Central Memory Phenotype Research Articles

PD1-CD28 Fusion Protein Enables CD4+ T Cell Help for Adoptive T Cell Therapy in Models of Pancreatic Cancer and Non-hodgkin Lymphoma.

Background: Interaction of the programmed death receptor 1 (PD-1) and its ligand, PD-L1, suppresses T cell activity and permits tumors to evade T cell-mediated immune surveillance. We have recently demonstrated that antigen-specific CD8+ T cells transduced with a PD1-CD28 fusion protein are protected from PD-1-mediated inhibition. We have now investigated the potential of PD1-CD28 fusion protein-transduced CD4+ T cells alone or in combination with CD8+ T cells for immunotherapy of pancreatic cancer and non-Hodgkin lymphoma.Methods: OVA-specific CD4+ and CD8+ were retrovirally transduced with the PD1-CD28 fusion protein. Cytokine release, proliferation, cytotoxic activity, and phenotype of transduced T cells were assessed in the context of Panc02-OVA (murine pancreatic cancer model) and E.G7-PD-L1 (murine T cell lymphoma model) cells.Results: Stimulation of PD1-CD28 fusion protein-transduced CD4+ T cells with anti-CD3 and recombinant PD-L1 induced specific T cell activation, as measured by IFN-y release and T cell proliferation. Coculture with Panc02-OVA or E.G7-PD-L1 tumor cells also led to specific activation of CD4+ T cells. Cytokine release and T cell proliferation was most effective when tumor cells simultaneously encountered genetically engineered CD4+ and CD8+ T cells. Synergy between both cell populations was also observed for specific tumor cell lysis. T cell cytotoxicity was mediated via granzyme B release and mediated enhanced tumor control in vivo. Transduced CD4+ and CD8+ T cells in co-culture with tumor cells developed a predominant central memory phenotype over time. Different ratios of CD4+ and CD8+ transduced T cells led to a significant increase of IFN-y and IL-2 secretion positively correlating with CD4+ T cell numbers used. Mechanistically, IL-2 and MHC-I were central to the synergistic activity of CD4+ and CD8+ T cells, since neutralization of IL-2 prevented the crosstalk between these cell populations.Conclusion: PD1-CD28 fusion protein-transduced CD4+ T cells significantly improved anti-tumoral effect of fusion protein-transduced CD8+ T cells. Thus, our results indicate that PD1-CD28 fusion protein-transduced CD4+ T cells have the potential to overcome the PD-1-PD-L1 immunosuppressive axis in pancreatic cancer and non-Hodgkin lymphoma.

The Profile of T Cell Responses in Bacille Calmette-Guérin-Primed Mice Boosted by a Novel Sendai Virus Vectored Anti-Tuberculosis Vaccine.

The kinds of vaccine-induced T cell responses that are beneficial for protection against Mycobacterium tuberculosis (Mtb) infection are not adequately defined. We had shown that a novel Sendai virus vectored vaccine, SeV85AB, was able to enhance immune protection induced by bacille Calmette–Guérin (BCG) in a prime-boost model. However, the profile of T cell responses boosted by SeV85AB was not determined. Herein, we show that the antigen-specific CD4+ and CD8+ T cell responses were both enhanced by the SeV85AB boost after BCG. Different profiles of antigen-specific po T cell subsets were induced in the local (lung) and systemic (spleen) sites. In the spleen, the CD4+ T cell responses that were enhanced by the SeV85AB boost were predominately IL-2 responses, whereas in the lung the greater increases were in IFN-γ- and TNF-α-producing CD4+ T cells; in CD8+ T cells, although IFN-γ was enhanced in both the spleen and lung, only IL-2+TNF-α+CD8+ T subset was boosted in the latter. After a challenge Mtb infection, there were significantly higher levels of recall IL-2 responses in T cells. In contrast, IFN-γ-producing cells were barely boosted by SeV85AB. After Mtb challenge a central memory phenotype of responding CD4+ T cells was a prominent feature in SeV85AB-boosted mice. Thus, our data strongly suggest that the enhanced immune protection induced by SeV85AB boosting was associated with establishment of an increased capacity to recall antigen-specific IL-2-mediated T cell responses and confirms this Sendai virus vector system as a promising candidate to be used in a heterologous prime-boost immunization regimen against TB.

Rapid ex vivo expansion of highly enriched human invariant natural killer T cells via single antigenic stimulation for cell therapy to prevent graft-versus-host disease

