Here, we present a protocol to produce a bispecific antibody GPC3-S-Fab in Escherichia coli. The purified GPC3-S-Fab has potent cytotoxicity against GPC3 positive liver cancer cells.
This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes through their two different arms. bsAbs have been actively studied for their ability to directly recruit immune cells to kill tumor cells. Currently, the majority of bsAbs are produced in the form of recombinant proteins, either as Fc-containing bsAbs or as smaller bsAb derivatives without the Fc region. In this study, GPC3-S-Fab, an antibody fragment (Fab) based bispecific antibody, was designed by linking the Fab of anti-GPC3 antibody GC33 with an anti-CD16 single domain antibody. The GPC3-S-Fab can be expressed in Escherichia coli and purified by two affinity chromatographies. The purified GPC3-S-Fab can specifically bind to and kill GPC3 positive liver cancer cells by recruiting natural killer cells, suggesting a potential application of GPC3-S-Fab in liver cancer therapy.
Monoclonal antibodies are now broadly used for cancer treatment1. Due to the flexibility of antibodies, various antibody-based formats have been actively explored. Compared with monoclonal antibodies, bsAbs have two different antigen binding modules, enabling them to recognize two different targets simultaneously and efficiently trigger the recruitment of immune effector cells to target and kill tumor cells2.
Current recombinant bsAb formats can be generally assigned to two classes: Fc-containing bsAbs and bsAbs without an Fc region. Compared with Fc-containing formats that are mostly produced in mammalian cells, bsAbs without an Fc region have the advantages of smaller sizes, are more readily produced in microorganism expression systems, and can penetrate tumor tissues more efficiently3.
bsAbs without an Fc region are commonly formed by linking individual binding moieties, such as single-chain variable fragments (scFvs) or Fabs3. Without the stabilizing domains, bsAbs based on scFv fragments often have compromised thermal stability, low solubility, or an increased potential for aggregation4,5. In contrast, Fab-based bsAbs are more stable due to the heterodimerization of the CH1 and CL in the native Fab moiety4,6.
Variable domain from heavy-chain-only antibodies (VHHs, also referred to as single domain antibodies) are the active antigen-binding fragment of natural heavy chain antibodies7. VHHs have the characteristics of high affinity, specificity of conventional IgGs8, low immunogenicity, and high yields in bacterial expression9. Compared with Fv fragments, VHHs have higher thermal stability10. Compared with Fab moieties, VHHs have smaller sizes due to the lack of CH1 and CL. Thus, S-Fab, the bsAb format obtained by linking the Fab with a single domain antibody, VHH, was designed and studied for its anti-tumor effects11,12.
In this study, the construction of GPC3-S-Fab by linking the Fab of hGC3313 with an anti-CD16a VHH14 was described. The GPC3-S-Fab can be efficiently produced by periplasmic expression in Escherichia coli (E. coli). Functional studies of GPC3-S-Fab suggested that GPC3-S-Fab is a promising strategy for liver cancer therapy. Thus, the advantages of GPC3-S-Fab over alternative techniques with applicable references to previous studies include easy production and purification, and more stable bsAbs.
Mammalian expression systems and prokaryotic expression systems have been used to express various formats of BsAbs. In contrast to mammalian expression systems, E. coli-based protein expression systems have many benefits, including high yields, low cost and labor-saving, the ease of genetic manipulations, and high transformation efficiency15. For bsAbs expression in E. coli, there are two basic strategies: expression in the cytoplasm and expression in the periplasm between the cytoplasm and outer cell membranes15. Compared to the reducing environment of cytoplasm, the periplasm is a more oxidizing environment, which promotes the correct folding and co-expression of proteins16. Correct folding plays a key role in solubility, stability and function generation of bsAbs. Therefore, a signal sequence pelB was added to the N-terminus of the S-Fab to direct secretion to the periplasm of E. coli17. To ensure correct folding, solubility, thermal stability, and conformational stability, reducing the complexity and the size of an antibody is frequently employed16. The S-Fab format consists of one Fab and one VHH, which is expressed very well in bacterial systems likely due to the simple structure and small size.
GPC3 was chosen in this GPC3-S-Fab bispecific antibody format. Glypican-3 (GPC3) is a member of the heparin sulfate (HS) proteoglycan family that is anchored to the cell surface through glycosylphosphatidylinositol (GPI)18. GPC3 is overexpressed in 70% of hepatocellular carcinoma (HCC) cases, which account for the majority of liver cancers19,20,21,22. Because GPC3 is rarely expressed in normal tissues, GPC3 has been proposed as a potential target for HCC. Multiple mouse mAbs have been produced against GPC3. However, only GC33 exhibited limited anti-tumor activity 22, and it failed to exhibit clinical efficacy in patients. In this study, GPC3-S-Fab was shown to be able to recruit NK cells to kill GPC3 tumor cells14.
