Single-domain antibodies for targeting, detection andin vivoimaging of human CD4⁺cells

Abstract

The advancement of new immunotherapies for the treatment of cancers, infections, immune-mediated inflammatory diseases, and autoimmune diseases necessitates the co-development of appropriate probes to detect and monitor the distribution and infiltration of distinct immune cell populations. Considering the key role of CD4+ T cells in regulating immunological processes, we have developed a set of novel single-domain antibodies (nanobodies, Nbs) that specifically recognize the human CD4 co-receptor in its native state on various CD4+ cells. Following detailed characterization of binding properties, epitope mapping, and site-directed functionalization, we selected biologically inert Nbs that do not affect T cell proliferation or cytokine expression in vitro. We used fluorescently labeled Nbs to track the presence and location of CD4+ cells in a xenograft model, demonstrating a high signal-to-background ratio by in vivo optical imaging. In summary, this study reports for the first time the generation and application of human CD4-specific Nbs for the detection and in vivo imaging of CD4+ cells in a preclinical animal model. We anticipate that the Nbs presented in this study will be versatile probes, e.g. in immunoPET imaging for patient stratification and for monitoring individual immune responses during personalized immunotherapy.

Improved targeting of human CD4+ T cells by nanobody-modified AAV2 gene therapy vectors

“… (A) Amino acid alignment of the Nb sequences used in this study. Nb annotation is in accordance with the study by Traenkle and coworkers [ 39 ], which describes thorough Nb characterization and application for in vivo imaging. Of note, CD4-Nb1a is not specified in Traenkle et al, as its complementary determining region 3 (CDR3, black box) is identical to CD4-Nb1 (amino acids 101–117 in the alignment).…”

“…Recently, a set of five novel Nbs (amino acid sequence depicted in Fig 1A ) have been developed that specifically recognize the human CD4 receptor in its native state with high affinity [ 39 ]. Here, we assayed these Nbs in the context of AAV aiming for AAV capsid variants with tropism for CD4 + T lymphocytes.…”

“…We employed the alpaca derived Nbs CD4-Nb1 and CD4-Nb3, which were previously reported to display affinities (i.e. K D values) for human CD4 of ~5 nM and ~75 nM, respectively [ 39 ]. Interestingly, the presentation of these Nbs on the surface of the capsid as part of VP1 protein appeared to be more effective than their display as fusion to the amino-terminus VP2 (compare Figs 3 and 4 ).…”

“…These findings confirmed that this site in VP1 can indeed accommodate at least two Nb-encoding sequences, potentially allowing the insertion of heterologous Nb tandem repeats to target different receptors using the same AAV vector construct. In particular a combination of CD4-Nb1 and CD4-Nb3 might result in an even more CD4-specific AAV2 vector as it has been shown that both Nbs can bind CD4 in parallel, meaning their epitopes do not overlap [ 39 ].…”

“…The failure of CD4-Nb3-modified, as opposed to CD4-Nb1-modified VP2 capsid proteins to target the respective vector particles to CD4 + cells (Figs 3 and 4 ) may be explained by the obvious variability in the complementarity determining region 3 (CDR3) of these Nbs [ 39 ] (depicted in Fig 1A ), which may negatively affect the overall structure of the VP2 protein, and thus the appropriate surface presentation of the CD4-Nb3.…”