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PCR amplification of the functional immunoglobulin heavy chain variable gene from a hybridoma in the presence of two aberrant transcripts
Journal of Immunological Methods 336 (2008) 246–250
Contents lists available at ScienceDirect
Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j i m
Technical note
PCR amplification of the functional immunoglobulin heavy chain variable gene from a hybridoma in the presence of two aberrant transcripts Yazad Irani a,1, Melinda Tea a,1, Ronald G. Tilton b, Douglas J. Coster a, Keryn A. Williams a, Helen M. Brereton a,⁎ a b
Department of Ophthalmology, Flinders University of South Australia, Adelaide, SA 5042, Australia Division of Endocrinology and Stark Diabetes Center, University of Texas Medical Branch, Galveston, TX 77555-1060, USA
a r t i c l e
i n f o
Article history: Received 10 September 2007 Received in revised form 4 March 2008 Accepted 19 April 2008 Available online 16 May 2008 Keywords: scFv Hybridoma Myeloma fusion partner P3X63-Ag8.653 Unproductive immunoglobulin rearrangement MOPC21
a b s t r a c t Single chain antibody fragment genes are commonly created by splicing together the immunoglobulin light chain (VL) and heavy chain variable (VH) genes of a monoclonal antibody produced by a hybridoma. Selective PCR amplification of the functional immunoglobulin variable gene rearrangements can be complicated by the existence of other unproductive immunoglobulin gene rearrangements in the hybridoma. Here we report the detection and preferential amplification of aberrant transcripts from two unproductive VH gene rearrangements derived from the fusion partner of a hybridoma. The functional VH gene of the monoclonal antibody was successfully amplified by selective use of primers to individual JH segments. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Single chain antibody fragment (scFv) genes are commonly produced by splicing together the functional light (VL) and heavy chain variable (VH) genes amplified by reverse transcriptase (RT)-PCR from a hybridoma that secretes antibody of the required specificity (Plückthun et al., 1996). The presence of non-functional variable gene transcripts in the hybridoma is a recognised phenomenon that can cause considerable problems with the selective amplification of the
Abbreviations: VL, immunoglobulin light chain variable gene; VH, immunoglobulin heavy chain variable gene; scFv, single chain antibody fragment; CDR, complementarity determining region; VEGF, vascular endothelial growth factor; PNA, peptide nucleic acid. ⁎ Corresponding author. Department of Ophthalmology, Flinders University, Bedford Park, SA 5042, Australia. Tel.: +61 8 8204 4123; fax: +61 8 8277 0899. E-mail address: helen.brereton@flinders.edu.au (H.M. Brereton). 1 Contributed equally to this work. 0022-1759/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2008.04.014
functional transcripts (Krebber et al., 1997). Aberrant variable gene transcripts can arise either from an unproductively rearranged allele(s) present in the B cell (Vidarsson et al., 2001) or from the non-secreting myeloma fusion partner used in the production of the hybridoma, and can often be in much greater abundance than the functional variable gene transcripts (Carroll et al., 1988). A very well known example is the aberrant VLκ allele present in MOPC21 myeloma-derived fusion partners, which is frequently amplified by PCR in preference to the functional VL of the antibody, and several strategies have been devised to overcome this problem. The aberrant rearrangement of the MOPC21κ allele causes a frameshift that results in a premature stop codon. Nichols et al. (1993) screened scFv clones by expression in a coupled transcription/ translation system in vitro, and distinguished between those containing the functional and aberrant VL genes on the basis of the size of the protein produced. Ostermeier and Michel (1996) used a strategy involving antisense-directed RNase H digestion of the unproductive mRNA prior to PCR to improve recovery of the functional VL gene. In a similar vein, a peptide nucleic acid
Y. Irani et al. / Journal of Immunological Methods 336 (2008) 246–250
(PNA) clamp specific for the MOPC21κ allele was used during PCR to suppress amplification of the non-functional VL gene (Cochet et al., 1999). Yet another strategy involved the identification of a restriction enzyme that was used for selective degradation of MOPC21κ-derived product after PCR amplification and prior to cloning and sequencing (Juste et al., 2005). Amplification of aberrant VH genes has been reported (Kütemeier et al., 1992; Krebber et al., 1997; Vidarsson et al., 2001), however, the problem appears more diverse and not as widely recognised as is that of the MOPC21κ allele. Two general strategies have been proposed to avoid cloning aberrant V genes. Krebber et al. (1997) used phage display and selective panning against antigen to enrich for functional scFv clones, however, this is not a trivial exercise and requires considerable expertise. Alternatively a method for multiplex PCR screening of colonies using primers specific for the CDR3 region of aberrant V genes has been described (Vidarsson et al., 2001). In this case the sequence of the aberrant V gene rearrangement must be known or determined. During the recent generation of scFv from a specific hybridoma, the functional VL gene was readily amplified, however, amplification of the functional VH gene proved difficult. Here we report the presence of two aberrant VH transcripts derived from the fusion partner of the hybridoma and the strategy used to amplify the functional VH gene of the antibody. 