To recruit NK cells, anti-CD16 VHH was used. CD16a is a low affinity IgG receptor, expressed mainly on natural killer (NK) cells, macrophages, monocytes and some subtypes of T cells. It is involved in antibody-dependent cell cytotoxicity (ADCC) by NK cells23. Human NK cells can be categorized into two types, CD56-CD16+ and CD56+CD16-. In contrast to CD56+CD16− NK cells, CD56-CD16+ NK cells can release higher levels of perforin and granzyme B and thus present a strong cytotoxicity24. Kupffer cells (KCs), expressing CD16a, are the resident macrophages in liver. Kupffer cells play an important role in the suppression of liver cancer25. Thus, bsAbs targeting CD16a may be a more promising strategy than engaging T cells against liver cancer.
All of the procedures including human blood collection were approved by the Sun Yat-Sen University Ethics Committee.
1. GPC3-S-Fab Design Strategy
2. Transformation and Culture
3. GPC3-S-Fab Periplasmic Purification
4. SDS-PAGE and Western-blotting Analysis
5. Gel Filtration Analysis
6. Flow Cytometry Analysis
7. Cytotoxic Assays
GPC3-S-Fab purification
GPC3-S-Fab was purified from E. coli by a two-step affinity purification, first with Ni-NTA-agarose, followed by IgG-CH1 affinity purification. After the two-step affinity purification, GPC3-S-Fab was purified to homogeneity with the two chains close to 1:1 (Figure 2A). The presence of both VH-CH1-CD16 VHH and VL-CL polypeptides can be identified by their distinct C-terminal tags, anti-His for the VL-CL approximately 25 kDa, and anti-Flag for the VH-CH1-VHH approximately 38 kDa (Figure 2B). After the two-step affinity purification, ~1.2 mg protein can be obtained from 1 L of SB culture. To further characterize the purified GPC3-S-Fab, gel filtration was performed. Based on the standard markers (Figure 2C), GPC3-S-Fab was identified as a homogenous monomer in the form of a heterodimer with a molecular size of approximately 63 kDa (Figure 2C).
GPC3-S-Fab binding activities
To evaluate whether the GPC3-S-Fab recognizes the GPC3-positive cells and CD16-positive cells, flow cytometry analysis was conducted using the GPC3-positive liver cancer cell lines HepG2, Hep3B, Huh7, CHO/GPC3 and the GPC3-negative liver cancer cell line, MHCC-97H, and CHO cells. The GPC3-S-Fab could bind to all the GPC3-positive cells, although with weaker binding to Hep3B (Figure 3B) and Huh7 cells (Figure 3C) than to HepG2 (Figure 3A) and CHO/GPC3 (Figure 2F). No or minimal binding to GPC3-negative MHCC-97H and CHO cells was observed (Figure 3D, 3E). The GPC3-S-Fab also could bind to the CD16-positive cells, NK92/CD16 cells (Figure 3G). These results are consistent with previous results using other anti-GPC3 antibodies30, suggesting that GPC3-S-Fab can specifically bind GPC3-positive cell lines and CD16-positive cells.
To evaluate the cytotoxicity of GPC3-S-Fab, tumor cells were incubated with fresh isolated NK cells with different concentrations of GPC3-S-Fab. Without NK cells, GPC3-S-Fab had no cytotoxicity against tumor cells regardless of the GPC3 expression status (Figure 4). In the presence of NK cells, GPC3-S-Fab triggered strong cytotoxicity against HepG2, Hep3B, and Huh7 cells in a dose-dependent manner but showed no effect on GPC3-negative MHCC-97H cells (Figure 4), suggesting that the cytotoxicity of GPC3-S-Fab depends on GPC3 expression on the tumor cell surface.
Figure 1. Constructs of GPC3-S-Fab
(A) The bacterial expression constructs of GPC3-S-Fab. The constructs contain a pelB signal sequence, a humanized anti-GPC3 (GC33) VH-CH1-anti-CD16 VHH (heavy chain), or a VL-CL (light chain). To facilitate protein detection and purification, a Flag tag or a His8 tag was added to the C-terminus. (B) Diagram of GPC3-S-Fab after co-expression.
Figure 2. GPC3-S-Fab purification from E. coli.
(A) Left panel, after Ni-NTA affinity chromatography; Right panel, after anti-IgG-CH1 affinity chromatography; M, molecular weight ladder; Coomassie blue staining. (B) Left panel, after Ni-NTA affinity chromatography; Right panel, after anti-IgG-CH1 affinity chromatography; M, molecular weight ladder; Western-Blotting. (C) Gel filtration analysis of GPC3-S-Fab. Top panel, standard markers; bottom panel, GPC3-S-Fab. Please click here to view a larger version of this figure.