Background aimsCD1d-restricted invariant natural killer (iNK) T cells are rare regulatory T cells that may contribute to the immune-regulation in allogeneic stem cell transplantation (ASCT). Here, we sought to develop an effective strategy to expand human iNK T cells for use in cell therapy to prevent graft-versus-host disease (GVHD) in ASCT. MethodsHuman iNK T cells were first enriched from peripheral blood mononuclear cells (PBMCs) using magnetic-activated cell sorting separation, then co-cultured with dendritic cells in the presence of agonist glycolipids, alpha-galactosylceramide, for 2 weeks. ResultsThe single antigenic stimulation reliably expanded iNK T cells to an average of 2.8 × 107 per 5 × 108 PBMCs in an average purity of 98.8% in 2 weeks (N = 24). The expanded iNK T cells contained a significantly higher level of CD4+ and central memory phenotype (CD45RA−CD62L+) compared with freshly isolated iNK T cells, and maintained their ability to produce both Th-1 (interferon [IFN]γ and tumor necrosis factor [TNF]α) and Th-2 type cytokines (interleukin [IL]-4, IL-5 and IL-13) upon antigenic stimulation or stimulation with Phorbol 12-myristate 13-acetate/ionomycin. Interestingly, expanded iNK T cells were highly autoreactive and produced a Th-2 polarized cytokine production profile after being co-cultured with dendritic cells alone without exogenous agonist glycolipid antigen. Lastly, expanded iNK T cells suppressed conventional T-cell proliferation and ameliorated xenograft GVHD (hazard ratio, 0.1266; P < 0.0001). ConclusionWe have demonstrated a feasible approach for obtaining ex vivo expanded, highly enriched human iNK T cells for use in adoptive cell therapy to prevent GVHD in ASCT.

Abstract 5616: Rituximab chemotherapy induces a partial recovery from T-cell exhaustion in follicular lymphoma patients

Abstract For many years, the clinical responses observed in cancer patients treated with antitumor antibodies have been viewed only as a consequence of the recruitment and activation of innate immune cells. Several preclinical studies have indicated that anti-tumor antibodies can also induce a potent anti-tumor adaptive T-cell response capable of protecting animals from tumor challenge. Our studies have shown that CD4+ Th1 cells play a central role in the response to anti-CD20 tumor therapy by opposing Treg expansion and that CD20-specific memory T cells are responsible for anti-tumor protection observed in surviving animals subjected to tumor rechallenge. Whether an adaptive T-cell response is elicited in patients with Follicular Lymphoma (FL) receiving anti-CD20-based treatment is not known. In the present work, we analyzed peripheral T cells before and during anti-CD20-chemotherapy treatment of FL patients (R-CHOP) with high tumor burden. We first examined in ELISPOT assays the IFN-γ production by PBMC from 20 patients against human CD20-derived peptides previously selected by combining in silico and in vivo screening using transgenic human HLA-DR mice. Both FL patients before treatment and patients undergoing R-CHOP treatment exhibited peripheral T-cell responses to the selected CD20-derived peptides. However, these responses were markedly lower than those observed in healthy donors. Similar results were also obtained when T cell responses against viruses commonly infecting large numbers of individuals (CMV, EBV, influenza) were assessed, suggesting that high tumor burden FL patients exhibit an immune exhaustion of T cells. Multiparametric flow cytometry analyses suggested that their poor ability to respond to viral and CD20-derived peptides is related to the presence of an immunocompromised and exhausted blood T-cell peripheral compartment with higher percentages of TIGIT- and PD-1-expressing T cells, of peripheral Treg, and terminally differentiated CD8+ TEMRA. R-CHOP therapy induced a shift of CD4+ and CD8+ T cells toward a central memory phenotype (TCM), an additional shift of CD8+ T cells to a naive phenotype, and a decrease in the number of PD-1+ and TIGIT+ T cells, making these cells evolving toward a lesser degree of exhaustion. However, it did not restore a level of specific responses similar to those observed in healthy donors. These results support the use of anti-PD-1 and anti-TIGIT antibodies in combination with anti-CD20 in FL patients with high-tumor burden. Citation Format: Benoit Milcent, Pauline Brice, Catherine Thieblemont, Jean-Luc Teillaud, Sophie Siberil. Rituximab chemotherapy induces a partial recovery from T-cell exhaustion in follicular lymphoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5616.

Abstract LB-147: Comprehensive immune and molecular analysis of two metastatic melanoma patients treated with a personal neoantigen vaccine, NEO-PV-01, in combination with anti-PD1: A case study

