Improving Biologics’ Effectiveness in Clinical Oncology: From the Combination of Two Monoclonal Antibodies to Oligoclonal Antibody Mixtures

Monoclonal antibodies have revolutionized the treatment of many diseases, but their clinical efficacy remains limited in some other cases. Pre-clinical and clinical trials have shown that combinations of antibodies that bind to the same target (homo-combinations) or to different targets (hetero-combinations) to mimic the polyclonal humoral immune response improve their therapeutic effects in cancer. The approval of the trastuzumab/pertuzumab combination for breast cancer and then of the ipilimumab/nivolumab combination for melanoma opened the way to novel antibody combinations or oligoclonal antibody mixtures as more effective biologics for cancer management. We found more than 300 phase II/III clinical trials on antibody combinations, with/without chemotherapy, radiotherapy, small molecules or vaccines, in the ClinicalTrials.gov database. Such combinations enhance the biological responses and bypass the resistance mechanisms observed with antibody monotherapy. Usually, such antibody combinations are administered sequentially as separate formulations. Combined formulations have also been developed in which separately produced antibodies are mixed before administration or are produced simultaneously in a single cell line or a single batch of different cell lines as a polyclonal master cell bank. The regulation, toxicity and injection sequence of these oligoclonal antibody mixtures still need to be addressed in order to optimize their delivery and their therapeutic effects.

Cell Banking of HEK293T Cell Line for Clinical-Grade Lentiviral Particles Manufacturing

Background: Cell banks have been widely used to preserve cell properties as well as to record and control cell line access in research. However, the generation of cell banks involved in the manufacturing of Advanced therapy medicinal products such as cell or gene therapy products must comply with the current Good Manufacturing Practice regulation. Similarly, the quality of those cell lines used as starting materials in viral-vector manufacturing processes must be also evaluated.Methods: Three batches of both Master Cell Bank and Working Cell Bank of the HEK293T cell line were manufactured under the current Good Manufacturing Practices regulation. Quality control test were performed according to the product specifications. The process validation includes previous qualification of the manufacturing personnel by performing simulation tests as well as the continuous measure of environmental parameters during manufacturing such as air particles and microorganism. Cell number and viability of cryopreserved cells were periodically measured in order to define the stability of these cellular products.Results: All batches of Master Cell Bank and Working Cell Bank fulfilled the acceptance criteria of their specifications showing the robustness and homogeneity of the processes. In addition, both Master and Working cell bank maintain the defined viability and cell number 37 months after cryopreservation. Conclusions: Manufacturing cell banks under Good Manufacturing Practices regulation for its use as raw material or final cellular product is feasible. HEK293T cell banks have been used to manufacture clinical-grade lentiviral particles for Chimeric Antigen Receptor T-cell based clinical trials.

Cell Banking of HEK293T Cell Line for Clinical-Grade Lentiviral Particles Manufacturing

Background: Cell banks have been widely used to preserve cell properties as well as to record and control cell line access in research. However, the generation of cell banks involved in the manufacturing of Advanced therapy medicinal products such as cell or gene therapy products must comply with the current Good Manufacturing Practice regulation. Similarly, the quality of those cell lines used as starting materials in viral-vector manufacturing processes must be also evaluated.Methods: Three batches of both Master Cell Bank and Working Cell Bank of the HEK293T cell line were manufactured under the current Good Manufacturing Practices regulation. Quality control test were performed according to the product specifications. The process validation includes previous qualification of the manufacturing personnel by performing simulation tests as well as the continuous measure of environmental parameters during manufacturing such as air particles and microorganism. Cell number and viability of cryopreserved cells were periodically measured in order to define the stability of these cellular products.Results: All batches of Master Cell Bank and Working Cell Bank fulfilled the acceptance criteria of their specifications showing the robustness and homogeneity of the processes. In addition, both Master and Working cell bank maintain the defined viability and cell number 37 months after cryopreservation. Conclusions: Manufacturing cell banks under Good Manufacturing Practices regulation for its use as raw material or final cellular product is feasible. HEK293T cell banks have been used to manufacture clinical-grade lentiviral particles for Chimeric Antigen Receptor T-cell based clinical trials.

Abstract 260: Cardiosphere Derived Cells Generated from the Human Atria Exhibit Superior Potency Compared to the Left Ventricle

Currently, allogeneic cardiosphere-derived cells (CDCs) are being tested in a phase I/II clinical trial. Our manufacturing process utilizes the atria and other regions of the heart to build a master cell bank, but leaves the left ventricle (LV) intact thus eliminating a potential source to build our clinical stocks. In an effort to increase CDC yield per heart, we compared CDCs generated from the atria to the left ventricle (LV). To investigate this hypothesis we characterized CDCs by flow cytometry, growth kinetics and in vivo potency. The surface marker profiles for atrial CDCs and LV CDCs were very similar. Of the 17 markers used to characterize CDCs (c-kit, MDR-1, Sca-1, Abcg2, CD133, CD31, CD34, CD45, CD105, CD29, CD44, CD73, CD166, CD140b, CD90, DDR2 and α-SMA) only one demonstrated substantial differential expression in LV versus atria. PDGFR-β (CD140b) was upregulated in CDCs derived from the LV compared to the atria. Consistent with this finding, GEO2R analysis of DNA microarray data revealed increased PDGFR-β and PDGF-B expression in normal human LV tissue compared to atrial tissue. We have also observed that CDC expression of PDGFR-β negatively correlates with in vivo potency (R=0.899) suggesting that the high expression of this marker in CDCs derived from the LV may limit its regenerative performance. Indeed when calculated growth rates were compared, tissue samples from the atria yielded 4 to 6-fold more CDCs compared to the LV (atria=26±10 versus LV=5±5 Million CDCs/g/day). Also, CDCs derived from the atria and LV led to differential improvements in ejection fraction (EF) over three weeks. CDCs from the atria significantly outperformed the control group (atrial CDCs=Δ+12.6±10.8%, control=Δ-15.3±13.6%), while CDCs from the LV showed minimal treatment effects and failed to meet our minimal potency requirement (LV CDCs=Δ+0.0±6.2%). In conclusion, LV CDCs display limited potential for clinical use. This observation provides a unique opportunity to explore the mechanisms that govern functional potency and assist in understanding the basic processes involved in CDC mediated repair.