2. Materials and methods 2.1. Generation of hybridoma cDNA mRNA was isolated from a hybridoma secreting an antivascular endothelial growth factor (VEGF) antibody (Tilton et al., 1997) (isotype IgG1κ) using a mRNA Catcher™ PLUS Kit (Invitrogen, Carlsbad, CA, USA) and converted to cDNA using Superscript III™ First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. 2.2. PCR amplification of VL and VH genes from hybridoma cDNA Amplifications were carried out using Elongase enzyme mix (Invitrogen, Carlsbad, CA, USA), which contains a DNA polymerase with proof-reading activity. Unless otherwise indicated, the VL backward/VL forward (LB/LF) or VH backward/ VH forward (HB/HF) primer sets and thermal cycling conditions described by Plückthun et al. (1996) and Krebber et al. (1997) were employed to obtain the immunoglobulin VL or VH genes, respectively. The LB and HB sets are complex mixtures of degenerate oligonucleotides containing 143 and 98 different sequences, respectively. The LF and HF sets are simpler, containing 5 and 4 oligonucleotides, respectively, which are homologous to the immunoglobulin JL and JH segments. The VL gene was amplified using the LB and LF primer sets. A specific peptide nucleic acid (PNA) inhibitor was included in the reaction to block amplification of the aberrant MOPC21 VLκ gene (Cochet et al., 1999) and the DNA extension phase was carried out at 60 °C rather than 72 °C to maintain binding of the PNA oligomer. The VH sequences were amplified either
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by using the HB and HF primer sets and standard thermal cycling conditions or alternatively by a two-stage PCR protocol. The first round used the HB primer set and each of the primers, HF1, HF2, HF3 or HF4, in separate reactions for 30 cycles of high stringency amplification, 92 °C for 1 min, 65 °C for 1 min, 72 °C for 1 min for 30 cycles. The products from each first round reaction were diluted ten fold and individually amplified for a second round, with similar cycling conditions, using the same HF primer and the HB primer set. 2.3. Sequence analysis VL and VH PCR products were gel purified (QIAquick gel extraction kit, Qiagen, Valencia, CA, USA) and sequenced bidirectionally using BigDye Terminator v3.1 Cycle Sequencing Kit and resolved using the ABI 3100 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). The sequences were assembled and assessed for functional translation using Vector NTI Advance™10 software (Invitrogen, Carlsbad, CA, USA) and were aligned to immunoglobulin sequences in the GenBank database using IgBLAST. 3. Results 3.1. Amplification of light chain variable gene The anti-VEGF hybridoma was created by fusing spleen cells from BALB/c mice immunised with recombinant human VEGF with cells of the non-secreting myeloma cell line P3X63-Ag.8.653 (Tilton et al., 1997). A functionally rearranged VL gene product was readily amplified by PCR from cDNA of this hybridoma using the LB and LF primer sets (Plückthun et al., 1996; Krebber et al., 1997), by including a PNA oligomer inhibitor specific for the MOPC21 Vκ gene (Cochet et al., 1999). 3.2. Amplification of aberrant heavy chain variable genes Previously, we have found that amplification of the functional VH gene from a number of hybridomas has been straightforward, however, this was not the case for the antiVEGF hybridoma. Attempted amplification of the VH gene using the Plückthun HB and HF primer sets consistently produced a PCR product smaller that expected, which was identified by sequencing as an unproductive VH gene rearrangement containing a 50 bp deletion that resulted in a frame shift. Sequence analysis also indicated that the 3' primer that was incorporated into this aberrant product was HF4, and the product was designated abVH-HF4 (Fig. 1). An attempt was made to amplify the functional VH gene from this hybridoma by omitting the HF4 primer from the HF primer mix, employing a mixture of forward primers, HF1, HF2 and HF3 in conjunction with the HB primer set. A PCR product of approximately 400 bp was obtained. Sequence analysis revealed this VH gene product appeared to be derived from a different unproductive VH gene rearrangement containing a change in the reading frame in the VDJ joining region encoding CDR3. The 3' primer that was incorporated into this aberrant product was HF3, and the product was designated abVH-HF3 (Fig. 1).