Abstract Background: Neoantigens arise from DNA mutations and are critical targets that are presented on the surface of cancer cells for tumor-specific T cell responses. Vaccines targeting neoantigens have the potential to induce de novo and amplify pre-existing anti-tumor T cell responses. NEO-PV-01, a personal neoantigen vaccine that is designed based on a patient's tumor-specific mutations, is comprised of up to 20 long peptides, and administered with the immune adjuvant Poly-ICLC. Here we report a comprehensive immune and molecular analysis observed in two metastatic melanoma patients treated with NEO-PV-01 in combination with nivolumab (ClinicalTrials.gov: NCT02897765). Methods: Immune monitoring was performed from peripheral blood and tumor biopsies collected i) prior to any treatment, ii) after 12-weeks of nivolumab monotherapy and, iii) after NEO-PV-01 vaccination. Peripheral blood samples were evaluated for the presence of antigen-specific T cell responses by IFNγ ELISPOT, intracellular cytokine staining, multi-parameter surface and functional phenotyping by FACS, and presence of cytolytic properties. Tumor biopsies were analyzed for multiple immune and tumor markers by immunohistochemistry, gene expression, and whole exome sequencing. Results: IFNγ ELISPOT analysis with PBMCs revealed neoantigen-specific CD4 and CD8 T cell responses that were predominantly observed following vaccination. Vaccine-induced, neoantigen-specific CD8 T cells had an effector memory and central memory phenotype, secreted IFNγ, TNFα and IL2, and were cytolytic. Finally, induction of neoantigen-specific immunity by NEO-PV-01 led to epitope spreading of the immune response to neoantigens not included in the vaccine. Assessment of pre- and post-treatment tumor biopsies with repeat exome sequencing, gene expression, as well as immunohistochemistry and pathologic analysis will be presented. Conclusions: NEO-PV-01 is immunogenic and leads to multiple de novo neoantigen-specific immune responses in the peripheral blood. Furthermore, the detection of epitope spreading to additional neoantigen targets suggests vaccine-induced broadening of the immune response. Citation Format: Aung Naing, Patrick A. Ott, Samantha J. Gates, Jose G. Martinez, Riley R. Curran, Victoria L. Kohler, Meghan E. Bushway, Julian Scherer, Dominik Barthelme, Jesse Z. Dong, April Lamb, Lisa D. Cleary, Melissa Moles, Joel Greshock, Richard B. Gaynor, Matthew J. Goldstein, Lakshmi Srinivasan. Comprehensive immune and molecular analysis of two metastatic melanoma patients treated with a personal neoantigen vaccine, NEO-PV-01, in combination with anti-PD1: A case study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-147.

Abstract CT125: A personal neoantigen vaccine, NEO-PV-01, with anti-PD1 induces broad de novo anti-tumor immunity in patients with metastatic melanoma, NSCLC, and bladder cancer

Abstract Background: Neoantigens arise from DNA mutations in cancer cells and are critical targets for tumor-specific T cell responses. Adjuvant treatment with a personal neoantigen vaccine induced broad immune responses in melanoma patients and suggested the potential for synergy with an immune checkpoint inhibitor (Ott et al, Nature 2017). NEO-PV-01 is a personal neoantigen vaccine containing multiple peptide epitopes based on the genomic profile of each patient's tumor. We report the first clinical and immune data of NEO-PV-01 in combination with nivolumab for patients with metastatic cancer (ClinicalTrials.gov: NCT02897765). Methods: NT-001 is a single-arm, Phase 1b study of NEO-PV-01 with nivolumab in patients with advanced melanoma, non-small cell lung cancer, or transitional cell carcinoma of the bladder. NEO-PV-01 is designed for each patient using DNA and RNA sequencing from a recently biopsied tumor, selecting patient MHC class I- and II-restricted neoantigen epitopes, and designing and manufacturing up to 20 long peptides. Patients begin bi-weekly nivolumab at week 0. At week 12, NEO-PV-01 plus the immune adjuvant Poly-ICLC is administered subcutaneously in a prime-boost format. The primary endpoint of the study is safety; secondary endpoints include overall response rate (ORR), clinical benefit rate (CBR), and progression free survival (PFS). Comprehensive immune assessment before, during, and after therapy is performed for each patient. Results: Of 21 patients who received at least one vaccination by the time of data cutoff (15 NOV 2017), 14 have completed the full vaccination schedule (10 - Melanoma, 3 - Lung, 1 - Bladder). Vaccine-related adverse events were mild, including injection site reactions and flu-like symptoms, with no vaccine-related SAEs observed. In the first 14 patients who completed vaccination by the time of data cutoff, we observed 5 patients with partial response (PR), 5 with stable disease (SD), and 4 with progressive disease (PD) (2 of whom had progression prior to vaccination). Several patients with PR and SD had durable and evolving responses following vaccination. Across all three tumor types, neoantigen-specific and polyfunctional CD4 and CD8 T cell responses of effector memory and central memory phenotypes were predominantly observed following vaccination as measured by ELISPOT, MHC tetramer staining, and FACS. Epitope spreading (T cell responses to neoantigens not included in the vaccine but identified in the patient's tumor), was observed in 2 of 4 patients analyzed. Assessment of pre- and post-treatment tumor biopsies with repeat exome sequencing, gene expression, as well as immunohistochemistry and pathologic analysis will be presented. Conclusions: Treatment with NEO-PV-01 with nivolumab has demonstrated minimal toxicity and ongoing clinical activity. Further, NEO-PV-01 is immunogenic inducing broad de novo neoantigen-specific immune responses in patients with metastatic cancers. Citation Format: Patrick A. Ott, Ramaswamy Govindan, Aung Naing, Terence W. Friedlander, Kim Margolin, Jessica J. Lin, Nina Bhardwaj, Matthew D. Hellmann, Lakshmi Srinivasan, Joel Greshock, Melissa Moles, Richard B. Gaynor, Matthew J. Goldstein, Siwen Hu-Lieskovan. A personal neoantigen vaccine, NEO-PV-01, with anti-PD1 induces broad de novo anti-tumor immunity in patients with metastatic melanoma, NSCLC, and bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT125.

