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.

Spatial control of cell differentiation in Myxococcus xanthus.

Myxococcus xanthus develops species-specific multicellular fruiting bodies. Starting from a uniform mat of cells, some cells enter into nascent fruiting body aggregates, whereas other cells remain outside. The cells within the fruiting body differentiate from rods into spherical, heat-resistant spores, whereas the cells outside the aggregates, called peripheral cells, remain rod-shaped. Early developmentally regulated genes are expressed in peripheral cells as well as by cells in the fruiting bodies. By contrast, late developmental genes are only expressed by cells within the nascent fruiting bodies. The data show that peripheral cells begin to develop, but are unable to express genes that are switched on later than about 6 h after the start of development. All of the genes whose expression is limited to the fruiting body are dependent on C-signaling either directly or indirectly, whereas the genes that are equally expressed in peripheral rods and in fruiting body cells are not. One of the C-signal-dependent and spatially patterned operons is called dev, and the dev operon has been implicated in the process of sporulation. It is proposed that expression of certain genes, including those of the dev operon, is limited to the nascent fruiting body because fruiting body cells engage in a high level of C-signaling. Peripheral cells do less C-signaling than fruiting body cells, because they have a different spatial arrangement and are at lower density. As a consequence, peripheral cells fail to express the late genes necessary for spore differentiation.

Ectopic expression of a chimeric colony-stimulating factor-1/TrkB-receptor promotes CSF-1-dependent survival of cultured sympathetic neurons.

The regulation of the density of innervation and the promotion of survival of neurons are the original effects depending on neurotrophins. Here we analyse such effects evoked by trkB tyrosine kinase in transfected PC12 cells and transfected sympathetic neurons. In order to exclude the previously described modulation of trk kinase activity by the extracellular activation of the low-affinity p75 neurotrophin receptor, we applied a chimeric receptor approach: The extracellular domain of colony-stimulating factor-1 (CSF-1) receptor was fused to the transmembrane and cytoplasmic domain of the trkB tyrosine kinase receptor, allowing its selective activation by the heterologous ligand. Protein expression and CSF-1-induced tyrosine phosphorylation of the chimeric receptor protein was demonstrated in transfected COS cells. After stable transfection into nerve growth factor (NGF)-responsive PC12 cells, CSF-1 mediated the K252a-sensitive induction of fiber outgrowth. Furthermore, we were able to show by heterologous expression of the chimeric receptor, that activation of trkB tyrosine kinase activity is sufficient to promote survival of neurotrophin deprived sympathetic neurons.

Gliding movements in Myxococcus xanthus.

Prokaryotic gliding motility is described as the movement of a cell on a solid surface in the direction of the cell's long axis, but its mechanics are unknown. To investigate the basis of gliding, movements of individual Myxococcus xanthus cells were monitored by employing a video microscopy method by which displacements as small as 0.03 micron could be detected and speeds as low as 1 micron/min could be resolved. Single cells were observed to glide with speeds varying between 1 and 20 microns/min. We found that speed variation was due to differences in distance between the moving cell and the nearest cell. Cells separated by less than one cell diameter (0.5 micron) moved with an average speed of 5.0 micron/min, whereas cells separated by more than 0.5 micron glided with an average speed of 3.8 microns/min. The power to glide was found to be carried separately at both ends of a cell.

The effects of axial rotational alignment of the femoral component on knee stability and patellar tracking in total knee arthroplasty demonstrated on autopsy specimens.

Four fresh-frozen anatomic knee specimens were tested for knee stability, patellar tracking, and patellofemoral contact points with the femoral component positioned in 5 degrees internal, 5 degrees external, or neutral axial rotational alignment of the femoral component referenced on the posterior femoral condyles. The externally rotated specimens had varus-valgus stability of the knee that was closest to the normal control. The internally rotated specimens shifted into valgus alignment with flexion. Patellar tracking also was closest to normal in the externally rotated specimens. Patellofemoral contact was more evenly distributed between the medial and lateral contact areas in the externally rotated specimens than in the internally rotated or in the neutral specimens. Internal rotation of the femoral component in the knee with perpendicular resection of the tibia causes undesirable changes in knee stability, patellar tracking, and patellofemoral contact points. Neutral positioning produces similar but less negative effects on knee stability and patellar kinematics. External rotation improves both patellar tracking and knee stability characteristics.