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Y. Irani et al. / Journal of Immunological Methods 336 (2008) 246–250
Fig. 1. Alignment of the DNA sequences (A), and predicted peptide sequences (B) of the non-functional VH gene rearrangements, abVH-HF4 and abVH-HF3, identified in an anti-VEGF hybridoma, and VH gene rearrangements from MOPC21 myeloma derived-cell lines, P3X63-Ag.8 (J00522) and P3X63-Ag.8.653 (X58634). PCR primer regions are underlined (A), CDR regions are indicated in bold and unproductive peptide sequence in grey text (B). Gaps are represented by dashes, identical bases or amino acids by ⁎, conserved amino acids by :, semi-conserved amino acids by a dot. The alignments were obtained using the ClustalW multiple sequence alignment program. GenBank accession nos: abVH-HF3, EU121635; abVH-HF4, EU121634.
3.3. Amplification of the functional heavy chain variable gene The functional VH gene product was detected and successfully amplified by employing each of the Plückthun HF primers, HF1, HF2, HF3 and HF4, individually in conjunction with the HB primer set in separate PCR reactions. As there is considerable homology between the individual HF primers, PCR was carried out at higher than usual stringency using a primer annealing temperature of 65 °C, to avoid misprimed amplification of the aberrant VH genes that appeared to be present in the cDNA in greater abundance than the functional VH gene. Under these conditions, the yield of PCR product generated with primers HF1 and HF3 was low and the aberrant product (abVH-HF4, ~350 bp) was obtained as expected using primer HF4 (Fig. 2). Performing a second round PCR using the same HF primer in conjunction with HB primer set generated substantial amounts of VH PCR products of the
expected size (~ 400 bp) with primers HF1 and HF3. No specific product was obtained with HF2 (Fig. 2). Sequence analysis of the HF1 and HF3 PCR products revealed they were derived from different VH gene rearrangements. Primer HF3 amplified the 400 bp aberrant VH gene rearrangement (abVH-HF3) already described. Primer HF1 amplified a potentially functional VH product containing an open reading frame that showed characteristic VH region features on translation. Assembly PCR was then used to splice together the functional VL and VH gene products to create a scFv gene according to the method of Plückthun et al. (1996) and Krebber et al. (1997). Sequence analysis of multiple clones indicated a scFv gene with a full length open reading frame. ScFv protein expression was confirmed in E. coli by detection of the C-terminal 6-Histidine tag. Binding of the recombinant scFv to human VEGF was demonstrated by ELISA (data not shown)
Y. Irani et al. / Journal of Immunological Methods 336 (2008) 246–250
Fig. 2. Amplification of multiple VH gene rearrangements from cDNA of an anti-VEGF hybridoma by PCR with selective primers homologous to the individual JH gene segments. The HB primer set was used in all PCR reactions in conjunction with the HF primer set (mix) or individual primers HF1 (1), HF2 (2), HF3 (3) or HF4 (4). First round and second round PCR products are indicated. Products were separated on 1% agarose gel; DNA size marker (M) fragment sizes are indicated in base pairs (bp).
