Nanobodies in cancer.

For treatment and diagnosis of cancer, antibodies have proven their value and now serve as a first line of therapy for certain cancers. A unique class of antibody fragments called nanobodies, derived from camelid heavy chain-only antibodies, are gaining increasing acceptance as diagnostic tools and are considered also as building blocks for chimeric antigen receptors as well as for targeted drug delivery. The small size of nanobodies (∼15 kDa), their stability, ease of manufacture and modification for diverse formats, short circulatory half-life, and high tissue penetration, coupled with excellent specificity and affinity, account for their attractiveness. Here we review applications of nanobodies in the sphere of tumor biology.

A class II MHC-targeted vaccine elicits immunity against SARS-CoV-2 and its variants.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (SpikeRBD) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHHMHCII). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHHMHCII-SpikeRBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHHMHCII-SpikeRBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy.

Nanobody-based CAR NK cells for possible immunotherapy of MICA+ tumors.

The glycoproteins MICA and MICB are upregulated on the surface of cells undergoing stress, for instance due to (viral) infection or malignant transformation. MICA/B are the ligands for the activating receptor NKG2D, found on cytotoxic immune cells like NK cells, CD8+ T cells, and γδ T cells. Upon engagement of NKG2D, these cells are activated to eradicate the MICA/B-positive targets, assisted by the secretion of cytokines. Nanobodies, or VHHs, are derived from the variable regions of camelid heavy-chain only immunoglobulins. Nanobodies are characterized by their small size, ease of production, stability, and specificity of recognition. We generated nanobodies that recognize membrane-bound MICA with high affinity. Here, we use these nanobodies as building blocks for a chimeric antigen receptor (CAR) to establish VHH-based CAR NK cells. These anti-MICA nanobody-based CAR NK cells recognize and selectively kill MICA-positive tumor cells in vitro and in vivo. We track localization of the VHH-based CAR NK cells to MICA-positive lung metastases by immuno-positron emission tomography imaging.

A monoclonal antibody that recognizes a unique 13-residue epitope in the cytoplasmic tail of HLA-E.

The Class I MHC molecule (MHC-I) HLA-E presents peptides that are derived from the signal sequences, either those of other MHC-I products, or of viral type I membrane glycoproteins. Monoclonal antibodies with proven specificity for HLA-E, and with no cross-reactions with other MHC-I products, have yet to be described. To obtain anti-HLA-E-specific antibodies suitable for a range of applications, we generated monoclonal antibodies against a unique feature of HLA-E: its cytoplasmic tail. We created an immunogen by performing an enzymatically catalyzed transpeptidation reaction to obtain a fusion of the cytoplasmic tail of HLA-E with a nanobody that recognizes murine Class II MHC (MHC-II) products. We obtained a mouse monoclonal antibody that recognizes a 13-residue stretch in the HLA-E cytoplasmic tail. We cloned the genes that encode this antibody in expression vectors to place an LPETG sortase recognition motif at the C-terminus of the heavy and light chains. This arrangement allows the site-specific installation of fluorophores or biotin at these C-termini. The resulting immunoglobulin preparations, labeled with 4 equivalents of a fluorescent or biotinylated payload of choice, can then be used for direct immunofluorescence or detection of the tag by fluorescence or by streptavidin-based methods. We also show that the 13-residue sequence can serve as an epitope tag, independent of the site of its placement within a protein's sequence. The antibody can be used diagnostically to stain for HLA-E on patient tumor samples, it can be used as an antibody-epitope tag for extracellular proteins, and it enables research into the unique role of the cytoplasmic tail of HLA-E.

MICA-specific nanobodies for diagnosis and immunotherapy of MICA+ tumors.

MICA and MICB are Class I MHC-related glycoproteins that are upregulated on the surface of cells in response to stress, for instance due to infection or malignant transformation. MICA/B are ligands for NKG2D, an activating receptor on NK cells, CD8+ T cells, and γδ T cells. Upon engagement of MICA/B with NKG2D, these cytotoxic cells eradicate MICA/B-positive targets. MICA is frequently overexpressed on the surface of cancer cells of epithelial and hematopoietic origin. Here, we created nanobodies that recognize MICA. Nanobodies, or VHHs, are the recombinantly expressed variable regions of camelid heavy chain-only immunoglobulins. They retain the capacity of antigen recognition but are characterized by their stability and ease of production. The nanobodies described here detect surface-disposed MICA on cancer cells in vitro by flow cytometry and can be used therapeutically as nanobody-drug conjugates when fused to the Maytansine derivative DM1. The nanobody-DM1 conjugate selectively kills MICA positive tumor cells in vitro.

ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells.

Cancer stem cells (CSCs) may serve as the cellular seeds of tumor recurrence and metastasis, and they can be generated via epithelial-mesenchymal transitions (EMTs). Isolating pure populations of CSCs is difficult because EMT programs generate multiple alternative cell states, and phenotypic plasticity permits frequent interconversions between these states. Here, we used cell-surface expression of integrin ß4 (ITGB4) to isolate highly enriched populations of human breast CSCs, and we identified the gene regulatory network operating in ITGB4+ CSCs. Specifically, we identified ΔNp63 and p73, the latter of which transactivates ΔNp63, as centrally important transcriptional regulators of quasi-mesenchymal CSCs that reside in an intermediate EMT state. We found that the transcriptional program controlled by ΔNp63 in CSCs is largely distinct from the one that it orchestrates in normal basal mammary stem cells and, instead, it more closely resembles a regenerative epithelial stem cell response to wounding. Moreover, quasi-mesenchymal CSCs repurpose this program to drive metastatic colonization via autocrine EGFR signaling.

A potential role for CCN2/CTGF in aggressive colorectal cancer.

CCN2, also known as connective tissue growth factor (CTGF) is a transcriptional target of TGF-ß signaling. Unlike its original name ("CTGF") suggested, CCN2 is not an actual growth factor but a matricellular protein that plays an important role in fibrosis, inflammation and connective tissue remodeling in a variety of diseases, including cancer. In pancreatic ductal adenocarcinoma, CCN2 signaling induces stromal infiltration and facilitates a strong tumor-stromal interaction. In many types of cancer, CCN2 overexpression has been associated with poor outcome. CMS4 (Consensus Molecular Subtype 4) is a recently identified aggressive colorectal cancer subtype, that is characterized by up-regulation of genes involved in epithelial-to-mesenchymal transition, TGF-ß signaling, angiogenesis, complement activation, and extracellular matrix remodeling. In addition, a high influx of stromal fibroblasts contributes to the mesenchymal-like gene expression profile of this subtype. Furthermore, compared with the other three CMS groups, CMS4 tumors have the worst prognosis. Based on these observations, we postulated that CCN2 might contribute to colorectal cancer progression, especially in the CMS4 subtype. This review discusses the available literature on the role of CCN2 in colorectal cancer, with a focus on the 'fibrotic subtype' CMS4.