Affinity maturation of a GPCR binder using OXGENE’s novel self-labelling antibody discovery platform

This paid-for advertorial by OXGENE appeared in DDW Volume 22, Issue 1 – Winter 2020/21

Identification and affinity maturation of antibodies against membrane proteins in their native configuration remains challenging. Here, OXGENE uses its SLIM display antibody discovery platform for an affinity maturation project using a parental antibody against a GPCR target from DJS Antibodies.

Introduction

G-Protein coupled receptors (GPCRs) are a large family of cell surface receptors that regulate many cellular functions, and are involved in mediating tumour growth, invasion and migration. This makes GPCRs attractive targets for many drug discovery programmes, including the recently reported GPRC5D3 as a CAR-T therapeutic target1. However, finding antibodies against these proteins can be challenging. GPCRs consist of a seven transmembrane bundle with small extracellular loops above the cell membrane, so most of the protein is inaccessible to extracellular binders, apart from the large N-terminal extracellular domain found in family B/C GPCRs. Furthermore, even in cases where an antibody does bind to an extracellular epitope, it may not have any effect on receptor signalling, and therefore is likely to be therapeutically irrelevant.

Antibody discovery against purified GPCR proteins faces additional hurdles. First, there’s a high chance that any novel antibody will bind to the now-visible intracellular, rather than extracellular domain. Secondly, discovered antibodies that bind to linear epitopes within the extracellular domain may not bind when the protein is in its native conformation2.

A new antibody discovery platform

OXGENE’s self-labelling integral membrane (SLIM™) mammalian display system is designed to overcome these obstacles by discovering antibodies against complex membrane proteins in their native configuration. In SLIM display, a target membrane protein and a library of potential binders are expressed in the same mammalian cells, which then progress through multiple rounds of enrichment and purification to obtain a single clone expressing an scFv that can bind the target antigen3. Unlike other mammalian display systems, the SLIM platform is compatible with libraries of up to 10^9 variants. This is approximately a 100-fold increase compared to libraries used in traditional mammalian display. In contrast, phage display libraries, while often larger, frequently result in false positives so may require subsequent rounds of screening to confirm hits. SLIM display on the other hand implements a specific strategy to reduce false positives and ensures that confirmed binders only target native epitopes. Furthermore, by using Chinese hamster ovary (CHO) cells throughout discovery, binders are far more likely to be compatible with downstream manufacturing.

Adapting for affinity maturation: a case study with DJS Antibodies

DJS Antibodies is focussed on developing a new generation of highly potent, functional monoclonal antibodies (mAbs) against GPCR targets for the treatment of chronic inflammatory diseases. They provided OXGENE with a moderate affinity binder against a target GPCR and challenged us to adapt our SLIM display antibody discovery system for affinity maturation to identify a binder with an improved potency.

The first step was to establish a CHO cell line overexpressing the GPCR target on its surface. We then generated two custom built HA-tagged scFv lentiviral libraries for the light and heavy chain complementarity determining region 3 (CDR3), based on the parent scFv template.

We then transduced our GPCR expressing cell line with both libraries respectively. This resulted in a pool of cells that expressed both the target protein and putative higher-affinity binders. Cells that express an scFv variant specific to the GPCR target ‘self-label’ and can be isolated by FACS via the scFv HA tag (Figure 1).

To isolate potentially higher affinity binders, we selected cells that displayed a self-labelling signal above that of the parent template under stringent wash conditions (Figure 2), and confirmed specificity by testing each supernatant for cross labelling against target positive and target negative CHO cells. Clones that exhibited high self-labelling signal, high cross-labelling signal and a long half-life were then selected for sequencing.

We then reformatted the top five variants from both heavy and light chain libraries into IgG combinations, measured the binding signal and off-rate and took the top nine forward for medium scale purification and EC50 measurement. We returned the top improved binder to DJS Antibodies for further functional analysis.

DJS Antibodies then compared the parental antibody they initially supplied to OXGENE with an antibody incorporating the mutations discovered using SLIM in a functional second-messenger signalling assay using CHO cells overexpressing the GPCR of interest. The SLIM-matured variant showed improved function compared to the starting mAb (Figure 3).

Ultimately, through this partnership, we have demonstrated the capability and strengths of our SLIM display platform for affinity maturation of antibodies against hard-to-target membrane proteins. We identified a functionally improved binder for DJS and are excited to continue developing this technology through further collaborations.

 

Figure 1: We generated a humanised scFv library based on the original GPCR antibody template. We then transduced CHO cells over expressing the target GPCR with the scFv library, and enriched and isolated cells expressing an scFv that bound the target GPCR.

 

 

 

Figure 2: We generated two scFv libraries based on the template of the parental binder, sorted cells and screened clones for cells expressing a binder that displayed a self-labelling signal above that of the parent template under stringent wash conditions.

 

 

 

Figure 3: DJS Antibodies used a functional second-messenger assay in CHO cells over expressing the target GPCR to confirm that the antibody containing the mutations identified by OXGENE’s SLIM platform (right) showed improved function compared to the original parental template (left) at two different concentrations (red and blue bars).

 

References

  1.  Smith EL et.al. GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells. Science Translational Medicine 27 Mar 2019. DOI: 10.1126/scitranslmed.aau7746
  2. Hutchings CJ, Koglin M, Marshall FH. Therapeutic antibodies directed at G protein-coupled receptors. MAbs. 2010;2(6):594-606. doi:10.4161/mabs.2.6.13420
  3. Robertson N, Lopez-Anton N, Gurjar SA, Khalique H, Khalaf Z, Clerkin S, Leydon VR, Parker-Manuel R, Raeside A, Payne T, Jones TD, Seymour LW, Cawood R. Development of a novel mammalian display system for selection of antibodies against membrane proteins. J Biol Chem. 2020 Oct 30:jbc.RA120.015053. doi: 10.1074/jbc.RA120.015053. Epub ahead of print. PMID: 33127646.

 

About OXGENE

www.oxgene.com

OXGENE works with pioneering cell and gene therapy companies. We provide proven technologies that break through scientific boundaries to achieve the impossible; to accelerate development and reduce costs from discovery to biomanufacture of your cell and gene therapies.

We offer a range of discovery technologies, including antibody mammalian display, AAV capsid discovery and CRISPR screening, as well as cutting edge biomanufacturing solutions like our novel TESSA™ technology for AAV production, and lentiviral packaging and producer cell lines.

To find out more about our SLIM™ antibody discovery platform, or any other of our technologies, please email business@oxgene.com.

 

 

 

 

 

 

 

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