and confirmed the preservation of the antigen-binding specificity of the parental antibody. 4. Discussion Amplification of the functional VL gene rearrangement from the anti-VEGF hybridoma was achieved without difficulty. The myeloma cell line P3X63-Ag.8.653 (Kearney et al., 1979) was derived from the MOPC21 myeloma that is known to express an aberrantly rearranged Vκ gene (GenBank accession no. M35669). This aberrant transcript is often abundantly expressed in hybridomas generated with P3X63Ag.8.653 and other MOPC21-derived myeloma cell lines as the fusion partner, and is commonly amplified by PCR in preference to the functional VL gene when complex primer mixtures are used. We have found that the routine use of a PNA inhibitor specific for the aberrant MOPC21 Vκ gene has simplified amplification of the functional VL genes from many hybridomas generated using P3X63-Ag.8.653. Amplification of the functional VH gene rearrangement from the anti-VEGF hybridoma proved more difficult. This hybridoma contained three different VH gene rearrangements, which were detected by selective use of the individual Plückthun HF primers. The two unproductive VH gene rearrangements were amplified preferentially, possibly because they were in greater abundance than the functional VH transcript and, in the case of the aberrant product abVH-HF4, because of its shorter length. The unproductive rearrangement abVH-HF4 showed 99% homology to the active H-chain V-region of MOPC21γ1 (GenBank accession no. J00522, V(MOPC21)-D(SP2.5,-7-8)J4), except for a deletion of 50 bp extending from the middle of FWR3 to the VDJ joining region in CDR3 (Fig. 1A). The cell line P3X63-Ag.8, from which the P3X63-Ag.8.653 cell line was derived, secreted MOPC21γ1κ immunoglobulin (Kearney et al., 1979). GenBank contains one report of this heavy chain gene rearrangement, V(MOPC21)-D(SP2.5,-7-8)-J4, that was unproductive due to an out-of-frame DVJ joining, but this
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did not contain a deletion (GenBank accession no. M18314). However, we were surprised to find seven distinct GenBank entries homologous to the abVH-HF4 rearrangement that contained the same deletion that we observed. These sequences had been derived by PCR amplification from cDNA of hybridomas that secreted functional antibodies (GenBank accession nos. AF089740, AF019945, AF230099, DQ355823, X80944, X80954) and two had been incorporated into engineered scFvs (AF089742, AF039853). In some instances the homology to the active MOPC21γ1 VH had been recognised and discussed (X80944, X80954, AF039853). It seems unlikely that the same non-functional heavy chain rearrangement produced a protein that was functional in antibodies with diverse specificities. We suggest that it is likely that a nonfunctional VH rearrangement from the fusion partner was mistakenly amplified by PCR in most cases. The second unproductive rearrangement, abVH-HF3, showed 98% homology to a previously described aberrantly rearranged heavy chain gene (GenBank accession no. X58634) believed to be derived from the fusion partner P3X63Ag8.653 (Fig. 1). This aberrant rearrangement, V(Q52.8.22)D(?)-J3, has also been recognised by others as coming from a fusion partner (GenBank accession nos. D50398, S65377, Krebber et al., 1997). Again we discovered other VH sequences in the GenBank database, homologous to the abVH-HF3 with out-of-frame VDJ joining, that have been amplified by PCR from cDNA of hybridomas that secreted functional antibodies (GenBank accession nos. D14170, D14171, D14173). It is probable that both unproductive VH gene rearrangements detected in the anti-VEGF hybridoma derived from the P3X63-Ag.8.653 fusion partner that was used to generate this hybridoma. Indeed it seems that it is relatively common for the unproductive VH genes present in fusion partners derived from the MOPC21 myeloma to be amplified preferentially by PCR from hybridomas, and we suggest that in some instances these have been mistaken for the functional VH of an antibody. A number of strategies are used to circumvent the problem of preferential amplification of the aberrant MOPC21 VLκ gene present in myeloma fusion partners. Here we highlight a similar problem with two aberrant MOPC21 VH gene rearrangements and describe the simple strategy that we employed to obtain the functional VH gene from an antiVEGF hybridoma. This strategy is unlikely to be useful where the functional VH gene incorporates the same J segment as the aberrant VH genes. For such situations, strategies analogous to those used to suppress the amplification of the aberrant MOPC21 VLκ gene, described above, would be more useful for reliable identification of the functional VH gene. Acknowledgements This work was supported by the National Health and Medical Research Council of Australia, the Ophthalmic Research Institute of Australia, and the Flinders Medical Centre Foundation. The authors wish to thank Oliver van Wageningen for DNA sequencing. References Carroll, W.L., Mendel, E., Levy, S., 1988. Hybridoma fusion cell lines contain an aberrant kappa transcript. Mol. Immunol. 25, 991.
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Cochet, O., Martin, E., Fridman, W.H., Teillaud, J., 1999. Selective PCR amplification of functional immunoglobulin light chain from hybridoma containing the aberrant MOPC 21-derived Vκ by PNA-mediated PCR clamping. BioTechniques 26, 818. Juste, M., Muzard, J., Billiald, P., 2005. Cloning of the antibody κ light chain V-gene from murine hybridomas by bypassing the aberrant MOPC21derived transcript. Anal. Biochem. 349, 159. Kearney, J.F., Radbruch, A., Liesegang, B., Rajewsky, K., 1979. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J. Immunol. 123, 1548. Krebber, A., Bornhauser, S., Burmester, J.J., Honegger, A., Willuda, J., Bosshard, H.R., Plückthun, A., 1997. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J. Immunol. Methods 201, 35. Kütemeier, G., Harloff, C., Mocikat, R., 1992. Rapid isolation of immunoglobulin variable genes from cell lysates of rat hybridomas by polymerase chain reaction. Hybridoma 11, 23. Nichols, P.J., Johnson, V.G., Blanford, M.D., Andrew, S.M., 1993. An improved method for generating single-chain antibodies from hybridomas. J. Immunol. Methods 165, 81.