Abstract 5546: eFT508, a potent and highly selective inhibitor of MNK1 and MNK2, regulates T-cell differentiation promoting an antitumor immune response

Abstract An effective and durable T-cell response is a cornerstone of current immunotherapies. We show that eFT508, a potent, selective inhibitor of MNK1 and MNK2, establishes a regulatory program that promotes multiple steps in the cancer immunity cycle including expansion of memory T cells and prevention of T-cell exhaustion. Using OT-I and OT-II transgenic systems, we show that eFT508 shifts the distribution of T cells towards a CD62LhighCD44high central memory (CM) phenotype in both CD4 and CD8 T cells upon activation with SIINFEKL peptide in vitro without adverse effects on T-cell proliferation, interferon-γ production or cytotoxic function. Similar effects are seen in vivo, where eFT508 treatment also enriches the CM T-cell pool in a SIINFEKL vaccine-induced OT-I adoptive T-cell transfer model, which results in increased persistence as demonstrated by a higher memory-recall T-cell response upon rechallenge. In addition, the CM bias elicited by eFT508 remains dominant when combined with agonists of co-stimulatory molecules, such as 4-1BB, OX-40 and GITR, or checkpoint inhibitors, such as PD-1, PD-L1 and CTLA-4, suggesting that eFT508 can affect the rate of T-cell differentiation in these combinations. eFT508 treatment also reduces the expression of exhaustion markers such as PD-1, LAG3 and TIM3, leading to increased cytotoxic T-cell function. eFT508 is currently under evaluation as a single agent in two phase 1/2 clinical trials for patients with advanced solid tumors and patients with advanced lymphoma. In addition, a phase 2 study evaluating eFT508, alone or in combination with avelumab, a PD-L1 immune checkpoint inhibitor, in microsatellite stable relapsed or refractory CRC patients is ongoing. The preclinical studies presented here provide further evidence that eFT508 may combine well with additional immunotherapies beyond checkpoint blockade. Citation Format: Rajesh K. Sharma, Vikas K. Goel, Jocelyn Staunton, Maria Barrera, Ana Parra, Eric Sung, Gary G. Chiang, Kevin R. Webster. eFT508, a potent and highly selective inhibitor of MNK1 and MNK2, regulates T-cell differentiation promoting an antitumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5546.

Near complete response to Pembrolizumab in microsatellite-stable metastatic sebaceous carcinoma

BackgroundSebaceous carcinoma is an aggressive adnexal skin tumor with a predilection for the eyelids and sebaceous glands of the head and neck.Case presentationA 73 year-old man presented with confusion and was found to have widely disseminated sebaceous carcinoma with metastases to brain, lungs, liver, bowel, lymph nodes, and bone. Following initial treatment of the brain metastases with surgery he received post-operative radiosurgery. He then began systemic immunotherapy with pembrolizumab. After 6 months, he developed a near complete response to therapy by irRECIST and RECIST v.1.1. The response was associated with circulating CD8+ T cells with central memory (CM) and effector memory (EM) phenotype and mature CD16 + CD57+ NK cells. During treatment the patient developed adrenal insufficiency requiring high-dose systemic corticosteroids and later adrenal replacement therapy. After 12-months of follow-up he showed imaging evidence of progression in liver, mediastinum, and abdominal lymph nodes. Given persistent, strong PD-L1 expression he resumed pembrolizumab therapy and showed radiographic evidence of an ongoing response to therapy.ConclusionsThis is the first report describing objective clinical and radiographic responses following immunotherapy for widely metastatic sebaceous carcinoma. The dramatic therapeutic response to pembrolizumab was associated with peripheral blood circulating memory T cells and mature Natural Killer cells after 6 months (24 weeks) of therapy. This report supports prospective clinical trials of anti-PD1 checkpoint blockade for metastatic sebaceous carcinoma.

Establishing the HIV reservoir: HIV-susceptible cells and the signals that recruit them

Abstract HIV infection rapidly causes localized inflammation, which contributes to viral pathogenesis by recruiting cells to become infected. At the same time, the stable HIV reservoir forms swiftly and persists over the life of the individual, likely due to infection of long-lived central memory CD4 T cells. The elimination/control of the reservoir is critical to an HIV cure. We sought to understand how host inflammatory responses to HIV drive the formation of the viral reservoir. Our early studies found that CD4 T cells rapidly produce the chemoattractant CCL2 following HIV infection, and this host response is dependent on HIV reverse transcription but not integration of viral DNA. We proposed that CCL2 produced early in response to HIV lures cells bearing the receptor—CCR2—to the zone of infection. Using mass cytometry, we identified a novel population of human lymphoid CCR2+ CD4 T cells that co-express CCR5 and have a central memory phenotype, along with markers associated with the HIV reservoir. We found that CCR2/5+ cells migrate in response to CCL2, fuse to R5 and X4-tropic HIV and support productive and latent infection. To measure the HIV reservoir in vivo, we sorted CCR2/5+ cells from PBMCs of HIV-infected ART-suppressed individuals and measured integrated HIV proviral DNA. Our preliminary findings show that CCR2/5+ cells have significantly higher levels of integrated HIV than CCR-negative memory (12-fold, p = 0.046) or naïve cells (28-fold, p = 0.012). We propose that HIV hijacks the host response to the virus to recruit CCR2+ cells, which may become latently infected to form the long-lived reservoir, providing a link between inflammation and reservoir seeding. We are now testing if blockade of CCL2 disrupts formation of the HIV reservoir.

