“Stapling” scFv for multispecific biotherapeutics of superior properties

ABSTRACT

Single-chain fragment variable (scFv) domains play an important role in antibody-based therapeutic modalities, such as bispecifics, multispecifics and chimeric antigen receptor T cells or natural killer cells. However, scFv domains exhibit lower stability and increased risk of aggregation due to transient dissociation (“breathing”) and inter-molecular reassociation of the two domains (VL and VH). We designed a novel strategy, referred to as stapling, that introduces two disulfide bonds between the scFv linker and the two variable domains to minimize scFv breathing. We named the resulting molecules stapled scFv (spFv). Stapling increased thermal stability (Tm) by an average of 10°C. In multiple scFv/spFv multispecifics, the spFv molecules display significantly improved stability, minimal aggregation and superior product quality. These spFv multispecifics retain binding affinity and functionality. Our stapling design was compatible with all antibody variable regions we evaluated and may be widely applicable to stabilize scFv molecules for designing biotherapeutics with superior biophysical properties.

KEYWORDS:

• Multi-specific antibody
• Scfv
• Scfv stabilization
• Single chain fv
• Spfv
• Stapled scfv

Abbreviations

aSEC	=	Analytical size-exclusion chromatography
BLI	=	Bio-layer interferometry
CAR-NK	=	Chimeric antigen receptor NK-cells
CAR-T	=	Chimeric antigen receptor T-cells
CR	=	Constant region
DSC	=	Differential thermal calorimetry
dsFv	=	Disulfide Fv
ELISA	=	Enzyme-linked immunosorbent assay
Fab	=	Fragment of antigen-binding
FDA	=	U.S. Food and Drug Administration
Fv	=	Variable fragment
HC	=	Heavy chain
HL	=	Heavy-light orientation
LC	=	Light chain
LC-MS	=	Liquid chromatography-mass spectrometry
LH	=	Light-heavy orientation
mAb	=	Monoclonal antibody
mL	=	milliliter
scFv	=	Single-chain fragment variable
spFv	=	Stapled scFv
Tm	=	Thermal stability
VH	=	Variable heavy
VL	=	Variable light
VR	=	Variable region

Acknowledgments

The authors thank the following for their technical and other assistance. Daniel Grau, Michael Kane, Laetitia Eugene, Hau Truong, Sitilia Rencheli and Sidharth Mohan in protein expression and purification; Xiefan Lin-Schmidt for providing the BCMA sequence; and Advance Photon Source (APS) for X-ray data collection.

Disclosure statement

JL, LEB, MF, MD, AAA, AT & CH are co-inventors in a provisional patent application (US20210047435A1). This application covers the scFv stapling technology and its potential applications in multispecific antibodies and other therapeutic or diagnostic protein modalities and detection reagents wherever scFv can normally be used.

Data availability statement

Atomic coordinates and structure factors of spFv and scFv/spFc:antigen complexes have been deposited in the Protein Data Bank, www.rcsb.org, under the ID codes 8DY0, 8DY1, 8DY2, 8DY3, 8DY4 and 8DY5. All other data are included in the main text and SI.

Contributions

JL devised and supervised the project. LEB generated scFv/spFv constructs and crystallized and determined all spFv structures. EGP designed and characterized the BCMA/CD3 BsAbs. MF and MD generated scFv/spFv proteins for analysis by AG. FY, RN, S-JW, TL, ERL and SJ carried out biophysical studies. NK, BDR, BW, PA and RD generated all bispecific proteins. SH, JT and NM performed cell binding and killing studies. EG, TL, AM, HN and HG performed mass spectrometry analysis. AAA, AT, CH contributed to ideation. AAA crystallized and determined the structures of scFv/spFv antigen complexes. AZ, PC, WCC and JL supervised many activities. JL, LEB and EGP wrote the manuscript. PC provided extensive guidance and critical editing. All coauthors contributed to the manuscript preparation.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2195517

Additional information

Funding

The author(s) reported that there is no funding associated with the work featured in this article.