Development of Anti-LRRC15 Small Fragments for Imaging Purposes Using a Phage-Display ScFv Approach

Abstract

The human leucine-rich repeat-containing protein 15 (LRRC15) is a membrane protein identified as a marker of CAF (cancer-associated fibroblast) cells whose overexpression is positively correlated with cancer grade and outcome. Nuclear molecular imaging (i.e., SPECT and PET) to track LRRC15 expression could be very useful in guiding further therapeutic strategies. In this study, we developed an ScFv mouse phage-display library to obtain small fragment antibodies against human LRRC15 for molecular imaging purposes. Mice were immunized with recombinant human LRRC15 (hLRRC15), and lymph node cells were harvested for ScFv (single-chain variable fragment) phage-display analysis. The built library was used for panning on cell lines with constitutive or induced expression after transfection. The choice of best candidates was performed by screening various other cell lines, using flow cytometry. The selected candidates were reformatted into Cys-ScFv or Cys-diabody by addition of cysteine, and cloned in mammalian expression vectors to obtain batches of small fragments that were further used in site-specific radiolabeling tests. The obtained library was 1.2 × 107 cfu/µg with an insertion rate >95%. The two panning rounds performed on cells permittedenrichment of 2 × 10−3. Screening with flow cytometry allowed us to identify 28 specific hLRRC15 candidates. Among these, two also recognized murine LRCC15 and were reformatted into Cys-ScFv and Cys-diabody. They were expressed transiently in a mammalian system to obtain 1.0 to 4.5 mg of Cys fragments ready for bioconjugation and radiolabeling. Thus, in this paper, we demonstrate the relevance of the phage-display ScFv library approach for the fast-track development of small antibodies for imaging and/or immunotherapy purposes.

Phage Display Technology in Biomarker Identification with Emphasis on Non-Cancerous Diseases

“…The application of the phage display approach to create a vaccine against systemic candidiasis was recently proposed in a mouse model [114]. Several experiments have employed phage display to generate antibodies against bacterial toxins such as scFv antibodies against botulinum and alpha-toxin or VHH antibodies against anthrax toxin and Clostridioides Difficile toxin [115]. In a very recent study, Xu et al used phage display to produce a scFv-Fc antibody that neutralized the α-hemolysin toxin of Staphylococcus aureus.…”

New role of bacteriophages in medical oncology

“…They showed that by using the phage display technique, a fast-track development can be provided to obtain antibodies for imaging and diagnostic purposes. 53 In a 2013 study by Tsedev et al, they engineered an M13-derived small filamentous phage called "inho" by constructing a collection of small phage ssDNA packaged by M13 capsid proteins. 54 The 50 nm size of this phage makes it advantageous for use in the diagnosis, imaging and treatment of human tumors.…”

“…After biopanning, a suitable single‐chain variable fragment molecule was selected and radiolabeled through bioconjugation. They showed that by using the phage display technique, a fast‐track development can be provided to obtain antibodies for imaging and diagnostic purposes 53 . In a 2013 study by Tsedev et al., they engineered an M13‐derived small filamentous phage called “inho” by constructing a collection of small phage ssDNA packaged by M13 capsid proteins 54 .…”

New Anti-RSV Nucleoprotein Monoclonal Antibody Pairs Discovered Using Rabbit Phage Display Technology

“…A chimeric Fab library (the variable part from the rabbit and the constant Fab part from the mouse Kappa/IgG1 domain) was built in our proprietary phagemid [17]. The histidine tag was followed by a Myctag in the C-terminal part of the construction (Scheme 1).…”

“…The rabbit variable IgG VH and VL sequences were amplified following 2 rounds of successive PCRs using a set of primers adapted from Nguyen et al [18]. The PCR protocol was a modification of the method used by Baurand et al [17]. The first PCR rounds were performed to amplify the VH or VL sequences starting from the cDNA.…”