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Applications of the immunoglobulin Cw fragment (IgCw) composed of the constant regions of heavy and light (CH and CL) chains
Biochemical and Biophysical Research Communications 512 (2019) 571e576
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Applications of the immunoglobulin Cw fragment (IgCw) composed of the constant regions of heavy and light (CH and CL) chains Minjae Kim a, b, Juho Choi a, b, Youngsil Seo a, b, Myung-Hee Kwon a, b, c, * a
Dept. of Biomedical Sciences, Graduate School, Ajou University, 206 World Cup-ro, Yeongtong-gu, Suwon, 16499, Gyeonggi-do, South Korea Dept. of Microbiology, Ajou University School of Medicine, 206 World Cup-ro, Yeongtong-gu, Suwon, 16499, Gyeonggi-do, South Korea c Genome Stability Institute, 206 World Cup-ro, Yeongtong-gu, Suwon, 16499, Gyeonggi-do, South Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 March 2019 Accepted 17 March 2019 Available online 23 March 2019
We report the production and application of a recombinant IgCw molecule, which is composed of only the constant domains of the heavy (CH) and light (CL) chains, lacking a variable (V) domain. We produced IgCw, especially human IgCw-gk (98 kDa), composed of two human Cg chains (37 kDa each) and two Ck chains (12 kDa each), using HEK293F cell culture. We found that the yield of IgCw-gk protein was ~20 mg/L, which was comparable to that of full-size IgG; it bound to Fcg receptor-positive cells with a low background noise on Fcg receptor-negative cells; and IgCw-gk can be used as a reference for measurement of Ig concentration. Moreover, Cg and Ck chains were easily isolated from IgCw-gk by a single step of affinity chromatography in the presence of a reducing agent. These results demonstrate that the IgCw molecule has the potential to be used for certain in vitro and in vivo applications as an alternative to an irrelevant isotype control IgG, and to be used a favorable antigen for acquiring isotypespecific antibodies by immunizing animals. © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: Antibody fragment IgCw Constant region Isotype control Reference antibody
1. Introduction Recombinant antibodies (Abs) and their fragments are now immensely valuable in biomedical science and clinical applications, because of the advantages of easy Ab gene manipulation in optimizing affinity, stability, productivity, and clinical efficacy [1,2]. Specifically, IgG isotype Abs, which are engineered to target clinically important proteins, are produced at a high yield by wellestablished manufacturing processes involving the use of mammalian cells [3], and are preferred over other isotypes in the production of recombinant Abs. This is because, aside from Fcmediated effector functions, the Fc region of IgG isotype Ab enables IgG Ab to bind to the neonatal Fc receptor (FcRn), which is expressed on vascular endothelial cells and mediates the recycling
abbreviations: Ab, antibody; AP, alkaline phosphatase; CH, constant domain of heavy chain; CL, constant domain of light chain; DTT, dithiothreitol; ELISA, enzymelinked immunosorbent assay; ER, endoplasmic reticulum; FcgR, receptor for Fcg; g chain, gamma chain; IgCw, Ig constant domain fragment; k chain, kappa chain; PBS, phosphate-buffered saline; PEI, polyethyleneimine; RT, room temperature; VH, variable domain of heavy chain; VL, variable domain of light chain. * Corresponding author. Department of Microbiology, Ajou University School of Medicine, 206 World Cup-ro, Yeongtong-gu, Suwon, 16499, South Korea. E-mail address: [email protected] (M.-H. Kwon).
and transcytosis of IgG, thereby increasing the plasma half-life of IgG in vivo [4]. To validate the in vitro and in vivo efficacy of recombinant human IgG, the reference human Abs (irrelevant isotype control Abs) are indispensable. Reference Abs could be isolated from human polyclonal Abs, or produced from the culture of cells transfected with expression constructs coding for the H and L chains of Abs. Although reference Abs are not expected to bind to a target molecule, the possibility for cross-reactivity resulting in noisy positive signal cannot be completely disregarded, as long as reference Ig possesses the variable (V) domains responsible for antigen-binding. Here, we report a human Ig fragment called IgCw-gk (~98 kDa), which is composed of gamma 1 constant regions of the heavy (CH) and kappa light (CL) chains. The overall structure is similar to that of natural IgG1 (~150 kDa), except for the absence of the variable domains of the heavy (VH) and light (VL) chains. We anticipated the advantages of IgCw-gk to be as follows: (1) it will give consistent production yield, unlike full-size IgG, for which the production yield varies depending on the amino acid sequence of V regions, (2) it can be a reference molecule for Ab activities under certain research conditions, on behalf of the full-size IgG isotype control, and (3) it can be used as an immunogen to acquire isotype-specific Abs by excluding the possibility for unwanted generation of anti-
https://doi.org/10.1016/j.bbrc.2019.03.108 0006-291X/© 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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idiotypic Abs, which are raised against V regions. We produced IgCw-gk protein using HEK293F cell culture and examined the possibility for its use as an alternative to irrelevant full-size IgG in biomedical research, diagnostic testing, and immunogen preparation for generating anti-isotypic Abs. This work shows that IgCw-gk could have varied applications in the field of biomedical science. 2. Materials and methods 2.1. Plasmid vectors The DNA fragment encoding human Cg (Cg1-Cg2-Cg3) was cloned into the expression vector KV10-HL, which encodes the gene for MluI-bacterial ccdB-NheI-human Cg-BamHI and the gene for DraIII-LacZa-BstWI human Ck-EcoRI, between restriction sites MluI and BamHI, resulting in the construction of vector KV10-Cg. Then DNA fragment encoding human Ck gene was cloned into the KV10Cg between restriction sites DraIII and EcoRI, generating vector KV10-IgCw-gk, which contains human Cg1-3 and human Ck genes under the control of two individual CMV promoters (PCMV), which allow simultaneous expression of Cg and Ck chains with a leader sequence. To construct the plasmid vector for IgG (chimeric 6C406 and 3D8) expression, DNA fragments encoding the VH and VK domains were cloned into the KV10-HL between restriction sites MluI and NheI, and between DraIII and BstWI, respectively. 