Soluble anti-CD3 Research Articles

Construction and Optimization of CD19 Chimeric Antigen Receptor T Cells Derived from C57BL/6J Mice

To explore the stimulation conditions, optimal culture time and infection time of C57BL/6J mice CD3+ T cells in vitro, so as to improve the infection efficiency of CD19 chimeric antigen receptor T cells (mCD19 CAR-T). Purified C57BL/6J mice CD3+ T cells were cultured in anti-CD3/CD28 coated, anti-CD3 coated+soluble anti-CD28 and anti-CD3 coated, respectively. The cells were stimulated in above three conditions for 12 h and 24 h, following with 24 h, 48 h and 72 h incubation and then the number of cell clones was recorded. C57BL/6J mice CD3+ T cells were stimulated for 12 h, 24 h, and 36 h under the above three conditions, then interleukin (IL)-2 (100 U/ml) was added. The number of cell clones was recorded under microscope at 24 h, 48 h, and 72 h of culture. After 24 h of stimulation, CD3+ T cells derived from C57BL/6J mice were infected with retrovirus for 48 h to establish mCD19 CAR-T cells, and the percentage of GFP+ CAR-T cells was detected by flow cytometry. The infection efficiency of mCD19 CAR-T cells derived from C57BL/6J mice was only 5.23% under the optimized conditions of mCD19 CAR-T cells derived from BALB/c mice. The number of clones of C57BL/6J mice CD3+ T cells was the highest in anti-CD3 coated+soluble anti-CD28 group after stimulated for 24 h and followed cultured for 48 h. After 24 hours of stimulation under the above conditions and 48 hours of culture with IL-2, the number of T cell proliferating clones in the anti-CD3 coated+soluble anti-CD28 group was significantly increased compared with the same group without IL-2, and the infection efficiency of CAR-T cells in this group reached 17.63%±4.17%. The optimal conditions for constructing CAR-T cells from C57BL/6J mice CD3+ T cells are different from those of BABL/c mice. T cells stimulated by anti-CD3 coated+soluble anti-CD28+IL-2 can obtain mCD19 CAR-T cells with the highest efficiency after retrovirus infection.

Abstract 7473: Flotillin-2 ablation in T cells enhances antigen sensitivity and functionality

Abstract The membrane scaffolding protein flotillin-2 (Flot2) has been proposed to support receptor signaling, but its involvement in T cell receptor (TCR) signaling and T cell responses remains unclear. Here, we show that the deletion of Flot2 increases antigen sensitivity along with changes in surface TCR clustering, resulting in enhanced TCR signaling and effector function to weak TCR stimulation. T cell-specific Flot2-deficient mice showed enhanced proliferation and effector cytokine production in both CD4+ and CD8+ T cells, resulting in better control of tumor growth and infection. However, in vitro TCR stimulation with plate-bound anti-CD3 and soluble anti-CD28 showed increased TCR signaling and early activation of CD4+, but not CD8+ Flot2-deficient T cells upon weak stimulation. In addition, Flot2-deficient CD4+ T cells showed better in vitro polarization toward T helper 1 cells upon weak TCR stimulation, likely due to increased sensitivity of the TCR-triggering threshold. Changes in TCR clustering on the membrane have been suggested to influence the early detection of foreign stimuli and the formation of immune synapses. Flot2 ablation indeed increased the number of membrane TCR nanoclusters on naïve CD4+ T cells. Collectively, this study suggests a mechanism for increasing T cell functionality to weak TCR stimulation by modulating the membrane protein Flot2, which affects surface TCR clustering. These results may have important implications for improving T cell reactivity in diseases with poor antigenicity, such as anti-tumor immune responses. Citation Format: Sookjin Moon, Peer W. Karmaus, Michael B. Fessler. Flotillin-2 ablation in T cells enhances antigen sensitivity and functionality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7473.

A Heterozygous Nonsense Variant of NFKBIA Leading to Gain-of-Function

The NFKBIA gene encodes the protein IκBα, an important regulatory protein that interacts with nuclear factor kappa B family of transcription factors required for innate and adaptive immune responses and normal ectodermal development. Heterozygous nonsense and missense variants of the NFKBIA gene have been reported in a total of 19 individuals with autosomal dominant ectodermal dysplasia with immunodeficiency (EDA-ID) since 2003. Here, we report a patient with a novel de novo heterozygous nonsense variant in the N-terminal of NFKBIA. This patient initially presented to Immunology due to an abnormal presentation of IgA nephropathy (IgAN) with solely kappa staining and a notable history of recurrent AOM, PNA and viral infections including septic shock, diffuse watershed ischemic injury and new seizure onset due to influenza A. Immunologic assessment revealed lymphocytosis of T- and B-cells, particularly CD4+ and CD8+ memory, and CD27-IgD+ cells. In contrast, class-switched, non-class-switched memory B-cells, CD21lo, and plasmablasts were low. A distinct B-cell population expressing less CD19 with negative surface IgD, IgG, and IgM was also observed. Roughly 50% of these cells had IgM expression without surface IgD and were CD24 and CD27 negative, CD21 lo, and CD28 mid. Functionally, T-cells had poor response and proliferation to PHA as well as soluble anti-CD3, anti-CD3+anti-CD28, and anti-CD3+IL-2. Vaccine response was diminished, particularly to polysaccharides. Furthermore, TLR function of PBMCs were assessed revealing decreased cytokine response to soluble ligand exposure. Physical exam findings consistent with EDA-ID included several absent and conical teeth, xerosis, and sparse hair with a history notable for heat intolerance. In line with prior reports of patients with early truncating variants in NFKBIA, the infectious manifestations in these patients have been mild, particularly in comparison to those with missense variants that affect serine 32. In this patient, HSCT was not undertaken, but rather IgG replacement therapy was initiated resulting in absence of any further serious infections. Additionally, glucocorticoid therapy was initiated for IgAN with subsequent sustained remission. With an increase in published data, this may help broaden our scope of what phenotypical presentation can be expected, which may help increase the rate of diagnosis and early identification.

