Abstract
Nucleolin arises as a relevant target for cancer therapy, as it is overexpressed at the surface of cancer and angiogenic endothelial cells thus enabling a dual cellular targeting strategy. Immunotherapeutic strategies, albeit of proven therapeutic relevance, have been scarcely explored against this target. Therefore, this work aimed at engineering an anti-nucleolin VHH-based antibody capable of triggering anticancer immune responses. Herein, anti-nucleolin VHHs have been generated upon grafting F3 peptide-derived nucleolin-binding sequences onto a VHH CDR1 or CDR3. One of these nucleolin-binding CDR3-grafted VHH was subsequently fused to a human IgG1 Fc region, enabling a significant antibody-dependent cell-mediated cytotoxicity (ADCC). The generated anti-nucleolin VHH revealed increased binding and antiproliferative effects against cancer cells, relative to the parental VHH, while the VHH-Fc counterpart presented increased cytotoxicity relative to the corresponding VHH. This VHH-Fc also triggered an ADCC effect, in the nanomolar range, against a nucleolin-overexpressing cancer cell line. This effect was evidenced by a 2 or 1.7-fold increase of cell death, in the presence of PBMCs, relative to the parental VHH-Fc or the VHH counterpart, respectively. Overall, these formats represent the first anti-nucleolin VHHs and the first anti-nucleolin antibody with ADCC activity that have been successfully developed.
Highlights
Nucleolin is a multifunctional protein expressed in the nucleus of exponentially growing eukaryotic cells, where it participates in rRNA synthesis and ribosome biogenesis[1]
The nucleolin-binding VHHs grafted onto CDR1 or CDR3 presented a 2- or 3-fold increased binding to nucleolin, respectively, relative to the parental VHH, without engraftment of the F3 peptide-derived sequence (Fig. 1b)
As human IgG1 Fc region enables antibody-dependent cell-mediated cytotoxicity (ADCC) responses[30], we evaluated cancer cell death upon incubation with anti-nucleolin VHH-Fc antibody and effector cells (PBMCs)
Summary
Nucleolin is a multifunctional protein expressed in the nucleus of exponentially growing eukaryotic cells, where it participates in rRNA synthesis and ribosome biogenesis[1]. Antibodies are nowadays one of the major classes of therapeutics and are currently used against several malignancies These proteins combine a high affinity to their targets through the variable domains (VH and VL) of the antigen binding fragment (Fab), with the capacity to trigger cell death by several mechanisms. These include direct cell death (upon interfering with the signalling pathways in which the target is involved) and immune responses, mediated by the Fc region. It is hypothesized that generation of anti-nucleolin antibodies, generated upon grafting of the nucleolin binding domain from the F3 peptide, will enable increased cytotoxicity against nucleolin-overexpressing cells and, upon further fusion to a human IgG1 Fc region, will induce an ADCC effect
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