H-Tg

Radioassays

The binding of ¹²⁵I-labeled scFv to H-Tg was assessed by radioassays. All assays were performed in triplicates. scFv was labeled with ¹²⁵I using iodogen as described by Fraker and Speck.⁽²⁶⁾ Monoclonal antibody B10IV was also radiolabeled with ¹²⁵I for comparative studies. Radiolabeled scFv/B10IV was incubated overnight at room temperature (RT) with H-Tg coated (200 ng/100 μL/well) onto the microtiter plate. At the end of the incubation period, the wells were washed and radioactivity measured in a multi-well gamma counter (Riastar, Packard, Prospect, CT).

Inhibition radioassays

Varying concentrations of either unlabeled scFv (0.5–3 μg) or unlabeled B10IV (2.5 − 100 ng) were incubated for 3 h at RT with 200 ng of H-Tg coated on the microtiter plate. After incubation, the wells were washed with PBS (pH 7.4) followed by incubation with radiolabeled B10IV and radiolabeled scFv, respectively, overnight at RT. The wells were then washed and radioactivity measured in a multi-well gamma counter (Riastar). In another set of experiments, specificity of binding of radiolabeled scFv to H-Tg was assessed by incubating unlabeled scFv (1 and 2 μg) with H-Tg, followed by incubation with radiolabeled scFv for 3 h at 37°C.

Results

Construction and purification of scFv

Anti-H-Tg monoclonal antibody

Anti-H-Tg monoclonal antibody B10IV belonged to isotype IgG2a/κ and exhibited a high affinity of 2.9 × 10¹⁰ M⁻¹ to H-Tg. It did not show any cross-reactivity with human thyroid hormones or bovine or porcine Tg.

Sequence of V_H and V_L

To generate scFv fragment, the V_L and V_H genes were cloned using cDNA from hybridoma cells secreting anti-H-Tg MAb B10IV and joined by a peptide linker. Figure 1 shows the nucleotide and deduced amino acid sequences of the V_L and V_H genes, respectively. The complementarity determining regions of the V_L and V_H genes were identified and are shown. The light chain of scFv showed ∼96% homology with other available sequences of mouse kappa light chain, and the heavy chain showed ∼98% homology with the existing mouse variable heavy chain sequences. The DNA sequences of V_L and V_H are now registered in the GeneBank Database (accession nos. AM072962 and AJ508533, respectively).

FIG. 1.

(A) Nucleotide and deduced amino-acid sequence of B10IV variable light chain. Complementarity-determining regions (CDR) are highlighted. The cDNA of the V_L region of MAb B10IV has been deposited in the EMBL database (accession no. AM072962). (B) Nucleotide and deduced amino acid sequence of B10IV variable heavy chain. Complementarity-determining regions (CDR) H1, H2, and H3 are highlighted. Cloning sites (SalI and BamHI) are underscored. The cDNA of the V_H region of MAb B10IV has been deposited in the EMBL database (accession no. AJ508533).

Restriction analysis

The assembled scFv was inserted into the bacterial expression vector pET28a. Twenty randomly selected recombinant clones on restriction digestion released an insert of 900 bp. Figure 2 shows restriction analysis of plasmids from 12 recombinant bacterial clones harboring assembled scFv.

FIG. 2.

Restriction analysis of plasmids from recombinant bacterial clones harboring assembled scFv. Lane 1, 1 kb DNA ladder; lanes 2–13, plasmids from randomly selected colonies digested with NdeI and BamHI.

IPTG induction and Western blot analysis

Small-scale expression of scFv was initially checked by Western blot analysis using anti-His-tag antibody. A protein band obtained at 30 kDa confirmed the presence of recombinant scFv in the bacterial extract (Fig. 3).

FIG. 3.

Western blot analysis of scFv protein preparation with anti-His-tag antibody from randomly selected recombinant bacterial clones. Lane 1, molecular weight makers; lanes 2–7, colonies screened for production of scFv.

Purification of scfv

The scFv was expressed as a His-tagged protein. Hence the affinity purification of scFv was carried out by immobilized metal affinity chromatography using Ni-NTA agarose that binds to the His-tag. The yield of scFv in bacterial culture obtained was 7 mg/L.

Specificity of scFv against H-Tg

The specificity of scfv against H-Tg was further judged by the binding of affinity purified scFv to H-Tg in Western blotting. As shown in Figure 4, Western blot analysis revealed a single band at the expected molecular mass of 30 kDa.

FIG. 4.

Western blot showing specific binding of purified scFv to H-Tg. Lane 1, molecular weight makers; lanes 2 and 3, purified scFv protein.

Immunoreactivity of scFv

Immunoreactivity of scFv was tested against H-Tg by radioassay using radiolabeled scFv.

