Car Construction Research Articles

Response to CAR-T Therapy Can be Monitored Using Genome-Wide Sequencing of Cell-Free DNA in Patients with DLBCL

Response to CAR-T Therapy Can be Monitored Using Genome-Wide Sequencing of Cell-Free DNA in Patients with DLBCL

Development of CAR-T Cell Constructs with Broad Anti-Tumor Tropism

Chimeric antigen receptor T (CAR-T) cell therapy is a transformative approach to cancer eradication. CAR-T is expensive in part due to the restricted use of each CAR construct for a specific set of tumors such as B cell lymphoma targeted with CD19 and multiple myeloma targeted with BCMA. A CAR construct with broad anti-tumor activity can be advantageous due to wide applicability and scalability of production. We show that CD126, the IL-6 receptor alpha, is an antigen that is expressed by many hematologic and solid malignancies including multiple myeloma, non-Hodgkin lymphoma, acute myeloid leukemia, pancreatic and prostate adenocarcinoma, non-small cell lung cancer and malignant melanoma amongst others. High CD126 expression is a negative prognostic marker in many malignancies. The two CD126 targeting CAR-T cell constructs contain the CD28 anchoring domain followed by 4-1BB and CD3 zeta signaling domain. Lentiviral vectors were generated with triple plasmid (CAR, psPAX2 and pMD2.G) transfection of 293T cells and the vector concentrated by ultracentrifugation and used to transduce human T cells. T cells were isolated from leuko-reduction cones using negative selection with magnetic beads. The transduction efficiency was around 60%. The T cells were activated with anti-CD3/CD28 beads and expanded for two weeks before using for downstream experiments. CD126 CAR-T cells are able to kill many tumor cells in an antigen specific manner and with an efficiency that is directly proportional to the cell surface expression of CD126 expression (rho = 0.6, p = 0.0019). The presence of soluble CD126 in the culture media did not interfere with CAR-T cell killing. The CAR-T constructs bind murine CD126. However, injection of CD126 targeting CAR-T cells in NSG mice did not lead to any evidence of hepatotoxicity and weight loss despite possible expression of this antigen on hepatocytes. In vivo studies in NSG mice with multiple myeloma (RPMI-8226) and prostate adenocarcinoma (DU-145) xenograft models (n=10 tumors per group) showed that the intravenously injected CD126 targeted CAR-T cells (107) infiltrated the tumors, expanded, produced human interferon gamma and killed the tumor cells (p<0.001). Bioluminescence imaging showed control of tumor growth in the actively treated tumors compared to the controls (p<0.05). At post mortem, mice injected with CD126 targeted CAR-T cells had smaller residual tumors compared to controls injected with non-engineered human T cells from the same donor. Binding of sIL-6R by CAR-T cells could mitigate cytokine release syndrome. In support of this, murine SAA-3 levels (the equivalent of human CRP) were lower in mice injected with CD126 CAR-T compared to controls (p<0.05), suggesting that binding of sIL-6R by CAR-T cells could mitigate cytokine release syndrome. CD126 provides a novel therapeutic for CAR-T cells in a broad variety of tumors with low risk of toxicity. Disclosures Dingli: Apellis: Consultancy; Millenium: Consultancy; Janssen: Consultancy; Bristol Myers Squibb: Research Funding; Sanofi-Genzyme: Consultancy; Alexion: Consultancy; Rigel: Consultancy; Karyopharm Therapeutics: Research Funding.

Generation of CAR-NK Cells Using a Notch-Mediated Cell Expansion and Differentiation Platform