Ostermeier, C., Michel, H., 1996. Improved cloning of antibody variable regions from hybridomas by an antisense-directed RHase H digestion of the P3-X63-Ag8.653 derived pseudogene mRNA. Nucleic Acids Res. 24, 1979. Plückthun, A., Krebber, A., Krebber, C., 1996. Producing antibodies in Escherichia coli: from PCR to fermentation. In: McCafferty, J., Hoogenboom, H.R., Chiswell, D.J. (Eds.), Antibody engineering: a practical approach. Oxford University Press, Oxford, p. 203. Tilton, R.G., Kawamura, T., Chang, K.C., Ido, Y., Bjercke, R.J., Stephan, C.C., Brock, T.A., Williamson, J.R., 1997. Vascular dysfunction induced by elevated glucose levels in rats is mediated by vascular endothelial growth factor. J. Clin. Invest. 99, 2192. Vidarsson, G., van de Winkel, J.G.J., van Dijk, M.A., 2001. Multiplex screening for functionally rearranged immunoglobulin variable regions reveals expression of hybridoma-specific aberrant V-genes. J. Immunol. Methods 249, 245.
Contents lists available at ScienceDirect
Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j i m
Technical note
PCR amplification of the functional immunoglobulin heavy chain variable gene from a hybridoma in the presence of two aberrant transcripts Yazad Irani a,1, Melinda Tea a,1, Ronald G. Tilton b, Douglas J. Coster a, Keryn A. Williams a, Helen M. Brereton a,⁎ a b
Department of Ophthalmology, Flinders University of South Australia, Adelaide, SA 5042, Australia Division of Endocrinology and Stark Diabetes Center, University of Texas Medical Branch, Galveston, TX 77555-1060, USA
a r t i c l e
i n f o
Article history: Received 10 September 2007 Received in revised form 4 March 2008 Accepted 19 April 2008 Available online 16 May 2008 Keywords: scFv Hybridoma Myeloma fusion partner P3X63-Ag8.653 Unproductive immunoglobulin rearrangement MOPC21
a b s t r a c t Single chain antibody fragment genes are commonly created by splicing together the immunoglobulin light chain (VL) and heavy chain variable (VH) genes of a monoclonal antibody produced by a hybridoma. Selective PCR amplification of the functional immunoglobulin variable gene rearrangements can be complicated by the existence of other unproductive immunoglobulin gene rearrangements in the hybridoma. Here we report the detection and preferential amplification of aberrant transcripts from two unproductive VH gene rearrangements derived from the fusion partner of a hybridoma. The functional VH gene of the monoclonal antibody was successfully amplified by selective use of primers to individual JH segments. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Single chain antibody fragment (scFv) genes are commonly produced by splicing together the functional light (VL) and heavy chain variable (VH) genes amplified by reverse transcriptase (RT)-PCR from a hybridoma that secretes antibody of the required specificity (Plückthun et al., 1996). The presence of non-functional variable gene transcripts in the hybridoma is a recognised phenomenon that can cause considerable problems with the selective amplification of the
Abbreviations: VL, immunoglobulin light chain variable gene; VH, immunoglobulin heavy chain variable gene; scFv, single chain antibody fragment; CDR, complementarity determining region; VEGF, vascular endothelial growth factor; PNA, peptide nucleic acid. ⁎ Corresponding author. Department of Ophthalmology, Flinders University, Bedford Park, SA 5042, Australia. Tel.: +61 8 8204 4123; fax: +61 8 8277 0899. E-mail address: helen.brereton@flinders.edu.au (H.M. Brereton). 1 Contributed equally to this work. 0022-1759/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2008.04.014
functional transcripts (Krebber et al., 1997). Aberrant variable gene transcripts can arise either from an unproductively rearranged allele(s) present in the B cell (Vidarsson et al., 2001) or from the non-secreting myeloma fusion partner used in the production of the hybridoma, and can often be in much greater abundance than the functional variable gene transcripts (Carroll et al., 1988). A very well known example is the aberrant VLκ allele present in MOPC21 myeloma-derived fusion partners, which is frequently amplified by PCR in preference to the functional VL of the antibody, and several strategies have been devised to overcome this problem. The aberrant rearrangement of the MOPC21κ allele causes a frameshift that results in a premature stop codon. Nichols et al. (1993) screened scFv clones by expression in a coupled transcription/ translation system in vitro, and distinguished between those containing the functional and aberrant VL genes on the basis of the size of the protein produced. Ostermeier and Michel (1996) used a strategy involving antisense-directed RNase H digestion of the unproductive mRNA prior to PCR to improve recovery of the functional VL gene. In a similar vein, a peptide nucleic acid
Y. Irani et al. / Journal of Immunological Methods 336 (2008) 246–250
