Ultra-Rare Internalizing HCAbs

Finding cross-reactive, internalizing, heavy chain-only antibodies for single-domain discovery

Single-domain antibodies (VHH) can reach targets conventional antibodies cannot. Technological challenges, including sourcing and finding ultra-rare heavy chain-only antibodies (HCAb) from nature, have limited their development as antibody medicines. 

With technologies intentionally built to break the barriers of conventional discovery, we’re helping partners unlock new opportunities to bring treatments to patients sooner.

The Challenge

A clinically validated single-pass transmembrane protein that is expressed in cancer cells, but not in most normal tissues.

The Goal

Find antibodies that are: 

• Quickly internalized
• Cross-reactive to human, cynomolgus (cyno), and mouse homologs 
• Heavy chain-only

The Outcome

Eight HCAbs with favorable developability properties, binding affinities comparable to clinical benchmarks, and internalization function when reformatted as VHH-Fc fusions.

Our Approach

SOURCE

We used proprietary immunization strategies to induce robust immune responses in two different camelid species.

SEARCH

We screened 2.5 million single antibody-producing cells for cross-reactive and internalizing HCAbs from camelid samples. By combining cell- and bead-based screens into a two-step assay, we identified antibodies with both desired properties upfront. 

We recovered 1,049 single-cell hits, 53% of which produced antibodies that internalized and showed cross-reactivity to human, cyno, and mouse homologs during screening.

DECODE

We used single-cell sequencing to identify 208 unique antibody sequences. To maximize diversity of antibodies selected for expression, we selected 46 antibodies for reformatting as Fc fusions based on:

ANALYZE

We used high-throughput antibody expression and characterization to validate binding and internalization of reformatted antibodies. 82% of produced antibodies were internalizers when reformatted as Fc fusions.

High-throughput antibody assessment revealed that, among these 28 internalizers, eight were high-affinity binders with favorable developability properties, including: 

• High solubility and stability
• Low aggregation and hydrophobicity