Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction

doi:10.1016/0022-1759(92)90092-8

Journal of Immunological Methods

Volume 152, Issue 1, 31 July 1992, Pages 89-104

https://doi.org/10.1016/0022-1759(92)90092-8 Get rights and content

Abstract

A new family of vectors has been produced which facilitates the cloning and expression of immunoglobulin variable regions cloned by polymerase chain reaction (PCR). The vectors are designed to express the cloned variable regions joined to human constant regions and take advantage of priming in the leader sequence so that no amino acid changes will be introduced into the mature antibody molecule.

Both the heavy chain and light chain vectors utilize a murine V₁₁ promoter provided with an EcoRV restriction site so that the amplified variable regions can be directly cloned into a functional promoter. For the heavy chain an NheI restriction site has been generated at the first two amino acids of C₁₁l and the cloned leader and variable region are fused directly to the C₁₁l domain of the constant region. When the leader and variable regions of the light chain were fused directly to C_κ, no expression was observed. Therefore the light chain expression vector was designed with a Sall restriction site for cloning into a splice junction 3′ of the variable region; V_L then is joined to C_κ by splicing. Both vectors direct the expression of functional. fully assembled immunoglobulin molecules with the expected molecular weight. Use of redundant oligomers to prime the PCR permits the cloning and expression of recombinant antibodies without any prior information as to their sequence and makes it possible to rapidly generate recombinant antibodies from any monoclonal antibody producing cell line.

References (31)

P. Chomczynski et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction
Anal. Biochem.
(1987)
S.D. Gillies et al.
High-level expression of chimeric antibodies using adapted cDNA variable region cassettes
J. Immunol. Methods
(1989)
S.N. Ho et al.
Site-directed mutagenesis by overlap extension using the polymerase chain reaction
Gene
(1989)
J.W. Larrick et al.
Rapid cloning of rearranged immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction
Biochem. Biophys. Res. Commun.
(1989)
R.D. LeBoeuf et al.
Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxy-ribonucleotides and polymerase chain reaction
Gene
(1989)
S.-U. Shin et al.
Production and properties of chimeric antibody molecules
Methods Enzymol.
(1989)
J. Xiang et al.
Production of murine V-human Cr1 chimeric anti-Tag72 antibody using V region cDNA amplified by PCR
Mol. Immunol.
(1990)
A.G. Amit et al.
Three-dimensional structure of an antigen-antibody complex at 2.8 Å resolution
Science
(1986)
W.L. Caroll et al.
Hybridoma fusion cell lines contain an aberrant kappa transcript
Mol. Immunol.
(1988)
V.K. Chaudhary et al.
A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins

N.C. Chien et al.

Significant structural and functional change of an antigen-binding site by a distant amino acid substitution: Proposal of a structural mechanism

M.J. Coloma et al.

Primer design for the cloning of immunoglobulin heavy-chain leader-variable regions from mouse hybridoma cells using the PCR

BioTechniques

(1991)

J.V. Gavilondo-Cowley et al.

Specific amplification of rearranged immunoglobulin variable region genes from mouse hybridoma cells

Hybridoma

(1990)

S.D. Gillies et al.

Expression of human anti-tetanus toxoid antibody in transfected murine myeloma cells

BioTechnology

(1989)

S.C. Hartman et al.

Two dominant-acting selectable markers for gene transfer studies in mammalian cells

Cited by (164)

Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen
2018, Vaccine
Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 2040 patients and caused 712 deaths since its first appearance in 2012, yet neither pathogen-specific therapeutics nor approved vaccines are available. To address this need, we are developing a subunit recombinant protein vaccine comprising residues 377–588 of the MERS-CoV spike protein receptor-binding domain (RBD), which, when formulated with the AddaVax adjuvant, it induces a significant neutralizing antibody response and protection against MERS-CoV challenge in vaccinated animals. To prepare for the manufacture and first-in-human testing of the vaccine, we have developed a process to stably produce the recombinant MERS S377-588 protein in Chinese hamster ovary (CHO) cells. To accomplish this, we transfected an adherent dihydrofolate reductase-deficient CHO cell line (adCHO) with a plasmid encoding S377-588 fused with the human IgG Fc fragment (S377-588-Fc). We then demonstrated the interleukin-2 signal peptide-directed secretion of the recombinant protein into extracellular milieu. Using a gradually increasing methotrexate (MTX) concentration to 5 μM, we increased protein yield by a factor of 40. The adCHO-expressed S377-588-Fc recombinant protein demonstrated functionality and binding specificity identical to those of the protein from transiently transfected HEK293T cells. In addition, hCD26/dipeptidyl peptidase-4 (DPP4) transgenic mice vaccinated with AddaVax-adjuvanted S377-588-Fc could produce neutralizing antibodies against MERS-CoV and survived for at least 21 days after challenge with live MERS-CoV with no evidence of immunological toxicity or eosinophilic immune enhancement. To prepare for large scale-manufacture of the vaccine antigen, we have further developed a high-yield monoclonal suspension CHO cell line.
Over-expression of a human CD62L ecto-domain and a potential role of RNA pseudoknot structures in recombinant protein expression
2017, Protein Expression and Purification
Citation Excerpt :
We used the recently developed MSX-driven GS-system to amplify recombinant CD62L expression [8,9], similar to the DHFR gene amplification method [10,11]. Signal peptides are known to affect gene expression and some, such as those from rat serum albumin (RSA) and immunoglobin G (IgG), have been used to enhance recombinant protein expressions [8,12]. In attempts to over-express CD62L, we generated recombinant CD62L expression constructs using four different signal sequences in the same GS vector.
L-selectin (CD62L) is an extracellular protein with a lectin-like domain that mediates rolling adhesion of leukocytes to vascular endothelial cell surfaces. Currently, there are no solved structures for the ectodomain of CD62L, nor of CD62L in complex with its ligand. We have developed a rapid mammalian recombinant protein expression system using an amplifiable glutamine synthase based vector. Here, we further developed and applied this method to express and purify the entire extracellular region of CD62L. This resulted in excess of 20 mg/L yield of recombinant CD62L. In an attempt to understand the different expression levels among four similar CD62L constructs that differ primarily in signal sequences, we calculated the presence of potential RNA pseudoknots in their signal sequences. The results showed the presence of pseudoknots involving the start codon and between the signal sequence and gene in the mRNA of the non-expressing constructs, suggesting a potential inhibitory role of RNA pseudoknots in recombinant protein expression.
Overproduction of recombinant human transforming growth factor beta 3 in Chinese hamster ovary cells
2015, Protein Expression and Purification
Transforming growth factor beta 3 (TGFβ3) is an important cytokine, functioning in cell proliferation and differentiation, and has been considered to have therapeutic potential for treating various diseases and for scar reduction in adult wound healing. In the current study, a Chinese hamster ovary (CHO) cell line overexpressing recombinant human TGFβ3 (rhTGFβ3) was established. Through a 15-day fed-batch culture process in a 7.5-l bioreactor (5-l working volume) using chemically defined medium, the established cells could produce over 133 mg/l of rhTGFβ3 protein. The rhTGFβ3 was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure, resulting in a recovery rate of approximately 65%, with protein purity greater than 97%. The N-terminal amino acid sequences of the purified rhTGFβ3 were confirmed by N-terminal sequencing analysis. The purified rhTGFβ3 was further demonstrated to be functionally active by measuring the inhibition of growth of HT-2 cells, revealing a half-maximal effective concentration of 42.11 pg/ml and specific activity of 1.84 × 10⁷ U/mg.
Mesenchymal stromal cell delivery of full-length tumor necrosis factor-related apoptosis-inducing ligand is superior to soluble type for cancer therapy
2015, Cytotherapy
Citation Excerpt :
To create the flT vector, the flT-encoding complementary DNA (cDNA) was amplified by means of PCR with the use of our previously constructed inducible flT plasmid [10] as a template and inserted into the backbone in place of the green fluorescent protein (GFP) sequence through the use of BamHI and SalI sites; the resulting new plasmid is designated pCCL-CMV-flT. To create the sT vector, an open reading frame encoding an N-terminal–truncated extracellular portion of human TRAIL (amino acids 95–281) was amplified by means of PCR, which was then used as template for sequential PCRs to fuse the isoleucine zipper (IZ) (MKQIEDKIEEILSKIYHIENEIARIKKLIGERE) [30] in-frame and the murine immunoglobulin К-chain (IgК; 5′-ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGAC-3′) leader sequence [31] to its N-terminal. The obtained sT sequence was inserted into the pCCL-CMV-flT in place of flT through the BamHI and SalI sites, creating the sT vector designated pCCL-CMV-sT.
Mesenchymal stromal cell (MSC) delivery of pro-apoptotic tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an attractive strategy for anticancer therapy. MSCs expressing full-length human TRAIL (flT) or its soluble form (sT) have previously been shown to be effective for cancer killing. However, a comparison between the two forms has never been performed, leaving it unclear which approach is most effective. This study addresses the issue for the possible clinical application of TRAIL-expressing MSCs in the future.
MSCs were transduced with lentiviruses expressing flT or an isoleucine zipper-fused sT. TRAIL expression was examined and cancer cell apoptosis was measured after treatment with transduced MSCs or with MSC-derived soluble TRAIL.
The transduction does not adversely affect cell phenotype. The sT-transduced MSCs (MSC-sT) secrete abundant levels of soluble TRAIL but do not present the protein on the cell surface. Interestingly, the flT-transduced MSCs (MSC-flT) not only express cell-surface TRAIL but also release flT into medium. These cells were examined for inducing apoptosis in 20 cancer cell lines. MSC-sT cells showed very limited effects. By contrast, MSC-flT cells demonstrated high cancer cell-killing efficiency. More importantly, MSC-flT cells can overcome some cancer cell resistance to recombinant TRAIL. In addition, both cell surface flT and secreted flT are functional for inducing apoptosis. The secreted flT was found to have higher cancer cell-killing capacity than either recombinant TRAIL or MSC-secreted sT.
These observations demonstrate that MSC delivery of flT is superior to MSC delivery of sT for cancer therapy.
A cytotoxic humanized anti-ganglioside antibody produced in a murine cell line defective of N-glycolylated-glycoconjugates
2011, Immunobiology
Gangliosides containing the N-glycolyl (NGc) form of sialic acid are tumor-associated antigens and promising candidates for cancer therapy. We previously generated the murine 14F7 monoclonal antibody (mAb), specific for the N-glycolyl-GM3 ganglioside (NGcGM3), which induced an oncosis-like type of cell death on malignant cell lines expressing this antigen and recognized breast carcinoma by immunoscintigraphy in cancer patients. As humanization is expected to enhance its use for human cancer therapy, herein we describe the design and generation of two humanized versions of the 14F7 mAb by disrupting potential human T cell epitopes on its variable region. No differences in antigen reactivity or cytotoxic properties were detected among the variants tested and with respect to the chimeric counterpart. Humanized 14F7 genes were transfected into the NGcGM3-expressing NS0 cell line. Therefore, in the industrial scaling-up of the transfectoma in serum-free medium, cell viability was lost due to the cytotoxic effect of the secreted antibody. This shortcoming was solved by knocking down the CMP-N-acetylneuraminic acid hydroxylase enzyme, thus impairing the synthesis of NGc-glycoconjugates. Humanized 14F7 mAb is of potential value for the therapy of NGcGM3-expressing tumors.
Selective cell-surface labeling of the molecular motor protein prestin
2011, Biochemical and Biophysical Research Communications
Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity.