Radioassay

The recombinant scFv protein and MAb B10IV were labeled with ¹²⁵I using iodogen to yield specific activities of 3.114 μ Ci/μg and 8.6 μ Ci/μg, respectively. The radiolabeled scFv revealed immunoreactivity of only 10.33% compared to the parental MAb B10IV, which showed immunoreactivity of 37.27%.

Inhibition radioassay

In inhibition radioassay the binding of radiolabeled B10IV to H-Tg was partially inhibited by scFv. The inhibition obtained was only 12%, with a very high concentration of scFv used (Fig. 5A). In contrast, MAb B10IV significantly inhibited binding of the radiolabeled scFv to H-Tg by up to 45% (Fig. 5B). As shown in Figure 5C, more than 57% inhibition was observed when the binding of radiolabeled scFv was inhibited by cold scFv. Non-immune mouse IgG, when used as a control, failed to inhibit the binding of radiolabeled scFv to H-Tg, indicating its specificity against H-Tg.

FIG. 5.

Immunoreactivity of scFv to H-Tg as tested by inhibition radioassays. (A) Inhibition of binding of labeled MAb B10IV in the presence of varying concentrations of unlabeled scFv. (B) Inhibition of binding of labeled scFv in the presence of unlabeled MAb B10IV and (C) inhibition of binding of labeled scFv to H-Tg in the presence of unlabeled scFvB10IV.

Discussion

The advent of genetic engineering has facilitated rapid and inexpensive production of recombinant antibody molecules. scFv is one such man-made antibody fragment resulting from the recent development in antibody engineering.⁽⁸⁾ In this study we have reported the production of scFv against human Tg using anti-Tg B10IV hybridoma cells as a source of genetic starting material and PCR-based cloning. In PCR based cloning, amplification of a specific gene by PCR uses 5′ and 3′ oligonucleotides primers complementary to the respective ends of the gene. This genetically applicable approach to clone V genes using oligonucleotides based on framework1 (FR1) and J segments was developed by Orlandi and colleagues.⁽²⁷⁾ The restriction sites within a primer can be devised to allow the forced cloning of the amplified DNA.⁽²⁷⁾

Since then, PCR is commonly used to clone the genes of individual MAbs, as well as whole antibody gene repertoires of immunized or non-immunized animals, including humans. Chapal and colleagues⁽²⁸⁾ have described the use of in-cell PCR together with Cre-recombination for amplification and recombination of the VH and VL genes within CD19+ B cells isolated from human thyroid tissue to obtain scFv. Another report describes isolation of scFvs that bind bovine Tg comprised of the complete variable regions from shark light and heavy chains.⁽²⁹⁾ Thyroglobulin was used as the selecting antigen, as both sharks and humans express natural antibodies to mammalian Tg in the absence of purposeful immunization.

In our study, the primers for scFv generation based on N-terminal amino acid sequence and J region on 5′ and 3′ sides, respectively, were adapted from Chaudhary and colleagues.⁽²⁵⁾ Using these primers amplification of V_H genes was successful. However amplification of the V_L genes from hybridoma cells gave different PCR products. It has been reported that although hybridomas secreting high affinity antibodies are good sources of mRNA for cloning of desired V genes, sequences of non-functional rearranged V genes of the hybridoma cDNA are often obtained by PCR.^(27,30) Secondly, the non-secreting cell line P3-X63-Ag8.653 or SP2/0-Ag14 is used for the establishment of hybridoma cell lines by the standard fusion techniques.⁽³¹⁾ In many cases these hybridoma cells not only transcribe the desired MAb DNA, but also bear high levels of non-functionally rearranged mRNAs. These mRNAs represent pseudogenes and can greatly exceed the level of normal antibody mRNA, thus resulting in non-specific gene amplification.^(32,33) The V_L genes from B10IV hybridoma cells were then amplified using first the degenerate set of primers to amplify light chain genes. The V_L gene was then pulled out from the amplified product and the linker peptide added sequentially.

The scFv antibody fragments generated in different studies have different expression levels.^(34–36) Differences in the translation machinery and folding pathways of eukaryotic and bacterial cells can lead to poor expression.⁽³⁷⁾ In our study optimal expression levels of scFv reached up to 7 mg/L of culture and corresponds to ∼22% of the total protein, which is comparable to protein yields reported by Sanchez and colleagues.⁽³⁸⁾ Huston and colleagues reported, after performing affinity chromatography, a yield of 12.6% of the renatured active anti-digoxin scFv protein produced in E. coli.⁽³⁹⁾