An important challenge in the generation of therapeutic chimeric antigen receptor expressing natural killer (CAR-NK) cells is poor gene transfer efficiencies in mature NK cells requiring large quantities of viral vectors, significantly increasing the production cost. We have an established clinical stem cell expansion platform used to generate off-the-shelf hematopoietic stem/progenitor cell products that have been evaluated in 6 clinical trials to date. This proprietary Notch-mediated expansion platform has now been optimized to generate large numbers of functional NK cells by first ex vivo expansion and priming of CD34+ cells isolated from cord blood (CB), followed by directed differentiation to NK cells. The final NK cell product can be cryopreserved with post-thaw maintenance of viability (≥ 80%) and function. With respect to generation of CAR-NK cells, this manufacturing process is scalable and cost-effective and importantly, allows transduction using a viral vector early in the expansion phase of culture without compromising the robust expansion or phenotype of the developing cells. This significantly reduces both the number of cells to be transduced and the amount of viral vector needed to <5% of that required to transduce the final NK product. As proof of concept, we developed CB-derived CAR NK (CB-CAR-NK) cells with specificity against mesothelin (MSLN). Mesothelin is a cell surface adhesion molecule that is highly upregulated in 30-50% of AML cases in pediatric and adult cohorts and lacks expression in normal hematopoiesis. The AML-restricted expression of mesothelin makes it an ideal tumor-associated antigen for immunotherapeutic targeting in AML. CB derived CD34+ cells are transduced with a mesothelin-directed CAR vector during the expansion phase in serum-free expansion media supplemented with cytokines. Transduction efficiencies were assessed periodically in bulk, CD34+ and CD56+ cells throughout the culture duration by flow cytometry analysis of truncated CD19, which is linked to CAR expression by a cleavable T2A. We measured cytolytic activity of CB-CAR-NK and CB-NK cells against NOMO-1 AML cells, which express endogenous MSLN, or NOMO-1 MSLN knockout cells by flow cytometry analysis of cell viability in the target cells. Our manufacturing process generates over 2000 CD56+ NK cells per CD34+ cell initiated in culture. Following transduction and differentiation, we achieve a robust cell product that is >75% CD56+ NK cells and ~22% CD56+CAR+ using a multiplicity of infection of 10 virus particles per cell. The ex vivo generated CAR-NK cells demonstrate an immunophenotype similar to the unmodified NK cells, expressing the NK canonical surface markers NKG2A, NKG2D, NKp46, CD16, NKp44 and NKp30. Despite a modest transduction efficiency, the CB-CAR-NK cells demonstrate superior cytotoxicity against NOMO-1 cells over the unmodified CB NK cells (~4-fold increase). This enhanced killing is highly specific as CB-CAR-NK and CB-NK exhibited similar tumor-killing efficiency in NOMO-1 MSLN KO cells. Having demonstrated the functional potential of our CAR-NK cells in vitro, we are now working toward assessing their function in vivo in a mouse xenograft model. We will also explore other optimal leukemia-specific antigens as targets for our CAR-NK cells and optimize our CAR construct to further enhance transduction efficiency. The vast majority of anti-cancer cellular immunotherapies are patient specific, require apheresis, high volumes of viral vector and complex manufacturing processes that are expensive with a risk of delayed or no treatment if the production fails, all contributing to poor clinical adoption despite transformative outcomes. We have developed our platform to overcome these hurdles with the goal of delivering safe and cost-effective allogeneic off-the-shelf immunotherapies for improved clinical access. Disclosures Chandrasekaran: Deverra Therapeutics: Current Employment, Current equity holder in private company. Prieve:Deverra Therapeutics: Current Employment, Current equity holder in private company. Stoltzman:Deverra Therapeutics: Current Employment, Current equity holder in private company. Cook:Deverra Therapeutics: Current Employment, Current equity holder in private company. Blake:Deverra Therapeutics: Current Employment, Current equity holder in private company. Massey:Deverra Therapeutics: Current Employment, Current equity holder in private company. Delaney:Deverra Therapeutics: Current Employment, Current equity holder in private company.

Preclinical Development of Anti-FLT3 CAR-T Therapy for the Treatment of Acute Myeloid Leukemia

FMS-like tyrosine kinase 3 (FLT3) is a class III transmembrane receptor tyrosine kinase involved in survival, proliferation, and differentiation of hematopoietic stem/progenitor cells. It is preferentially expressed on the leukemic cells of myeloid lineage including acute myeloid leukemia (AML) and is mutated in approximately one-third of patients with AML, resulting in constitutive signaling associated with poor disease prognosis. Although small molecule inhibitors targeting FLT3 have shown some success in clinical trials, they only work transiently while resistance develops in virtually all patients. The only proven curative treatment for the relapsed or refractory (R/R) AML is allogenic hematopoietic stem cell transplantation (HSCT) which requires highly toxic conditioning regimens often associated with fatal side effects. Thus, there still remains an urgent need for the development of safe yet effective new therapies for the treatment of AML. We developed a novel chimeric antigen receptor modified T (CAR-T) cell therapy targeting FLT3 to eliminate FLT3+ R/R AML leukemia via cytotoxic T lymphocytes (CTL)-mediated cytolysis. Since FLT3 is also expressed on hematopoietic stem cells (HSCs) as well as on early hematopoietic progenitors (HPs), we evaluated the conditioning efficacy of our anti-FLT3 CAR-T in addition to its anti-leukemic activity. We first discovered a novel mouse monoclonal antibody that binds to the extracellular domain of human FLT3 with high affinity (0.8 nM EC50 in FLT3+ leukemic cell line REH) while not competing with FLT3 ligand in order to achieve unobstructed and efficient binding to FLT3. We next generated humanized single-chain variable fragment (scFv) antibodies and characterized their binding affinities. The scFv clone that exhibited highest binding to FLT3 (3.42 nM EC50 in REH cells) was used to design a third generation CAR construct with CD28 and 4-1BB costimulatory and CD3ζ activation domains. T cells isolated from peripheral blood (PB) were transduced with a lentiviral vector encoding the FLT3-CAR. Transduced cells exhibited stable expression of CAR protein and expanded over 120-fold after 18 days in culture. We demonstrated high cytotoxicity of FLT3-CAR-T cells towards AML-derived cell lines in co-culture experiments, even at effector-to-target cells ratios as low as 1:10. In vivo functionality of FLT3-CART was determined by flow cytometry analysis of leukemia burden in the peripheral blood of mice engrafted with GFP+ MOLM-13 (FLT3+ AML cell line) and treated with two doses of 4x106 control or FLT3-CAR-T cells. Compared to control, the appearance of MOLM-13 cells in peripheral blood was significantly delayed in FLT3-CAR-T treated mice. AML progression in mice was also assessed by detection of physical symptoms such as cachexia and hind-leg paralysis in terminal stages. FLT3-CAR-T treatment extended the median survival to 47 days compared to 24 days in control. Moreover, to test if our CAR-T therapy can also efficiently eliminate FLT3+ HSCs and HPs, humanized mice generated by engrafting human cord blood CD34+ cells were injected with autologous control or FLT3-CAR-T cells. Analysis of bone marrow 18 days post treatment, showed that mice that received FLT3-CAR-T cells exhibited dramatically lower frequencies (by 57% in CD38+ and 86% in CD38-) of human CD34+ hematopoietic stem and progenitor cells than control mice, suggesting the potential of CAR-T therapy for HSCT conditioning. In conclusion, our CAR-T therapy shows robust cytolytic activity against FLT3+ cells, demonstrates high efficacy in eradicating FLT3+ R/R AML leukemia in vivo and enables bone marrow conditioning for potentially curative HSCTs by specifically targeting FLT3+ HSCs and early HPs. To prevent the potentially harmful side effects associated with CAR-T therapies, such as cytokine release syndrome and cytotoxicity towards newly transplanted HSCs post conditioning, we are currently testing FLT3-CAR-T cells equipped with inducible caspase9 or EGFRT expression based safety switch to specifically eliminate CAR-Ts by administering FDA-approved small molecules or biologics. Disclosures Shrestha: Hemogenyx Pharmaceuticals LLC: Current Employment. Liang:Hemogenyx Pharmaceuticals LLC: Current Employment. Sirochinsky:Hemogenyx Pharmaceuticals LLC: Current Employment. Ben Jehuda:Hemogenyx Pharmaceuticals LLC: Current Employment. Sandler:Hemogenyx Pharmaceuticals LLC: Current Employment, Current equity holder in publicly-traded company.