2.2. Preparation of Ig proteins using HEK293F cells The FreeStyle™ 293-F cell line (Thermo Fisher Scientific), a cell line adapted to grow in a suspension culture in serum-free conditions, was used for Ab production. FreeStyle HEK293F cells (100 ml of 1 106 cells/ml) were seeded 24 h prior to transfection in a 500 ml flask (Corning Cat# 431145) to ensure that cells reached the appropriate cell density (2 106 cells/ml) at the time of transfection. FreeStyle 293-F cells were cultured in serum-free FreeStyle 293 media (Invitrogen Cat# 12338) at 8% CO2 and 37 C, with shaking at 130 rpm. KV10 plasmids encoding 6C407 IgG1, IgCw-gk, Cg, and Ck were transiently introduced into 100 ml of FreeStyle 293-F cells using polyethylenimine (PEI) reagent (Polyscience Cat# 23966e2). Briefly, PEI reagent (400 mg) was incubated with plasmid DNA (200 mg) at room temperature for 10 min, and then inoculated into 100 ml of cells to achieve a final PEI concentration of 4 mg/ml. After 7 days, the culture supernatant was harvested by centrifugation. From the supernatants, 6C407 IgG1, IgCw-gk, and Cg proteins were purified by affinity chromatography using Protein A (GE Healthcare; cat# 17-1279-02) according to the manufacturer's guide. The purification of Ck was attempted using a CaptureSelect™ KappaXL column (Thermo Fisher Scientific; cat# 194321005). If necessary, IgCw-g/k was also purified by affinity chromatography using a CaptureSelect™ KappaXL column. The concentrations of the purified proteins were determined by absorbance at 280 nm and the extinction coefficients of 1.43 for 6C407 IgG1, 1.19 for IgCw-gk, 1.27 for Cg, and 0.97 for Ck, in units of (mg ml)1 cm1 at 280 nm, were calculated from the respective amino acid sequences (http:// web.expasy.org/protparam/). 2.3. Cell culture Human epithelial cervical carcinoma-derived cell line HeLa (ATCC® number: CCL-2™) and human embryonic kidney 293 cell line HEK293T (ATCC® number: CRL-3216™) were maintained in Dulbecco's Modified Eagle's Medium (DMEM). Human monocytic cell line THP-1 (ATCC® number: TIB-202) and human Burkitt's lymphoma cell line Raji (ATCC® number: CCL-86™) were maintained in RPMI 1640 medium. DMEM and RPMI 1640 were
supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. All cells were cultured at 37 C/5% CO2. 2.4. Flow cytometry To detect the binding of IgCw-gk to FcgR expressed on the cell surface, FcgR HEK293T, FcgR HeLa, FcgRsþ THP-1, and FcgRIIBþ Raji cells (1 106 cells) were treated for 30 min at 4 C with Ig proteins (final concentration, 3 mM). After washing with cold phosphate-buffered saline (PBS), cells were fixed for 10 min at room temperature (RT) with 4% paraformaldehyde in PBS. The pair of HEK293T and THP-1 cells was incubated with a primary goat anti-human kappa chain Ab (Thermo Fisher Scientific; cat# 31129), followed by a secondary FITC-conjugated rabbit anti-goat IgG Ab (Thermo Fisher Scientific; cat# 31533) diluted in buffer S (0.5% BSA and 2 mM EDTA prepared in PBS, pH 8.5). Both HeLa and Raji cells were incubated with a primary rabbit anti-human kappa chain Ab (Abcam; cat# 134083), followed by a secondary FITC-conjugated donkey anti-rabbit IgG-FITC (Millipore; cat# AP182F) diluted in buffer S. Incubation with each antibody was conducted for 1 h at 4 C. After three washes with cold PBS, cells were finally analyzed by flow cytometry using a FACS Canto II cytometer (BD Biosciences). 2.5. Separation of CH and CL chains from IgCw-gk protein Reducing reagent DTT was added to IgCw-gk protein (0.5 mg/ml in PBS) to achieve a final DTT concentration of 20 mM. The protein solution was directly loaded to the Protein A column to trap the Cg chain. The flow-through containing the Ck chain was collected in a tube. The column was washed four times with PBS containing 1 mM DTT, to remove the residual Ck chain that can be co-purified in the elution step. Cg protein was eluted from the Protein A column with 100 mM glycine HCl at pH 3.0. The eluted fractions were pooled, and the buffer was changed to PBS, pH 7.4, using 3 kDa molecular mass cutoff membrane filtration tubes (Sartorius; #cat VS0691). The obtained proteins were run on an SDS-polyacrylamide gel under reducing and non-reducing conditions, followed by Coomassie Blue staining or immunoblotting. 2.6. ELISA to assess IgG concentration Enzyme-linked immunosorbent assay (ELISA) was performed to generate standard curves using known concentrations of full-size IgG (Sigma-Aldrich; cat# I8640) and IgCw-gk, respectively. Wells of a 96-well polystyrene plate were coated with 100 ml (2 mg/ml) of goat anti-human IgG/Fc Ab (Abcam; cat# ab97221) for 1 h at RT, washed three times with TRIS-buffered saline (TBS, 50 mM TRIS-Cl, 50 mM NaCl, pH 7.4) containing 0.05% Tween-20 (TBST), and blocked with 3% BSA for 1 h at RT. Wells were subsequently incubated with a two-fold dilution with a starting concentration of 250 ng/ml of polyclonal human IgG and IgCw-gk, respectively, for 1 h at RT, followed by alkaline phosphatase (AP)-conjugated goat anti-human Ck Ab F (ab')2 (Rockland; cat# 709e1510). Each incubation step was followed by washing three times with TBST. Finally, p-nitrophenyl phosphate (Sigma-Aldrich; cat# N2765) solution (1 mg/ml in 0.1 M glycine, 1 mM ZnCl2, and 1 mM MgCl2, pH 10.3) was added to each well, and the absorbance at 405 nm was measured using a microplate reader (Molecular Devices). Otherwise, the plate coated with goat anti-human IgG/Fc Ab was incubated with an IgG sample, such as monoclonal 6C407 IgG1 and 3D8 IgG1, followed by AP-conjugated goat anti-human Ck Ab F (ab')2. The concentrations of IgG samples were determined by interpolating Y-axis values on two different standard curves, which were generated using known concentrations of human IgG and IgCw-gk, respectively.
M. Kim et al. / Biochemical and Biophysical Research Communications 512 (2019) 571e576
Fig. 1. Schematic representation of the Ig types produced using HEK293F cell culture.
