Towards a commercial process for the manufacture of genetically modified T cells for therapy.

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.

Expression of gangliosides GM3 (NeuAc) and GM3 (NeuGc) in myelomas and hybridomas of mouse, rat, and human origin.

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.

Rejection prophylaxis with interleukin-2 receptor antibody BT 563: mechanisms of action on human cells.

We investigated the in vitro immunosuppressive effect of BT 563, a monoclonal antibody, against the alpha-chain 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 BT563-treated kidney transplant patients. In mixed lymphocyte cultures BT 563 caused a reduction of T 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.

[Lung imaging in intensive medicine. A comparison of digital storage-screen imaging and conventional film-screen imaging]. / Lungenaufnahmen in der Intensivmedizin. Ein Vergleich von digitalen Speicherfolien- und konventionellen Film-Folien-Aufnahmen.

Luminescence radiography is a new digital imaging modality, which can be used instead of conventional film radiography for many examinations. Imaging capabilities of luminescence radiography were compared with film radiography by test images and examinations especially in intensive care radiology. The results demonstrate, that luminescence radiography is an advantageous imaging method for intensive care radiology.

[Cerebral blood flow scintigraphy using 99mTc-HM-PAO in the diagnosis of brain death]. / Hirnblutflussszintigraphie mit 99mTc-HM-PAO zur Diagnose des Hirntodes.

In 9 patients with the clinical diagnosis of brain death, a HM-PAO-SPECT was performed to document the arrest of cerebral circulation. Besides brain-stem areflexia, negative apnea-test and an observation time of at least 12 hours neurophysiological examinations (EEG and evoked potentials) were performed. In 4 cases, an additional arterial digital subtraction angiography proved the arrest of cerebral circulation shown in SPECT. In 5 cases, there was cortical low activity still present in EEG with negative SPECT. HM-PAO-SPECT seems to be a reliable noninvasive radiological method in the determination of brain death. It is especially helpful in cases with persisting focal cortical EEG-activity.

Forssman glycolipid, an antigenic marker for a major subpopulation of macrophages from murine spleen and peripheral lymph nodes.

Three hybridoma clones were isolated after hybridization of a mouse myeloma line with splenocytes from rats immunized with Forssman glycosphingolipid (Fo). Two of these clones produced Fo-specific monoclonal antibodies (MAB) of the IgM class, one MAB of the IgG2c class. In complement-dependent depletion experiments and immunofluorescence studies on the nature of Fo-positive leukocytes in CBA/J mice the following results were obtained: whereas blood monocytes, polymorphonuclear leukocytes, and lymphocytes were Fo negative, 5 to 10% of suspended spleen cells were positive. The majority of these were macrophage-like, glass- and nylon-adherent, nonspecific esterase-positive phagocytizing cells carrying Ia and globoside markers. These cells participated as accessory cells in the mixed lymphocyte culture reaction. In cell suspensions from axillary and inguinal lymph nodes, 2% were Fo positive. They were enriched up to 70% in the glass-adherent, esterase-positive population from this source. In contrast, no Fo-positive cells were detected in mesenteric lymph nodes, and less than 0.1% of the resident peritoneal macrophages bore this marker. The percentage of Fo-positive cells increased to 1% in thioglycollate-elicited peritoneal cells. Immunostaining of cryosections of lung and liver tissue showed alveolar macrophages and Kupffer cells, respectively, to be Fo negative.

An improved semi-quantitative enzyme immunostaining procedure for glycosphingolipid antigens on high performance thin layer chromatograms.

An immunoassay is described which allows the detection of glycosphingolipid (GSL) antigens on high performance thin layer chromatograms (HPTLC). The method involves: (1) the separation of GSL on HPTLCs; (2) incubation with specific antibodies against carbohydrate structures of GSL, and (3) the detection of specifically bound antibodies with alkaline phosphatase-conjugated second antibodies and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) as substrate. Using a monoclonal rat IgG2c antibody against Forssman GSL, a BALB/c monoclonal antibody against asialo GM2, and polyclonal rabbit antibodies against asialo GM1, it was shown that as little as 3 ng GSL antigen could be detected in a procedure taking detected in a procedure taking only 4 h to perform. The assay should be useful for screening mono- and polyclonal antibodies with potential specificity for GSL antigens, for the detection and quantification of GSL-antigens in tissue extracts, and for defining the specificity of anti-GSL antibodies.

The glycosphingolipid globoside as a serological marker on cytolytic T lymphocyte precursors and alloantigen-responsive proliferating T lymphocytes in murine spleen.