A newborn with multiple intestinal atresias found to have variants in tetratricopeptide repeat domain 7A gene

We report a male infant born with multiple intestinal atresias (MIA) prompting early genetic testing. Bi-allelic, compound heterozygous variants in tetratricopeptide repeat domain 7A (TTC7A) gene were identified. TTC7A is involved in transporting phosphatidylinositol 4-kinase IIIα into the plasma membrane (1). Mutations in this gene disrupt intestinal development and polarity, causing intestinal diseases such as atresia, enteropathy, and very early onset inflammatory bowel disease (VEO-IBD), as well as variable degrees of immunodeficiency (1).The identification of variants in TTC7A gene prompted an immune evaluation since as many as 75% of patients with TTC7A mutations reported in the literature have an underlying immunodeficiency (2). Flow cytometry was relatively unremarkable except for a slightly low memory T-cell count and percentage. NK-cell and B-cell numbers and percentages were appropriate for age. His phytohemagglutinin response was relatively low, but still significant, suggesting a possible component of combined immunodeficiency. To further characterize his T-cell function, we assessed his T-cell proliferation response to anti-CD3, which showed moderately decreased response of CD45+ lymphocytes and CD3+ T-cells to soluble anti-CD3 alone. However, co-stimulation with anti-CD3+anti-CD28 and anti-CD3+IL-2 normalized his T-cell proliferative response. Immunoglobulins were all low. Given his subtle T-cell defect and hypogammaglobulinemia, he was classified as having MIA with combined immunodeficiency (MIA-CID) phenotype of TTC7A.Cobalamin was tried in this patient as it has a low side effect profile and showed benefits in gut motility and intestinal tract luminal narrowing in a TTC7A zebrafish model (3). We considered investigational use of leflunomide, which showed the most promising results in the same zebrafish model (3). Bowel and hematopoietic stem-cell transplantation were considered too. Enterectomy was also offered, as this was shown to be effective in a case report of two patients with similar MIA-CID TTC7A phenotype (4). Unfortunately, the patient did not respond to steroids or cobalamin. His clinical condition worsened, and he passed away at four months of age.Mutations in the TTC7A gene result in various gastrointestinal pathologies with varying degrees of immunodeficiency in approximately 75% of patients (2). All patients with MIA or VEO-IBD should have an immune evaluation and genetic testing.

PD17-10 DISCOVERY OF A NOVEL MONOCLONAL PD-L1 ANTIBODY H1A THAT PROMOTES T-CELL MEDIATED TUMOR KILLING ACTIVITY IN RENAL CELL CARCINOMA

You have accessJournal of UrologyCME1 Apr 2023PD17-10 DISCOVERY OF A NOVEL MONOCLONAL PD-L1 ANTIBODY H1A THAT PROMOTES T-CELL MEDIATED TUMOR KILLING ACTIVITY IN RENAL CELL CARCINOMA Reza Nabavizadeh, Rodrigo Rodrigues Pessoa, Zesheng An, Joanina Gicobi, Susan Harrington, Henan Zhang, Tianxiao Xu, Jacob Hirdler, Christine Lohse, Bradley Leibovich, Haidong Dong, and Fabrice Lucien Reza NabavizadehReza Nabavizadeh More articles by this author , Rodrigo Rodrigues PessoaRodrigo Rodrigues Pessoa More articles by this author , Zesheng AnZesheng An More articles by this author , Joanina GicobiJoanina Gicobi More articles by this author , Susan HarringtonSusan Harrington More articles by this author , Henan ZhangHenan Zhang More articles by this author , Tianxiao XuTianxiao Xu More articles by this author , Jacob HirdlerJacob Hirdler More articles by this author , Christine LohseChristine Lohse More articles by this author , Bradley LeibovichBradley Leibovich More articles by this author , Haidong DongHaidong Dong More articles by this author , and Fabrice LucienFabrice Lucien More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003272.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Immune checkpoint inhibitors (ICI) have shown conflicting outcomes in the adjuvant setting for high-risk clear cell renal cell carcinoma (ccRCC) patients. There is an unmet need to identify patients who would benefit from ICI treatment and to develop new therapies for those who progress on current anti PD1/PD-L1 treatments. Our group has recently created a novel monoclonal antibody (H1A) that induces PD-L1 destabilization at the cell surface resulting in its degradation. In this study, we evaluated the T-cell killing rate in response to current FDA approved treatments as well as H1A. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 13 subjects 3 months after nephrectomy for ccRCC. For treatment with ICI, antibodies were added at the time of activation with soluble anti-CD3 and at 20 μg/ml concentration. Human 786-O ccRCC cells was used as target cells and killing rates were measured by calcein release assay. Paired ANOVA test was used to compare efficacy of ICI. RESULTS: The median increase in T-cell killing rate for pembrolizumab, nivolumab and atezolizumab were 37.8% (range 17.4%-57.7%), 36.6% (range 6 22.7%-65.4%) and 29.9% (range 10%-52.5%), respectively. Individual response to immune checkpoint inhibitors was heterogeneous with some patients showing stronger response with nivolumab (n=9) while others had better killing activity with pembrolizumab (n=3) and one patient with no difference between different treatments. No best response was observed with atezolizumab across all patients. Median killing rate for H1A was 41.7% (range 25%-71.2%). Overall, median killing rate was significantly higher with H1A compared to the pembrolizumab (p=0.0003) and nivolumab (p=0.003) (Figure 1). In terms of individual response, all H1A-treated patients showed improved tumor cell killing efficiency compared to other treatment groups. CONCLUSIONS: In this proof-of-concept, we showed differential T-cell cytotoxic activity in response to various FDA-approved ICI. Additionally, we demonstrated the therapeutic value of a newly developed PD-L1 monoclonal antibody that induces stronger cytotoxic tumor cell killing from ccRCC patient-derived CD8 T cells compared to other ICI. Source of Funding: This work was supported by NIH R01CA256927 (HD) and R01AI095239 (HD) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e499 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Reza Nabavizadeh More articles by this author Rodrigo Rodrigues Pessoa More articles by this author Zesheng An More articles by this author Joanina Gicobi More articles by this author Susan Harrington More articles by this author Henan Zhang More articles by this author Tianxiao Xu More articles by this author Jacob Hirdler More articles by this author Christine Lohse More articles by this author Bradley Leibovich More articles by this author Haidong Dong More articles by this author Fabrice Lucien More articles by this author Expand All Advertisement PDF downloadLoading ...