A Phase I Study of CD19-Targeted 19(T2)28z1xx CAR T Cells in Adult Patients with Relapsed or Refractory B-Cell Malignancies

Background: Autologous CAR T cell therapy targeting the B-cell specific surface antigen CD19 has demonstrated favorable clinical responses in relapsed or refractory (R/B) B-cell lymphomas (BCL). However, despite 40-60% initial complete response (CR) rates, only a subset of patients experience durable remissions, and there is a need to further improve the efficacy of CAR therapies by preventing relapse and attaining a deeper CR. We hypothesized that the redundancy of CD28 and CD3V signaling in a CAR design incorporating all 3 CD3Vimmunoreceptor tyrosine-based activation motifs (ITAMs) might foster counterproductive T cell differentiation and exhaustion, and therefore created a new CD19 CAR construct with calibrated CAR activation potential by mutating 2 of the 3 ITAMs, termed 1XX. In systemic ALL mouse models, 19-28z1XX CAR induced effective tumor eradication at low CAR T cell doses with improved survival compared to conventional 19-28z CAR. Further preclinical studies demonstrated that the enhanced therapeutic benefit resulted from the reduced strength of activation mediated by the 19-28z1XX CAR, achieving a favorable balance of effector and memory functions, thereby enhancing persistence of functional CAR T cells and promoting effective elimination of CD19+ leukemia at lower T cell doses than needed with 19-28z CAR T cells (Feucht J et al. Nat Med 2019). To further improve the persistence of functional CAR T cells, we screened different humanized CD19-directed scFv in the context of a 19-28z1XX CAR design and proved high specificity and functionality of 19-28z1XX CARs containing a novel humanized scFv T2 - termed 19(T2)28z1XX. Study Design and Methods: This study is a single center Phase I clinical trial of 19(T2)28z1XX in patients with R/R B-cell malignancies at Memorial Sloan Kettering Cancer Center (NCT04464200). Key disease eligibility criteria include R/R diffuse large B cell lymphoma (DLBCL), high grade BCL, primary mediastinal BCL, indolent BCL and chronic lymphocytic leukemia (CLL). Patients with prior CD19 CAR therapies are eligible as long as expression of CD19 is confirmed. Key exclusion criteria include ongoing immunosuppression such as systemic GvHD therapy and active CNS disease. The study uses a 3+3 dose-escalation design to identify the maximum tolerated dose for BCL. There are 5 planned flat-dose levels. Patients will receive conditioning chemotherapy consisting of 3 days of fludarabine and cyclophosphamide followed by a single infusion of 19(T2)28z1XX CAR T cells. In the dose-escalation phase, patients with DLBCL, high grade BCL, and primary mediastinal BCL are eligible to participate. Once the recommended phase 2 dose (RP2D) is determined, the study will open to dose expansion phase with two cohorts. Cohort 1 includes DLBCL, high grade BCL and primary mediastinal BCL (i.e. same eligibility criteria as the dose-escalation phase). Cohort 2 will include patients with indolent BCL, CLL, and Richter's transformation. The dose-expansion part of the trial is designed to further characterize the safety, efficacy, and pharmacokinetics of 19(T2)28z1XX CAR in multiple indications. The primary objective of the trial is to evaluate safety and tolerability and determine the recommended Phase 2 dose of 19(T2)28z1XX. Key secondary objectives include evaluation of 19(T2)28z1XX's efficacy and cellular kinetics. Exploratory objectives include assessment of B cell aplasia, and analysis of serum cytokines. The trial has begun enrollment in August 2020. The investigators are hopeful this study will lead to development of improved CD19 CAR T cell therapy with enhanced efficacy and favorable toxicity profiles with lower infused T cell dose. Disclosures Park: AstraZeneca: Consultancy; Servier: Consultancy, Research Funding; Autolus: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Novartis: Consultancy; Minverva: Consultancy; Artiva: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Research Funding; Kite: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Genentech/Roche: Research Funding; Juno Therapeutics: Research Funding; GSK: Consultancy; Intellia: Consultancy; Allogene: Consultancy. Riviere:Fate Therapeutics Inc.: Consultancy, Other: Ownership interest , Research Funding; FloDesign Sonics: Consultancy, Other: Ownership interest; Juno Therapeutics: Other: Ownership interest, Research Funding; Takeda: Research Funding; Atara: Research Funding. Palomba:Genentech: Research Funding; Juno Therapeutics, a Bristol-Meyers Squibb Company: Honoraria, Research Funding; Regeneron: Research Funding; Novartis: Honoraria; Merck: Honoraria; Celgene: Honoraria; Pharmacyclics: Honoraria. Brentjens:BMS: Research Funding; Gracell Therapeutics: Consultancy; Juno Therapeutics (a Bristol Myers Squibb company): Patents & Royalties. Sadelain:Atara: Patents & Royalties, Research Funding; Fate Therapeutics: Patents & Royalties, Research Funding; Minerva: Other: Biotechnologies , Patents & Royalties; Mnemo: Patents & Royalties; Takeda: Patents & Royalties, Research Funding. OffLabel Disclosure: Cyclophosphamide and fludarabine will be used as conditioning therapy prior to 19(T2)28z1XX CAR T cell administration.

