Construction of a Large Size Human Immunoglobulin Heavy Chain Variable (VH) Domain Library, Isolation and Characterization of Novel Human Antibody VH Domains Targeting PD-L1 and CD22

Abstract

Phage display is a well-established technology for in vitro selection of monoclonal antibodies (mAb), and more than 12 antibodies isolated from phage displayed libraries of different formats have been approved for therapy. We have constructed a large size (10^11) human antibody VH domain library based on thermo-stable, aggregation-resistant scaffolds. This diversity was obtained by grafting naturally occurring CDR2s and CDR3s from healthy donors with optimized primers into the VH library. This phage-displayed library was used for bio-panning against various antigens. So far, panels of binders have been isolated against different viral and tumor targets, including the SARS-CoV-2 RBD, HIV-1 ENV protein, mesothelin and FLT3. In the present study, we discuss domain library construction, characterize novel VH binders against human CD22 and PD-L1, and define our design process for antibody domain drug conjugation (DDC) as tumoricidal reagents. Our study provides examples for the potential applications of antibody domains derived from library screens in therapeutics and provides key information for large size human antibody domain library construction.

Discovery of a novel human antibody VH domain with potent activity against mesothelin expressing cancer cells in both CAR T-cell and antibody drug conjugate formats

“…Domain drug conjugates to cytotoxic payloads (DCC) have been evaluated recently in different tumor models [12]. Exposed lysine-based conjugation is a widely used non-specific conjugation strategy for antibody drug conjugates with two lysine-based ADCs having been approved by the FDA [15–17].…”

“…Despite donor heterogeneity, 3C9-CAR transduced T cells mediated superior antigen-specific killing of MSLN positive AsPC-1 and NCI-H2452 cells compared with untransduced T cells and did not kill MSLN negative 293T cells nonspecifically (Figure 3B). Domain drug conjugates have been discussed recently in different tumor models [18]. Exposed lysine based conjugation is a widely used non-specific conjugation strategy in DDC development with two lysine based ADCs having been approved by the FDA [22][23][24].…”

“…We designed human recombinant MSLN constructs, which were expressed and used as antigens to isolate MSLN-specific binders from a large size human antibody VH domain library by phage display [12]. This approach yielded a panel of unique domains with the affinities ranging from 0.5 nM to 200 nM for human MSLN.…”

“…This approach yielded a panel of unique domains with the affinities ranging from 0.5 nM to 200 nM for human MSLN. We extensively characterized one VH domain, 3C9, with high affinity and favorable non-aggregation profile ( [12], and Figure 1A). In an ELISA assay, 3C9 exhibited an EC50 less than 10 nM (Figure 1B).…”

“…Previously, we constructed a large-scale human antibody VH domain library based on thermo-stable anti-aggregation scaffolds for phage display [11, 12]. Panels of binders were isolated from this antibody VH domain library against different cancer targets, demonstrating the quality and diversity of the library.…”