Soluble and Immobilized Anti-CD3/28 Distinctively Expand and Differentiate Primary Human T Cells: An Implication for Adoptive T Cell Therapy.

Cell-based cancer therapies have led to a paradigm shift in the treatment of patients with various cancers. To date, a vast majority of cancer immunotherapies have used genetically engineered T cells to target tumors. Stimulation and ex vivo expansion of T cells, as one of the crucial starting materials for T cell manufacturing, have always been a critical part of adoptive T-cell therapy (ACT). Typically, anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) along with interleukin-2 (IL-2), through transducing signals one, two, and three, respectively, are essential for in vitro T cell activation. Terminal differentiation and replicative senescence are the main barriers of the ACTs during the manufacturing of engineered T cells ex vivo.In this study, we aimed to compare the T cell activation protocol that we developed in our lab (soluble anti-CD3/28 mAbs) with a common T cell activation protocol (immobilized anti-CD3/soluble anti-CD28) in terms of T cell expansion, activation, immunophenotype, and cellular fate. We observed that T cells were equally expanded in both protocols. Notably, our modified protocol promoted the outgrowth of CD8+ T cells postactivation. Concerning the low concentrations of both soluble anti-CD3 and anti-CD28, the modified protocol could significantly enrich memory T cell subsets. In conclusion, our data demonstrated that the soluble CD3/28 mAbs protocol is cost-effective and more efficient for generating more potent T cells, thereby expecting a better therapeutic outcome.

Optimization of CD19 chimeric antigen receptor T cell establishment and observation of the killing effect in vitro and in vivo

Objective: To optimize the stimulation and activation system of mouse CD3(+) T cells in vitro and explore the optimal infection time of CD3(+) T cells to establish mouse CD19 chimeric antigen receptor T cells (mCD19 CAR-T) , and to also verify its killing effect in vivo and in vitro. Method: Splenic CD3(+)T cells were isolated and purified using magnetic beads, and the cells were cultured in Soluble anti-CD3/CD28, PMA+Ionomycin, and Plated anti-CD3/CD28. Cell activation and apoptosis were assessed by flow cytometry after 8, 24, 48, and 72 hours. ScFv plasmid of mouse CD19 antibody was transfected to plat-E cells to package retrovirus. Activated CD3(+) T cells were infected to construct mouse-specific CD19 chimeric antigen receptor T cells (mCD19 CAR-T) , and mCD19 CAR-T cells were co-cultured with B-cell lymphoma cell line A20 in vitro. The specific toxicity of A20 was detected by flow cytometry, and mCD19 CAR-T cells were infused into the lymphoma mouse model to detect its killing effect and distribution. Results: The activation effect of Plated anti-CD3/CD28 on CD3(+) T cells was superior, with the cells exhibiting good viability 24-48 hours after stimulation. Established mCD19 CAR-T cells with stable efficiency[ (32.27±7.56) % ] were specifically able to kill A20 tumor cells (The apoptosis rate was 24.3% at 48 h) . In vivo detection showed a non-significant decrease in the percentage[ (1.83±0.58) % ] of splenic CD19(+) cells 6 days after mCD19 CAR-T cell infusion. A marked clearance in bone marrow and spleen appeared on day 12 compared with the A20 group, and this difference was statistically significant[spleen: (0.36±0.04) % vs (47.00±13.46) % , P<0.001; bone marrow: (1.82±0.29) % vs (37.30±1.44) % , P<0.0001]. Moreover, mCD19 CAR-T cells were distributed in high proportions in the peripheral blood, spleen, and bone marrow[ (2.90±1.12) % , (4.96±0.80) % , (13.55±1.56) % ]. Conclusion: This study demonstrated an optimized activation system and the optimal infection time of CD3(+) T cells. Furthermore, stable constructed mCD19 CAR-T cells showed a remarkable killing ability in vitro and in vivo.

