Function-focused antibody (FFmab) screening

AN ANTIBODY-BASED PHENOTYPIC SCREENING PLATFORM DRIVEN BY FUNCTION

FFmab SCREENING PLATFORM OVERVIEW

WHAT IS FFmab SCREENING?

FFmab screening is a platform for identifying novel functional antibodies that change disease-modifying phenotypes of immune cells. The platform identifies innovative therapeutic antibodies that mediate differentiated mechanisms-of-action by binding either novel targets or new functional epitopes on known targets.

Figure 9: Overview of FFmab screening platform

HOW DOES IT WORK?

The FFmab screening workflow consists of three main phases preluded by identifying an immune cell type as a key mediator of a disease phenotype. In Phase 1 adequate amounts of the primary human immune cell are generated, immunisations therewith are performed and antibodies that bind selectively to this immune cells type are identified. These cells may be purified from peripheral blood samples of healthy donors and patients or derived and expanded from a precursor cell type e.g. iPSCs. For a robust FFmab campaign, 2 x 10₉ cells of the immune cell type of interest are required. Immunising mice with these cells provokes a broad range of antibodies against cell surface antigens in vivo.

Figure 10: Using proprietary immunisation protocols, robust antibody response with selectivity for memory T cells was achieved. Antibodies against multiple immune modulatory targets were detectable in serum.

The success of the immunisation is monitored. Here, in addition to classical titre tests that measure the quantity of antibody response, we perform assays to qualify the response of individual animals. After verifying the success of the immunisation campaign, we generate antibodies from selected responder animals by state-of-the-art hybridoma technology. Using high-throughput flow cytometry-based screening of ~10,000 hybridoma clones, we identify those antibodies that bind selectively to the cell type of interest. This is accomplished by cell-multiplexing. Selective antibody clones are isolated and sequenced.

Figure 11: Screening for binding to memory T cells and simultaneous counter-screening against naïve T cells enrich for effector memory/effector T cell-selective antibodies.

After identification of a large panel of adequately diverse and developable antibodies, the project enters into the second phase. Phase 2 involves functional assessment of the antibodies in relevant in vitro assays using primary immune cells.

Figure 12: Screening for binding to memory T cells and simultaneous counter-screening against naïve T cells enrich for effector memory/effector T cell-selective antibodies.

For this purpose, we convert the antibodies into a human isotype-matched format to exclude confounding effects mediated by different Fc types. Depending on the desired mode-of-action, we design the recombinant antibodies to exert normal, attenuated or enhanced Fc effects. Optimised processes for generating expression constructs and a high-throughput transient CHO antibody expression platform provide high quality material for the functional screens. Identified functional antibodies undergo further characterisation by epitope binning and assessment of their binding to a panel of known pre-clinical and clinical targets in the field of the disease indication in question.

The third phase of the project focuses on target deconvolution using the functional antibodies identified in the previous phase. To increase the likelihood of identifying novel targets, selected functional antibodies are first counter screened against “usual suspect” targets in the indication area. Then, using the functional antibodies as basis for identification of their respective targets, we employ the services of Retrogenix and DualSystems for target deconvolution. We perform in-house validation of any candidate target identified in this way by verifying that the respective antibody binds to the target expressed in cells recombinantly.

Figure 13

At this stage, the project team may decide to generate an additional panel of antibodies with comparable functional properties using a target-specific discovery campaign. For selected clinical candidate antibodies, we perform further experiments demonstrating their therapeutic mechanism-of-action in vitro and in vivo in order to generate a strong pre-clinical data package.

WHAT HAS BEEN DONE SO FAR?

The first project run on the FFmab screening platform focused on effector memory T cells. We selected effector memory T cells (TEM) as this T cell subset plays a key pathologic role in cancer and autoimmune diseases. For this pilot project, we immunised mice with TEM cells collected from PBMCs of 10 healthy donors. Immunisations resulted in serum responses against known immune modulatory targets, like PD-1, CD2, and OX-40 (Figure 10). We screened the generated hybridoma clones for TEM-selective antibodies versus antibodies that also bound to naïve T cells. We functionally tested recombinant antibodies in a TEM-based platebound TCR co-stimulation assay and a mixed lymphocyte reaction (MLR) assay. In this pilot project, we have identified an agonistic antibody against a novel target belonging to the immunoglobulin superfamily and an antibody against a new epitope on a known, clinically validated target. The unique epitope of the latter antibody enables its use in a different disease indication with a highly differentiated mechanism-of-action. Both antibodies are currently in pre-clinical development as first-in-class opportunities.

Figure 14: Pipeline arising from pilot project using effector memory T cells

The pilot study delivered a robust workflow optimised for speed and yield and provided proof-of-concept for the platform. This has encouraged initiating another screen focused on a different immune cell population that is currently underexploited for immune oncology.

WHAT IS IN PROGRESS?

Currently we are using the FFmab screening platform to identify novel functional antibodies directed against myeloid-derived suppressor cells (MDSCs). MDSCs are key suppressor cells in the tumour microenvironment. Patients with high MDSCs have poorer survival rates. Circulating MDSCs predict advanced cancer stage and increased risk of resistance to immune checkpoint inhibitor (ICI) therapy. MDSCs have hence emerged as a pivotal hurdle for more widespread clinical success of current ICI approaches. Nevertheless, MDSC-targeting therapeutic approaches are currently limited due to the dynamic and poorly defined phenotype of these cells.

Figure 15

We have developed protocols to generate and expand MDSCs and characterise their suppressive phenotype in T cell co-culture assays. By immunising mice, we have generated hundreds of MDSC-selective antibodies with high sequence diversity.

The first recombinant antibodies are currently entering functional screening. We will focus on selecting antibodies that:

• block the suppressive activity of MDSCs and/or
• reprogram MDSCs from an immune-suppressive to an immune-stimulatory phenotype

Highly MDSC-selective antibodies that have neither of those two functional properties may still be functionalised by conjugating them to a payload for MDSC depletion.