Figure 3. GPC3-S-Fab recognizes GPC3 positive cells and CD16-positive cells.
Flow cytometry analysis of HepG2 (A), Hep3B (B), Huh7 (C), MHCC-97H (D), CHO (E), CHO/GPC3 (F) and NK92/CD16 (G) cells was performed as described in the protocol. Red line: tumor cells only; Green line: isotype control, tumor cell + human IgG1 + anti-human IgG(H+L)-488; blue line: tumor cell + GPC3-S-Fab + anti-human IgG (H+L)-488. Please click here to view a larger version of this figure.
Figure 4. GPC3-S-Fab promotes the cell death of GPC3-positive cancer cells.
Dose-dependent cytotoxicity assays were performed as described in the protocol for HepG2 (A), Hep3B (B), Huh7 (C), and MHCC-97H (D). The concentrations of GPC3-S-Fab were from 0.0045 µg/mL to 30 µg/mL. The data are the mean of triplicates with error bars representing the standard deviation. Solid circle, only GPC3-S-Fab; solid square, GPC3-S-Fab+ NK, NK cells (50,000 per well) and target cells (5,000 per well). The mixtures were incubated for 72 h before cytotoxicity measurement. Please click here to view a larger version of this figure.
In this study, we present a strategy to construct a new format of bsAbs, GPC3-S-Fab, which can recruit NK cells targeting GPC3 positive tumor cells. The S-Fab is based on the natural Fab format by adding an anti-CD16 VHH11,12. Compared with the bsAbs containing Fc region, GPC3-S-Fab can easily be produced in the periplasm of bacteria on a large scale.
Using the expression and purification strategy described in the protocol, we obtained a soluble and functional GPC3-S-Fab in large quantities. To facilitate periplasmic expression, a signal sequence pelB was added to the N-terminus to direct secretion to the periplasm of E. coli17. In contrast to the cytoplasm, the more oxidizing environment in the periplasmic space between the cytoplasmic and outer membranes is equipped with a number of proteins important for protein folding and assembly and thus promotes the correcting folding and solubility of recombinant proteins16. However, the machinery for protein folding and export to the periplasm in E. coli has limited capacity. High expression of recombinant proteins often results in the accumulation of insoluble product in the periplasm16. To avoid the high expression of protein over a short period of time, low IPTG (0.2 mM) and low temperature (16 °C) in nutritious medium were applied in this protocol to avoid the accumulation of insoluble protein. In this study, ~1.2 mg protein was obtained from 1 L of medium, improving the yields at least 2-fold compared with previous works12.
To avoid protein degradation, all fractions containing GPC3-S-Fab in various steps should be kept on ice. With the His tag at the C-terminal of VL-CL, Ni-NTA-agarose was used for purification. However, single Ni-NTA-agarose purification is not sufficient to remove unwanted proteins, such as unpaired VL-CL protein. To purify the homogenous heterodimeric GPC3-S-Fab, the second step, anti-IgG-CH1 affinity purification, was applied. The purified protein had high purity and two polypeptides close to 1:1 (Figure 2A-2B).
Gel filtration chromatography can determine the molecular weights of proteins based on their molecular sizes and shapes; analyze the degree of purity; and determine whether the purified protein is a monomer, dimer, or composed of aggregates15. By gel filtration chromatography, GPC3-S-Fab was identified with the expected molecular size of approximately 63 kDa, which is in the form of a monomer with heterodimerization of GPC3-S-Fab (Figure 2C).
Flow cytometry can determine whether an antibody can bind its antigen on the cell membrane, compared with traditional western-blotting and ELISA, which only detect antigen-antibody binding reactions. By flow cytometry analysis, GPC3-S-Fab recognized the GPC3 on the cell membrane, suggesting the GPC3-Fab moiety was functional. When performing flow cytometry analysis, it is critical to keep all of the steps on ice or at 4 °C after the antibody is added. When the antibody binds to the cell surface antigen, the antibody-antigen complex will initiate internalization. By keeping it cold, the internalization process is slowed significantly.
PBMCs and NK cells were efficiently isolated from human peripheral blood by centrifugation using lymphocyte separation medium, followed by magnetic-activated cell sorting strategies. GPC3-S-Fab showed potent cytotoxicity against the GPC3 positive cells when the ratio of NK cells to target cells (tumor cells) was 10:1, and the ratio of NK cells to target cells could vary from 50:1 to 5:1.