Alcohol Impairs Immunometabolism and Promotes Naïve T Cell Differentiation to Pro-Inflammatory Th1 CD4+ T Cells.

CD4+ T cell differentiation to pro-inflammatory and immunosuppressive subsets depends on immunometabolism. Pro-inflammatory CD4+ subsets rely on glycolysis, while immunosuppressive Treg cells require functional mitochondria for their differentiation and function. Previous pre-clinical studies have shown that ethanol (EtOH) administration increases pro-inflammatory CD4+ T cell subsets; whether this shift in immunophenotype is linked to alterations in CD4+ T cell metabolism had not been previously examined. The objective of this study was to determine whether ethanol alters CD4+ immunometabolism, and whether this affects CD4+ T cell differentiation. Naïve human CD4+ T cells were plated on anti-CD3 coated plates with soluble anti-CD28, and differentiated with IL-12 in the presence of ethanol (0 and 50 mM) for 3 days. Both Tbet-expressing (Th1) and FOXP3-expressing (Treg) CD4+ T cells increased after differentiation. Ethanol dysregulated CD4+ T cell differentiation by increasing Th1 and decreasing Treg CD4+ T cell subsets. Ethanol increased glycolysis and impaired oxidative phosphorylation in differentiated CD4+ T cells. Moreover, the glycolytic inhibitor 2-deoxyglucose (2-DG) prevented the ethanol-mediated increase in Tbet-expressing CD4+ T cells but did not attenuate the decrease in FOXP3 expression in differentiated CD4+ T cells. Ethanol increased Treg mitochondrial volume and altered expression of genes implicated in mitophagy and autophagosome formation (PINK1 and ATG7). These results suggest that ethanol impairs CD4+ T cell immunometabolism and disrupts mitochondrial repair processes as it promotes CD4+ T cell differentiation to a pro-inflammatory phenotype.

Targeting the Cytosolic Branched Chain Aminotransferase (BCATc) for T Cell Driven Anti‐Lymphoma Immunotherapy

The lymphoma microenvironment is a dynamic network between lymphoma cells and non‐malignant components that may promote tumor growth and consequently drug resistance. Progress in T cell metabolism has demonstrated that T cells experience metabolic disadvantage in the tumor microenvironment, which commonly manifests in T cell exhaustion and jeopardizes their potential to destroy cancer cells. We identified the cytosolic branched chain aminotransferase (BCATc) as a novel metabolic checkpoint of T cell activation. BCATc catalyzes the first step in the degradation of the essential amino acid leucine, which is a well‐known activator of complex 1 of the mammalian target of rapamycin (mTORC1). In T cells, BCATc is induced upon T cell activation and is a part of a negative feedback loop regulation of mTORC1. Based on extensive earlier research, we proposed that BCATc exerts immunosuppressive properties in the tumor microenvironment and we hypothesized that deleting BCATc from T cells renders them effective in eradicating lymphoma tumors.By using our newly designed mouse model with BCATc deleted from CD4+ and CD8+ T cells (T‐BCATcKO mice), we performed tumor studies where male and female mice were subcutaneously injected with 2.5x105 murine EL‐4 lymphoma cells [n=8] or phosphate‐buffered saline [n=4‐6]. As controls, mice expressing the floxed BCATc gene (T‐BCATcfl/flmice, n=9) were used. Tumor growth, body weight, and food intake were monitored for up to 15 days followed by collection of tumor tissues and organs. In addition, CD4+ T cells were isolated from spleens of T‐BCATcfl/fl and T‐BCATcKO mice, activated with a plate‐bound anti‐CD3 and soluble anti‐CD28 and left untreated or treated with 5 mM gabapentin (a competitive inhibitor of BCATc) or 10 mM N‐acetyl‐leucine amide (NALA, a structural analog of leucine) for 24‐72 hours to measure changes in the secretion of interferon gamma (IFNγ) from T helper 1 (Th1) cells or the expression of Foxp3, a master activator of regulatory T cells (CD4+CD25+ T reg cells).Deletion of BCATc from T cells or inhibition of the BCATc activity by gabapentin caused a significant increase in the secretion of IFNγ from Th1 cells after 24‐72 hours of activation, while CD4+CD25+ T reg cells expressed reduced levels of Foxp3 in response to gabapentin. In contrast, NALA blocked the IFNγ secretion and caused an increase in the expression of Foxp3. Following tumor inoculation, the T‐BCATcKO mice demonstrated a significant delay in tumor appearance and growth (~50 % reduction in tumor volumes) and the final tumor masses were reduced by 25% compared to control mice. None of the animals differed in body, organ weights or food intake and deletion of BCATc from T cells did not lead to systemic overactivation of the immune system. This study revealed that deleting BCATc from T cells may offer an advantage to better fight lymphoma and identified BCATc as a new therapeutic target to improve the efficiency of checkpoint inhibitors and help increase the low rates of remission seen in lymphoma patients.

