Identification of CD4 and transferrin receptor antibodies by CXCR4 antibody-guided Pathfinder selection

Area of Science:

• Immunology
• Virology
• Biotechnology

Background:

• CXCR4 is a crucial chemokine receptor and a primary coreceptor for HIV-1 entry.
• Developing specific human antibodies against CXCR4 is vital for potential therapeutic interventions against HIV-1.
• Phage display technology offers a powerful platform for antibody discovery.

Purpose of the Study:

• To generate human antibodies targeting the CXCR4 receptor using advanced phage display selection techniques.
• To evaluate the efficacy and specificity of Pathfinder and Step-back selection methods for whole-cell antibody discovery.
• To identify limitations in current phage display strategies for selecting antibodies against cell surface receptors.

Main Methods:

• Utilized Pathfinder and Step-back selection strategies on a large phage display antibody library.
• Employed Jurkat cells for selection, using anti-CXCR4 monoclonal antibodies or biotinylated phage antibodies as guide molecules.
• Characterized over 100 pan-Jurkat-cell-positive antibodies through extensive screening.

Main Results:

• No CXCR4-specific antibodies were successfully generated despite extensive selection efforts.
• Several antibodies targeting CD4 and the transferrin receptor were identified, indicating off-target binding.
• The selection process demonstrated limitations in achieving target specificity on whole cells.

Conclusions:

• Pathfinder and Step-back selection are viable for whole-cell phage antibody selection but face challenges with target specificity.
• Inaccessibility of extracellular target structures and limitations of guide molecules likely contributed to the lack of specific CXCR4 antibodies.
• Refinements in phage display selection techniques are necessary to enhance target specificity and selectivity for complex cell surface receptors.