Mitochondrial Ca2+ transport-dependent intercellular crosstalk between CD8 T and natural killer cells prevents tumor development and escape

Abstract Unraveling the complexity of lymphocyte interactions is key to understanding immune network and improving cancer immunotherapy. By designing various adoptive cell transfer protocols in solid tumors established in RAG−/−, RAG−/−gc−/− and RAG−/−GzmB-Tom mice and tracing lymphocytes specific to a self-tumor antigen P1A encoded by an X-linked cancer-germline gene Trap1a, we explored the functional dynamics between T and NK cells. We found that activated CD8+T cells augmented NK cell effector function in a pre-tumor T cell protocol that prevented the development of antigen-escape tumor variants. Using a silica nanofiber 3D matrix to control cellular mobility, confocal imaging showed intercellular contacts of activated CD8+T cells (CD69highCD25high) with multiple naïve NK cells, while naïve CD8+T cells formed single or no contact with NK cells. This interaction led to crossregulation of mitochondrial Ca2+ (mitoCa2+) oscillations in both cells whereby activated CD8+T cells induced differentiation of NK cell effector phenotype and NK cells polarized CD8+T cells towards a central memory phenotype. Intracellularly, CD8+T cells increased JAK1, JAK3, TYK2, STAT2 and STAT6 phosphorylation and oxidative signaling in NK cells. In turn, NK cells restrained IL-2 signaling in CD8+T cells by dampening activation-induced STAT5-dependent expression of IL-2-receptor-a chain. These effects were abrogated following the blockade of mitoCa2+ uptake. Mice deficient in mitoCa2+ handling-regulatory gene Fus1 showed increased incidence of a range of spontaneous sarcomas, lymphomas and leukemia. Data suggest that mitoCa2+ transport-guided intercellular crosstalk between CD8+T and NK cells is critical to prevent tumor development and escape.

Eomesodermin promotes a central memory phenotype in CD8+ T cells through the induction of IL-10.

Abstract Following an immune response, a fraction of antigen-specific T cells persist as memory cells. Of these, central memory CD8+ T cells (TCM), which express secondary lymphoid organ-homing molecules and self-renew efficiently, are critical for maintaining long term memory. High levels of the T-box transcription factor Eomesodermin (Eomes) are associated with the TCM phenotype, but the mechanisms by which Eomes controls different aspects of TCM function are poorly understood. Using Eomes-knockout (EKO) CD8+ T cells, we demonstrate that Eomes regulates the expression of a number of TCM-associated markers including Bcl6, Ly6C and CD62L. In addition, we found that Eomes promotes the expression of IL-10 in activated CD8+ T cells. Interestingly, the addition of exogenous IL-10 was sufficient to upregulate CD62L expression in WT cells. Taken together, our data suggest that Eomes uses IL-10 to promote at least part of the central memory phenotype in CD8+ T cells. Consistent with this model, we find that TCM are secreting IL-10 in vivo, without additional re-stimulation. These findings warrant a modified model of TCM differentiation which includes distinct roles for Eomes and IL-10 in a T cell-intrinsic program driving TCM formation.

Induction of a central memory and stem cell memory phenotype in functionally active CD4+ and CD8+ CAR T cells produced in an automated good manufacturing practice system for the treatment of CD19+ acute lymphoblastic leukemia.

Relapsed/refractory B-precursor acute lymphoblastic leukemia (pre-B ALL) remains a major therapeutic challenge. Chimeric antigen receptor (CAR) T cells are promising treatment options. Central memory T cells (Tcm) and stem cell-like memory T cells (Tscm) are known to promote sustained proliferation and persistence after T-cell therapy, constituting essential preconditions for treatment efficacy. Therefore, we set up a protocol for anti-CD19 CAR T-cell generation aiming at high Tcm/Tscm numbers. 100ml peripheral blood from pediatric pre-B ALL patients was processed including CD4+/CD8+-separation, T-cell activation with modified anti-CD3/-CD28 reagents and transduction with a 4-1BB-based second generation CAR lentiviral vector. The process was performed on a closed, automated device requiring additional manual/open steps under clean room conditions. The clinical situation of these critically ill and refractory patients with leukemia leads to inconsistent cellular compositions at start of the procedure including high blast counts and low T-cell numbers with exhausted phenotype. Nevertheless, a robust T-cell product was achieved (mean CD4+ = 50%, CD8+ = 39%, transduction = 27%, Tcm = 50%, Tscm = 46%). Strong proliferative potential (up to > 100-fold), specific cytotoxicity and low expression of co-inhibitory molecules were documented. CAR T cells significantly released TH1 cytokines IFN-γ, TNF-α and IL-2 upon target-recognition. In conclusion, partly automated GMP-generation of CAR T cells from critically small blood samples was feasible with a new stimulation protocol that leads to high functionality and expansion potential, balanced CD4/CD8 ratios and a conversion to a Tcm/Tscm phenotype.