Manufacturing Anti-CD19 CAR-Tscm Cells for Immunotherapy Using Innovative Microbubble-Based Technologies for Precision Cell Processing

INTRODUCTIONChimeric antigen receptor (CAR) T cell therapy provides a potential cure for patients who don't respond to standard treatments. One of the major challenges for current mainstream CAR-T therapy is lack of in vivo persistence of transferred cells. Intriguingly, more differentiated effector T cells that acquire enhanced in vitro anti-tumor properties were found to be paradoxically less effective for in vivo tumor treatment. It has become clear that the persistence of adoptive transferred T cells is crucial for in vivo activities. A significant linear correlation was found between the differentiation status of infused T cells and the potency of tumor regression, in the order of Tscm > Tcm >Tem. While promising, CAR-Tscm cell therapy demands technical innovation for precision cell processing and production. Here, we show CAR-Tscm cells generated by microbubble-based technologies for precision T cell sorting and activation and viral transduction are more robust than those generated by the conventional method in in vitro assays and in vivo animal study using the NSG mouse model.METHODSNaïve T cells were isolated by sequential sorting with 3 targeted lipid shell microbubbles (conjugated with anti-CD8, anti-CD45RA, and anti-CD62L). Briefly, CD8+ T cells were floated by anti-CD8 conjugated microbubbles and separated from nontargeted cells in PBMC. The floated CD8+ cells returned to solution after the microbubbles had undergone dissolution by increasing ambient pressure. These cells were subsequently floated by anti-CD45RA and anti-CD62L microbubbles sequentially, as described earlier. To generate CD19-CAR-modified Tscm-enriched cells, isolated naïve T cells were activated by anti-CD3/CD28 conjugated microbubbles and transduced by CD19-CAR retrovirus via Retronectin conjugated microbubbles. Transduced naïve T cells were cultured in media with IL-7, IL-21, and TWS119. Control CD19-CAR T cells were produced from same donors' PBMCs using standard culture conditions (soluble IL-2 and anti-CD3/CD28 coated culture flasks). Raji cells were co-cultured with effector cells (Tscm-enriched and standard CD19 CAR T cells) at a gradient ET ratio for in vitro cytotoxicity assay. Tscm-enriched and standard CD19-CAR T cells were injected into Raji-luc-bearing NSG mice. Tumor burden was measured by the XenogenIVIS system.RESULTSNaïve CD8+ T lymphocytes were enriched by sequential isolation of CD8+, CD62L+, and CD45RA+ cells from healthy donor PBMCs using respective antibody conjugated microbubbles. The CD8, CD62L, and CD45RA triple positive population was enriched from initial 10.08%± 2.4% to 71.84%±1.9% (n=4). We discovered that Retronectin conjugated microbubbles are advantageous to replace spinoculation technique to simplify viral transduction procedures. The average transduction efficiency was 27.71%± 8.7% using unconcentrated retroviral vectors. The Tscm-like cells were significantly enriched after two-week culture, compared to the standard method (37.78± 14.5% vs. 4.38± 0.7%). Tscm-enriched CD19-CAR T cells exhibited a stronger in vitro cytotoxicity towards Raji cells in comparison with standard CD19-CAR T cells (Figure 1). Consistently, in the NSG mice engrafted with Raji-Luc cancer cells, the CD19-CAR-modified Tscm-enriched cells showed longer-lasting antitumor responses than the CD19-CAR-T cells generated by the standard manufacturing process (Figure 2).CONCLUSIONWe have developed a bead-free, multi-positive selection system for CAR-Tscm production, and demonstrated that these Tscm cells effectively cause tumor regression and prolong diseased animal lifespan. A closed, automated, microbubble-based CAR-T cell manufacturing system, combining T cell isolation, activation, and transduction will be developed to improve performance and reduce costs. [Display omitted] DisclosuresFu: Diagnologix LLC: Current Employment. Shi: Diagnologix LLC: Current Employment. Liu: Diagnologix LLC: Current Employment.

Controlling T cells spreading, mechanics and activation by micropatterning

We designed a strategy, based on a careful examination of the activation capabilities of proteins and antibodies used as substrates for adhering T cells, coupled to protein microstamping to control at the same time the position, shape, spreading, mechanics and activation state of T cells. Once adhered on patterns, we examined the capacities of T cells to be activated with soluble anti CD3, in comparison to T cells adhered to a continuously decorated substrate with the same density of ligands. We show that, in our hand, adhering onto an anti CD45 antibody decorated surface was not affecting T cell calcium fluxes, even adhered on variable size micro-patterns. Aside, we analyzed the T cell mechanics, when spread on pattern or not, using Atomic Force Microscopy indentation. By expressing MEGF10 as a non immune adhesion receptor in T cells we measured the very same spreading area on PLL substrates and Young modulus than non modified cells, immobilized on anti CD45 antibodies, while retaining similar activation capabilities using soluble anti CD3 antibodies or through model APC contacts. We propose that our system is a way to test activation or anergy of T cells with defined adhesion and mechanical characteristics, and may allow to dissect fine details of these mechanisms since it allows to observe homogenized populations in standardized T cell activation assays.

A soluble activator that favors the ex vivo expansion of CD8+CD27+ T cells.