In summary, GPC3-S-Fab, which can be produced in a microorganism expression system, provides a new avenue to create functional formats of bsAbs against tumors.
The authors have nothing to disclose.
This work was financially supported by the R&D Plan of Guangdong Province (PR China) (2016A050503028).
Shaking incubator | Thermo Fisher | MAXQ 4000 | |
Shaking incubator | Zhicheng | ZWYR-D2402 | |
Centrifuge | Cence | GL-10MD | |
Centrifuge | Beckman coulter | Avanti j-26S XPI | |
Centrifuge | eppendorf | 5810R | |
Ultraviolet spectrophotometer | Thermo Fisher | Nanodrop | |
Analytical polyacrylamide gel electrophoresis apparatus | Mini-PROTEAN® Tetra | Bio-rad | |
Trans-blot apparatus | Criterion | Bio-rad | |
Imaging system | Bio-rad | chemidoc tm XRS+ | |
Fast Protein Liquid chromatogram | GE Healthcare | AKTA avant | |
GF column | GE Healthcare | 28-9909-44 Superdex 200 Increase 10/300 GL | |
Flow Cytometer | Beckman coulter | FC500 | |
Centrifuge | eppendorf | 5702R | |
Envision plate reader | TECAN | Infinite F50 | |
Anti His-tag | eBioscience | 14-6657-82 | |
anti-Flag-tag | Sigma | F1804 | |
anti-human(H&L)-488 | A11013 | Invitrogen | |
Anti-mouse IgG HRP-linked antibody | Cell Signaling | 7076S | |
Ni-NTA-Agarose | Tribioscience | TBS9202-100 | |
IgG-CH1 affinity resin | Thermo Fisher | 194320005 | |
Ficoll-Plaque Plus | GE Healthcare | 17-1440-03 | |
NK cell enrichment kit | Stemcell | 19055 | |
Magnet | Stemcell | 18000 | |
CCK8 kit | Dojindo | CK04 | |
DMEM | Gibco | C11995500CP | |
RPMI-1640 | Gibco | C11875500CP | |
Fetal Bovine Serum (FBS) | Sigma | F2442 | |
Trypsin | Gibco | 15050-057 | |
Penicillin-Streptomycin | Gibco | 15140-122 | Cell culture |
Standard marker | Sigma Aldrich | MWGF200 | Gel filtration |
Isopropyl-b-D-thio-galactopyranoside (IPTG) | VWR chemicals | VWRC0487-100G | |
Dialysis tubing | Sigma Aldrich | D0655-100FT | |
Knanamycine | VWR | VWRC0408-100G | |
Ampicillin | VWR | VWRC0339-100G | |
Tryptone | Thermo Fisher | LP0042B | |
Yeast Extract | Thermo Fisher | LP0021B | |
NaCl | Sangon Biotech | A100241 | |
Trizma base | Sigma Aldrich | T6791-1KG | |
EDTA | Sigma Aldrich | V900106 | |
Glycine | aladdin | A110752-500g | |
KCl | aladdin | P112134-500g | |
MgCl | Sigma Aldrich | V900020 | |
Agar | Sangon Biotech | A505255-0250 | |
Potassium Phosphate, Monobasic Anhydrous (KH2PO4) | VWR | 7778-77-0 | |
Sodium Phosphate, Dibasic, Anhydrous (Na2HPO4) | VWR | 7558-79-4 | |
2-Mercaptoethanol | VWR | 60-24-2 | |
Phenylmethyl Sulfonyl Fluoride (PMSF) | VWR | 329-98-6 | |
Lysozyme | Sigma Aldrich | L6876-25G | |
Coomassie Brilliant Blue R250 | VWR | VWRC0472-50G | |
Bromophenol blue | Sangon Biotech | A500922-25G | |
Bovine Serum Albumin (BSA) | VWR | VWRC0332-100G | |
Glycerol | Sigma Aldrich | V900122 | |
100mm x 20mm plastic dish | Corning | 430167 | |
25cm2 flask | Corning | 430639 | |
96 well cell culture cluster | Corning | 3599 | |
Sucrose | Sangon Biotech | A610498-0005 | |
CHO | the Type Culture Collection of the Chinese Academy of Sciences | GNHa 3 | |
MHCC-97H | the Type Culture Collection of the Chinese Academy of Sciences | SCSP-528 | |
HepG2 | the Type Culture Collection of the Chinese Academy of Sciences | TCHu 72 | |
Huh7 | the Type Culture Collection of the Chinese Academy of Sciences | TCHu182 | |
Hep3B | the Type Culture Collection of the Chinese Academy of Sciences | TCHu106 | |
NK92 | ATCC | CRL2408 |