Evaluation of the effect of the femoral articular surface material on the wear of a metal-backed patellar component.

Wear characteristics of metal-backed, polyethylene patellar components were tested using cobalt-chromium, titanium alloy (Ti), and ion-implanted titanium alloy (IITi) articular surfaces. Patellar components were cycled in a bovine serum bath at 3 Hz for 1 million cycles, under a compressive load that varied from 343 N at 0 degree flexion to 2255 N at 120 degrees flexion. After testing, the polyethylene articular surfaces of the patellar components were evaluated for wear and graded using a subjective numbering system. Overall wear damage to the polyethylene surface was much worse with both Ti and IITi than with cobalt-chromium. Differences in mean wear scores were statistically significant when cobalt-chromium was compared with either Ti or IITi, but there were no statistically significant differences between Ti and IITi. Polyethylene surfaces that articulated against Ti femoral surfaces had more severe scratching. The IITi test group had areas of delamination not observed in the other test groups. Subjective evaluation of the metal surfaces showed evidence of wear damage as well. The metal articular surface of IITi resisted scratching as long as the treated surface was intact. In the high-stress areas, however, such as the edges of the intercondylar notch, the ion-implanted surface quickly wore away, exposing the untreated titanium alloy. The cobalt-chromium femoral articular surface had the least amount of scratching and no evidence of loss of metal.

The effect of screws and pegs on the initial fixation stability of an uncemented unicondylar knee replacement.

Two uncemented unicompartmental tibial components were examined for initial fixation stability. A conventional design that employed a single posteriorly angled peg was compared with a new design that was held in place by cancellous bone screws. The components were implanted into the medial condyles of 12 preserved human tibiae, and a cyclic load was first applied anteromedially and then posteromedially. The screwed implants failed at significantly higher loads (1634.8 +/- 121.6 N, mean +/- standard error of the mean) than the pegged implants (1103.3 +/- 152.0 N). On application of a 19.6-N preload, the screwed implants moved significantly less than the pegged implants. Although the differences in micromotion and subsidence were not always significant, there were definite trends. The screwed implants had much lower levels of temporary and permanent displacement compared with the pegged implants for all load levels from the initial load of 245.2 N up to and including the failure load. When the motion that resulted from moving the load from the anterior position to the posterior position was examined, the screwed implant's average total motion was less than 10 microns compared with almost 135 microns for the pegged implant after the 245.2-N load cycle. For the cycle before failure, the screwed implant's average motion increased to less than 29 microns, whereas the pegged implant's average total motion was almost 354 microns. From this information it appears clear that screws provide better initial fixation stability than angled pegs for uncemented unicondylar tibial components.

An analysis of screw fixation of the femoral component in cementless hip arthroplasty.

A cementless hip stem that allows screw fixation of the collar to cortical bone in the calcar region was found to achieve enhanced rotational stability when implanted in preserved cadaveric human femora. Although the implants with screws showed less tendency for subsidence than the implants without screws, rotational micromotion was not found to be statistically different under light loading conditions. When implanted in composite bone, the addition of screws in the configuration tested was associated with significant metal-on-metal wear during combined compression and rotational cyclic loading. This finding is of concern due to potential wear particle toxicity and possible lowered fatigue life of the prosthesis. Therefore, specific design changes are recommended.

Examination of rotational fixation of the femoral component in total hip arthroplasty. A mechanical study of micromovement and acoustic emission.