3. Results 3.1. IgCw-gk is produced at a yield comparable to that of a full-size IgG We compared the yields of Ig molecules produced by suspension culture of the HEK293F cells transiently transfected with KV10IgCw-gk, KV10-6C407, KV10-Cg, and KV10-Ck plasmid, respectively. The expected structures of the expressed proteins are shown in Fig. 1. IgCw-gk, 6C407 IgG, and Cg proteins were purified from culture media using Protein A-Sepharose that binds to the Fc region of IgG on day 7 post-transfection, whereas Ck was purified using Kappa XL-Agarose that binds to human Ck domain. The average yield of IgCw-gk was estimated to be ~20 mg/L, which is equivalent to ~20 mg/L of a control 6C407 IgG. In contrast, the yield was much lower: ~0.6 mg/L for Cg and ~4 mg/L for Ck (summarized in Table 1). It shows the significant advantage of IgCw-gk over Cg protein only and Ck protein only in terms of the production yield. 3.2. Cg and Ck chains can be prepared easily from IgCw-gk On the SDS-PAGE gel, IgCw-gk are of the expected sizes of ~37 kDa and ~12 kDa under the reducing condition and ~98 kDa
Table 1 The yield of Ig molecules purified from the supernatant of HEK293F cell culture. Expression vector
Purification
Yield (mg/L)
KV10-6C407 IgG KV10-IgCw-gk KV10-Cg KV10-Ck
Protein A Protein A Protein A Kappa XL
~20 ~20 ~0.6 ~4
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under the non-reducing condition, indicating that the IgCw-gk preparation took a dimeric form, much like a full-size IgG. We hypothesized that Cg and Ck chains could be beneficial immunogens for acquiring isotype-specific Abs (anti-Cg and anti-Ck Abs, respectively). Thus, we set up the process for preparing each g and k chain from the purified IgCw-gk molecule. Each Cg and Ck chain was separately obtained by just two steps, 20 mM DTT treatment to reduce disulfide bonds and Protein A affinity chromatography to capture the g chain (Fig. 2A). One set of proteins of each step after SDS-PAGE was stained by Coomassie Blue (Fig. 2B), while the other sets were subjected to immunoblotting (Fig. 2C). The Ck chain was detected only in the flow-through fraction, and the Cg chain was detected only in the eluate (Fig. 2B and C), indicating complete chain separation. Thus IgCw-gk can be used to simultaneously and easily obtain both Cg and Ck chains by a single action of protein expression. 3.3. IgCw-gk binds to FcgR-positive cells with a low background noise on FcgR-negative cells We performed flow cytometric analyses to examine the IgCw-
gk binding to FcgRs expressed on the surface of human immune cells. IgCw-gk bound the immune cells THP-1 and Raji as full-size
IgGs do. No binding to HEK293T and HeLa cells that do not express any FcgRs was observed (Fig. 3AeD). Human monocytes/macrophages express FcgRI, and very low percentages or rare subsets express FcgRIIA or FcRn, whereas human B cells express only FcgRIIB [5]. This indicates that IgCw-gk can be recognized by various FcgRs, as full-size IgGs do. Moreover, the relative background signal from IgCw-gk on FcgR HEK293T cells against FcgRþ THP-1 cells was observed to be lower than that by full-size IgGs of monoclonal 6C407 IgG1 and polyclonal human IgGs (Fig. 3C and D). The lower background signal is likely to be due to the deficiency of V regions, which are responsible for antigen binding in the IgCw-gk structure. 3.4. IgCw-gk can be used as a reference for the quantification of IgG concentration We examined whether IgCw-gk can be an alternative to a reference full-size IgG that is used to determine IgG concentration. Two logarithmic standard curves were generated by ELISA using the data of known concentrations of human polyclonal IgG (Fig. 4A) and IgCw-gk (Fig. 4B). The same assay was then performed with
Fig. 2. Separation of CH and CL from the IgCw-gk molecule. (A) Representation of the chain separation steps. (B) SDS-PAGE for the proteins obtained from each step. (C) Western blotting for the proteins obtained from each step.
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Fig. 3. Binding of IgCw-gk to FcgRs expressed on the cell surface. (A, B) Flow cytometry. The Ig proteins were treated to the pair of FcgR HEK293T cells and FcgRþ THP-1 cells (A) and the pair of FcgR HeLa cells and FcgRIIBþ Raji cells (B) for 30 min at 4 C. Cell surface-bound Igs were detected using a primary goat anti-human kappa chain antibody, followed by a secondary FITC-conjugated rabbit anti-goat IgG antibody. ‘No protein’ indicates the cells treated with only primary and secondary antibodies. (C, D) Relative percentage of binding to FcgR cells compared with binding to FcgRþ cells, calculated from the absolute mean fluorescence intensity values of ‘A’ (C) and ‘B’ (D).
two monoclonal IgG samples (6C407 and 3D8) of unknown concentrations. The IgG concentration was determined by interpolation on the respective curves (Table 2), using the linear interpolation formula: x ¼ x1 þ (x2 - x1) (y - y1)/(y2 - y1) (https:// formulas.tutorvista.com/math/interpolation-formula.html). The IgG concentrations determined by the standard curve of IgCw-gk were multiplied by the ratio of the molecular weights (3:2 M ratio
of IgG:IgCw-gk) to normalize the values. The normalized concentrations of 6C407 IgG and 3D8 IgG were 30.6 ng/ml and 25.3 ng/ml, respectively, which are equivalent to the concentrations (29.7 ng/ ml for 6C407 IgG and 25.0 ng/ml for 3D8 IgG) determined by the standard curve using human IgG. This indicates that IgCw-gk can be used as a reference molecule for determining the human IgG concentration.
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Fig. 4. Standard curves generated using the respective human IgG and IgCw-gk. ELISA data from the right-hand panels were used to plot the log graphs. Human IgG and IgCw-g with known concentrations were placed in wells coated with goat anti-human IgG/Fc antibody as a capture antibody, and bound Ig molecules were detected with AP-conjugated goat anti-human Ck antibody F (ab')2 as a detection antibody. Data are presented as mean ± SD, n ¼ 3.
Table 2 IgG concentration determined using two standard curves. Samples of unknown concentration
Absorbance at 405 nm
Concentration (ng/ml) determined by interpolating y values on the standard curve using Human IgG
6C407 IgG 3D8 IgG a
0.323 0.288
29.7 25.0
IgCw-gk Not normalized
Normalized ( 1.5)a
20.4 16.9
30.6 25.3
Normalized by multiplying the original concentration with the ratio of the molecular weights [3:2 ratio of IgG (~150 kDa): IgCw-gk (~100 kDa)].