A new approach to CAR T-cell gene engineering and cultivation using piggyBac transposon in the presence of IL-4, IL-7 and IL-21

Background aimsClinical-grade chimeric antigenic receptor (CAR)19 T cells are routinely manufactured by lentiviral/retroviral (LV/RV) transduction of an anti-CD3/CD28 activated T cells, which are then propagated in a culture medium supplemented with interleukin (IL)-2. The use of LV/RVs for T-cell modification represents a manufacturing challenge due to the complexity of the transduction approach and the necessity of thorough quality control. MethodsWe present here a significantly improved protocol for CAR19 T-cell manufacture that is based on the electroporation of peripheral blood mononuclear cells with plasmid DNA encoding the piggyBac transposon/transposase vectors and their cultivation in the presence of cytokines IL-4, IL-7 and IL-21. ResultsWe found that activation of the CAR receptor by either its cognate ligand (i.e., CD19 expressed on the surface of B cells) or anti-CAR antibody, followed by cultivation in the presence of cytokines IL-4 and IL-7, enables strong and highly selective expansion of functional CAR19 T cells, resulting in >90% CAR+ T cells. Addition of cytokine IL-21 to the mixture of IL-4 and IL-7 supported development of immature CAR19 T cells with central memory and stem cell memory phenotypes and expressing very low amounts of inhibitory receptors PD-1, LAG-3 and TIM-3. ConclusionsOur protocol provides a simple and cost-effective method for engineering high-quality T cells for adoptive therapies.

Abstract IA13: Novel therapeutic targets in multiple myeloma

Abstract Multiple myeloma (MM) is a model for using in vitro and in vivo models of the tumor in its bone marrow (BM) microenvironment to target tumor cells, tumor-host interactions, and the BM milieu in novel therapeutics. To date proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), histone deacetylase (HDAC) inhibitors, and monoclonal antibodies (MoAbs) have translated from the bench to the bedside and FDA approval, extending the survival of patients 3- to 4-fold. Further progress is advancing in novel strategies to target protein homeostasis, novel immune therapies, and treatments targeting genomic anomalies. PIs trigger apoptosis in MM cells, modulate adhesion molecule expression on tumor and BM cells, and regulate cytokine secretion in the BM milieu. They also promote osteoclast apoptosis and promote new bone formation. Ubiquitin proteasome receptor inhibitor and ubiquitylating agent inhibitors target the ubiquitin proteasome pathway upstream of the proteasome and can overcome PI resistance. HDAC6 selective inhibitors block aggresomal protein degradation and synergize with PIs. IMiDs bind and activate cereblon ubiquitin 3 ligase activity, with degradation of IZF1/3 in MM cells. Excitingly, prototype degronimids bind to ubiquitin ligase complexes including cereblon, VHL, or MDM2, as well as bind and selectively target substrates for degradation. Multiple immune therapies are promising in MM. IMiDs activate T, NK, and NK-T cells and target regulatory T cells, at least in part related to degradation of alios/ikaros and thereby stimulating IL-2 transcription and secretion. IMiDS also bind a novel substrate p-53 related protein kinase (PRPK) and mediate cereblon-independent MM cytotoxicity. MoAbs targeting SLAMF7 and CD38 have moved rapidly from the bench to the bedside, with enhanced activity in combination with IMiDs or PIs. B-cell maturation antigen (BCMA) is a selective plasma cell antigen currently targeted with immunotoxin and bispecific antibodies, as well as by targeting its ligands BAFF and APRIL to downregulate growth and survival signaling. Checkpoint inhibitors are expressed on MM cells, as well as immune effector cells and accessory cells (myeloid-derived suppressor cells and plasmacytoid dendritic cells), which promote MM cell growth and drug resistance. Importantly, checkpoint inhibitors can overcome the effect of these accessory cells and trigger autologous cytotoxicity of patient MM cells. Moreover, IMiDs can enhance checkpoint inhibitor-induced cytotoxicity. Vaccination of patients with smoldering MM with peptide-based vaccines can trigger autologous anti-MM immunity, which is enhanced by IMiDs and shifted to a central memory phenotype by HDAC 6 inhibitor. Finally, BCMA CART cell therapies are achieving complete responses even in MM refractory to all other therapies. Combinations of these immune approaches offer the most promise. Finally, MM is very genetically heterogeneous, with ongoing DNA damage. RAS mutations are most common, with transient responses to MEK/ERK inhibitor therapy. Patients with t(11:14) MM and overexpression of BCL-2 can respond to venetoclax. Ongoing efforts are presently delineating mechanisms underlying the extensive constitutive and ongoing DNA damage, including homologous recombination, APEX nuclease activity, pan-nuclease activity, and APOBEC activity, in order to develop selective inhibitors. Other strategies target the consequences of the genetic heterogeneity, such as targeting STK4 activity to restore p73-mediated apoptotic signaling in MM with decreased YAP1 copy number. Additionally, MM with MYC amplification has high levels of replicative stress and reactive oxygen species (ROS); ATR inhibitors to block replicative stress response in combination with agents to increase ROS trigger synergistic MM cytotoxicity. Finally, targeting fast-forward loops or regulatory loops in MM, such as KDM3A-KLF2-IRF4 axis, offers great promise to target both the tumor and its microenvironment. Citation Format: Kenneth Anderson. Novel therapeutic targets in multiple myeloma [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr IA13.