Rotational loosening has recently emerged as an important cause of failure of the femoral component of total hip arthroplasties. This study was designed to investigate the role played by torsional loads in loosening of cementless femoral components and to evaluate three cementing techniques involving a combination of canal irrigation, manual insertion, and vacuum mixing combined with pressure injection of the cement for their ability to improve rotational fixation. Rotational micromotion and subsidence were measured in 24 preserved human anatomic specimen femora. Acoustic emission (AE) technique was applied as a non-destructive method for evaluating material failure during loading. From the micromovement data, torque to 50 mu subsidence and torque to failure were surprisingly low with cementless fixation and with poor cement technique but were markedly improved with pulsed irrigation. Further improvement was achieved by pressure injection and vacuum mixing of the cement. However, AE was detected even in the most carefully performed cement specimens under torsional-loading conditions commonly occurring in daily activities. These signs of microfailure of the cement mantle at relatively low torsional loads suggest that the mode of failure of deeply penetrated cement is by microfracture of the cement mantle. The poor performance suggests that cementless fixation of intramedullary stems provides unsatisfactory fixation against torsional loading. There is need for major improvements in fixation mechanisms and techniques. The signs of failure of the cement mantle at normally occurring torsional loads suggest that even the best cement technique is prone to failure in torsion when exposed to normal daily use.

Bidirectional transcription and the regulation of Phage lambda repressor synthesis.

There are two promoters for transcription of gene cI in phage lambda, the gene that codes for phage repressor. The promoters, called pre and prm, are located on the distal (pre) and proximal (prm) sides of gene cro, which itself is adjacent to cI. Since cI and cro are transcribed in opposite directions, cI transcription initiating at pre gives rise to an antisense transcript of cro, while cI transcription initiating at prm does not. Pre, active after infection of a sensitive cell, is stimulated by products of phage genes cII and cIII, and may be located at the site defined by the mutant cY. Prm is active in an established lysogen. These conclusions are based on measurements of the rates of synthesis of antisense cro RNA, cI RNA, and repressor protein in infected and lysogenic cells. To measure antisense RNA, an assay based on the formation of nuclease-resistant, double-stranded RNA, specific to the cro region, was developed. These results raise the possibility that bidirectional transcription of cro has a regulatory function in phage lambda.

Control of lambda repressor synthesis.

Direct measurements of the intracellular level of lambda repressor have been made by a DNA-filter assay and a radioimmune assay. Transcription of cI, the structural gene for repressor, appears to initiate at two different promoters, prm and pre. Promoter pre is activated during the establishment of lysogeny by the action of cII and cIII proteins at the DNA site cY. Phage mutated in cII, cIII, or cY do not make a normal burst of repressor after infection and do not efficiently lysogenize the cell. Cro product stops repressor synthesis midway in the infective cycle. Promoter prm maintains the repressor level in established lysogens. Delection mapping places it very near the right operator (Or). Prm is activated by repressor bound to the right operator. In the absence of cII or cIII protein, repressor synthesis requires active repressor and only proceeds on genomes able to bind repressor at Or.

Association of autoantibodies to different nuclear antigens with clinical patterns of rheumatic disease and responsiveness to therapy.

Using a hemagglutination test which can detect antibodies to (a) native and denatured deoxyribonucleic acid (DNA) and (b) an extractable nuclear antigen (ENA), a comparative study of patterns of autoantibody formation has been done in systemic lupus erythematosus (SLE) and related rheumatic diseases. Antibody to native DNA was present in the serum in 96% of patients with active SLE and disappeared during remissions. Antibody to ENA was found in 86% of those patients with SLE nephritis who responded to treatment but in only 8% of those who did not. The highest titers of antibody to ENA were found in patients having a mixed connective tissue disease syndrome with features of SLE, scleroderma, and myositis. The latter syndrome was notable for the absence of renal disease and for a striking responsiveness to corticosteroid therapy. Hemagglutination testing of 277 sera from normal persons and patients with a wide variety of acute diseases other than SLE revealed the presence of antibody to native DNA in only 1.4% and antibody to ENA in only 0.4%. These results yield significant correlations among the pattern of autoimmune reactivity, the clinical form of the rheumatic disease, and responsiveness to treatment. They implicate the qualitative nature of the patient's immune response as a conditioning factor in the type of disease. Together with other correlations they may allow classification of rheumatic diseases into more biologically meaningful groups and lead to more selective methods of therapy.