Clinical Response of CD19 CAR-T Cells (relmacabtagene autoleucel, relma-cel) in Adults with Heavily-Pre-Treated Relapsed/Refractory (r/r) Large B-Cell Lymphoma in China

Currently, no CAR-T product has been approved for use in China despite having hundreds of active CAR-T trials. Relma-cel (JWCAR029) is a CAR-T product with a CAR construct comprised of a binding domain, derived from a murine CD19-specific mAb (FMC63), a CD3ζ activation domain, and a 4-1BB costimulatory domain, and manufactured in China with a commercial-ready, highly automated, single train process to select, activate, transduce and expand both CD4 and CD8 cells to a wide range of doses with consistent product attributes. Relma-cel was evaluated in the first prospective, single-arm, multi-center, pivotal study (RELIANCE Trial) of CAR-T therapy conducted under Chinese IND to support an NMPA-accepted BLA submission in r/r large B-cell lymphoma (LBCL) (NCT 04089215) and the preliminary results are presented herein. Eligible adult patients had measurable, histologically confirmed r/r LBCL, failed at least 2 prior therapies, including anthracycline and anti-CD20 agents, and not had allogeneic transplant within 90 days or primary CNS lymphoma. Patients were randomized to receive either 100×106 (low dose) or 150×106 (high dose) CAR+ T cells as a single infusion following fludarabine 25 mg/m2 & cyclophosphamide 250 mg/m2 daily×3 as lymphodepletion. Patients were evaluated for efficacy (Lugano criteria, 2014), safety (CRS by Lee 2014, and all other events by CTCAE v4.03), and PK (by qPCR and flow cytometry). The protocol pre-specified and regulatory authority-agreed primary endpoint was 3 month ORR by landmark analysis to exclude a 3-month 20% ORR (Michael, 2017). Secondary endpoints included best overall response rate (BOR), duration of response (DOR), progression free survival (PFS), overall survival (OS), frequency/severity of AEs and CAR-T cell expansion. Investigators adjudicated disease response and progression based on imaging studies, pathology and clinical data; a sensitivity analysis was also conducted using an independent review committee adjudication. Between May 2018 and Dec 2019, 69 patients were screened, and 59 enrolled and treated with relma-cel, with median age of 56.0 years (18-75), 59.3% ECOG 1-2, and included several LBCL subtypes (DLBCL NOS 69.5%, TFL 15.3%, DHL/THL 5.1%, and PMBCL 6.8%) and nearly all with poor risk disease (81.4% treatment refractory, 45.8% ≥3 lines of prior therapies, 42.4% requiring bridging therapy, 39% IPI≥3). The median doses administered in low and high dose groups were 99.7×106 (range, 80.1-101.3) and 150.0×106 (range, 120.0-156.4) CAR+ T cells, respectively. Among the 58 efficacy evaluable patients, the primary analysis demonstrated a 3 month ORR of 58.6% (95% CI, 44.9-71.4) (the excluded patient had product that did not meet viability threshold criterion, but achieved CR at D29 that is ongoing for >1 year). As of Jun 17, 2020 data cut-off, BOR was 75.9% (95% CI, 62.8-86.1) with CR rate of 51.7% (95% CI, 38.2-65.1). With a median follow-up of 8.9 months, median OS were not reached, and 6 month DOR, PFS and OS were 60.0%, 54.2% and 90.8%, respectively. No improvement in efficacy outcomes was observed in the high dose group. Of 59 treated patients, grade ≥3 AEs occurring in >5% of patients were cytokine release syndrome (CRS), febrile neutropenia and lung infection (all, 5.1%). Grade ≥3 lab abnormalities in >10% of patients were neutrophil count decreased (30.5%), white blood cell count decreased (13.6%), leukopenia (10.2%) and neutropenia (10.2%). The rates of CRS, neurotoxicity (NT), death, and the use of tocilizumab/steroids are shown in Table 1. All events of CRS and NT resolved except for 1 patient with grade 4 CRS ongoing at the time of death as the result of sepsis at day 8. As of data cut off, no cases of severe cytopenia or severe infections occurred beyond 30 days of infusion. The two dose groups did not differ significantly in PK parameters (see Table 1). The RELIANCE Trial provided the first demonstration of licensure-quality CAR-T manufacturing and clinical trial data generation in r/r patients originating in China. These results with relma-cel demonstrate similar preliminary response rates and PK profiles while providing the potential for an improved toxicity profile in heavily-pre-treated patients with r/r DLBCL having poor risk features relative to other CD19-specific CAR-Ts approved in the US and EU. Figure Disclosures Zhang: JW Therapeutics (Shanghai) Co. Ltd: Current Employment, Current equity holder in private company. Yang:JW Therapeutics (Shanghai) Co. Ltd: Current Employment, Current equity holder in private company. Zhou:JW Therapeutics (Shanghai) Co. Ltd: Current Employment, Current equity holder in private company. Zheng:JW Therapeutics (Shanghai) Co. Ltd: Current Employment, Current equity holder in private company.