(PNA) clamp specific for the MOPC21κ allele was used during PCR to suppress amplification of the non-functional VL gene (Cochet et al., 1999). Yet another strategy involved the identification of a restriction enzyme that was used for selective degradation of MOPC21κ-derived product after PCR amplification and prior to cloning and sequencing (Juste et al., 2005). Amplification of aberrant VH genes has been reported (Kütemeier et al., 1992; Krebber et al., 1997; Vidarsson et al., 2001), however, the problem appears more diverse and not as widely recognised as is that of the MOPC21κ allele. Two general strategies have been proposed to avoid cloning aberrant V genes. Krebber et al. (1997) used phage display and selective panning against antigen to enrich for functional scFv clones, however, this is not a trivial exercise and requires considerable expertise. Alternatively a method for multiplex PCR screening of colonies using primers specific for the CDR3 region of aberrant V genes has been described (Vidarsson et al., 2001). In this case the sequence of the aberrant V gene rearrangement must be known or determined. During the recent generation of scFv from a specific hybridoma, the functional VL gene was readily amplified, however, amplification of the functional VH gene proved difficult. Here we report the presence of two aberrant VH transcripts derived from the fusion partner of the hybridoma and the strategy used to amplify the functional VH gene of the antibody. 2. Materials and methods 2.1. Generation of hybridoma cDNA mRNA was isolated from a hybridoma secreting an antivascular endothelial growth factor (VEGF) antibody (Tilton et al., 1997) (isotype IgG1κ) using a mRNA Catcher™ PLUS Kit (Invitrogen, Carlsbad, CA, USA) and converted to cDNA using Superscript III™ First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. 2.2. PCR amplification of VL and VH genes from hybridoma cDNA Amplifications were carried out using Elongase enzyme mix (Invitrogen, Carlsbad, CA, USA), which contains a DNA polymerase with proof-reading activity. Unless otherwise indicated, the VL backward/VL forward (LB/LF) or VH backward/ VH forward (HB/HF) primer sets and thermal cycling conditions described by Plückthun et al. (1996) and Krebber et al. (1997) were employed to obtain the immunoglobulin VL or VH genes, respectively. The LB and HB sets are complex mixtures of degenerate oligonucleotides containing 143 and 98 different sequences, respectively. The LF and HF sets are simpler, containing 5 and 4 oligonucleotides, respectively, which are homologous to the immunoglobulin JL and JH segments. The VL gene was amplified using the LB and LF primer sets. A specific peptide nucleic acid (PNA) inhibitor was included in the reaction to block amplification of the aberrant MOPC21 VLκ gene (Cochet et al., 1999) and the DNA extension phase was carried out at 60 °C rather than 72 °C to maintain binding of the PNA oligomer. The VH sequences were amplified either
247
by using the HB and HF primer sets and standard thermal cycling conditions or alternatively by a two-stage PCR protocol. The first round used the HB primer set and each of the primers, HF1, HF2, HF3 or HF4, in separate reactions for 30 cycles of high stringency amplification, 92 °C for 1 min, 65 °C for 1 min, 72 °C for 1 min for 30 cycles. The products from each first round reaction were diluted ten fold and individually amplified for a second round, with similar cycling conditions, using the same HF primer and the HB primer set. 2.3. Sequence analysis VL and VH PCR products were gel purified (QIAquick gel extraction kit, Qiagen, Valencia, CA, USA) and sequenced bidirectionally using BigDye Terminator v3.1 Cycle Sequencing Kit and resolved using the ABI 3100 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). The sequences were assembled and assessed for functional translation using Vector NTI Advance™10 software (Invitrogen, Carlsbad, CA, USA) and were aligned to immunoglobulin sequences in the GenBank database using IgBLAST. 3. Results 3.1. Amplification of light chain variable gene The anti-VEGF hybridoma was created by fusing spleen cells from BALB/c mice immunised with recombinant human VEGF with cells of the non-secreting myeloma cell line P3X63-Ag.8.653 (Tilton et al., 1997). A functionally rearranged VL gene product was readily amplified by PCR from cDNA of this hybridoma using the LB and LF primer sets (Plückthun et al., 1996; Krebber et al., 1997), by including a PNA oligomer inhibitor specific for the MOPC21 Vκ gene (Cochet et al., 1999). 3.2. Amplification of aberrant heavy chain variable genes Previously, we have found that amplification of the functional VH gene from a number of hybridomas has been straightforward, however, this was not the case for the antiVEGF hybridoma. Attempted amplification of the VH gene using the Plückthun HB and HF primer sets consistently produced a PCR product smaller that expected, which was identified by sequencing as an unproductive VH gene rearrangement containing a 50 bp deletion that resulted in a frame shift. Sequence analysis also indicated that the 3' primer that was incorporated into this aberrant product was HF4, and the product was designated abVH-HF4 (Fig. 1). An attempt was made to amplify the functional VH gene from this hybridoma by omitting the HF4 primer from the HF primer mix, employing a mixture of forward primers, HF1, HF2 and HF3 in conjunction with the HB primer set. A PCR product of approximately 400 bp was obtained. Sequence analysis revealed this VH gene product appeared to be derived from a different unproductive VH gene rearrangement containing a change in the reading frame in the VDJ joining region encoding CDR3. The 3' primer that was incorporated into this aberrant product was HF3, and the product was designated abVH-HF3 (Fig. 1).
