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

Area of Science:

• Biotechnology
• Molecular Imaging
• Immunology

Background:

• Leucine-rich repeat-containing protein 15 (LRRC15) is a membrane protein and a marker for cancer-associated fibroblasts (CAFs).
• Overexpression of LRRC15 correlates with cancer grade and patient outcomes.
• Molecular imaging techniques like SPECT and PET can track LRRC15 expression for therapeutic guidance.

Purpose of the Study:

• To develop small antibody fragments targeting human LRRC15 for molecular imaging.
• To establish a rapid method for generating antibodies using phage-display technology.

Main Methods:

• Construction and screening of a single-chain variable fragment (ScFv) mouse phage-display library against human LRRC15.
• Immunization of mice with recombinant hLRRC15 and harvesting lymph node cells.
• Panning the library on cell lines, followed by flow cytometry screening.
• Reformatting selected ScFv candidates into Cys-ScFv or Cys-diabody for radiolabeling.

Main Results:

• A phage-display library of 1.2 × 10^7 cfu/µg with >95% insertion rate was generated.
• Flow cytometry screening identified 28 specific hLRRC15 candidates.
• Two candidates recognizing both human and murine LRRC15 were reformatted and expressed as Cys-ScFv and Cys-diabody fragments (1.0-4.5 mg).

Conclusions:

• Phage-display ScFv library approach is effective for rapid development of small antibodies.
• The generated antibody fragments are suitable for bioconjugation and site-specific radiolabeling.
• This work facilitates the development of novel imaging agents and potential immunotherapies targeting LRRC15.