CD22-Directed CAR T-Cell Therapy Mediates Durable Complete Responses in Adults with Relapsed or Refractory Large B-Cell Lymphoma after Failure of CD19-Directed CAR T-Cell Therapy and High Response Rates in Adults with Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia

CD22-Directed CAR T-Cell Therapy Mediates Durable Complete Responses in Adults with Relapsed or Refractory Large B-Cell Lymphoma after Failure of CD19-Directed CAR T-Cell Therapy and High Response Rates in Adults with Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia

CRISPR-Cas9 Knock out of CD5 Enhances the Anti-Tumor Activity of Chimeric Antigen Receptor T Cells

CRISPR-Cas9 Knock out of CD5 Enhances the Anti-Tumor Activity of Chimeric Antigen Receptor T Cells

Chimeric Antigen Receptor T-Cell Therapy in Glioblastoma: Current and Future.

Glioblastoma (GBM) is a highly aggressive glioma with an extremely poor prognosis after conventional treatment. Recent advances in immunotherapy offer hope for these patients with incurable GBM. Our present review aimed to provide an overview of immunotherapy for GBM, especially chimeric antigen receptor T-cell (CAR T) therapy. CAR T-cell immunotherapy, which involves the engineering of T cells to kill tumors by targeting cell surface-specific antigens, has been successful in eliminating B-cell leukemia by targeting CD19. IL-13Rα2, EGFRvIII, and HER2-targeted CAR T cells have shown significant clinical efficacy and safety in phase 1 or 2 clinical trials conducted in patients with GBM; these findings support the need for further studies to examine if this therapy can ultimately benefit this patient group. However, local physical barriers, high tumor heterogeneity, and antigen escape make the use of CAR T therapy, as a treatment for GBM, challenging. The potential directions for improving the efficacy of CAR T in GBM are to combine the existing traditional therapies and the construction of multi-target CAR T cells.

Study of strength characteristics of the long wheelbase flat cars

Transportation of goods in large containers with loading length of 80 feet is one of the promising directions of railway transport development. However, despite considerable experience in construction of long-wheelbase flat cars, occasionally problems with the strength of load-bearing elements arise at the calculation, design and manufacture of such products. The article presents the technical characteristics of a long wheelbase flat car, as well as the results of experimental studies of fatigue tests of its load-bearing elements. The optimization of the design of the long wheelbase flat car was achieved both by increasing the sizes and shapes, and by using materials of increased strength. An analysis of the study’s results of an improved design showed the compliance of the values of the safety factor of fatigue strength and the service life of the flat car with regulatory and technical documentation. The conducted studies allow us to make recommendations regarding the design, rebuilding and testing of long wheelbase flat cars. The primary results of fatigue tests of the main load-bearing structural elements within the scope of experimental studies turned out to be mostly below acceptable values. This article contains facts confirming it. The results of experimental studies to determine the fatigue strength proved that the design elements require improvement and strengthening.

Lattice Gas Cellular Automata Fluid Dynamics Case Study

The Navier–Stokes equations are the basis for describing the flow of a viscus material and are used to model fluid motion from weather to air flow over a wing. These equations, however, tend to be notoriously difficult to solve, whether analytically or computationally, even for small systems due to their highly nonlinear nature. Thus, less complicated methods that are more computationally tractable are desirable in domains as varied as hydroelectric power to race car construction. At a fundamental level, fluids are composed of interacting molecules. Lattice Gas Cellular Automata (LGCA) represents an efficient way to simulate these interacting fluid particles on a lattice. LGCA captures the microscopic behavior of the fluid by applying simple collision and propagation rules at each lattice site. This leads to realistic macroscopic behavior that can be used to build insight about real fluid flow. Here, we show the Hardy, Pomeau, and de Pazzis model for simulating a lattice gas. The computational framework presented in this case study can also be expanded to more complicated LGCA.

Advances in chimeric antigen receptor T cells.

To discuss the important advances in CAR T cell therapy over the past year, focusing on clinical results where available. Approximately 30 years after they were first conceived of and 15 years after the first small-scale single-center clinical trials, the past 3 years represent a major milestone in the development of CAR T cells. In the United States, the Food and Drug Administration (FDA) approved Tisagenlecleucel for the treatment of relapsed/refractory B-ALL and Axicabtagene Ciloleucel, for adults with relapsed/refractory diffuse large B cell lymphoma (R/R DLBCL) in 2017. Tisagenlecleucel received a second indication in adults with R/R DLBCL in 2018. Regulatory approval for CAR T cells was then granted in Europe, Canada, Australia, and Japan. Most recently, in July 2020 the FDA granted regulatory approval to a third CAR T cell product, Brexucabtagene Autoleucel for mantle cell lymphoma. All products target the CD19 antigen but differ in the costimulatory molecule within the CAR construct. Currently, it is unknown whether there are any differences in clinical activity or toxicity between these products. The CAR T cell the platform is evolving at a rapid pace and is expected to further improve the therapeutic outcomes of hematological malignancies.