4. Discussion In this study, we report the applications of a previously undescribed IgCw-gk molecule, an Ig fragment composed of whole constant regions (Cg and Ck). Our data indicate the potential use of the IgCw-gk as an alternative to the IgG isotype used in biomedical research fields. Recombinant Abs and their derivatives have enormous potential as research reagents and therapeutics. So far, variable (V) domaincontaining Ab fragments that offer antigen-binding site (s), such as the fragment antigen-binding (Fab), the single-chain variable Ab fragment (scFv), and the single domain Ab (sdAb), have been generated [6]. V domain-containing Ab fragments could undergo further modification by functional moiety (drugs) conjugation to confer diagnostic and therapeutic benefits [7], and by signaling moieties of T cell receptors to develop chimeric antigen receptor T cells (CAR-T cells) [8]. However, the Fc fragment of IgG Ab, composed of hinge-CH2-CH3, has been used to produce Fc-fusion proteins in which the Fc domain is linked to a protein of interest to increase halflife in vivo [4]. It is worth noting that our study suggests the potential uses of the IgCw-gk reagent, a previously undescribed Ab format. Recombinant Ab formats have frequently faced difficulty with poor production yields, which have an impact on their utility and affordability. Production yields of Abs are dependent on the properties (amino acid sequences) of VH and VL regions. Thus, the production yield of recombinant IgG can be quite variable, depending on the amino acid sequence of V domains, even in a well-established process. It was possible to efficiently produce IgCw as disulfidelinked 2 heavy and 2 light chains, which is observed in natural IgG in HEK293F cell cultures. IgCw lacking in V regions should give an invariant production yield in the optimized host cell setting. The production yield of IgCw-gk (20 mg/L) at 7 days post-transfection was equivalent to a productive model IgG (6C407 Ab), approximately 30-fold and 5-fold higher than those of the Cg-only and Ckonly domains, respectively (Table 1). The production yield of IgCwgk is comparable to that of other Abs [9]. This suggests that IgCw-gk would be structurally stable during the folding and assembly process in the endoplasmic reticulum (ER), given the existence of the
ER-quality control system that permits the secretion of only correctly assembled Ig molecules [10]. Productive folding of CH1 occurs only after the interaction with the folded CL domain, i.e., the CL-induced folding of the CH1 domain [11,12]. This quality control system, first identified in B lineage cells [13], is known to function in Chinese hamster ovary (CHO) cells used for Ab production [14,15]. The ER quality control system would be expected to also work in HEK293F cells during the expression of IgCw-gk, though it allows the secretion of Cg-only proteins at low levels. IgCw-gk can be used as a much more favorable antigen for the production of anti-isotype Abs d which are raised against C regions (CH and CL) of Abs from a different species by immunizing animals d compared to full-size IgG. This is because IgCw-gk does not allow the generation of undesirable anti-idiotype Abs (raised against VH and VL domains), whereas immunization with full-size allows the generation of undesirable anti-idiotype Abs, as well as desired antiisotype Abs. Abs generated by immunization with IgG are usually affinity purified, and cross-adsorbed to remove any Abs that crossreacts with V regions. Using IgCw-gk, the high quality of antiisotype Abs with specific windows can be produced, by eliminating the chance to elicit Abs against V domains. Moreover, easy preparation of Cg and Ck chains from IgCw-gk (Fig. 2) makes antigen preparation for anti-Cg Ab and anti-Ck Ab straightforward. To validate certain effects by antigen-specific Abs, reference (or irrelevant isotype control) Abs that have no relevant specificity to a target antigen are commonly used as negative controls in many applications, to distinguish non-specific background signal from antigen-specific Ab signal. However, classical reference Abs could still give undesired background signals due to cross-reactivity not being fully defined. IgCw-gk could be a better reference in many experimental settings and situations, compared to an irrelevant IgG control, because it has no antigen binding capacity and can therefore be expected to exclude the possibility for unpredictable crossreactivity. Practically, the background signal for the binding to cell surface FcgRs was shown to be lower with IgCw-gk than with classical reference IgG (Fig. 3). Moreover, we showed that IgCw-gk is also sufficient to be used as a reference for Ig concentration measurement in biological samples (Fig. 4).
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If we showed the examples for several applications of the IgCw-
gk as an alternative to a reference IgG, it is possible to exploit other IgCw forms that have different isotypes of H and L chains. These include IgCw-gl composed of Cg and Cl, IgCw-εk composed of Cε and Ck, IgCw-εl composed of Cε and Cl, and so on. These IgCw molecules could also be considered as valuable reagents for in vitro and in vivo research. Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article. Author contributions MK, JC, and YS conducted the experiments and analyzed the results. M HK coordinated the study and wrote the paper. Funding source This work was supported by the Cooperative Research Program (PJ012534) of the Rural Development Administration of Korea. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.03.108. References [1] R.E. Kontermann, Alternative antibody formats, Curr. Opin. Mol. Ther. 12
(2010) 176e183. [2] S.J. Demarest, S.M. Glaser, Antibody therapeutics, antibody engineering, and the merits of protein stability, Curr. Opin. Drug Discov. Dev 11 (2008) 675e687. [3] R. Kunert, D. Reinhart, Advances in recombinant antibody manufacturing, Appl. Microbiol. Biotechnol. 100 (2016) 3451e3461. [4] D.C. Roopenian, S. Akilesh, FcRn: the neonatal Fc receptor comes of age, Nat. Rev. Immunol. 7 (2007) 715e725. [5] P. Bruhns, Properties of mouse and human IgG receptors and their contribution to disease models, Blood 119 (2012) 5640e5649. [6] T. Schirrmann, L. Al-Halabi, S. Dubel, M. Hust, Production systems for recombinant antibodies, Front. Biosci. 13 (2008) 4576e4594. [7] A. Beck, L. Goetsch, C. Dumontet, N. Corvaia, Strategies and challenges for the next generation of antibody-drug conjugates, Nat. Rev. Drug Discov. 16 (2017) 315e337. [8] C.H. June, R.S. O'Connor, O.U. Kawalekar, S. Ghassemi, M.C. Milone, CAR T cell immunotherapy for human cancer, Science 359 (2018) 1361e1365. [9] T.S. Dodev, P. Karagiannis, A.E. Gilbert, D.H. Josephs, H. Bowen, L.K. James, H.J. Bax, R. Beavil, M.O. Pang, H.J. Gould, S.N. Karagiannis, A.J. Beavil, A tool kit for rapid cloning and expression of recombinant antibodies, Sci. Rep. 4 (2014) 5885. [10] P.E. Mains, C.H. Sibley, The requirement of light chain for the surface deposition of the heavy chain of immunoglobulin M, J. Biol. Chem. 258 (1983) 5027e5033. [11] M.J. Feige, S. Groscurth, M. Marcinowski, Y. Shimizu, H. Kessler, L.M. Hendershot, J. Buchner, An unfolded CH1 domain controls the assembly and secretion of IgG antibodies, Mol. Cell 34 (2009) 569e579. [12] M. Vanhove, Y.K. Usherwood, L.M. Hendershot, Unassembled Ig heavy chains do not cycle from BiP in vivo but require light chains to trigger their release, Immunity 15 (2001) 105e114. [13] M.J. Feige, L.M. Hendershot, J. Buchner, How antibodies fold, Trends Biochem. Sci. 35 (2010) 189e198. [14] N. Borth, D. Mattanovich, R. Kunert, H. Katinger, Effect of increased expression of protein disulfide isomerase and heavy chain binding protein on antibody secretion in a recombinant CHO cell line, Biotechnol. Prog. 21 (2005) 106e111. [15] C.L. Stoyle, P.E. Stephens, D.P. Humphreys, S. Heywood, K. Cain, N.J. Bulleid, IgG light chain-independent secretion of heavy chain dimers: consequence for therapeutic antibody production and design, Biochem. J. 474 (2017) 3179e3188.
