Manufacturing of patient specific novel T cell therapies using the Cocoon® Platform automated system

Engineered T cell therapies, particularly chimeric antigen receptor T cell (CAR-T) immunotherapies, have proved effective against hematologic cancers. However, CAR-T therapies can potentiate immune responses causing cytokine release syndrome (CRS; “cytokine storm”) leading to adverse events in patients. Additionally, CAR-T has shown sporadic success in solid tumor indications. Novel therapies which activate T cells via the native T cell receptors (TCR) have shown greater tumor antigen recognition providing an alternative therapy which may prove effective against solid tumors. Utilizing novel cell immunotherapy modalities is only part of the solution as challenges remain to scale manufacturing to meet commercial demand. Scaling out commercial patient-specific cell therapy manufacturing for large populations using current methods will be expensive (cleanrooms and FTEs) and complex (logistics). Innovative manufacturing solutions will be required to manufacture patient-specific therapies in a robust and cost-effective manner. The Cocoon® Platform is one such innovation, a functionally-closed, automated, scalable cell therapy manufacturing platform. This abstract highlights a therapeutic T cell process translated from an open, manual process to the Cocoon® Platform. During process translation, the functionally-closed Cocoon® Platform was used to automate cell seeding, activation, transduction, feeding, real-time process monitoring, washing, and final product harvest using the single-use Cocoon cassette. During process development and translation, important process parameters were identified, optimized, and programmed enabling multiple process step automation removing the need for manual intervention. For the process, 200 million CD4+ and CD8+ isolated T cells were inoculated with TransActTM activator. The following day, cells were transduced with HER-2 lentivirus vector at various multiplicities of infection (MOI). Cells were expanded with a predefined feeding strategy in media supplemented with IL-2 until final product harvest. Following harvest, cells were assessed for cell yield, viability, transduction efficiency, and VCN. T cell phenotype and functionality was assessed via flow cytometry. The Cocoon manufacturing processes yielded 2.7 x 109 viable cells on average with viability \u3e85%. The Cocoon processes supported both CD4+ and CD8+ T cell expansion with 68% CD4+ T cells and 31% CD8+ T cells on average. The final product exhibited high T cell purity and viability (i.e. \u3e90% abTCR+ and 89% abTCR+, respectively) with transduction efficiencies varied from ~30% to \u3e65% depending on the process MOI. Vector copy number (VCN) was evaluated after each process and found to be ≤5 copies/transduced T cell. In summary, a gene-modified T cell process was successfully translated to the Cocoon and the harvested final products met all pre-defined acceptable criteria. The Cocoon represents a tool for manufacturing cell therapies in a robust manner, while maintaining comparability, and lowering manufacturing costs via increased automation. Ultimately the Cocoon will enable and accelerate development of cell therapies to address solid tumor indications and meet a critical patient need

An automated and closed system for patient specific CAR-T cell therapies

Autologous cell therapies, particularly chimeric antigen receptor T-cell (CAR-T) immunotherapies, are becoming a promising treatment option for difficult diseases. Immunotherapies for blood cancers have dominated the pipeline, while treatments for solid tumors have started to become more successful. However, as the market continues to grow and more clinical trials begin globally, the challenge of manufacturing autologous cell therapies remains significant. A greater number of patients will lead to an increase in cost, labor, and the complexity of logistics for scaling out the commercial production of patient specific therapies. To enable clinical and commercial success, novel manufacturing platforms, such as closed and automated systems, will be required to produce cost effective and robust therapies. This abstract highlights a successful CAR-T process translation from a manual process to an automated patient scale system. To accomplish a CAR-T process translation, we utilized a platform that automates cell seeding, activation, transduction, real time process monitoring, feeding, washing and concentration, and harvesting. In order to mimic a therapeutic CAR-T cell process, manual research scale processes were optimized, scaled up, and then programmed to run automatically without manual intervention. In these processes, 100 million peripheral blood mononuclear cells (PBMC) were first inoculated with CD3/CD28 activation beads. The following day, cells were transduced with HER-2 lentivirus vector. Cells were then expanded with a defined feeding strategy and IL-2 supplements until harvested when target yields were reached. After harvest, cells were analyzed for cell yield, viability, transduction efficiency, and an array of cell phenotype, potency and functionality via FACS and killing assays. Specifically, CAR-T cells were analyzed for the presence of naïve T cells, T stem cell memory, T central memory, T effector memory, and T effector cells. We show here how we optimized, scaled up, and automated manual processes to reach clinical requirements. Automated runs using the above process with cells transduced by HER-2 virus yielded an average of 2 x 109 cells post harvest with a viability \u3e 90%. Automated runs and associated controls were able to support the expansion of both CD4+ and CD8+ T cells with 73% CD4+ T cells and 20% CD8+ T cells. Harvested cells yielded approximately 80% NGFR+ cells with a higher detection of NGFR in the CD4+ fraction than in the CD8+ fraction for all samples. Both CD4+ and CD8+ subsets demonstrated T cell phenotype such as naïve T cells, T stem cell memory, T central memory, T effector memory, and T effector cells. Both subsets also only expressed between 15-20% of immunosuppressive regulatory T cells. Cell health was evaluated by the levels of exhaustion marker, PD-1, which was 19% in CD4+ T cells and \u3c 1% in CD8+ T cells. Furthermore, there was a negligible amount of senescent T cells and anergic cells and \u3c 10% expression of the apoptotic marker, Caspase-3. Subsequently, cells from multiple automated runs showed the specific killing of NGFR+ tumor line were correlated with high levels of effector cytokines: TNF-alpha (~34%) and IFN-gamma (20-25%) as compared to a manual control. In summary, automated CAR-T process in the Cocoon system yields a healthy populations of T cell subsets. This system is a viable solution to translate labor-intensive CAR-T process into a fully automated system, thus allowing scalability, high yield, reduction of manufacturing cost, and better process control to yield high quality CAR-T cells

Peristaltic Transport of a Couple Stress Fluid: Some Applications to Hemodynamics

The present paper deals with a theoretical investigation of the peristaltic transport of a couple stress fluid in a porous channel. The study is motivated towards the physiological flow of blood in the micro-circulatory system, by taking account of the particle size effect. The velocity, pressure gradient, stream function and frictional force of blood are investigated, when the Reynolds number is small and the wavelength is large, by using appropriate analytical and numerical methods. Effects of different physical parameters reflecting porosity, Darcy number, couple stress parameter as well as amplitude ratio on velocity profiles, pumping action and frictional force, streamlines pattern and trapping of blood are studied with particular emphasis. The computational results are presented in graphical form. The results are found to be in good agreement with those of Shapiro et. al \cite{r25} that was carried out for a non-porous channel in the absence of couple stress effect. The present study puts forward an important observation that for peristaltic transport of a couple stress fluid during free pumping when the couple stress effect of the fluid/Darcy permeability of the medium, flow reversal can be controlled to a considerable extent. Also by reducing the permeability it is possible to avoid the occurrence of trapping phenomenon

Peristaltic Transport of a Physiological Fluid in an Asymmetric Porous Channel in the Presence of an External Magnetic Field

The paper deals with a theoretical investigation of the peristaltic transport of a physiological fluid in a porous asymmetric channel under the action of a magnetic field. The stream function, pressure gradient and axial velocity are studied by using appropriate analytical and numerical techniques. Effects of different physical parameters such as permeability, phase difference, wave amplitude and magnetic parameter on the velocity, pumping characteristics, streamline pattern and trapping are investigated with particular emphasis. The computational results are presented in graphical form. The results are found to be in perfect agreement with those of a previous study carried out for a non-porous channel in the absence of a magnetic field

PreImplantation factor (PIF) protects cultured embryos against oxidative stress: relevance for recurrent pregnancy loss (RPL) therapy

Recurrent pregnancy loss (RPL) affects 2-3% of couples. Despite a detailed work-up, the etiology is frequently undefined, leading to non-targeted therapy. Viable embryos and placentae express PreImplantation Factor (PIF). Maternal circulating PIF regulates systemic immunity and reduces circulating natural killer cells cytotoxicity in RPL patients. PIF promotes singly cultured embryos' development while anti-PIF antibody abrogates it. RPL serum induced embryo toxicity is negated by PIF. We report that PIF rescues delayed embryo development caused by 2 fold the number of embryos reaching the blastocyst stage. Mechanistically, PDI-inhibitor preferentially binds covalently to oxidized PDI over its reduced form where PIF avidly binds. PIF by targeting PDI/TRX at a distinct site limits the inhibitor's pro-oxidative effects. The >3kDa RPL serum increased embryo demise by three-fold, an effect negated by PIF. However, embryo toxicity was not associated with the presence of putative anti-PIF antibodies. Collectively, PIF protects cultured embryos both against ROS, and higher molecular weight toxins. Using PIF for optimizing in vitro fertilization embryos development and reducing RPL is warranted

PreImplantation Factor (PIF) correlates with early mammalian embryo development-bovine and murine models

<p>Abstract</p> <p>Background</p> <p>PreImplantation Factor (PIF), a novel peptide secreted by viable embryos is essential for pregnancy: PIF modulates local immunity, promotes decidual pro-adhesion molecules and enhances trophoblast invasion. To determine the role of PIF in post-fertilization embryo development, we measured the peptide's concentration in the culture medium and tested endogenous PIF's potential trophic effects and direct interaction with the embryo.</p> <p>Methods</p> <p>Determine PIF levels in culture medium of multiple mouse and single bovine embryos cultured up to the blastocyst stage using PIF-ELISA. Examine the inhibitory effects of anti-PIF-monoclonal antibody (mAb) added to medium on cultured mouse embryos development. Test FITC-PIF uptake by cultured bovine blastocysts using fluorescent microscopy.</p> <p>Results</p> <p>PIF levels in mouse embryo culture medium significantly increased from the morula to the blastocyst stage (ANOVA, P = 0.01). In contrast, atretic embryos medium was similar to the medium only control. Detectable - though low - PIF levels were secreted already by 2-cell stage mouse embryos. In single bovine IVF-derived embryos, PIF levels in medium at day 3 of culture were higher than non-cleaving embryos (control) (P = 0.01) and at day 7 were higher than day 3 (P = 0.03). In non-cleaving embryos culture medium was similar to medium alone (control). Anti-PIF-mAb added to mouse embryo cultures lowered blastocyst formation rate 3-fold in a dose-dependent manner (2-way contingency table, multiple groups, X2; P = 0.01) as compared with non-specific mouse mAb, and medium alone, control. FITC-PIF was taken-up by cultured bovine blastocysts, but not by scrambled FITC-PIF (control).</p> <p>Conclusions</p> <p>PIF is an early embryo viability marker that has a direct supportive role on embryo development in culture. PIF-ELISA use to assess IVF embryo quality prior to transfer is warranted. Overall, our data supports PIF's endogenous self sustaining role in embryo development and the utility of PIF- ELISA to detect viable embryos in a non-invasive manner.</p

Weak pairwise correlations imply strongly correlated network states in a neural population

Biological networks have so many possible states that exhaustive sampling is impossible. Successful analysis thus depends on simplifying hypotheses, but experiments on many systems hint that complicated, higher order interactions among large groups of elements play an important role. In the vertebrate retina, we show that weak correlations between pairs of neurons coexist with strongly collective behavior in the responses of ten or more neurons. Surprisingly, we find that this collective behavior is described quantitatively by models that capture the observed pairwise correlations but assume no higher order interactions. These maximum entropy models are equivalent to Ising models, and predict that larger networks are completely dominated by correlation effects. This suggests that the neural code has associative or error-correcting properties, and we provide preliminary evidence for such behavior. As a first test for the generality of these ideas, we show that similar results are obtained from networks of cultured cortical neurons.Comment: Full account of work presented at the conference on Computational and Systems Neuroscience (COSYNE), 17-20 March 2005, in Salt Lake City, Utah (http://cosyne.org

Critical behaviour of the Random--Bond Ashkin--Teller Model, a Monte-Carlo study

The critical behaviour of a bond-disordered Ashkin-Teller model on a square lattice is investigated by intensive Monte-Carlo simulations. A duality transformation is used to locate a critical plane of the disordered model. This critical plane corresponds to the line of critical points of the pure model, along which critical exponents vary continuously. Along this line the scaling exponent corresponding to randomness $\phi=(\alpha/\nu)$ varies continuously and is positive so that randomness is relevant and different critical behaviour is expected for the disordered model. We use a cluster algorithm for the Monte Carlo simulations based on the Wolff embedding idea, and perform a finite size scaling study of several critical models, extrapolating between the critical bond-disordered Ising and bond-disordered four state Potts models. The critical behaviour of the disordered model is compared with the critical behaviour of an anisotropic Ashkin-Teller model which is used as a refference pure model. We find no essential change in the order parameters' critical exponents with respect to those of the pure model. The divergence of the specific heat $C$ is changed dramatically. Our results favor a logarithmic type divergence at $T_{c}$, $C\sim \log L$ for the random bond Ashkin-Teller and four state Potts models and $C\sim \log \log L$ for the random bond Ising model.Comment: RevTex, 14 figures in tar compressed form included, Submitted to Phys. Rev.