General principles of expanding the speed range of operation of autonomous power supply systems for passenger cars

Authors address the issues of expanding the speed range for operation of autonomous power supply systems for air-conditioned passenger cars. For the first time in the domestic car construction, the issue of reducing the minimum start-up speed of a generator-drive unit arose when the passenger fleet of the Sakhalin Railway was renewed, where the speed of passenger cars is much lower than the overall network speed. Research carried out by CJSC SO TIV and JSC TVZ has shown that the use of V-belts in the modernization of the textropic-reduction-cardan drive allows solving this problem. Kinematic diagram of the drive was adopted without changes, but with the replacement of the V-belts C (B)-2360T with new narrow belts XPC-2360 without working surface wrap and with a formed tooth on the lower base. In the tensioner, the main spring with a stiffness of 75 N/mm was replaced with a spring of increased stiffness (100 N/mm). The main change was the replacement of the 2GV003 generator with a 2GV008 low-speed generator, which has a minimum rotation at full power (700 rpm). This modernization of the drive made it possible to turn on the generator 2GV008 at rated power, starting at a speed of 30 km/h. The article sets the task of expanding the speed range of operation of the EV-7 and EV-10 autonomous power supply systems for cars without air conditioning. It is solved by forcing the excitation of the generators. Analysis of modern systems of autonomous power supply of passenger cars showed that the expansion of the speed range of its operation can be achieved in three ways: • raising the gear ratio of the gearbox to ensure it is switched on to the nominal mode at a carriage speed of 15 - 20 km/h without changing the parameters of the generator and the entire system of autonomous power supply; • modernization of the electrical equipment complex for passenger cars to ensure the operation of the main power consumers and charge the battery while driving at low speeds (over 18 km/h) without changing the mechanical parameters and composition of the serial generator-drive unit; • modernization of the generator in order to reduce its turnon revolutions to the power required to provide the main power consumers without changing the mechanical parameters of the drive. All three methods have its advantages and disadvantages, the choice of any of them is determined by the technical requirements for a specific car model.

Abstract 6595: Targeting T cell lymphomas with CRISPR/Cas9-generated anti-CD70 allogeneic CAR-T cells

Abstract T cell lymphomas account for 10% to 15% of non-Hodgkin lymphomas and are diverse biologically and clinically. Unlike B-cell lymphomas, T cell lymphomas are rather resistant to conventional therapies, such as chemotherapy and antibody-based therapeutics. This resistance coupled with the diversity of the T cell diseases means that there is a significant unmet need across the T cell lymphoma subtypes. CD70 (CD27 ligand) is a candidate target antigen for T cell lymphomas and has been is the subject of clinical trials using an enhanced ADCC antibody (ARGX-110). Using flow cytometry and immunohistochemistry (IHC) methods, we analyzed the expression of CD70 in cell lines and clinical samples representing T cell lymphomas and found significant expression of CD70 in multiple types of T cell lymphoma, but at highly variable antigen density. CAR-T cells targeting CD70 may thus be a potent new therapy to tackle these diseases. However, the use of autologous CAR-T therapy against T cell lymphomas is complicated by the likelihood of creating lymphoma cells carrying the CAR construct. Thus, we sought to examine the potency of our allogeneic anti-CD70 CAR-T cells (CTX130) against T cell lymphoma cells. CTX130 has previously been shown to be active across a range of CD70 expression levels in other cell types representing other malignancies. Consistent with these prior observations, CTX130 exhibited high potency in vitro and in vivo against T cell lymphoma cells across a range of CD70 antigen density and representing different types of T cell lymphomas such as Sezary syndrome and cutaneous T cell lymphoma (CTCL). CTX130 may thus be a valid therapeutic to evaluate in T cell lymphoma patients. Citation Format: Sushant Karnik, Minh Thu Pham, Pooja Keerthipati, Zinkal Padalia, Julie Carson, Tony Ho, Ewelina Morawa, Matthias Will, Jonathan Terrett, Mary-Lee Dequeant. Targeting T cell lymphomas with CRISPR/Cas9-generated anti-CD70 allogeneic CAR-T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6595.

Abstract 2188: Generation and characterization of human placental-derived CD19 CAR-T cells using viral vectors

