A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

Summary

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.

Abstract

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.

Introduction

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....

Protocol

All of the procedures including human blood collection were approved by the Sun Yat-Sen University Ethics Committee.

1. GPC3-S-Fab Design Strategy

1. Design GPC3-S-Fab by linking a Fab of anti-GPC3 (humanized GC33¹³) with an anti-CD16 VHH¹⁴ (Figure 1).
2. Synthesize and clone the VH-CH1-CD16-VHH and VL-CL into the pET26b and pET21a vectors as previously reported¹².

2. Transformation and Culture

1. Add 100 ng of pET26b containing VH-CH1-CD16 (Figure 1A)....

Results

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 .......

Discussion

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 VHH^11,12. 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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