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VHH Discovery with Alicanto versus Phage Display

Oct 28, 2024 | Science, Services

Project Summary

Camelids such as llamas and alpacas produce conventional antibodies as well as heavy-chain only antibodies (HCAbs) from which the small, stable, antigen-binding domain (VHH) can be extracted. Target-specific VHHs can be identified from immunized animals using several different technologies.

1. Phage display was the first technology used to isolate heavy chain only antibodies.  In this process, VHH genes are cloned into a phage library. Primers for this process can introduce bias, and incorporate conventional antibody heavy chains, as discussed in a previous blog post comparing phage libraries to starting PBMC repertoires. The VHHs are expressed on the phagemids, bound to the selected target, eluted, and the bound phage genes are amplified in bacteria. This process is repeated several times to enrich for VHHs that bind the target, however, biases may be introduced as a result of bacterial expression.

2. More recently, single cell screening techniques have gained popularity as they’ve become widely used in other species.  One challenge for a B cell-focused approach is the relative infrequency of HCAbs.  The immune response of llamas is dominated by conventional antibodies, with the proportion of B cells producing HCAbs can be anywhere from 10%-30% of the total population. 

3. A third approach, and one which we’ve adopted at Abterra, is to focus on serum antibodies using Alicanto. We’ve talked a great deal about Alicanto (see case studies on our Literature page), and in this blog post we provide a head to head comparison with traditional phage display. From the same immunized animal, we identify candidates via both Alicanto and phage display to identify differences and similarities.

Methods:

A single llama was immunized with a protein immunogen over the course of 14 weeks. Peripheral blood mononuclear cells (PBMCs) were collected at week 9 and week 13, and serum was collected at week 14. We identified three sets of VHH candidates using three approaches in parallel: Phage display, Phage library enrichment, and Alicanto.

Phage Display Hits: A phage library was constructed from RNA extracted at both time points. The library underwent three rounds of panning.  Colonies were selected after the third round of panning, screened via ELISA, and then sequenced.

VHH Discovery by Phage Display

Overview of the phage library process: From an immunized llama, a phage library was constructed from PBMCs collected during the immunization. A VHH library was constructed, and three rounds of panning were performed to arrive at a set of candidate VHHs (Phage Display Hits)

Phage Enrichment Hits: Candidate VHHs were selected using our antibody repertoire analysis platform, Reptor. We identified VHHs that were enriched across panning rounds from the same phage library and panning process as the phage display hits. Details on the analysis and selection of these candidates are included in a previous blog post. The top VHH sequences ranked by enrichment between the round 3 and pre-panning libraries were selected as the Phage Enrichment Hits. We chose to select the top VHH sequences (as opposed to top CDR3 sequences) in order to more closely match phage display. In phage display the sequence is not known prior to colony picking and diversity of CDR3 sequence is not enforced.

Alicanto Hits: Alicanto was used to analyze the serum antibody repertoire and reported candidate VHHs with reactivity to the target.  Unlike the previous two approaches described, Alicanto is ‘sequence-aware’ and selects candidate antibodies that broadly sample the diversity of the immune response.  Thus, Alicanto does not oversample large expanded clones, and ensures rare clones are tested.

    VHH Discovery with Alicanto workflow

    Overview of the Alicanto process: From an immunized llama, an in silico database was constructed from repertoire sequencing of PBMCs collected during the immunization. HCAbs that were reactive to the target antigen were enriched from serum using affinity chromatography followed by mass spectrometry analysis. The Alicanto Discovery Engine Software integrates the proteomics and genomics data to identify a diverse set of VHH candidates.

    Results:

    Alicanto and phage display resulted in completely distinct sets of VHHs.

    From phage display, 88 phage colonies were selected for sequencing after positive ELISA results. The 88 colonies collapsed to 25 unique clones. We define a clone as a unique CDR3 amino acid sequence.

    Alicanto identified 320 clones present in serum. The Alicanto candidate set was completely disjoint from the Phage Display candidateset.

    Phage enrichment analysis yielded 20 distinct VHHs across 13 unique CDR3s. Eight of the these CDR3s also appeared in the Alicanto candidate list, but none of the phage enrichment clones overlapped with the phage display clones.

    A Venn Diagram of clones found across three VHH discover methods.

    Alicanto and phage enrichment hits expressed well as VHH-Fc fusions.

    Up to 24 VHH sequences from each of the three approaches were reformatted into VHH-Fc with a human IgG1 Fc and transiently expressed in CHO cells. All methods showed variable expression levels, with Phage Display candidates showing the widest variation in expression levels, and 6 VHHs (25%) resulting in insufficient expression for binding analysis.

    Expression profiles of VHHs discovered across three strategies.

    Phage enrichment hits exhibited binding to the target, but reduced CDR3 diversity compared to other methods.

    The recombinant VHHs from each discovery method were evaluated via ELISA.

    Out of the 18 phage display candidates that had sufficient expression, 4 showed good binding. All of the phage display candidates had distinct CDR3s.

    Of the phage enrichment candidates, all 20 showed good binding to the target, but exhibited a small set of CDR3 families (11 families).

    Alicanto candidates were selected for sequence diversity by default, and therefore had high CDR3 diversity. Out of the 24 selected VHHs, 16 showed good binding to the target.

    Summary of VHH binding and sequence diversity

    Summary of the binding data and CDR3 diversity of candidates across the three discovery methods: For each discovery method, the list of candidates is shown with binding data as determined by ELISA. Black: no expression, white: no binding, green: binding. Candidates with CDR3s sharing at least 80% identity were clustered into CDR3 families. The diversity of the candidates is shown via the coloring of CDR3 family.

    Take home message:

    • Alicanto and phage display yielded distinct sets of candidate VHHs from the same immunization. Combining the two approaches yields a larger candidate set.

    • Surprisingly, phage enrichment analysis and traditional phage display produced distinct sets of candidates. Phage enrichment analysis is a fast approach to expand candidate sets found by phage panning.

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