Abstract Introduction: Celularity, Inc. is developing a CD19 CAR-T cell therapy using an allogeneic platform derived from postpartum human placental cells. To augment the CD19 CAR expression and activity of Placental-derived T (P-T) cells, we have evaluated the use of various lentiviral (LV) vectors, as well as a retroviral (RV) CD19 CAR construct and assessed the effects of each virus on the phenotypic characteristics and activity of our cells. Fully humanized scFv CD19 constructs have been shown to be less immunogenic with improved persistence and efficacy in patients (Sommermeyer, et. al. Leukemia, 2017), so human (Hu) vs. mouse (Ms) scFv CD19 CAR constructs were also evaluated. Experimental Procedures: Isolated P-T cells were activated and transduced with various Ms and Hu scFv CD19 CAR viral vectors, including two Ms scFv LV sequences (containing 4-1BB costimulatory domains), three different Hu scFv LV sequences (two containing 4-1BB and one containing CD28 costimulatory domains), and one Ms scFv RV sequence (containing 4-1BB costimulatory domain). In vitro functional activity of these P-T CD19 CAR cells was assessed against CD19+ Burkitt's Lymphoma (Daudi) and Acute lymphoblastic Leukemia (NALM6) cell lines in cytotoxicity assays. In vivo anti-tumor activity of P-T Ms scFv CD19 CAR LV vs. RV cells was assessed using a disseminated lymphoma xenograft model in NSG mice. Results: All CD19 CAR constructs containing the 4-1BB costimulatory domain resulted in high transduction efficiency in P-T cells: Ms LV: 32-70% (n=4); Hu LV: 26-42% (n=2); Ms RV: 22-52% (n=6). P-T cells transduced with LV resulted in lower frequency of CD8+ T cells (9-42%), as compared to cells transduced with RV (46-59%), and consequently had lower CD8+ CD19 CAR+ (of CD3+) expression (LV: 2.8-6.4%; RV: 14-30%). In vitro, all CD19 CAR constructs specifically lysed CD19+ Daudi/ Nalm6 targets in cytotoxicity assays. In vivo, both P-T Ms scFv CD19 CAR LV and RV cells significantly reduced tumor burden and improved survival over the vehicle control. P-T CD19 CAR RV cells demonstrated superior anti-tumor activity over P-T CD19 CAR LV with 100% survival out to day 120, while the CD19 CAR LV treated group succumbed to 100% mortality by day 70. Summary: We have demonstrated the ability to generate P-T CD19 CAR cells using a variety of viral vectors containing the 4-1BB costimulatory domain. Transduction of P-T cells using Hu vs. Ms scFv CD19 CAR sequences resulted in comparable CD19 CAR expression and in vitro cytotoxic activity, whereas use of LV vs. RV had an impact on the phenotypic characteristic of the cells and resulted in differences in the in vivo tumor activity of the cells. Citation Format: Kathy Karasiewicz, Shuyang He, Kristina Tess, Weifang Ling, Kevin Jhun, Gunnar Kaufmann, Jerome B. Zeldis, Henry Ji, Robert Hariri, Xiaokui Zhang. Generation and characterization of human placental-derived CD19 CAR-T cells using viral vectors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2188.

Abstract 3243: Allogeneic anti-PTK7 CAR-T cells for the treatment of solid tumors

Abstract Protein Tyrosine Kinase 7 (PTK7), also known as colon cancer carcinoma kinase 4 (CCK-4), is a highly conserved catalytically inactive tyrosine kinase with inherent signal transduction activity. PTK7 is a member of the Wnt signaling pathway and thought to function in cell proliferation, adhesion, migration and apoptosis. PTK7 has been shown to be highly expressed in certain cancer types including colon, breast, lung, pancreatic, renal and ovarian. Downregulation of PTK7 by shRNA has been shown to reduce tumor growth and metastases development in xenograft mouse models. Furthermore, high PTK7 expression in triple negative breast cancer and NSCLC patients has been linked to poor prognosis. PTK7 therefore appears to be an attractive target for solid cancer therapeutic intervention and has been targeted clinically with an Antibody Drug Conjugate (PF-06647020). However, the protein is also expressed to a lesser degree in some normal adult tissues, particularly in the stromal cells of the ovary, placenta, uterus and lung. We have developed a human/murine cross-reactive anti-PTK7 CAR construct and assessed the ability of allogeneic anti-PTK7 CAR-T cells bearing that construct to target cancer cells and elicit toxicity in mice. These anti-PTK7 CAR-T cells were shown to be efficacious in vitro and in immunocompromised mouse xenograft models of breast, lung, colon, pancreatic and ovarian cancer. However, consistent in all xenograft models studied, a drop in body weight was observed shortly after injection from which all mice rapidly recovered to above baseline. Latent toxicity that was more variable amongst the xenograft experiments was also observed. Understanding and developing appropriate mitigation strategies to address the observed toxicities may be required to develop further a safe and efficacious allogeneic anti-PTK7 CAR-T cell therapy for solid tumor cancers. Citation Format: Katie Levitsky, Zejun Li, Minh Thu Pham, Kelly Maeng, Luke Hanley, Jason Sagert, Jon Terrett, Jui Dutta-Simmons, Melanie Allen. Allogeneic anti-PTK7 CAR-T cells for the treatment of solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3243.

Abstract 3234: Multi-antigen targeting of CD19, CD22 and TSLPR to prevent Ph-like ALL resistance