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.

[Anesthesia in concomitant bronchopulmonary diseases]. / Anästhesie bei bronchopulmonalen Nebenerkrankungen.

The goal of any anaesthesiologist delivering anaesthesia to patients with pre-existing lung disease has to be the prevention of intra- and postoperative complications. Changes in gas exchange imposed by anaesthesia and surgery can cause dangerous disturbances in oxygen transport and in acid-base balance. Uptake and elimination of volatile and gaseous anaesthetics depend upon normal respiratory function. Any marked impairment can considerably modify the kinetics of these processes.

[The need for a specific postoperative, total parenteral nutrition therapy after various intra-abdominal procedures]. / Zur Frage der Notwendigkeit einer spezifischen postoperativen, totalen parenteralen Ernährungstherapie nach unterschiedlichen intraabdominellen Eingriffen.

119 metabolically healthy surgical patients, who had to undergo elective intraabdominal surgery, were separated into upper -- cholecystectomy, gastric resection -- and lower -- colonic and sigma surgery -- intraabdominal procedures and divided into 8 groups with different infusion regimes. During the early postoperative period patients with upper abdominal surgery had significantly higher postoperative enzyme changes compared with patients with lower abdominal surgery. Patients with cholecystectomy had the significantly highest postoperative enzyme changes. On postoperative day 6 the enzyme changes showed a significant dependence from the duration of total parenteral nutrition, the chosen calorie-nitrogen-ratio, and the chosen energy substrate, xylitol or glucose. We could show a significant negative correlation between the extent of the stimulation of hepatic lipid synthesis and the protein parameters. Of all chosen infusion regimens xylitol in a dose of 0.11 g/kg BW X h together with the high amino acid infusion rate of 1.76 g/kg BW X day had the most favourable effect on the extent of stimulation of hepatic lipid synthesis, the late postoperative enzyme changes and the synthesis rate of visceral proteins. This study could demonstrate, that different intraabdominal surgical procedures need a more specific nutritional therapy.

[Comparison of xylose and glucose as energy sources in hypocaloric, postoperative parenteral nutrition]. / Vergleich von Xylit und Glukose als Energieträger im Rahmen der hypokalorischen postoperativen parenteralen Ernährungstherapie.

After a 5 day preoperative preparing period 24 metabolically healthy patients, who had to undergo gastric resection, were fed postoperatively by hypocaloric total parenteral nutrition for a 5 day period. Group I (n = 13) received 0,11 g Xylitol/kg BW X h; Group II (n = 11) received 0,11 g Glucose/kg BW X h; Both groups received 1,71 g L-crystalline amino acids/kg BW X day. During the whole postoperative period group II had significantly higher serum Glucose and Insulin levels. Due to the high postoperative Insulin concentration in group II. Free Fatty Acids, Acetate, beta-Hydroxybutyrate and Branched Chained Amino Acids were significantly lower. The augmented release of Muscle Amino Acids and the covering of a part of the energy expenditure by increased Fatty Acid oxidation in group I led to a higher postoperative synthesis rate of visceral proteins. From postoperative day 3 on Transferrin and from postoperative day 6 on Albumin and Total Protein were significantly higher in group I. This study could demonstrate, that due to its special role in the intermediary metabolism during the postoperative period Xylitol leads to a significantly higher regeneration rate of visceral proteins compared to Glucose during total parenteral nutrition.

[Psychosomatic effects of intramuscular premedication wtih flunitrazepam, innovar and a placebo in combination with atropine (author's transl)]. / Psychische und somatische Auswirkungen der Prämedikation mit Rohypnol, thalamonal und Plazebo in Kombination mit Atropin.

A double-blind technique was used for studying the psychic and somatic effects of premedication with flunitrazepam, innovar and a placebo in 116 patients. Innovar and the placebo produced the same results. Flunitrazepam reduced anxiety and apprehension and had a significantly higher amnesic effect. Premedication with Innovar increased not only a feeling of fatigue and heaviness but also anxiety and tension in the patients. When assessing the psychic effects of premedication attention must be paid to the subjective impressions on the part of the patient.

[Differences in postoperative metabolism after pre- and postoperative beginning of total parenteral nutrition]. / Unterschiede im postoperativen Stoffwechselverhalten bei prä- und postoperativem Beginn der totalen parenteralen Ernährung. II. Mitteilung.