Adoptive cell therapy involves the infusion of tumor-reactive T cells into patients with cancer to provide antitumor immunity. The ex vivo expansion and differentiation of such T cells are key parameters that affect their therapeutic potential. Human T cells are presently expanded in culture through the use of anti-CD3 and anti-CD28 mAbs immobilized on beads, expressed on cells, or assembled in the context of soluble antibody complexes. Here we report the design of a small, bispecific single-chain variable fragment construct agonizing both CD3 and CD28 pathways. This soluble T cell expansion protein, termed T-CEP, activates, expands, and differentiates human T cells ex vivo at concentrations in the femtomolar range. Importantly, T-CEP promotes the preferential growth of human CD8+ T cells over the course of 12 days in comparison with methods involving immobilized anti-CD3 mAb/soluble anti-CD28 mAb or soluble anti-CD3/CD28 mAb complexes. The differentiation profile of the resulting human T cell population is also singularly affected by T-CEP, favoring the expansion of a preferred CD8+CD27+ T cell phenotype. The activity profile of T-CEP on human T cells ex vivo suggests its use in generating human T cell populations that are more suited for adoptive cell therapy.

A Comparison of Automated Perfusion- and Manual Diffusion-Based Human Regulatory T Cell Expansion and Functionality Using a Soluble Activator Complex

Absence or reduced frequency of human regulatory T cells (Tregs) can limit the control of inflammatory responses, autoimmunity, and the success of transplant engraftment. Clinical studies indicate that use of Tregs as immunotherapeutics would require billions of cells per dose. The Quantum® Cell Expansion System (Quantum system) is a hollow-fiber bioreactor that has previously been used to grow billions of functional T cells in a short timeframe, 8–9 d. Here we evaluated expansion of selected Tregs in the Quantum system using a soluble activator to compare the effects of automated perfusion with manual diffusion-based culture in flasks. Treg CD4+CD25+ cells from three healthy donors, isolated via column-free immunomagnetic negative/positive selection, were grown under static conditions and subsequently seeded into Quantum system bioreactors and into T225 control flasks in an identical culture volume of PRIME-XV XSFM medium with interleukin-2, for a 9-d expansion using a soluble anti-CD3/CD28/CD2 monoclonal antibody activator complex. Treg harvests from three parallel expansions produced a mean of 3.95 × 108 (range 1.92 × 108 to 5.58 × 108) Tregs in flasks (mean viability 71.3%) versus 7.00 × 109 (range 3.57 × 109 to 13.00 × 109) Tregs in the Quantum system (mean viability 91.8%), demonstrating a mean 17.7-fold increase in Treg yield for the Quantum system over that obtained in flasks. The two culture processes gave rise to cells with a memory Treg CD4+CD25+FoxP3+CD45RO+ phenotype of 93.7% for flasks versus 97.7% for the Quantum system. Tregs from the Quantum system demonstrated an 8-fold greater interleukin-10 stimulation index than cells from flask culture following restimulation. Quantum system–expanded Tregs proliferated, maintained their antigenic phenotype, and suppressed effector immune cells after cryopreservation. We conclude that an automated perfusion bioreactor can support the scale-up expansion of functional Tregs more efficiently than diffusion-based flask culture.

Streamlined production of genetically modified T cells with activation, transduction and expansion in closed-system G-Rex bioreactors

BackgroundGas Permeable Rapid Expansion (G-Rex) bioreactors have been shown to efficiently expand immune cells intended for therapeutic use, but do not address the complexity of the viral transduction step required for many engineered T-cell products. Here we demonstrate a novel method for transduction of activated T cells with Vectofusin-1 reagent. Transduction is accomplished in suspension, in G-Rex bioreactors. The simplified transduction step is integrated into a streamlined process that uses a single bioreactor with limited operator intervention. MethodsPeripheral blood mononuclear cells (PBMCs) from healthy donors were thawed, washed and activated with soluble anti-CD3 and anti-CD28 antibodies either in cell culture bags or in G-Rex bioreactors. Cells were cultured in TexMACS GMP medium with interleukin (IL)-7 and IL-15 and transduced with RetroNectin in bags or Vectorfusin-1 in the G-Rex. Total viable cell number, fold expansion, viability, transduction efficiency, phenotype and function were compared between the two processes. ResultsThe simplified process uses a single vessel from activation through harvest and achieves 56% transduction with 29-fold expansion in 11 days. The cells generated in the simplified process do not differ from cells produced in the conventional bag-based process functionally or phenotypically. DiscussionThis study demonstrates that T cells can be transduced in suspension. Further, the conventional method of generating engineered T cells in bags for clinical use can be streamlined to a much simpler, less-expensive process without compromising the quality or function of the cell product.

The phosphorylation kinetics of the key regulators in T cell receptor signaling pathway

Abstract T cell receptor (TCR) signaling pathway regulates T cell homeostasis and adaptive immune responses. Clinically, the activation of TCR is indispensable to the success of T cell-based immunotherapy. The engagement of TCR phosphorylates immunoreceptor tyrosine-based activation motifs (ITAM) on CD3, which is followed by a protein phosphorylation cascade. Protein phosphorylation, regulated by kinases and phosphatases, changes protein conformation and determines protein function. A better understanding of TCR signaling phosphorylation events is crucial to both decipher the T cell regulation mechanism and boost the efficacy of T cell immunotherapy. Here, we developed a panel of monoclonal antibodies that target several of the TCR signaling regulators and are applicable for both intracellular flow cytometry (ICFC) and western blot (WB). Using these antibodies, we performed a kinetic analysis of the TCR signaling pathway. Jurkat T cells were cross-linked by soluble anti-CD3 and anti-CD28 antibodies, and the phosphorylation kinetics of the key TCR signaling regulators, such as phospho-ZAP-70 and phospho-PLCg1, were evaluated by ICFC and WB. We show that TCR signaling activation induced by cross-linking CD3 and CD28 is a relatively transient event and the optimal activation of the key TCR signaling regulators varies from 1 to 15 minutes. Our data profiled the phosphorylation kinetics of the key regulators in TCR signaling pathway.