Specific immunoreactivity of scFv to purified H-Tg could be demonstrated in vitro using immunoassays. Solid phase radioassay revealed only10.3% immunoreactivity by scFv compared to 37% by MAb B10IV. This may be due to the differences in glycosylation states or to other modifications, which needs to be investigated. Our results are in agreement with Huston and colleagues,⁽³⁹⁾ who also reported production of low binding affinity anti-digoxin scFv compared to the parental MAb. Lower binding of scFvs can also result from sequence changes and conformation differences between scFv and MAb. Sequence changes may occur during the scFv construction procedures, including RT-PCR, fusion PCR, enzyme digestion, and ligation. Their conformations may also be different as scFv molecule has a flexible peptide linker coupled to the variable genes while a natural Ig molecule does not.⁽³⁹⁾ It is therefore advisable to produce scFv from the highest affinity parental MAb available.⁽⁴⁰⁾

To ensure that scFv recognizes the same epitope on Tg molecule as the parental B10IV antibody, an inhibition experiment was conducted using the two antibodies. Inhibition assay demonstrated that unlabelled scFv inhibited the binding of labeled MAb B10IV to H-Tg by 12%. This partial inhibition may be due to the fact that scFv does not saturate the binding sites on the Tg molecule. However, inhibition of binding of labeled scFv to H-Tg by unlabeled MAb B10IV was comparatively more (i.e., 45%). Taken together, this indicates that both the scFv and parental antibody recognize the same epitope on Tg molecule. Similar findings have also been reported by Li and associates by scfv mimicking human ovarian cancer antigen CA125.⁽⁴¹⁾

Specific targeting to tumor cells has been a major goal of in vivo use of MAbs and their fragments. As mentioned earlier advantages of scFv over whole antibodies are being explored for radioimmunodetection and for radiotherapy of cancer. Anti-Tg MAbs, either whole Ig or Fab have been attempted for in vivo immunolocalization.^(18–21,42) However scFv against H-Tg for immunodetection has not been reported thus far in the literature for in vivo detection of thyroid tumor. Although we have been successful in producing scFv against Tg, whether scFv exhibiting low immunoreactivity, which results in low binding affinity (as in our study), is suitable for in vivo studies is a moot point. It is possible that it may show even lower tumor uptake, thereby resulting in inconsequential tumor localization.

As reported in the literature, expectations to have a scFv with binding characteristics equal to those of other antibodies with improved pharmacokinetic profiles due to improved tissue penetration and faster serum clearance are often not fulfilled.^(43–45) Furthermore the monovalent nature of the scFv molecule also limits its utility for applications in fields of cancer imaging and therapy.⁽⁴⁵⁾ Recently, a number of strategies, including affinity maturation and modification of size and valence, are being explored for improving the in vivo efficacy of scFv molecules. Most of the approaches to optimize the size of the antibody for the desired pharmacokinetics are based on developing multivalent antibody fragments. This typically generates a molecule with an increased molecular weight, which leads to reduced renal uptake and longer circulating half-lives.^(44,45) Also, affinity of these scFv antibodies to the target antigen can further be improved by chain shuffling or by mutation of the V_H and V_L of scFv.^(46,47) It has been demonstrated that restriction of mutagenesis to the CDRs located in the center of the antibody combining site can yield increase in affinity.⁽⁴⁷⁾ For instance, mutation of the V_L and V_H CDR3 of C6.5 scFv yielded a scFv with a 1230-fold increased affinity, which was comparable to values previously reported either for in vivo or in vitro affinity maturation of antibodies.⁽⁴⁷⁾

In conclusion, we have cloned the anti-H-Tg MAb B10IV under the form of scFv. The scFv recognizes the same epitope as the parental antibody but with a lower affinity than would be expected for single-chain antibodies. Hence its biophysical characteristics could not be studied in depth. However once cloned, by mimicking the strategies of immune system, repertoire selection technologies may allow the creation of scFvs with high specificities and affinities for in vivo applications.

Footnotes

Acknowledgments

The authors thank Dr. Chakraborthy of Shanta Biotechnics (Hyderabad, India) for his valuable suggestions and for providing the degenerate set of primers.

Author Disclosure Statement

The authors have no financial interests to disclose.

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A Single-Chain Antibody Fragment Against Human Thyroglobulin: Construction and Evaluation of Immunoreactivity

Abstract

Introduction

Materials and Methods

Production of anti-human Tg MAb B10IV

RNA isolation, cDNA cloning, and nucleotide sequencing

Expression vector construction and screening of small expression cultures

Overexpression and purification

Specificity against H-Tg

Immunoreactivity against H-Tg

Radioassays

Inhibition radioassays

Results

Construction and purification of scFv

Anti-H-Tg monoclonal antibody

Sequence of VH and VL

Restriction analysis

IPTG induction and Western blot analysis

Purification of scfv

Specificity of scFv against H-Tg

Immunoreactivity of scFv

Radioassay

Inhibition radioassay

Discussion

Footnotes

Acknowledgments

Author Disclosure Statement

References

Sequence of V_H and V_L