In this study we intended to investigate the influence of a 24-h-preoperative total parenteral nutrition (TPN) therapy--10 surgical patients, group 1--compared with the postoperative beginning of TPN--9 surgical patients, group 2--on the postoperative metabolism. Most probably due to preoperatively depleted glycogen storages in G 2, the urinary carbohydrate losses declined more rapidly postoperatively compared with G 1. 45 mMol/l of sodium did not cover the postoperative requirements of G 1. Although we administered 90 mMol/l of potassium/24 h, several patients of G 1 needed an additional substitution. No additional phosphate substitution was necessary in either group. Except for a slight positive balance on the operation day, fluid balance was well balanced on all the other days in G 1. From postoperative day 2 on, G 2 developed a deficit of free water. It could be demonstrated very clear in this study that the postoperative fluid and electrolyte requirements are strongly influenced even by a short-term preoperative fasting period.

[Postoperative metabolism--differences between pre- and postoperative start of total parenteral nutrition (author's transl)]. / Der postoperative Stoffwechsel--Unterschiede zwischen prä- und postoperativem Beginn der totalen parenteralen Ernährung.

We investigated the postoperative metabolism of patients undergoing gastric operation by beginning total parenteral nutrition pre- (group 1) and postoperatively (group 2). A third group remained fasting until 3 h after the surgical intervention. Because of the high serum glucose concentrations and the high glucose losses in the 24-h-urine on the operation day in group 1 and 2 only the basic glucose requirement (150--250 mg/24h) should be given in the early postoperative period. The low blood glucose concentration in group 3 and the elevated lactate values in group 2 underline this recommendation. Compared with group 2 and 3 group 1 had low free fatty acid concentrations of 250 muVal/l because of the continuous pre-, intra-, and postoperative infusion. Compared with normal metabolic conditions the free fatty acids don't seem to have the same insulinantagonising effect in the posttraumatic state. The fall of cholesterol after the surgical trauma reached its maximum after 12--15 h and amounted to about 40mg% in the first two groups. Albumin and total protein fell continuously in group 1 and 2. The electrophoresis in group 1 showed a rise of alpha 1-, and alpha 2-globulin to more than double the initial value, the beta-globulin showed only slight changes, the gamma-globulin dropped only slightly up to the 4th postoperative day. As a sign of an augmented catabolism the serum urea concentration rose during the postoperative state. Group 1 and 2 had a favourable nitrogen balance. The postoperative bilirubin rise could be held lower in group 1 compared to group 2.

[Differences in postoperative metabolism in reference to pre- and postoperative initiation of total parenteral nutrition. I]. / Unterschiede im postoperativen Stoffwechselverhalten bei prä- und postoperativem Beginn der totalen parenteralen Ernährung. I. Mitteilung.

We investigated the postoperative metabolism of patients undergoing gastric resection by beginning total parenteral nutrition pre- (group 1) and postoperatively (group 2). A third group remained fasting until 3 h after the surgical intervention. Because of the high serum glucose concentrations and the high glucose losses in the 24-h urine on the operation day in group 1 and 2 only the base glucose requirement (150--250 g/24/h) should be given in the early postoperative period. The low blood glucose concentration in group 3 and the elevated lactate values in group 2 underline this recommandation. Compared with group 2 and 3, group 1 had low free fatty acid concentrations of 250 micronVal/l because of the continuous pre-, intra-, and postoperative infusion. Compared with normal metabolic conditions, the free fatty acids do not seem to have the same insulin-antagonizing effect in the posttraumatic state. The fall of cholesterol after the surgical trauma reached its maximum after 12--15 h and amounted to about 40 mg% in the first two groups. Albumin and total protein fell continuously in group 1 and 2. The electrophoresis in group 1 showed a rise of alpha 1- and alpha 2-globulin to more than double the initial value, the beta-globulin showed only slight changes, the gamma-globulin dropped only slightly up to the 4th postoperative day. As a sign of an augmented catabolism the serum urea concentration rose during the postoperative state. Group 1 and 2 had a favourable nitrogen balance. The postoperative bilirubin rise could be held lower in group 1 compared to group 2.

[The sedative and amnesic effects of flunitrazepam during regional anaesthesia (author's transl)]. / Rohypnol (Flunitrazepam) als Sedativum bei Leitungsanästhesien unter besonderer Berücksichtigung der amnestischen Wirkung.

The sedative, hypnotic and amnesic effects of flunitrazepam, a new benzodiazepine, were tested in doses of 0 (placebo), 0,4 mg and 0,8 mg. These doses were sufficient to produce a satisfying sedative effect on patients, being operated on in regional anaesthesia. In few cases we found undesirable effects. Positive and negative aspects of amnesia will be discussed.