View all citing articles on Scopus

View full text

Article preview

Journal of Immunological Methods

Abstract

References (31)

Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction

Anal. Biochem.

High-level expression of chimeric antibodies using adapted cDNA variable region cassettes

J. Immunol. Methods

Site-directed mutagenesis by overlap extension using the polymerase chain reaction

Gene

Rapid cloning of rearranged immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction

Biochem. Biophys. Res. Commun.

Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxy-ribonucleotides and polymerase chain reaction

Gene

Production and properties of chimeric antibody molecules

Methods Enzymol.

Production of murine V-human Cr1 chimeric anti-Tag72 antibody using V region cDNA amplified by PCR

Mol. Immunol.

Three-dimensional structure of an antigen-antibody complex at 2.8 Å resolution

Science

Hybridoma fusion cell lines contain an aberrant kappa transcript

Mol. Immunol.

A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins

Significant structural and functional change of an antigen-binding site by a distant amino acid substitution: Proposal of a structural mechanism

Primer design for the cloning of immunoglobulin heavy-chain leader-variable regions from mouse hybridoma cells using the PCR

BioTechniques

Specific amplification of rearranged immunoglobulin variable region genes from mouse hybridoma cells

Hybridoma

Expression of human anti-tetanus toxoid antibody in transfected murine myeloma cells

BioTechnology

Two dominant-acting selectable markers for gene transfer studies in mammalian cells

Cited by (164)

Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen

Over-expression of a human CD62L ecto-domain and a potential role of RNA pseudoknot structures in recombinant protein expression

Overproduction of recombinant human transforming growth factor beta 3 in Chinese hamster ovary cells

Mesenchymal stromal cell delivery of full-length tumor necrosis factor-related apoptosis-inducing ligand is superior to soluble type for cancer therapy

A cytotoxic humanized anti-ganglioside antibody produced in a murine cell line defective of N-glycolylated-glycoconjugates

Selective cell-surface labeling of the molecular motor protein prestin

Research reportNovel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction

Abstract

Anal. Biochem.

J. Immunol. Methods

Gene

Biochem. Biophys. Res. Commun.

Gene

Methods Enzymol.

Mol. Immunol.

Three-dimensional structure of an antigen-antibody complex at 2.8 Å resolution

Science

Hybridoma fusion cell lines contain an aberrant kappa transcript

Mol. Immunol.

A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins

Significant structural and functional change of an antigen-binding site by a distant amino acid substitution: Proposal of a structural mechanism

Primer design for the cloning of immunoglobulin heavy-chain leader-variable regions from mouse hybridoma cells using the PCR

BioTechniques

Specific amplification of rearranged immunoglobulin variable region genes from mouse hybridoma cells

Hybridoma

Expression of human anti-tetanus toxoid antibody in transfected murine myeloma cells

BioTechnology

Two dominant-acting selectable markers for gene transfer studies in mammalian cells

Research report
Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction