Development of Novel Antibody-Based Immunotherapies Targeting Human Chondroitin Sulfate Proteoglycan 4
Chondroitin sulfate proteoglycan 4 (CSPG4) is a promising target for cancer immunotherapy due to its high level of expression in a number of malignant tumors, and its essential role in tumor growth and progression. Clinical application of CSPG4-targeting immunotherapies is hampered by the lack of fully human CSPG4 antibodies or antibody fragments. In addition, the efficacy of cytotoxic monotherapies, such as the CSPG4-targeting immunotoxins (ITs), is limited by hyperactive anti-apoptotic pathways prevalent in tumor cells. Therefore, there is a need to discover novel, fully human antibodies for CSPG4-targeting immunotherapies and to develop new strategies that sensitize resistant CSPG4-expressing tumor cells to IT therapies.
To discover fully human antibodies that can be developed into potential CSPG4-targeting therapeutics, my first aim is to develop novel human single-chain variable fragments (scFvs) with high binding affinity and specificity to the CSPG4 antigen. Affinity maturation was performed on a novel, fully human anti-CSPG4 scFv using the random mutagenesis approach. A yeast display library was constructed for the mutant clones, and screened using a modified whole-cell panning method followed by fluorescence-activated cell sorting (FACS). After six rounds of panning and sorting, the top seven mutant scFvs were isolated and their binding affinities were characterized by flow cytometry and surface plasmon resonance. These mutant clones were highly specific to the CSPG4 antigen, and displayed nanomolar to picomolar binding affinities. While each of them harbored only two to six amino acid substitutions, they represented approximately 270-3000-fold improvement in affinity compared to the parental clone. These affinity-matured scFvs can be potentially developed into diagnostic or therapeutic agents for evaluation and treatment of CSPG4-expressing tumors.
To facilitate the screening of scFv libraries targeting CSPG4, my second aim is to develop a cell-based fluorescent assay for high-throughput analysis of antibody affinity (KD) in the nanomolar range. In this method, fluorescently labelled antibodies were added to antigen-positive and antigen-negative cell lines fixed on 96-well plates. The fluorescent signals from nonspecific binding to negative control cell lines is subtracted from the specific binding to the antigen-positive cell lines. The results confirmed that the KD values obtained using this method were comparable with values obtained by the conventional flow cytometry and radioactive (I125) scatchard assays. This demonstrates that the cell-based fluorescent method allows for accurate and efficient identification of therapeutically relevant leads.
Finally, to improve the efficacy of ITs targeting CSPG4, especially in the IT-resistant tumor cells, my third aim is to evaluate a multi-pathway therapy that combines anti-CSPG4 ITs and small molecule Bcl-2 inhibitors. To enhance sensitivity of cancer cells to ITs, we combined ITs (9.2.27-PE38KDEL or Mel-14-PE38KDEL) targeting CSPG4 with a Bcl-2 inhibitor (ABT-737, ABT-263, or ABT-199) against patient-derived glioblastoma xenografts, melanoma cell lines, and breast cancer cell lines. Results from the in vitro cytotoxicity assays demonstrated that the addition of the ABT compounds, specifically ABT-737, sensitized all three tumors to the IT treatment, and in some cases improved the IC50 values of 9.2.27-PE38KDEL by over 1000-fold. Mechanistic studies using 9.2.27-PE38KDEL and ABT-737 revealed that the rate of IT internalization and the efficiency of cleaved exotoxin accumulation in the cytosol correlated with the enhanced sensitivity of the tumor cells to the combination treatment. Furthermore, the synergistic effect of 9.2.27-PE38KDEL and ABT-737 combination therapy was confirmed in an orthotopic GBM xenograft model and a model of melanoma metastasized to the brain. For the first time, our study compares the efficacy of ABT-737 and 9.2.27-PE38KDEL combination therapy in GBM and a different brain metastases model, providing insights into overcoming IT resistance in brain tumors.
In conclusion, I discovered novel human scFvs with high binding affinities to CSPG4, developed a cell-based fluorescent method for accurate and efficient affinity analysis of antibodies, and investigated combination immunotherapies that utilized Bcl-2 inhibitors to sensitize tumor cells to treatment by CSPG4-targeting ITs. The results from these studies helped to facilitate the development of novel antibody-based immunotherapies and combination immunotherapies for CSPG4-expressing tumors.
Fluorescent affinity assay
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