Abstract Background: Despite high remission induction rates after CD19 chimeric antigen receptor T cell (CART) therapy in patients with B-acute lymphoblastic leukemia (ALL), many patients eventually relapse with loss of CD19 antigen. Strategies to overcome antigen-loss relapse include co-targeting of multiple antigens and selection of alternative functionally important surface targets. The thymic stromal lymphopoietin receptor (TSLPR), encoded by CRLF2 (cytokine receptor-like factor 2), is overexpressed in >50% of patients with Ph-like ALL and is associated with constitutively active JAK/STAT signaling targetable by ruxolitinib. Our groups previously demonstrated potent activity of TSLPRCART against TSLPR+ ALL (Qin Blood 2015). Our current study aims to develop (1) multi-specific CAR T cells targeting TSLPR and CD19 or CD22 and (2) TSLPRCART and ruxolitinib combinatorial approaches to decrease antigen-negative relapse potential after CART therapy. Methods: Lentiviral transduction of activated healthy donor T cells was used to introduce monovalent TSLPR or CD19 CARs and bivalent CD19xTSLPR or CD22xTSLPR CARs. CAR expression was quantified via flow cytometry. CRISPR/Cas9-modified Nalm-6 (human B-ALL) cells were transduced with a TSLPR expression vector to generate cell lines expressing combinations of CD19, CD22, and TSLPR antigens. In vitro functionality was assessed using enzyme linked immunosorbent assays to detect IL-2 and IFN-γ production following CART co-culture with Nalm-6. In vivo evaluation was performed against luciferase-transduced Nalm-6 murine xenograft models treated with 5e6 CART and monitored by bioluminescent imaging. TSLPRCART was also tested against MUTZ5 (TSLPR-overexpressing Ph-like ALL) cells in vitro and in vivo in combination with ruxolitinib to determine effects on CAR T cells. Results: TSLPRCART and CD19xTSLPRCART induced in vitro cytokine production when co-cultured with CD19+/TSLPR+ Nalm-6 cells with higher levels than those produced by CD19CART. Greater cytokine production was observed with co-incubation of TSLPR+ Nalm-6 cells with CD22xTSLPRCART vs TSLPRCART. In vivo studies demonstrated equivalent leukemia eradication by TSLPRCART and CD19CART compared to CD19xTSLPRCART in CD19+/TSLPR+ Nalm-6 xenograft models. Additional in vivo studies in MUTZ5 xenograft models co-treated with TSLPRCART and ruxolitinib demonstrated impaired T cell expansion and cytokine production, which could be overcome via higher CART cell dose or delayed ruxolitinib administration. Conclusions: TSLPRCART is a robust immunotherapeutic strategy for CRLF2-rearranged Ph-like ALL. We demonstrate potent in vitro and in vivo functionality of novel bivalent CD19xTSLPRCART and CD22xTSLPRCART. Ongoing studies are also evaluating bicistronic CD19xTSLPR CAR constructs. We further demonstrate that combining TSLPRCART with ruxolitinib moderately blunts T cell expansion and cytokine production, which could be an attractive strategy for management of CAR T cell-associated toxicities such as cytokine release syndrome. Citation Format: Savannah Ross, Asen Bagashev, Dina Schneider, Peirong Hu, Sarah Tasian, Terry Fry. Multi-antigen targeting of CD19, CD22 and TSLPR to prevent Ph-like ALL resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3234.

Automated Manufacture of Autologous CD19 CAR-T Cells for Treatment of Non-hodgkin Lymphoma.

Chimeric antigen receptor T cells (CAR-T cell) targeting CD19 are effective against several subtypes of CD19-expressing hematologic malignancies. Centralized manufacturing has allowed rapid expansion of this cellular therapy, but it may be associated with treatment delays due to the required logistics. We hypothesized that point of care manufacturing of CAR-T cells on the automated CliniMACS Prodigy® device allows reproducible and fast delivery of cells for the treatment of patients with non-Hodgkin lymphoma. Here we describe cell manufacturing results and characterize the phenotype and effector function of CAR-T cells used in a phase I/II study. We utilized a lentiviral vector delivering a second-generation CD19 CAR construct with 4-1BB costimulatory domain and TNFRSF19 transmembrane domain. Our data highlight the successful generation of CAR-T cells at numbers sufficient for all patients treated, a shortened duration of production from 12 to 8 days followed by fresh infusion into patients, and the detection of CAR-T cells in patient circulation up to 1-year post-infusion.

Accurate In-Vivo Quantification of CD19 CAR-T Cells after Treatment with Axicabtagene Ciloleucel (Axi-Cel) and Tisagenlecleucel (Tisa-Cel) Using Digital PCR.

Immunotherapy with CD19-specific chimeric antigen receptor (CAR-) T cells has shown excellent efficacy in relapsed/refractory B-cell cancers. The in vivo expansion and persistence of CAR-T cells after infusion are important response- and toxicity-determining variables, but diagnostic tools are largely missing. We showed previously for axi-cel that digital PCR (dPCR) is excellently suited to monitoring CAR-T cells in vivo. Here, we aimed to develop an analogous dPCR assay for tisa-cel. To do so, we cloned and sequenced the CAR construct from the lentiviral tisa-cel vector and designed primers and Black hole quencher (BHQ) probes complimentary to sequences present in the FMC63 scFv part of axi-cel (assay A), tisa-cel (T), and both constructs (U = “universal”). In conjunction with excellent specificity, all assays have a detection limit of one single CAR copy, corresponding to a sensitivity of approximately 1 in 5000 cells (0.02%) for 100 ng genomic DNA (for one vector copy per transduced cell). The new universal assay was first validated using patient samples previously quantified with the axi-cel-specific dPCR and thereafter applied to quantify and monitor adoptively transferred axi-cel and tisa-cel T cells in post-infusion samples (peripheral blood, bone marrow, liquor, and ascites). Actual CAR-T counts per µl were calculated, taking into account vector copy and peripheral blood mononuclear cell (PBMC) numbers, and showed very good correlation with flow cytometry results. We conclude that our novel dPCR assay is optimally suited to monitoring tisa-cel and axi-cel CAR-T cells in real-time in various body fluids.

CAR-T Therapy for Solid Tumors: Development of New Strategies

The recent approval of two CAR-T therapies by US Food and Drug Administration (FDA) marks a very significant development in cell-based cancer immunotherapy. This milestone was demonstrated by the effectiveness of eradicating hematologic cancers using CD19-specific CARs. The success spurred development of immune cell therapies for other cancers, especially solid tumors. The generation of novel CAR constructs for these cancer types represents a major challenge in bringing the technology ‘from-bench-to-bedside‘.In this review, we outline some new technologies we have developed to equip CAR-T cells to enhance efficiency while decreasing toxicity of CAR-T therapies in solid tumors.