Altered Expression of miR-326 in T Cell-derived Exosomes of Patients with Relapsing-remitting Multiple Sclerosis

Invasion of auto-reactive CD4+ T cells especially Th17 into central nervous system (CNS) is an underlying pathogenic mechanism in multiple sclerosis (MS). CD4+ T cells release exosomes which are enriched in microRNAs, reflective of cell's physiological or pathological condition. Thus exosomes could be potent agents to provide quantitative and qualitative information about involved cells in MS. We investigated the expression of pathogenic microRNAs in T cells-derived exosomes of MS patients or healthy controls. Conventional T cells (Tconv) derived from relapsing-remitting (RR) MS patients (n=10) and healthy controls (n=10) were purified and cultured for 3 days by soluble anti-CD3/CD28. Exosomes were purified from cultured-T cells supernatants. The expression levels of exosomal miR-146a, miR-29a, miR-155, and miR-326 were quantified by real-time PCR. A statistically significant increased expression of miR-326 in Tconv-derived exosomes was observed in RRMS patients as compared with controls (7.5±1.88vs 2.51±0.9 p=0.03), On the contrary, no differences were found in the expression levels of miR-155, miR-146a, and miR-29a, in Tconv-derived exosomes of patients as compared with controls (p>0.05).Our results point to altered expression in exosome-derived microRNAs. MiR-326 was previously shown to play a role in the immunopathogenesis of MS by inducing TH17 differentiation and maturation. Therefore, miR-326 containing exosomes might also be a potential clinical target in course of MS. Moreover, the deregulation of this miRNA in exosomes may serve as a diagnostic and prognostic biomarker.

Effects of starting cellular material composition on chimeric antigen receptor T-cell expansion and characteristics.

When manufacturing chimeric antigen receptor (CAR) T cells using anti-CD3/anti-CD28 beads, ex vivo T-cell expansion is dependent on the composition of leukocytes used in the manufacturing process. We investigated the effects of leukocyte composition on CAR T-cell expansion and characteristics using an alternative manufacturing method. Anti-B-cell maturation antigen and CD19-CAR T cells were manufactured using autologous peripheral blood mononuclear cell (PBMNC) concentrates. The PBMNCs were enriched for lymphocytes using density gradient separation, which were used for CAR T-cell culture initiation. T-cell expansion was stimulated with soluble anti-CD3 and interleukin-2. Fifty-one CAR T-cell products were evaluated; 28 anti-B-cell maturation antigen (BCMA) CAR T cells produced for 24 patients and 27 CD19 CAR T cells produced for 24 patients. CAR T-cell expansion was reduced when greater quantities of monocytes were present in the post-density gradient separation PBMNCs. In addition, the ratio of CD4 to CD8 cells in the CAR T-cell products after 7 days of culture was dependent on the quantity of monocytes, RBCs, and neutrophils in the post-density gradient separation PBMNCs. Greater quantities of monocytes and RBCs were associated with a greater proportion of CD4+ cells and greater quantities of neutrophils were associated with a greater proportion of CD8+ cells. The composition of leukocytes used to manufacture CAR T cells can affect cell expansion and the composition of CAR T-cell products. More uniform or complete lymphocyte enrichment of PBMNCs improves the consistency of final CAR T-cell products.

A Simplified Method for Transduction and Expansion of T Cells for Clinical Application

