The recent successes of adoptive T-cell immunotherapy for the treatment of hematologic malignancies have highlighted the need for manufacturing processes that are robust and scalable for product commercialization. Here we review some of the more outstanding issues surrounding commercial scale manufacturing of personalized-adoptive T-cell medicinal products. These include closed system operations, improving process robustness and simplifying work flows, reducing labor intensity by implementing process automation, scalability and cost, as well as appropriate testing and tracking of products, all while maintaining strict adherence to Current Good Manufacturing Practices and regulatory guidelines. A decentralized manufacturing model is proposed, where in the future patients' cells could be processed at the point-of-care in the hospital.

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In this study gangliosides from various myelomas and hybridomas of mouse, rat, and human origin were characterized by thin-layer and high-performance liquid chromatography, immunological methods (overlay technique) and fast atom bombardment mass spectrometry. Exclusively GM3 substituted with C24:1- and C16:0-fatty acid, was found in all B cell-derived cell lines. C18 sphingosine was the single long chain base in each GM3 ceramide portion. The mouse myeloma (NS-1) and all hybridomas, obtained by fusion of mouse, rat, or human B lymphocytes with murine myelomas, showed high GM3 (NeuGc) content (> 75%) and low GM3 (NeuAc) expression. Absolute amounts of GM3 ranged from 0.2 up to 0.8 mg x 10(-9) cells. Normally, human cells do not express NeuGc, and an Epstein-Barr virus-transformed human B lymphocyte line analyzed in this study retained this sialylation status, expressing exclusively GM3 (NeuAc) (100%). The fusion of human B lymphocytes with mouse myelomas led to high GM3 (NeuGc) expression (average about 85%) in all mouse/human heterohybridomas examined. Our results indicate the chromosomal gene "transfer" and/or the activation of enzymes involved in NeuGc-biosynthesis due to the somatic cell fusion process, which might explain the mouse dominance in the manifestation of the NeuGc-phenotype in hybridomas of human origin.

Biochemical analyses of murine lymphocytes have shown that the glycosphingolipid globoside (Glo) is present exclusively on alloantigen-stimulated murine T lymphocytes (Gruner, K. R., Van Eijk, R. V. W. and Mühlradt, P. F., Biochemistry 1981. 20: 4518). An anti-Glo antibody has now been raised in rabbits immunized with purified antigen. Most activity was recovered in the IgM fraction. The specificity of the antibody was ascertained in an enzyme-linked immunosorbent assay with purified glycosphingolipids bound to the solid phase. In antibody-dependent complement lysis experiments the anti-Glo eliminated about 20% of nylon wool-nonadherent splenic T cells of CBA/J mice. To determine the functional identity of these Glo+ cells, the effects of Glo+ cell elimination on mitogen stimulation with concanavalin A and lipopolysaccharide, as well as the effects on the mixed lymphocyte culture (MLC) reaction and cell-mediated lympholysis with mitomycin-treated DBA/2 splenocytes as stimulator cells were studied. Whereas lipopolysaccharide stimulation was not affected by elimination of Glo+ cells, there was a slight inhibitory effect on the concanavalin A stimulation, and a severe inhibition of the MLC reaction and the generation of H-2d-specific cytolytic T lymphocytes. Addition of interleukin 2 increased the MLC reaction, but interleukin 2-saturated cultures were also severely inhibited by anti-Glo and complement treatment. Combined treatment with anti-Glo and anti-Lyt-1 or anti-Lyt-2 antibodies, and determination of cytolytic T lymphocyte precursor frequencies in limiting dilution cultures after Glo+ cell elimination showed that a large proportion of T cells proliferating in a primary MLC are Lyt-1+,2+,3+Glo+, whereas in secondary MLC they are Lyt-1+,2-,3-,Glo+. Fifty % of the cytolytic T lymphocyte precursors in primary as well as secondary MLC are Glo+. The Glo marker is lost upon differentiation to cytolytic T lymphocyte effector cells. It is discussed herein that Glo is a marker for alloantigen-stimulated precursor T lymphocytes of both helper and cytolytic T cells.

We investigated the in vitro immunosuppressive effect of BT 563, a monoclonal antibody, against the achain of the human interleukin-2 (IL-2) receptor (p 55), which has been used to prevent transplant rejection in several clinical trials. We also measured the proliferative T cell alloresponse and pCTL frequencies of BT 563treated kidney transplant patients. In mixed lymphocyte cultures BT 563 caused a reduction ofT cell proliferation to about 50%. This could not be reversed by the addition of exogenous IL-2. A more effective reduction (80%) was seen in the generation of cytotoxic T cells from CML cultures and at the clonal level. The specific T cell response after preincubation with antigen and BT 563 was not reduced so that BT 563 did not induce tolerance. The in vitro findings indicated that BT 563 had a significant but incomplete immunosuppressive effect. This correlated with the clinical course and ex vivo analysis of PBL from BT 563-treated patients after kidney transplantation.The human interleukin-2 (IL-2} receptor consists of two glycoproteins, p55 and p75, which by themselves display low and intermediate affinity for IL-2 and combine to form a high affinity receptor complex.BT 563 is an IgG 1 mouse monoclonal antibody, developed by Wijdenes [6], which is directed against the p55 chain of the IL-2 receptor. It blocks ligand binding to both low and high affinity receptors. Since only activated T lymphocytes express p55 following organ transplantation, the suppressive effect should concentrate on transplant reactive cells. In several clinical trials BT 563 has been used to prevent rejection after solid organ transplantation and to treat graft-versus-host disease after bone marrow transplantation.Offprint requests to: Jorg Steinmann, Inst.Initial experiments have revealed that BT 563 does not deplete target cells and that the blocking of IL-2 binding is noncompetitive. To increase our understanding of the clinically observed immunosuppressive effect, we investigated the mode of action of BT 563 in various T cell assays in vitro. Additionally, anti-donor T cell reactivity in kidney transplant patients was measured before, during and after BT 563 rejection prophylaxis. Materials and methodsBT 563. BT 563 is a mouse IgG 1 monoclonal antibody against the human IL-2 receptor. It is produced and purified by Biotext Pharma according to the guidelines of the European Communities: "On the production and quality control of monoclonal antibodies of murine origin intended for usc in man".Cells. Peripheral blood lymphocytes (PBL) from healthy donors were obtained by density gradient centrifugation of heparinized blood.MLR. One-way mixed lymphocyte reactions (MLR) were carried out with PBL from different donors. We seeded 5 x 104 responder cells and 5 x 104 irradiated stimulator cells per well into 96-well round-bottomed microculuture plates in 200111 RPMI 1640, supplemented with 15% AB serum, glutamine and penicillin/streptomycin. After 96 h the microcultures were pulsed with 37 kBq/well JH-methylthymidine for 24 hand har...