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Applications of the immunoglobulin Cw fragment (IgCw) composed of the constant regions of heavy and light (CH and CL) chains Minjae Kim a, b, Juho Choi a, b, Youngsil Seo a, b, Myung-Hee Kwon a, b, c, * a
Dept. of Biomedical Sciences, Graduate School, Ajou University, 206 World Cup-ro, Yeongtong-gu, Suwon, 16499, Gyeonggi-do, South Korea Dept. of Microbiology, Ajou University School of Medicine, 206 World Cup-ro, Yeongtong-gu, Suwon, 16499, Gyeonggi-do, South Korea c Genome Stability Institute, 206 World Cup-ro, Yeongtong-gu, Suwon, 16499, Gyeonggi-do, South Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 March 2019 Accepted 17 March 2019 Available online 23 March 2019
We report the production and application of a recombinant IgCw molecule, which is composed of only the constant domains of the heavy (CH) and light (CL) chains, lacking a variable (V) domain. We produced IgCw, especially human IgCw-gk (98 kDa), composed of two human Cg chains (37 kDa each) and two Ck chains (12 kDa each), using HEK293F cell culture. We found that the yield of IgCw-gk protein was ~20 mg/L, which was comparable to that of full-size IgG; it bound to Fcg receptor-positive cells with a low background noise on Fcg receptor-negative cells; and IgCw-gk can be used as a reference for measurement of Ig concentration. Moreover, Cg and Ck chains were easily isolated from IgCw-gk by a single step of affinity chromatography in the presence of a reducing agent. These results demonstrate that the IgCw molecule has the potential to be used for certain in vitro and in vivo applications as an alternative to an irrelevant isotype control IgG, and to be used a favorable antigen for acquiring isotypespecific antibodies by immunizing animals. © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: Antibody fragment IgCw Constant region Isotype control Reference antibody
1. Introduction Recombinant antibodies (Abs) and their fragments are now immensely valuable in biomedical science and clinical applications, because of the advantages of easy Ab gene manipulation in optimizing affinity, stability, productivity, and clinical efficacy [1,2]. Specifically, IgG isotype Abs, which are engineered to target clinically important proteins, are produced at a high yield by wellestablished manufacturing processes involving the use of mammalian cells [3], and are preferred over other isotypes in the production of recombinant Abs. This is because, aside from Fcmediated effector functions, the Fc region of IgG isotype Ab enables IgG Ab to bind to the neonatal Fc receptor (FcRn), which is expressed on vascular endothelial cells and mediates the recycling
abbreviations: Ab, antibody; AP, alkaline phosphatase; CH, constant domain of heavy chain; CL, constant domain of light chain; DTT, dithiothreitol; ELISA, enzymelinked immunosorbent assay; ER, endoplasmic reticulum; FcgR, receptor for Fcg; g chain, gamma chain; IgCw, Ig constant domain fragment; k chain, kappa chain; PBS, phosphate-buffered saline; PEI, polyethyleneimine; RT, room temperature; VH, variable domain of heavy chain; VL, variable domain of light chain. * Corresponding author. Department of Microbiology, Ajou University School of Medicine, 206 World Cup-ro, Yeongtong-gu, Suwon, 16499, South Korea. E-mail address: [email protected] (M.-H. Kwon).
and transcytosis of IgG, thereby increasing the plasma half-life of IgG in vivo [4]. To validate the in vitro and in vivo efficacy of recombinant human IgG, the reference human Abs (irrelevant isotype control Abs) are indispensable. Reference Abs could be isolated from human polyclonal Abs, or produced from the culture of cells transfected with expression constructs coding for the H and L chains of Abs. Although reference Abs are not expected to bind to a target molecule, the possibility for cross-reactivity resulting in noisy positive signal cannot be completely disregarded, as long as reference Ig possesses the variable (V) domains responsible for antigen-binding. Here, we report a human Ig fragment called IgCw-gk (~98 kDa), which is composed of gamma 1 constant regions of the heavy (CH) and kappa light (CL) chains. The overall structure is similar to that of natural IgG1 (~150 kDa), except for the absence of the variable domains of the heavy (VH) and light (VL) chains. We anticipated the advantages of IgCw-gk to be as follows: (1) it will give consistent production yield, unlike full-size IgG, for which the production yield varies depending on the amino acid sequence of V regions, (2) it can be a reference molecule for Ab activities under certain research conditions, on behalf of the full-size IgG isotype control, and (3) it can be used as an immunogen to acquire isotype-specific Abs by excluding the possibility for unwanted generation of anti-
https://doi.org/10.1016/j.bbrc.2019.03.108 0006-291X/© 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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idiotypic Abs, which are raised against V regions. We produced IgCw-gk protein using HEK293F cell culture and examined the possibility for its use as an alternative to irrelevant full-size IgG in biomedical research, diagnostic testing, and immunogen preparation for generating anti-isotypic Abs. This work shows that IgCw-gk could have varied applications in the field of biomedical science. 2. Materials and methods 2.1. Plasmid vectors The DNA fragment encoding human Cg (Cg1-Cg2-Cg3) was cloned into the expression vector KV10-HL, which encodes the gene for MluI-bacterial ccdB-NheI-human Cg-BamHI and the gene for DraIII-LacZa-BstWI human Ck-EcoRI, between restriction sites MluI and BamHI, resulting in the construction of vector KV10-Cg. Then DNA fragment encoding human Ck gene was cloned into the KV10Cg between restriction sites DraIII and EcoRI, generating vector KV10-IgCw-gk, which contains human Cg1-3 and human Ck genes under the control of two individual CMV promoters (PCMV), which allow simultaneous expression of Cg and Ck chains with a leader sequence. To construct the plasmid vector for IgG (chimeric 6C406 and 3D8) expression, DNA fragments encoding the VH and VK domains were cloned into the KV10-HL between restriction sites MluI and NheI, and between DraIII and BstWI, respectively. 