September 1, 2015 (Vol. 35, No. 15)

Improving Surface Expression and Stability for Antibody and Vaccine Discovery

Monoclonal antibodies (mAbs) capable of targeting therapeutically important membrane proteins such as G protein-coupled receptors (GPCRs), ion channels, transporters, and viral envelope proteins are becoming harder to isolate. The problem isn’t a dearth of targets. Rather, increasingly difficult targets are being pursued to address unmet medical needs.

Few mAbs have been derived against any ion channel or transporter, and only a fraction of GPCRs have been successfully targeted by mAbs. Complex viral-membrane proteins also pose a unique set of challenges for vaccine discovery, and the induction of broadly reactive, potently neutralizing antibodies against viral envelope proteins remains a daunting task for pathogens such as HIV and HCV.

Underlying the difficulty in deriving useful mAbs is the structural complexity of membrane proteins and the need to present the target antigens within eukaryotic cell membranes to maintain structural integrity. Many membrane proteins span the lipid bilayer multiple times, form oligomeric structures, and have complex post-translational modifications.

The most effective mAbs usually target conformational epitopes on membrane proteins, so the use of structurally correct antigens is a crucial element in the process. Additionally, large quantities of cell-surface-expressed proteins are usually required for antibody isolation and vaccine discovery, where biochemically relevant levels of protein are essential for immunization, panning, screening, purification, crystallization, and other downstream studies.

With the requirement for high yield and native conformation, wild-type versions of membrane proteins are not always suitable. However, membrane proteins can often be engineered to achieve these requirements.

Challenges in Optimizing Membrane Protein Expression

Most often, the primary bottleneck in mAb discovery against membrane proteins is obtaining sufficient cell surface expression, since low levels of antigen can preclude a robust immune response in animals, limit the number of hits in phage or yeast display screens, and reduce the sensitivity of downstream immunoassays.

Limited surface expression can be attributed to multiple underlying factors. Some membrane proteins are poorly transcribed or translated, in some cases due to incompatible promoters, cells, or growth conditions. Other membrane proteins have intrinsic folding or subunit assembly limitations, and attempts at overexpression can overwhelm the capacity of the cell and result in premature degradation.

Once produced, some membrane proteins fail to traffic to the cell surface because of retention motifs, poor leader sequences, or the unavailability of protein chaperones. In other cases, proteins do successfully traffic to the cell surface, but are then actively internalized.

Finally, overexpressed membrane proteins can be toxic to cells due to their biological function, constitutive activity, or activation by serum components. Complicating these effects, all of these limitations can vary with cell type and cell growth conditions.

Engineering Strategies for Optimal Expression

To overcome the bottleneck of obtaining sufficient membrane protein surface expression, Integral Molecular has developed a suite of tools, termed the MPO Toolbox™, which is designed to optimize the expression and conformational stability of human and viral membrane proteins (Figure 1). MPO incorporates complementary strategies that optimize expression, trafficking, and protein stability, all designed to increase surface expression while retaining native conformation.

MPO begins with traditional optimizations that can increase expression, such as high-expression plasmids, codon optimization, and optimized leader sequences. Cell types and growth conditions are optimized for each target, and proprietary expression elements are added to plasmid constructs. MPO takes into account that some membrane proteins traffic better to the cell surface when expressed at lower levels that allow slower and more native folding of the protein.

The MPO algorithm next assesses opportunities for enhanced protein trafficking by scanning for sequence motifs that affect internalization and retention away from the cell surface, and alters these sequences accordingly. A panel of chaperones, signaling partners (such as alpha and beta subunits), and proprietary trafficking elements are tested for each target. These partner proteins can also help maintain the correct conformational state of the protein, a key factor in isolating the most desirable mAbs.

Finally, the MPO strategy screens through hundreds to thousands of mutants for more stable variants that enable higher surface expression levels while retaining native protein conformation. Typically, panels of point mutations, di-cysteine scans, or chimeras are screened for expression and conformational integrity, and the most effective changes are combined for additive benefits. These strategies rely on Integral Molecular’s proprietary high-throughput mutagenesis capabilities, which can rapidly generate and screen thousands of individual mutants in human cells.

Together, these tiered strategies allow assessment of both rational changes and large-scale mutagenesis scans to identify variants that increase stability and surface expression.

Figure 1. The MPO Toolbox uses a combination of strategies to optimize the expression, trafficking, and stability of challenging human and viral membrane proteins in order to increase their cell surface expression.

Case Studies

The MPO Toolbox has enabled the engineering of a variety of intractable targets for vaccine and antibody discovery projects. Vaccine projects, for example, have engineered proteins from dengue virus, respiratory syncytial virus (RSV), and HIV:

  • Dengue virus: an envelope protein  (prM/E) was engineered for enhanced viral particle production in combination with attenuated infection by assessment of 1,400 random mutations and 2,000 alanine-scan mutants (Figure 2A).
  • RSV: 1,000 random mutations and 400 alanine-scan mutants were screened in human cells to identify variants with increased expression (Figure 2B).
  • HIV: 900 envelope mutants with proline changes, cavity-filling substitutions, and novel disulfide bonds were used to identify individual variants with up to eightfold greater trimer stability (Figure 2C).

Therapeutic antibody discovery projects have also had the benefit of MPO-engineered receptors and ion channels:

  • The CB1 GPCR and the Nav1.7, Kv1.3, and TRP ion channels: These targets, which could advance drug discovery efforts for pain and autoimmune disorders, have been historically difficult to work with due to extremely poor expression. However, modifications to these targets with MPO, using strategies ranging from single-point mutants to chimeras with better-expressing family members (and combinations thereof), have improved their surface expression 10- to 100-fold to enable successful antibody campaigns (Figure 2D).
  • Claudin-4: MPO enabled the screening of 400 variants of the tetra-spanning membrane protein claudin-4 to identify mutations that confer higher detergent solubility in its native conformation (Figure 2E).
  • GPCR TAS2R16: A screen of 600 mutants of the GPCR TAS2R16 identified variants with up to a sevenfold improvement in EC50 for ligand signaling. In this case, MPO was used to generate proteins with enhanced functionality (Figure 2F). As increasingly difficult therapeutic targets are being considered to address unmet medical needs, new technologies and strategies are required to enable mAb and vaccine discovery. The MPO Toolbox offers access to previously intractable targets through the application of high-throughput, parallel protein optimization strategies to improve the surface expression of membrane proteins.

Figure 2. The MPO Toolbox enables antibody and vaccine discovery of challenging membrane protein targets by enabling high levels of surface expression. (A) Comprehensive mutation libraries of the dengue prM/E envelope protein identified individual mutants that provided a threefold increase in viral particle production while eliminating virus infectivity. (B) Mutation libraries of the RSV envelope protein identified individual mutants that demonstrated a fourfold increase in expression. (C) Mutations of HIV envelope were assessed using a trimer-specific mAb to identify individual mutants that displayed eightfold greater stability. (D) The GPCR CB1 and ion channels Nav1.7, Kv1.3, and TRP were engineered to enable high levels of expression 10–100 times their wild-type levels. (E) A mutation library of claudin-4 was screened for increases in detergent solubility while retaining conformation. (F) A mutation library of the GPCR TAS2R16 was screened to identify a highly active variant with a sevenfold increase in functional activity (EC50) in a cell-based calcium flux assay.

Benjamin J. Doranz, Ph.D. ([email protected]), is president and CSO and Soma S. R. Banik is the project leader for scientific communications at Integral Molecular.

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