Introduction: Genetically modified T cells are being investigated to treat a variety of disorders and have been particularly successful in treating B cell cancers. As more effort is poured into new targets, molecular switches, and various other modifications, development of processes to quickly manufacture new products must keep up. Current manufacturing processes often require highly skilled operators and specialized equipment. Here, we demonstrate a simplified, novel method for transduction of T cells, followed by robust expansion in the G-Rex bioreactor with no need for intervention until harvest. A scaled-up, closed-version of the same process, including a closed harvest step with the GatheRex is currently under evaluation.Methods: Frozen peripheral blood mononuclear cells (PBMCs) from healthy donors were used as starting material. PBMCs were thawed, washed, and activated with soluble anti-CD3 and anti-CD28 antibodies either in cell culture bags (32-C, Saint-Gobain Cell Therapy) or in G-Rex bioreactors (Wilson Wolf Corporation). Cells were cultured in TexMACS GMP medium (Miltenyi Biotec) with IL-7 and IL-15 throughout. For the transduction step, activated PMBCs and retroviral supernatant were incubated in cell culture bags coated with Retronectin (Takara) or in G-Rex bioreactors with vectofusin-1 (Miltenyi Biotec). Viral constructs contained either a CD34 or CD19 marker detectable by antibody staining. For transduction in the G-Rex, various cell densities, volumes, constructs, and multiplicity of infection (MOIs) were tested. Where applicable, the GatheRex device (Wilson Wolf Corporation) was used for volume reduction and harvest. Transduction efficiency and T cell phenotype were measured by flow cytometry. Cell count and viability were assessed with the NC-3000 (Chemometic). Glucose and lactate concentrations were checked daily for in-processing monitoring.Results: Overall transduction efficiency ranged from 30-90% depending on the experimental conditions. Incubating 1x107 activated PBMCs in 10 ml of medium in a 10-cm2 G-Rex (1.0 ml/cm2) with retrovirus at an MOI of 1 resulted in 3% transduced cells. Addition of vectofusin-1 to the same condition yielded transduction efficiency of 44%. Increasing the MOI to 10 lead to 86% transduced cells. Decreasing the transduction volume from 1.0 ml/cm2 to 0.4 ml/cm2 increased transduction efficiency from 34% to 55%. Reducing the volume further to 0.2 ml/cm2 did not improve efficiency, and rather had a negative impact compared to the 0.4 ml/cm2 condition (38%). 16-24 hrs after transduction, the volume of medium was increased to 10.0 ml/cm2 without a wash step. The dilution in place of a wash step had no negative impact on cell viability. 10.0 ml/cm2 medium supported high viability (>90%) and expansion (30-50 fold) over an additional 9 days without operator intervention. The phenotype of cells expanded in the G-Rex contained a mixed population of CD45RO+ and CD45RA+ cells, with a similar distribution of naive and memory cell subsets in G-Rex and bag cultures. Harvest of cells with the GatheRex was efficient; a 1L volume was reduced 10-fold in 5 minutes, and 95% of cells were recovered.Summary: T cells can be transduced with retroviral vectors in the G-Rex bioreactor. Clinically relevant levels of transgene expression can be achieved by combining reagents in the G-Rex, without complicated coating steps or time-consuming spinning steps. This simplified procedure reduces the hands-on time of the T cell transduction to minutes rather than hours. Transgenic cells can be expanded 30-50-fold in the G-Rex with limited operator intervention and without specialized equipment. DisclosuresKhalil:Bellicum Pharmaceuticals: Employment. Foster:Bellicum: Employment, Equity Ownership. Gagliardi:Bellicum Pharmaceuticals: Employment.

Low 17β-estradiol Levels Are Better Inducers of Regulatory Conditioned T Cells In-Vitro.

17β-estradiol (E2) has been known to modulate immune response. Recent studies indicate that E2 at pregnancy level plays a role in regulating T cell response. To investigate the optimum dose of E2 (from 10-9 to 10-7 M) in mediating the generation of regulatory T cells (Tregs), using naive human CD4+ T cells from healthy women. Naive peripheral T cells were purified and conditioned with soluble anti-CD28 in anti-CD3-coated plates in the presence or absence of E2. Flow cytometry was employed to assess the expression pattern of forkhead boxP3 (FOXP3) and programmed death-1 (PD-1). Proliferation and cytokine secretions were analyzed, using XTT and ELISA assays. In the presence of different doses of E2, the expression levels of anti-CD3/CD28 antibody-stimulated CD25/ FOXP3 and FOXP3/PD-1 in conditioned T cells (cT) were peaked at 1 ng/ml (early pregnancy level, E2(1)) (47.14% (37.3-74.9) and 32% (27.7-52.5), respectively) and a slight, but not significant, increase after declining at 36 ng/ml (late pregnancy/pharmaceutical, E2(36)) (19.4% (15.2-24.5) and 15.8% (10.6-26.8), respectively). E2(1) cT showed a significantly reduced proliferation capacity (p<0.05) and secretion of IL-10 was enhanced in supernatants of E2(1 and 36) cT (p<0.05). In contrast to decreased TNF-α and IFN-γ secretions in E2(1) cT supernatants, E2(36) stimulated TNF-α and IFN-γ secretions (p<0.05 and p<0.01, respectively). Our results indicate that the differential effect of E2 on generation of Tregs is consistent with the possibility that lower levels of pregnancy E2 are most efficient in induction of Tregs.

Functional stability of ACD anticoagulated blood samples stored for 72 hours at room temperature.

Abstract Characterization of the immune system in remote locations is challenging, requiring technical expertise and specific analytical equipment. Immune functional assessments require live cells, and the window of viability following blood donation is limited. To test the ability of ACD blood collection tubes to expand this window of viability, cellular function was analyzed over 4 consecutive days, beginning on the date of blood collection. T-cell activation, characterized by CD69 and CD25 expression, was measured following a 24-hour stimulation with soluble anti-CD3 and anti-CD28 antibodies (CD3/CD28) or with staphylococcus enterotoxins A and B (SEA+SEB). Mitogen stimulated cytokine profiles were determined following a 48-hour stimulation with CD3/CD28, phytohemagglutinin and ionomycin (PMA+I), or lipopolysaccharide (LPS). T-cell activation following stimulation with SEA+SEB was not altered by the processing delay; however, following stimulation with CD3/CD28, T-cell activation increased over the 72 hour period. The processing delay did not affect cytokine production following CD3/CD28 stimulation. Following stimulation with LPS and PMA+I, results were varied. While some cytokine responses were unaltered following the processing delay, others were impaired. These findings confirm the general viability of whole blood stored at room temperature (68 to 72°F) for 48 to 72 hours in ACD tubes and suggest many phenotypic and functional assays are feasible on blood samples aged as described. Investigators using this protocol should confirm their specific culture technique and measured output. This protocol may assist studies of subjects in remote locations with no immediate access to laboratory instrumentation.