Reversible Transgene Expression Reduces Fratricide and Permits 4-1BB Costimulation of CAR T Cells Directed to T-cell Malignancies.

T cells expressing second-generation chimeric antigen receptors (CARs) specific for CD5, a T-cell surface marker present on normal and malignant T cells, can selectively kill tumor cells. We aimed to improve this killing by substituting the CD28 costimulatory endodomain (28.z) with 4-1BB (BB.z), as 28.z CD5 CAR T cells rapidly differentiated into short-lived effector cells. In contrast, 4-1BB costimulation is known to promote development of the central memory subpopulation. Here, we found BB.z CD5 CAR T cells had impaired growth compared with 28.z CD5.CAR T cells, due to increased T-cell-T-cell fratricide. We demonstrate that TRAF signaling from the 4-1BB endodomain upregulated the intercellular adhesion molecule 1, which stabilized the fratricidal immunologic synapse between CD5 CAR T cells. As the surviving BB.z CD5 CAR T cells retained the desired central memory phenotype, we aimed to circumvent the 4-1BB-mediated toxicity using a regulated expression system that reversibly inhibits CAR expression. This system minimized CAR signaling and T-cell fratricide during in vitro expansion in the presence of a small-molecule inhibitor, and restored CAR expression and antitumor function of transduced T cells in vivo These studies reveal a mechanism by which 4-1BB costimulation impairs expansion of CD5 CAR T cells and offer a solution to mitigate this toxicity. Cancer Immunol Res; 6(1); 47-58. ©2017 AACR.

Closed-system manufacturing of CD19 and dual-targeted CD20/19 chimeric antigen receptor T cells using the CliniMACS Prodigy device at an academic medical center

Background aimsMultiple steps are required to produce chimeric antigen receptor (CAR)-T cells, involving subset enrichment or depletion, activation, gene transduction and expansion. Open processing steps that increase risk of contamination and production failure are required. This complex process requires skilled personnel and costly clean-room facilities and infrastructure. Simplified, reproducible CAR-T-cell manufacturing with reduced labor intensity within a closed-system is highly desirable for increased availability for patients. MethodsThe CliniMACS Prodigy with TCT process software and the TS520 tubing set that allows closed-system processing for cell enrichment, transduction, washing and expansion was used. We used MACS-CD4 and CD8-MicroBeads for enrichment, TransAct CD3/CD28 reagent for activation, lentiviral CD8 TM-41BB-CD3 ζ-cfrag vectors expressing scFv for CD19 or CD20/CD19 antigens for transduction, TexMACS medium-3%-HS-IL2 for culture and phosphate-buffered saline/ethylenediaminetetraacetic acid buffer for washing. Processing time was 13 days. ResultsEnrichment (N = 7) resulted in CD4/CD8 purity of 98 ± 4.0%, 55 ± 6% recovery and CD3+ T-cell purity of 89 ± 10%. Vectors at multiplicity of infection 5–10 resulted in transduction averaging 37%. An average 30-fold expansion of 108 CD4/CD8-enriched cells resulted in sufficient transduced T cells for clinical use. CAR-T cells were 82–100% CD3+ with a mix of CD4+ and CD8+ cells that primarily expressed an effector-memory or central-memory phenotype. Functional testing demonstrated recognition of B-cells and for the CAR-20/19 T cells, CD19 and CD20 single transfectants were recognized in cytotoxic T lymphocyte and interferon-γ production assays. DiscussionThe CliniMACS Prodigy device, tubing set TS520 and TCT software allow CAR-T cells to be manufactured in a closed system at the treatment site without need for clean-room facilities and related infrastructure.

C-MPL provides tumor-targeted T-cell receptor-transgenic T cells with costimulation and cytokine signals

AbstractAdoptively transferred T-cell receptor (TCR)-engineered T cells depend on host-derived costimulation and cytokine signals for their full and sustained activation. However, in patients with cancer, both signals are frequently impaired. Hence, we developed a novel strategy that combines both essential signals in 1 transgene by expressing the nonlymphoid hematopoietic growth factor receptor c-MPL (myeloproliferative leukemia), the receptor for thrombopoietin (TPO), in T cells. c-MPL signaling activates pathways shared with conventional costimulatory and cytokine receptor signaling. Thus, we hypothesized that host-derived TPO, present in the tumor microenvironment, or pharmacological c-MPL agonists approved by the US Food and Drug Administration could deliver both signals to c-MPL-engineered TCR-transgenic T cells. We found that c-MPL+ polyclonal T cells expand and proliferate in response to TPO, and persist longer after adoptive transfer in immunodeficient human TPO-transgenic mice. In TCR-transgenic T cells, c-MPL activation enhances antitumor function, T-cell expansion, and cytokine production and preserves a central memory phenotype. c-MPL signaling also enables sequential tumor cell killing, enhances the formation of effective immune synapses, and improves antileukemic activity in vivo in a leukemia xenograft model. We identify the type 1 interferon pathway as a molecular mechanism by which c-MPL mediates immune stimulation in T cells. In conclusion, we present a novel immunotherapeutic strategy using c-MPL-enhanced transgenic T cells responding to either endogenously produced TPO (a microenvironment factor in hematologic malignancies) or c-MPL-targeted pharmacological agents.

