Preparing clinical grade Ag-specific T cells for adoptive immunotherapy trials

Abstract

The production of clinical-grade T cells for adoptive immunotherapy has evolved from the ex vivo numerical expansion of tumor-infiltrating lymphocytes to sophisticated bioengineering processes often requiring cell selection, genetic modification and other extensive tissue culture manipulations, to produce desired cells with improved therapeutic potential. Advancements in understanding the biology of lymphocyte signaling, activation, homing and sustained in vivo proliferative potential have redefined the strategies used to produce T cells suitable for clinical investigation. When combined with new technical methods in cell processing and culturing, the therapeutic potential of T cells manufactured in academic centers has improved dramatically. Paralleling these technical achievements in cell manufacturing is the development of broadly applied regulatory standards that define the requirements for the clinical implementation of cell products with ever-increasing complexity. In concert with academic facilities operating in compliance with current good manufacturing practice, the prescribing physician can now infuse T cells with a highly selected or endowed phenotype that has been uniformly manufactured according to standard operating procedures and that meets federal guidelines for quality of investigational cell products. In this review we address salient issues related to the technical, immunologic, practical and regulatory aspects of manufacturing these advanced T-cell products for clinical use.

Figure 1

Schematic of relative propagation strategies to expand T cells numerically through TCR complex or CAR (shown as second generation capable of Ag-dependent signaling through chimeric CD3-ζ and CD28). (A) REP for robust and rapid Ag-independent T-cell proliferation by cross-linking CD3ε using OKT3 (and also if needed cross-linking CD28) bound to PBMC or aAPC. (B) Selective stimulation of (CD8⁺) T cells for Ag-dependent proliferation through TCR recognizing Ag in the context of HLA class I on APC or aAPC. A co-stimulatory signal initiated through B7 molecules (or anti-CD28) through CD28 is shown. (C) REP of genetically modified T cells co-expressing dominant selection marker (e.g. Escherichia coli hygromycin phosphotransferase) for Ag-independent proliferation by cross-linking CD3 (and CD28 if necessary) resulting in selection for outgrowth of CAR⁺ T cells in the presence of cytocidal concentrations of drug, e.g. hygromycin B. Incomplete CAR expression in stable transfectants may occur if CAR and drug-selection transgenes are under the control of separate promoters. (D) Selective stimulation of genetically modified T cells, in the presence of IL-15, for Ag-dependent proliferation through introduced CAR recognizing cell-surface Ag (e.g. CD19) independent of HLA class I on aAPC. The aAPC are typically modified to enforce expression of desired co-stimulatory molecules, e.g. 4-1BBL, CD80/86. (Figure not to scale and showing selected disulfide bonds).

Figure 1

Schematic of relative propagation strategies to expand T cells numerically through TCR complex or CAR (shown as second generation capable of Ag-dependent signaling through chimeric CD3-ζ and CD28). (A) REP for robust and rapid Ag-independent T-cell proliferation by cross-linking CD3ε using OKT3 (and also if needed cross-linking CD28) bound to PBMC or aAPC. (B) Selective stimulation of (CD8⁺) T cells for Ag-dependent proliferation through TCR recognizing Ag in the context of HLA class I on APC or aAPC. A co-stimulatory signal initiated through B7 molecules (or anti-CD28) through CD28 is shown. (C) REP of genetically modified T cells co-expressing dominant selection marker (e.g. Escherichia coli hygromycin phosphotransferase) for Ag-independent proliferation by cross-linking CD3 (and CD28 if necessary) resulting in selection for outgrowth of CAR⁺ T cells in the presence of cytocidal concentrations of drug, e.g. hygromycin B. Incomplete CAR expression in stable transfectants may occur if CAR and drug-selection transgenes are under the control of separate promoters. (D) Selective stimulation of genetically modified T cells, in the presence of IL-15, for Ag-dependent proliferation through introduced CAR recognizing cell-surface Ag (e.g. CD19) independent of HLA class I on aAPC. The aAPC are typically modified to enforce expression of desired co-stimulatory molecules, e.g. 4-1BBL, CD80/86. (Figure not to scale and showing selected disulfide bonds).

Figure 2

Schematic of an example of the physical infrastructure needed to manufacture clinical-grade T cells for phase I/II trials in compliance with current GMP. Not shown are the environmental control systems. The physical plant is staffed by dedicated and highly trained personnel following standard operating procedures (SOP) that conform to cGMP guidelines (not to scale).

Figure 3

Outline of single-institution reporting structure and requirements for manufacture and release of clinical-grade genetically modified T cells in compliance with cGMP for phase I/II trials.