2.2. Preparation of Ig proteins using HEK293F cells The FreeStyle™ 293-F cell line (Thermo Fisher Scientific), a cell line adapted to grow in a suspension culture in serum-free conditions, was used for Ab production. FreeStyle HEK293F cells (100 ml of 1 106 cells/ml) were seeded 24 h prior to transfection in a 500 ml flask (Corning Cat# 431145) to ensure that cells reached the appropriate cell density (2 106 cells/ml) at the time of transfection. FreeStyle 293-F cells were cultured in serum-free FreeStyle 293 media (Invitrogen Cat# 12338) at 8% CO2 and 37 C, with shaking at 130 rpm. KV10 plasmids encoding 6C407 IgG1, IgCw-gk, Cg, and Ck were transiently introduced into 100 ml of FreeStyle 293-F cells using polyethylenimine (PEI) reagent (Polyscience Cat# 23966e2). Briefly, PEI reagent (400 mg) was incubated with plasmid DNA (200 mg) at room temperature for 10 min, and then inoculated into 100 ml of cells to achieve a final PEI concentration of 4 mg/ml. After 7 days, the culture supernatant was harvested by centrifugation. From the supernatants, 6C407 IgG1, IgCw-gk, and Cg proteins were purified by affinity chromatography using Protein A (GE Healthcare; cat# 17-1279-02) according to the manufacturer's guide. The purification of Ck was attempted using a CaptureSelect™ KappaXL column (Thermo Fisher Scientific; cat# 194321005). If necessary, IgCw-g/k was also purified by affinity chromatography using a CaptureSelect™ KappaXL column. The concentrations of the purified proteins were determined by absorbance at 280 nm and the extinction coefficients of 1.43 for 6C407 IgG1, 1.19 for IgCw-gk, 1.27 for Cg, and 0.97 for Ck, in units of (mg ml)1 cm1 at 280 nm, were calculated from the respective amino acid sequences (http:// web.expasy.org/protparam/). 2.3. Cell culture Human epithelial cervical carcinoma-derived cell line HeLa (ATCC® number: CCL-2™) and human embryonic kidney 293 cell line HEK293T (ATCC® number: CRL-3216™) were maintained in Dulbecco's Modified Eagle's Medium (DMEM). Human monocytic cell line THP-1 (ATCC® number: TIB-202) and human Burkitt's lymphoma cell line Raji (ATCC® number: CCL-86™) were maintained in RPMI 1640 medium. DMEM and RPMI 1640 were
supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. All cells were cultured at 37 C/5% CO2. 2.4. Flow cytometry To detect the binding of IgCw-gk to FcgR expressed on the cell surface, FcgR HEK293T, FcgR HeLa, FcgRsþ THP-1, and FcgRIIBþ Raji cells (1 106 cells) were treated for 30 min at 4 C with Ig proteins (final concentration, 3 mM). After washing with cold phosphate-buffered saline (PBS), cells were fixed for 10 min at room temperature (RT) with 4% paraformaldehyde in PBS. The pair of HEK293T and THP-1 cells was incubated with a primary goat anti-human kappa chain Ab (Thermo Fisher Scientific; cat# 31129), followed by a secondary FITC-conjugated rabbit anti-goat IgG Ab (Thermo Fisher Scientific; cat# 31533) diluted in buffer S (0.5% BSA and 2 mM EDTA prepared in PBS, pH 8.5). Both HeLa and Raji cells were incubated with a primary rabbit anti-human kappa chain Ab (Abcam; cat# 134083), followed by a secondary FITC-conjugated donkey anti-rabbit IgG-FITC (Millipore; cat# AP182F) diluted in buffer S. Incubation with each antibody was conducted for 1 h at 4 C. After three washes with cold PBS, cells were finally analyzed by flow cytometry using a FACS Canto II cytometer (BD Biosciences). 2.5. Separation of CH and CL chains from IgCw-gk protein Reducing reagent DTT was added to IgCw-gk protein (0.5 mg/ml in PBS) to achieve a final DTT concentration of 20 mM. The protein solution was directly loaded to the Protein A column to trap the Cg chain. The flow-through containing the Ck chain was collected in a tube. The column was washed four times with PBS containing 1 mM DTT, to remove the residual Ck chain that can be co-purified in the elution step. Cg protein was eluted from the Protein A column with 100 mM glycine HCl at pH 3.0. The eluted fractions were pooled, and the buffer was changed to PBS, pH 7.4, using 3 kDa molecular mass cutoff membrane filtration tubes (Sartorius; #cat VS0691). The obtained proteins were run on an SDS-polyacrylamide gel under reducing and non-reducing conditions, followed by Coomassie Blue staining or immunoblotting. 2.6. ELISA to assess IgG concentration Enzyme-linked immunosorbent assay (ELISA) was performed to generate standard curves using known concentrations of full-size IgG (Sigma-Aldrich; cat# I8640) and IgCw-gk, respectively. Wells of a 96-well polystyrene plate were coated with 100 ml (2 mg/ml) of goat anti-human IgG/Fc Ab (Abcam; cat# ab97221) for 1 h at RT, washed three times with TRIS-buffered saline (TBS, 50 mM TRIS-Cl, 50 mM NaCl, pH 7.4) containing 0.05% Tween-20 (TBST), and blocked with 3% BSA for 1 h at RT. Wells were subsequently incubated with a two-fold dilution with a starting concentration of 250 ng/ml of polyclonal human IgG and IgCw-gk, respectively, for 1 h at RT, followed by alkaline phosphatase (AP)-conjugated goat anti-human Ck Ab F (ab')2 (Rockland; cat# 709e1510). Each incubation step was followed by washing three times with TBST. Finally, p-nitrophenyl phosphate (Sigma-Aldrich; cat# N2765) solution (1 mg/ml in 0.1 M glycine, 1 mM ZnCl2, and 1 mM MgCl2, pH 10.3) was added to each well, and the absorbance at 405 nm was measured using a microplate reader (Molecular Devices). Otherwise, the plate coated with goat anti-human IgG/Fc Ab was incubated with an IgG sample, such as monoclonal 6C407 IgG1 and 3D8 IgG1, followed by AP-conjugated goat anti-human Ck Ab F (ab')2. The concentrations of IgG samples were determined by interpolating Y-axis values on two different standard curves, which were generated using known concentrations of human IgG and IgCw-gk, respectively.
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Fig. 1. Schematic representation of the Ig types produced using HEK293F cell culture.