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Y. Irani et al. / Journal of Immunological Methods 336 (2008) 246–250
Fig. 1. Alignment of the DNA sequences (A), and predicted peptide sequences (B) of the non-functional VH gene rearrangements, abVH-HF4 and abVH-HF3, identified in an anti-VEGF hybridoma, and VH gene rearrangements from MOPC21 myeloma derived-cell lines, P3X63-Ag.8 (J00522) and P3X63-Ag.8.653 (X58634). PCR primer regions are underlined (A), CDR regions are indicated in bold and unproductive peptide sequence in grey text (B). Gaps are represented by dashes, identical bases or amino acids by ⁎, conserved amino acids by :, semi-conserved amino acids by a dot. The alignments were obtained using the ClustalW multiple sequence alignment program. GenBank accession nos: abVH-HF3, EU121635; abVH-HF4, EU121634.
3.3. Amplification of the functional heavy chain variable gene The functional VH gene product was detected and successfully amplified by employing each of the Plückthun HF primers, HF1, HF2, HF3 and HF4, individually in conjunction with the HB primer set in separate PCR reactions. As there is considerable homology between the individual HF primers, PCR was carried out at higher than usual stringency using a primer annealing temperature of 65 °C, to avoid misprimed amplification of the aberrant VH genes that appeared to be present in the cDNA in greater abundance than the functional VH gene. Under these conditions, the yield of PCR product generated with primers HF1 and HF3 was low and the aberrant product (abVH-HF4, ~350 bp) was obtained as expected using primer HF4 (Fig. 2). Performing a second round PCR using the same HF primer in conjunction with HB primer set generated substantial amounts of VH PCR products of the
expected size (~ 400 bp) with primers HF1 and HF3. No specific product was obtained with HF2 (Fig. 2). Sequence analysis of the HF1 and HF3 PCR products revealed they were derived from different VH gene rearrangements. Primer HF3 amplified the 400 bp aberrant VH gene rearrangement (abVH-HF3) already described. Primer HF1 amplified a potentially functional VH product containing an open reading frame that showed characteristic VH region features on translation. Assembly PCR was then used to splice together the functional VL and VH gene products to create a scFv gene according to the method of Plückthun et al. (1996) and Krebber et al. (1997). Sequence analysis of multiple clones indicated a scFv gene with a full length open reading frame. ScFv protein expression was confirmed in E. coli by detection of the C-terminal 6-Histidine tag. Binding of the recombinant scFv to human VEGF was demonstrated by ELISA (data not shown)
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Fig. 2. Amplification of multiple VH gene rearrangements from cDNA of an anti-VEGF hybridoma by PCR with selective primers homologous to the individual JH gene segments. The HB primer set was used in all PCR reactions in conjunction with the HF primer set (mix) or individual primers HF1 (1), HF2 (2), HF3 (3) or HF4 (4). First round and second round PCR products are indicated. Products were separated on 1% agarose gel; DNA size marker (M) fragment sizes are indicated in base pairs (bp).
and confirmed the preservation of the antigen-binding specificity of the parental antibody. 4. Discussion Amplification of the functional VL gene rearrangement from the anti-VEGF hybridoma was achieved without difficulty. The myeloma cell line P3X63-Ag.8.653 (Kearney et al., 1979) was derived from the MOPC21 myeloma that is known to express an aberrantly rearranged Vκ gene (GenBank accession no. M35669). This aberrant transcript is often abundantly expressed in hybridomas generated with P3X63Ag.8.653 and other MOPC21-derived myeloma cell lines as the fusion partner, and is commonly amplified by PCR in preference to the functional VL gene when complex primer mixtures are used. We have found that the routine use of a PNA inhibitor specific for the aberrant MOPC21 Vκ gene has simplified amplification of the functional VL genes from many hybridomas generated using P3X63-Ag.8.653. Amplification of the functional VH gene rearrangement from the anti-VEGF hybridoma proved more difficult. This hybridoma contained three different VH gene rearrangements, which were detected by selective use of the individual Plückthun HF primers. The two unproductive VH gene rearrangements were amplified preferentially, possibly because they were in greater abundance than the functional VH transcript and, in the case of the aberrant product abVH-HF4, because of its shorter length. The unproductive rearrangement abVH-HF4 showed 99% homology to the active H-chain V-region of MOPC21γ1 (GenBank accession no. J00522, V(MOPC21)-D(SP2.5,-7-8)J4), except for a deletion of 50 bp extending from the middle of FWR3 to the VDJ joining region in CDR3 (Fig. 1A). The cell line P3X63-Ag.8, from which the P3X63-Ag.8.653 cell line was derived, secreted MOPC21γ1κ immunoglobulin (Kearney et al., 1979). GenBank contains one report of this heavy chain gene rearrangement, V(MOPC21)-D(SP2.5,-7-8)-J4, that was unproductive due to an out-of-frame DVJ joining, but this
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did not contain a deletion (GenBank accession no. M18314). However, we were surprised to find seven distinct GenBank entries homologous to the abVH-HF4 rearrangement that contained the same deletion that we observed. These sequences had been derived by PCR amplification from cDNA of hybridomas that secreted functional antibodies (GenBank accession nos. AF089740, AF019945, AF230099, DQ355823, X80944, X80954) and two had been incorporated into engineered scFvs (AF089742, AF039853). In some instances the homology to the active MOPC21γ1 VH had been recognised and discussed (X80944, X80954, AF039853). It seems unlikely that the same non-functional heavy chain rearrangement produced a protein that was functional in antibodies with diverse specificities. We suggest that it is likely that a nonfunctional VH rearrangement from the fusion partner was mistakenly amplified by PCR in most cases. The second unproductive rearrangement, abVH-HF3, showed 98% homology to a previously described aberrantly rearranged heavy chain gene (GenBank accession no. X58634) believed to be derived from the fusion partner P3X63Ag8.653 (Fig. 1). This aberrant rearrangement, V(Q52.8.22)D(?)-J3, has also been recognised by others as coming from a fusion partner (GenBank accession nos. D50398, S65377, Krebber et al., 1997). Again we discovered other VH sequences in the GenBank database, homologous to the abVH-HF3 with out-of-frame VDJ joining, that have been amplified by PCR from cDNA of hybridomas that secreted functional antibodies (GenBank accession nos. D14170, D14171, D14173). It is probable that both unproductive VH gene rearrangements detected in the anti-VEGF hybridoma derived from the P3X63-Ag.8.653 fusion partner that was used to generate this hybridoma. Indeed it seems that it is relatively common for the unproductive VH genes present in fusion partners derived from the MOPC21 myeloma to be amplified preferentially by PCR from hybridomas, and we suggest that in some instances these have been mistaken for the functional VH of an antibody. A number of strategies are used to circumvent the problem of preferential amplification of the aberrant MOPC21 VLκ gene present in myeloma fusion partners. Here we highlight a similar problem with two aberrant MOPC21 VH gene rearrangements and describe the simple strategy that we employed to obtain the functional VH gene from an antiVEGF hybridoma. This strategy is unlikely to be useful where the functional VH gene incorporates the same J segment as the aberrant VH genes. For such situations, strategies analogous to those used to suppress the amplification of the aberrant MOPC21 VLκ gene, described above, would be more useful for reliable identification of the functional VH gene. Acknowledgements This work was supported by the National Health and Medical Research Council of Australia, the Ophthalmic Research Institute of Australia, and the Flinders Medical Centre Foundation. The authors wish to thank Oliver van Wageningen for DNA sequencing. References Carroll, W.L., Mendel, E., Levy, S., 1988. Hybridoma fusion cell lines contain an aberrant kappa transcript. Mol. Immunol. 25, 991.
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