Avoiding the Need for Clinical-Grade OKT3: Ex Vivo Expansion of T Cells Using Artificial Antigen Presenting Cells Genetically Modified to Cross-Link CD3 (2012)

T-cell immunotherapy protocols rely on activation for sustained proliferation in the ex vivo expansion of T cells to achieve clinically meaningful numbers for human applications. We have successfully expanded T cells using a robust platform technology based on K562 cells genetically modified to function as artificial antigen presenting cells (aAPC) for the numeric expansion of T cells. An aAPC clone (clone #4) was engineered to have enforced co-expression of CD64, CD86, membrane-bound interleukin (IL)-15, and CD137L (41BBL). Mouse anti-human CD3 (OKT3) monoclonal antibody can be loaded (bound by CD64) onto the aAPC to cross-link CD3 triggering ex vivo T-cell propagation. However, as clinical-grade Orthoclone OKT3 (muromonab from Ortho Biotech) is no longer available we generated a genetic approach to avoid the need to load OKT3. This was accomplished by heterologous fusion of the scFv region from OKT3 mAb to a drug resistance gene cassette (C3hygro) and enforcing expression under selection with hygromycin B. We initially expressed the C3hygro on tumor cells (e.g., MB-MDA-231 breast cancer cell line) and demonstrated with time-lapse video microscopy that C3hygro+ tumor cells could be efficiently killed by primary human T cells. This is similar in principle to the mechanism of action for bi-specific antibodies whereby T cells are brought into contact with antigen+ tumor cells, as our C3hygro+ tumor cells can be killed by previously unmodified T cells. We then adapted aAPC clone 4 to express C3hygro to generate a cellular platform for the non-specific propagation of T cells. These aAPC (K562OKT3) co-express the recognition domain of OKT3 in addition to the desired T-cell co-stimulatory molecules. T cells propagated using γ-irradiated K562OKT3 aAPC similar to OKT3-loaded clone #4 aAPC have comparable phenotype by flow cytometric analysis. The use of K562 is clinically appealing as we have generated master cell banks under INDs based on this cell line functioning as aAPC for human application. Indeed, the development of K562OKT3 aAPC appears to be a useful substitute for clinical protocols dependent on clinical-grade OKT3. In summary, we report our recent efforts to generate OKT3-based reagents K562OKT3 and C3hygro, useful for both clinical and research applications. T-cell immunotherapy protocols rely on activation for sustained proliferation in the ex vivo expansion of T cells to achieve clinically meaningful numbers for human applications. We have successfully expanded T cells using a robust platform technology based on K562 cells genetically modified to function as artificial antigen presenting cells (aAPC) for the numeric expansion of T cells. An aAPC clone (clone #4) was engineered to have enforced co-expression of CD64, CD86, membrane-bound interleukin (IL)-15, and CD137L (41BBL). Mouse anti-human CD3 (OKT3) monoclonal antibody can be loaded (bound by CD64) onto the aAPC to cross-link CD3 triggering ex vivo T-cell propagation. However, as clinical-grade Orthoclone OKT3 (muromonab from Ortho Biotech) is no longer available we generated a genetic approach to avoid the need to load OKT3. This was accomplished by heterologous fusion of the scFv region from OKT3 mAb to a drug resistance gene cassette (C3hygro) and enforcing expression under selection with hygromycin B. We initially expressed the C3hygro on tumor cells (e.g., MB-MDA-231 breast cancer cell line) and demonstrated with time-lapse video microscopy that C3hygro+ tumor cells could be efficiently killed by primary human T cells. This is similar in principle to the mechanism of action for bi-specific antibodies whereby T cells are brought into contact with antigen+ tumor cells, as our C3hygro+ tumor cells can be killed by previously unmodified T cells. We then adapted aAPC clone 4 to express C3hygro to generate a cellular platform for the non-specific propagation of T cells. These aAPC (K562OKT3) co-express the recognition domain of OKT3 in addition to the desired T-cell co-stimulatory molecules. T cells propagated using γ-irradiated K562OKT3 aAPC similar to OKT3-loaded clone #4 aAPC have comparable phenotype by flow cytometric analysis. The use of K562 is clinically appealing as we have generated master cell banks under INDs based on this cell line functioning as aAPC for human application. Indeed, the development of K562OKT3 aAPC appears to be a useful substitute for clinical protocols dependent on clinical-grade OKT3. In summary, we report our recent efforts to generate OKT3-based reagents K562OKT3 and C3hygro, useful for both clinical and research applications.