3. Results 3.1. IgCw-gk is produced at a yield comparable to that of a full-size IgG We compared the yields of Ig molecules produced by suspension culture of the HEK293F cells transiently transfected with KV10IgCw-gk, KV10-6C407, KV10-Cg, and KV10-Ck plasmid, respectively. The expected structures of the expressed proteins are shown in Fig. 1. IgCw-gk, 6C407 IgG, and Cg proteins were purified from culture media using Protein A-Sepharose that binds to the Fc region of IgG on day 7 post-transfection, whereas Ck was purified using Kappa XL-Agarose that binds to human Ck domain. The average yield of IgCw-gk was estimated to be ~20 mg/L, which is equivalent to ~20 mg/L of a control 6C407 IgG. In contrast, the yield was much lower: ~0.6 mg/L for Cg and ~4 mg/L for Ck (summarized in Table 1). It shows the significant advantage of IgCw-gk over Cg protein only and Ck protein only in terms of the production yield. 3.2. Cg and Ck chains can be prepared easily from IgCw-gk On the SDS-PAGE gel, IgCw-gk are of the expected sizes of ~37 kDa and ~12 kDa under the reducing condition and ~98 kDa
Table 1 The yield of Ig molecules purified from the supernatant of HEK293F cell culture. Expression vector
Purification
Yield (mg/L)
KV10-6C407 IgG KV10-IgCw-gk KV10-Cg KV10-Ck
Protein A Protein A Protein A Kappa XL
~20 ~20 ~0.6 ~4
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under the non-reducing condition, indicating that the IgCw-gk preparation took a dimeric form, much like a full-size IgG. We hypothesized that Cg and Ck chains could be beneficial immunogens for acquiring isotype-specific Abs (anti-Cg and anti-Ck Abs, respectively). Thus, we set up the process for preparing each g and k chain from the purified IgCw-gk molecule. Each Cg and Ck chain was separately obtained by just two steps, 20 mM DTT treatment to reduce disulfide bonds and Protein A affinity chromatography to capture the g chain (Fig. 2A). One set of proteins of each step after SDS-PAGE was stained by Coomassie Blue (Fig. 2B), while the other sets were subjected to immunoblotting (Fig. 2C). The Ck chain was detected only in the flow-through fraction, and the Cg chain was detected only in the eluate (Fig. 2B and C), indicating complete chain separation. Thus IgCw-gk can be used to simultaneously and easily obtain both Cg and Ck chains by a single action of protein expression. 3.3. IgCw-gk binds to FcgR-positive cells with a low background noise on FcgR-negative cells We performed flow cytometric analyses to examine the IgCw-
gk binding to FcgRs expressed on the surface of human immune cells. IgCw-gk bound the immune cells THP-1 and Raji as full-size
IgGs do. No binding to HEK293T and HeLa cells that do not express any FcgRs was observed (Fig. 3AeD). Human monocytes/macrophages express FcgRI, and very low percentages or rare subsets express FcgRIIA or FcRn, whereas human B cells express only FcgRIIB [5]. This indicates that IgCw-gk can be recognized by various FcgRs, as full-size IgGs do. Moreover, the relative background signal from IgCw-gk on FcgR HEK293T cells against FcgRþ THP-1 cells was observed to be lower than that by full-size IgGs of monoclonal 6C407 IgG1 and polyclonal human IgGs (Fig. 3C and D). The lower background signal is likely to be due to the deficiency of V regions, which are responsible for antigen binding in the IgCw-gk structure. 3.4. IgCw-gk can be used as a reference for the quantification of IgG concentration We examined whether IgCw-gk can be an alternative to a reference full-size IgG that is used to determine IgG concentration. Two logarithmic standard curves were generated by ELISA using the data of known concentrations of human polyclonal IgG (Fig. 4A) and IgCw-gk (Fig. 4B). The same assay was then performed with
Fig. 2. Separation of CH and CL from the IgCw-gk molecule. (A) Representation of the chain separation steps. (B) SDS-PAGE for the proteins obtained from each step. (C) Western blotting for the proteins obtained from each step.
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Fig. 3. Binding of IgCw-gk to FcgRs expressed on the cell surface. (A, B) Flow cytometry. The Ig proteins were treated to the pair of FcgR HEK293T cells and FcgRþ THP-1 cells (A) and the pair of FcgR HeLa cells and FcgRIIBþ Raji cells (B) for 30 min at 4 C. Cell surface-bound Igs were detected using a primary goat anti-human kappa chain antibody, followed by a secondary FITC-conjugated rabbit anti-goat IgG antibody. ‘No protein’ indicates the cells treated with only primary and secondary antibodies. (C, D) Relative percentage of binding to FcgR cells compared with binding to FcgRþ cells, calculated from the absolute mean fluorescence intensity values of ‘A’ (C) and ‘B’ (D).
two monoclonal IgG samples (6C407 and 3D8) of unknown concentrations. The IgG concentration was determined by interpolation on the respective curves (Table 2), using the linear interpolation formula: x ¼ x1 þ (x2 - x1) (y - y1)/(y2 - y1) (https:// formulas.tutorvista.com/math/interpolation-formula.html). The IgG concentrations determined by the standard curve of IgCw-gk were multiplied by the ratio of the molecular weights (3:2 M ratio
of IgG:IgCw-gk) to normalize the values. The normalized concentrations of 6C407 IgG and 3D8 IgG were 30.6 ng/ml and 25.3 ng/ml, respectively, which are equivalent to the concentrations (29.7 ng/ ml for 6C407 IgG and 25.0 ng/ml for 3D8 IgG) determined by the standard curve using human IgG. This indicates that IgCw-gk can be used as a reference molecule for determining the human IgG concentration.
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Fig. 4. Standard curves generated using the respective human IgG and IgCw-gk. ELISA data from the right-hand panels were used to plot the log graphs. Human IgG and IgCw-g with known concentrations were placed in wells coated with goat anti-human IgG/Fc antibody as a capture antibody, and bound Ig molecules were detected with AP-conjugated goat anti-human Ck antibody F (ab')2 as a detection antibody. Data are presented as mean ± SD, n ¼ 3.
Table 2 IgG concentration determined using two standard curves. Samples of unknown concentration
Absorbance at 405 nm
Concentration (ng/ml) determined by interpolating y values on the standard curve using Human IgG
6C407 IgG 3D8 IgG a
0.323 0.288
29.7 25.0
IgCw-gk Not normalized
Normalized ( 1.5)a
20.4 16.9
30.6 25.3
Normalized by multiplying the original concentration with the ratio of the molecular weights [3:2 ratio of IgG (~150 kDa): IgCw-gk (~100 kDa)].