Inhibition of AKT signaling uncouples T cell differentiation from expansion for receptor-engineered adoptive immunotherapy.

Adoptive immunotherapies using T cells genetically redirected with a chimeric antigen receptor (CAR) or T cell receptor (TCR) are entering mainstream clinical practice. Despite encouraging results, some patients do not respond to current therapies. In part, this phenomenon has been associated with infusion of reduced numbers of early memory T cells. Herein, we report that AKT signaling inhibition is compatible with CAR and TCR retroviral transduction of human T cells while promoting a CD62L-expressing central memory phenotype. Critically, this intervention did not compromise cell yield. Mechanistically, disruption of AKT signaling preserved MAPK activation and promoted the intranuclear localization of FOXO1, a transcriptional regulator of T cell memory. Consequently, AKT signaling inhibition synchronized the transcriptional profile for FOXO1-dependent target genes across multiple donors. Expression of an AKT-resistant FOXO1 mutant phenocopied the influence of AKT signaling inhibition, while addition of AKT signaling inhibition to T cells expressing mutant FOXO1 failed to further augment the frequency of CD62L-expressing cells. Finally, treatment of established B cell acute lymphoblastic leukemia was superior using anti-CD19 CAR-modified T cells transduced and expanded in the presence of an AKT inhibitor compared with conventionally grown T cells. Thus, inhibition of signaling along the PI3K/AKT axis represents a generalizable strategy to generate large numbers of receptor-modified T cells with an early memory phenotype and superior antitumor efficacy.

Dendritic cell vaccine induces antigen-specific CD8+ T cells that are metabolically distinct from those of peptide vaccine and is well-combined with PD-1 checkpoint blockade

ABSTRACTThe success of immune checkpoint blockade has unequivocally demonstrated that anti-tumor immunity plays a pivotal role in cancer therapy. Because endogenous tumor-specific T-cell responsiveness is essential for the success of checkpoint blockade, combination therapy with cancer vaccination may facilitate tumor rejection. To select the best vaccine strategy to combine with checkpoint blockade, we compared dendritic cell-based vaccines (DC-V) with peptide vaccines for induction of anti-tumor immunity that could overcome tumor-induced immunosuppression. Using B16 melanoma and B16-specific TCR-transgenic T-cells (pmel-1), we found that DC-V efficiently primed and expanded pmel-1 cells with an active effector and central memory phenotype that were not exhausted. Vaccine-primed cells were metabolically distinct from naïve cells. DC-V-primed pmel-1 cells contained the population that shifted metabolic pathways away from glycolysis to mitochondrial oxidative phosphorylation. They displayed better effector function and proliferated more than those induced by peptide vaccination. DC-V inhibited tumor growth in prophylactic and therapeutic settings. Only DC-V but not peptide vaccine showed augmented anti-tumor activity when combined with anti-PD-1 therapy. Thus, DC-V combined with PD-1 checkpoint blockade mediates optimal anti-cancer activity in this model.

The cathelicidin protein CRAMP is a potential atherosclerosis self-antigen in ApoE(-/-) mice.

Auto-immunity is believed to contribute to inflammation in atherosclerosis. The antimicrobial peptide LL-37, a fragment of the cathelicidin protein precursor hCAP18, was previously identified as an autoantigen in psoriasis. Given the reported link between psoriasis and coronary artery disease, the biological relevance of the autoantigen to atherosclerosis was tested in vitro using a truncated (t) form of the mouse homolog of hCAP18, CRAMP, on splenocytes from athero-prone ApoE(-/-) mice. Stimulation with tCRAMP resulted in increased CD8+ T cells with Central Memory and Effector Memory phenotypes in ApoE(-/-) mice, differentially activated by feeding with normal chow or high fat diet. Immunization of ApoE(-/-) with different doses of the shortened peptide (Cramp) resulted in differential outcomes with a lower dose reducing atherosclerosis whereas a higher dose exacerbating the disease with increased neutrophil infiltration of the atherosclerotic plaques. Low dose Cramp immunization also resulted in increased splenic CD8+ T cell degranulation and reduced CD11b+CD11c+ conventional dendritic cells (cDCs), whereas high dose increased CD11b+CD11c+ cDCs. Our results identified CRAMP, the mouse homolog of hCAP-18, as a potential self-antigen involved in the immune response to atherosclerosis in the ApoE(-/-) mouse model.