4. Discussion In this study, we report the applications of a previously undescribed IgCw-gk molecule, an Ig fragment composed of whole constant regions (Cg and Ck). Our data indicate the potential use of the IgCw-gk as an alternative to the IgG isotype used in biomedical research fields. Recombinant Abs and their derivatives have enormous potential as research reagents and therapeutics. So far, variable (V) domaincontaining Ab fragments that offer antigen-binding site (s), such as the fragment antigen-binding (Fab), the single-chain variable Ab fragment (scFv), and the single domain Ab (sdAb), have been generated [6]. V domain-containing Ab fragments could undergo further modification by functional moiety (drugs) conjugation to confer diagnostic and therapeutic benefits [7], and by signaling moieties of T cell receptors to develop chimeric antigen receptor T cells (CAR-T cells) [8]. However, the Fc fragment of IgG Ab, composed of hinge-CH2-CH3, has been used to produce Fc-fusion proteins in which the Fc domain is linked to a protein of interest to increase halflife in vivo [4]. It is worth noting that our study suggests the potential uses of the IgCw-gk reagent, a previously undescribed Ab format. Recombinant Ab formats have frequently faced difficulty with poor production yields, which have an impact on their utility and affordability. Production yields of Abs are dependent on the properties (amino acid sequences) of VH and VL regions. Thus, the production yield of recombinant IgG can be quite variable, depending on the amino acid sequence of V domains, even in a well-established process. It was possible to efficiently produce IgCw as disulfidelinked 2 heavy and 2 light chains, which is observed in natural IgG in HEK293F cell cultures. IgCw lacking in V regions should give an invariant production yield in the optimized host cell setting. The production yield of IgCw-gk (20 mg/L) at 7 days post-transfection was equivalent to a productive model IgG (6C407 Ab), approximately 30-fold and 5-fold higher than those of the Cg-only and Ckonly domains, respectively (Table 1). The production yield of IgCwgk is comparable to that of other Abs [9]. This suggests that IgCw-gk would be structurally stable during the folding and assembly process in the endoplasmic reticulum (ER), given the existence of the
ER-quality control system that permits the secretion of only correctly assembled Ig molecules [10]. Productive folding of CH1 occurs only after the interaction with the folded CL domain, i.e., the CL-induced folding of the CH1 domain [11,12]. This quality control system, first identified in B lineage cells [13], is known to function in Chinese hamster ovary (CHO) cells used for Ab production [14,15]. The ER quality control system would be expected to also work in HEK293F cells during the expression of IgCw-gk, though it allows the secretion of Cg-only proteins at low levels. IgCw-gk can be used as a much more favorable antigen for the production of anti-isotype Abs d which are raised against C regions (CH and CL) of Abs from a different species by immunizing animals d compared to full-size IgG. This is because IgCw-gk does not allow the generation of undesirable anti-idiotype Abs (raised against VH and VL domains), whereas immunization with full-size allows the generation of undesirable anti-idiotype Abs, as well as desired antiisotype Abs. Abs generated by immunization with IgG are usually affinity purified, and cross-adsorbed to remove any Abs that crossreacts with V regions. Using IgCw-gk, the high quality of antiisotype Abs with specific windows can be produced, by eliminating the chance to elicit Abs against V domains. Moreover, easy preparation of Cg and Ck chains from IgCw-gk (Fig. 2) makes antigen preparation for anti-Cg Ab and anti-Ck Ab straightforward. To validate certain effects by antigen-specific Abs, reference (or irrelevant isotype control) Abs that have no relevant specificity to a target antigen are commonly used as negative controls in many applications, to distinguish non-specific background signal from antigen-specific Ab signal. However, classical reference Abs could still give undesired background signals due to cross-reactivity not being fully defined. IgCw-gk could be a better reference in many experimental settings and situations, compared to an irrelevant IgG control, because it has no antigen binding capacity and can therefore be expected to exclude the possibility for unpredictable crossreactivity. Practically, the background signal for the binding to cell surface FcgRs was shown to be lower with IgCw-gk than with classical reference IgG (Fig. 3). Moreover, we showed that IgCw-gk is also sufficient to be used as a reference for Ig concentration measurement in biological samples (Fig. 4).
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If we showed the examples for several applications of the IgCw-
gk as an alternative to a reference IgG, it is possible to exploit other IgCw forms that have different isotypes of H and L chains. These include IgCw-gl composed of Cg and Cl, IgCw-εk composed of Cε and Ck, IgCw-εl composed of Cε and Cl, and so on. These IgCw molecules could also be considered as valuable reagents for in vitro and in vivo research. Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article. Author contributions MK, JC, and YS conducted the experiments and analyzed the results. M HK coordinated the study and wrote the paper. Funding source This work was supported by the Cooperative Research Program (PJ012534) of the Rural Development Administration of Korea. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.03.108. References [1] R.E. Kontermann, Alternative antibody formats, Curr. Opin. Mol. Ther. 12
(2010) 176e183. [2] S.J. Demarest, S.M. Glaser, Antibody therapeutics, antibody engineering, and the merits of protein stability, Curr. Opin. Drug Discov. Dev 11 (2008) 675e687. [3] R. Kunert, D. Reinhart, Advances in recombinant antibody manufacturing, Appl. Microbiol. Biotechnol. 100 (2016) 3451e3461. [4] D.C. Roopenian, S. Akilesh, FcRn: the neonatal Fc receptor comes of age, Nat. Rev. Immunol. 7 (2007) 715e725. [5] P. Bruhns, Properties of mouse and human IgG receptors and their contribution to disease models, Blood 119 (2012) 5640e5649. [6] T. Schirrmann, L. Al-Halabi, S. Dubel, M. Hust, Production systems for recombinant antibodies, Front. Biosci. 13 (2008) 4576e4594. [7] A. Beck, L. Goetsch, C. Dumontet, N. Corvaia, Strategies and challenges for the next generation of antibody-drug conjugates, Nat. Rev. Drug Discov. 16 (2017) 315e337. [8] C.H. June, R.S. O'Connor, O.U. Kawalekar, S. Ghassemi, M.C. Milone, CAR T cell immunotherapy for human cancer, Science 359 (2018) 1361e1365. [9] T.S. Dodev, P. Karagiannis, A.E. Gilbert, D.H. Josephs, H. Bowen, L.K. James, H.J. Bax, R. Beavil, M.O. Pang, H.J. Gould, S.N. Karagiannis, A.J. Beavil, A tool kit for rapid cloning and expression of recombinant antibodies, Sci. Rep. 4 (2014) 5885. [10] P.E. Mains, C.H. Sibley, The requirement of light chain for the surface deposition of the heavy chain of immunoglobulin M, J. Biol. Chem. 258 (1983) 5027e5033. [11] M.J. Feige, S. Groscurth, M. Marcinowski, Y. Shimizu, H. Kessler, L.M. Hendershot, J. Buchner, An unfolded CH1 domain controls the assembly and secretion of IgG antibodies, Mol. Cell 34 (2009) 569e579. [12] M. Vanhove, Y.K. Usherwood, L.M. Hendershot, Unassembled Ig heavy chains do not cycle from BiP in vivo but require light chains to trigger their release, Immunity 15 (2001) 105e114. [13] M.J. Feige, L.M. Hendershot, J. Buchner, How antibodies fold, Trends Biochem. Sci. 35 (2010) 189e198. [14] N. Borth, D. Mattanovich, R. Kunert, H. Katinger, Effect of increased expression of protein disulfide isomerase and heavy chain binding protein on antibody secretion in a recombinant CHO cell line, Biotechnol. Prog. 21 (2005) 106e111. [15] C.L. Stoyle, P.E. Stephens, D.P. Humphreys, S. Heywood, K. Cain, N.J. Bulleid, IgG light chain-independent secretion of heavy chain dimers: consequence for therapeutic antibody production and design, Biochem. J. 474 (2017) 3179e3188.