Allogeneic Dendritic Cells Research Articles

Generation of molecular-targeting helix-loop-helix peptides for inhibition of the interaction between cytotoxic T-lymphocyte-associated protein 4 and B7 in the dog

Blocking the interaction between CD28 and B7 by cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a potent immune checkpoint that prevents damage to host tissues from excessive immune responses. However, it also significantly diminishes immune responses against cancers and allows cancer cell growth. This study found that recombinant (r) human (h) CTLA-4 specifically binds to canine dendritic cells (DCs) and suppresses the responses of canine T cells to allogeneic DCs. ERY2-4, a peptide targeting rhCTLA-4 selected from a yeast-displayed library of helix-loop-helix (HLH) peptides and improved to have a binding affinity to rhCTLA-4 as strong as that of rhB7, inhibited the binding of rhCTLA-4 to canine DCs. Furthermore, the targeting peptide significantly enhanced the response of canine T cells to allogeneic DCs. These results suggest that the CTLA-4-targeting peptide enhances canine T cell activity by blocking the interaction between canine CTLA-4 on T cells and canine B7 on DCs. This study demonstrates the generation of a new type of immune checkpoint inhibitor, which may be applicable to cancer therapy in dogs.

Treatment with an Allogeneic Leukemia-Derived Dendritic Cell Vaccine in AML Patients Shows MRD Conversion and Improved Survival

Treatment with an Allogeneic Leukemia-Derived Dendritic Cell Vaccine in AML Patients Shows MRD Conversion and Improved Survival

Dual Inhibition of the MEK/ERK and PI3K/AKT Pathways Prevents Pulmonary Graft-Versus-Host Disease through Suppression of Arteriovenous Inflammation and Bronchiolitis

IntroductionDespite clinical development of novel immunosuppressants, the prognosis of pulmonary graft-versus-host disease (pGVHD) following allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still poor. It is clinically classified into obstructive and constrictive subtypes, but its pathophysiology is not fully elucidated due to difficulty in collecting human lung specimens. We have shown that the MEK inhibitors that target the MAP kinase pathway suppress gastrointestinal and cutaneous GVHD in mice. Therefore, other kinase inhibitors may have potentials to suppress pGVHD. We here explored key molecules in pGVHD patients who underwent lung transplantation and verified the potency of dual inhibition of the MEK/ERK and PI3K/AKT pathways.MethodsFor human histopathological analysis, we reviewed 45 lung specimens from the allo-HSCT recipients who underwent lung transplantation at our institute for pGVHD from 2008 through 2018. Single-cell level imaging mass cytometry of human pGVHD specimens was performed to visualize interactions of lymphocytes and monocytes around bronchioles. As for a murine pGVHD model, eight-week-old B10.BR (H2K k) mice were pretreated with 240 mg/kg cyclophosphamide and 7 Gy total body irradiation, followed by infusion of C57BL/6J (H2K b) bone marrow cells and splenocytes. Vehicles, the MEK inhibitors trametinib (tra) 0.1 mg/kg/day, cobimetinib (cobi) 0.1 mg/kg/day, the PI3K inhibitor taselisib (tase) 5 mg/kg/day and tacrolimus (tac) 1 mg/kg were given from day 0 through 28. Evaluation was made at day 42 following transplant: primary endpoint was survival rate and secondary endpoints included development of bronchiolitis and values of airway resistance and lung compliance. Immunosuppressive effects of those reagents for human cells were evaluated in vitro: CSFE dilution of T cells stimulated with allogeneic dendritic cells; expression of CD23/CD69 on B cells activated with IL-4/CD40L; TNF-α production of monocytes stimulated with lipopolysaccharide.ResultsThree pathological findings characterize human pGVHD: bronchiolitis obliterans, lymphocyte infiltration and wall thickening of bronchioles; pleuroparenchymal fibroelastosis (PPFE), proliferation of subpleural elastic fibers; peribronchitis, macrophage aggregation around the bronchi. Most cases with PPFE had subpleural arteriovenous inflammation, and immunostaining revealed that B cells and macrophages were abundantly infiltrated around the bronchioles. These results indicated that suppressing B cells and macrophages may be crucial to prevent pGVHD through avoiding vascular inflammation and bronchiolitis.In the murine pGVHD, cobi but not tra/tac improved survival rate (vehicle 31%; cobi 62%, tra 7%, tac 10% at day 42; p < 0.01). Cobi attenuated development of bronchiolitis, venulitis and peribronchial macrophage aggregation. Cobi improved respiratory functions, such as airway resistance (vehicle 1.65 ± 0.19, cobi 2.86 ± 0.75 cmH 2O.s/mL, p < 0.001) and lung compliance (vehicle 13.14 ± 0.71 cobi 8.99 ± 1.08 μL/cmH 2O, p < 0.05). Notably, T cell infiltration around bronchioles was inhibited by cobi and tra, but B cell infiltration was inhibited only by cobi.Whereas cobi inhibited phosphorylation of AKT, tra compensatory upregulated it. Cobi inhibited B cell activation more than tra (%CD23 +CD69 + B cells: 32.8% with cobi 1μM, 53.0% with tra 1μM, p < 0.05). In addition, cobi suppressed TNF-α production by monocytes more strongly than tra (%TNF-α + monocytes: 53.1% with cobi 1μM vs 71.6% with tra 1μM, p < 0.05). Dual inhibition of the MEK/ERK and PI3K/AKT pathways with combination of tra and tase strongly suppressed B cells in vitro, and improved survival rate compared with vehicle and monotherapy of tra or tase in mice (Figure). Finally, imaging mass cytometry of human pGVHD specimens revealed that lymphocytes around bronchioles and venules were positive for phospho-ERK1/2 and phospho-AKT.ConclusionspGVHD is associated with vascular inflammation and bronchiolitis in humans and mice. Furthermore, phosphorylation of ERK1/2 and AKT is upregulated in human pGVHD. Given that dual inhibition of MEK/ERK and PI3K/AKT signaling suppresses vascular inflammation and bronchiolitis thorough inhibition of B cells and monocytes in mice, this strategy may provide a novel treatment option against human pGVHD. [Display omitted] DisclosuresTakaori-Kondo: ONO PHARMACEUTICAL CO., LTD.: Research Funding; Bristol-Myers K.K.: Honoraria; Celgene: Research Funding.

Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines.

The adoptive transfer of alloantigen-specific regulatory T cells (alloTregs) has been proposed as a therapeutic alternative in kidney transplant recipients to the use of lifelong immunosuppressive drugs that cause serious side effects. However, the clinical application of alloTregs has been limited due to their low frequency in peripheral blood and the scarce development of efficient protocols to ensure their purity, expansion, and stability. Here, we describe a new experimental protocol that allows the long-term expansion of highly purified allospecific natural Tregs (nTregs) from both healthy controls and chronic kidney disease (CKD) patients, which maintain their phenotype and suppressive function under inflammatory conditions. Firstly, we co-cultured CellTrace Violet (CTV)-labeled Tregs from CKD patients or healthy individuals with allogeneic monocyte-derived dendritic cells in the presence of interleukin 2 (IL-2) and retinoic acid. Then, proliferating CD4+CD25hiCTV− Tregs (allospecific) were sorted by fluorescence-activated cell sorting (FACS) and polyclonally expanded with anti-CD3/CD28-coated beads in the presence of transforming growth factor beta (TGF-β), IL-2, and rapamycin. After 4 weeks, alloTregs were expanded up to 2,300 times the initial numbers with a purity of >95% (CD4+CD25hiFOXP3+). The resulting allospecific Tregs showed high expressions of CTLA-4, LAG-3, and CD39, indicative of a highly suppressive phenotype. Accordingly, expanded alloTregs efficiently suppressed T-cell proliferation in an antigen-specific manner, even in the presence of inflammatory cytokines (IFN-γ, IL-4, IL-6, or TNF-α). Unexpectedly, the long-term expansion resulted in an increased methylation of the specific demethylated region of Foxp3. Interestingly, alloTregs from both normal individuals and CKD patients maintained their immunosuppressive phenotype and function after being expanded for two additional weeks under an inflammatory microenvironment. Finally, phenotypic and functional evaluation of cryopreserved alloTregs demonstrated the feasibility of long-term storage and supports the potential use of this cellular product for personalized Treg therapy in transplanted patients.

Abstract LB163: Blocking adenosine production with ORIC-533, a CD73 inhibitor with best-in-class properties, reverses immunosuppression in high-AMP environments

Abstract Adenosine is recognized for its anti-inflammatory and immunosuppressive properties. Signaling through adenosine receptors expressed on immune cells initiates a cascade that diminishes activation and proliferation of cytotoxic T-cells, impairs activity of natural killer cells and CD4+ effector T-cells, and promotes the expansion of immunosuppressive cell types. CD73, a cell surface ecto-5'-nucleotidase, converts AMP to adenosine and is a major catalyst of adenosine generation in the tumor microenvironment. Overexpression of CD73 is observed in many solid tumors and may correlate with unfavorable clinical outcomes; therefore, reducing the level of adenosine via inhibition of CD73 is a strategy for enhancing antitumor immune responses and overcoming therapeutic resistance. We have developed ORIC-533, a potent, orally bioavailable, AMP-competitive, small molecule inhibitor of CD73. ORIC-533 is highly selective for CD73 and exhibits picomolar potency in biochemical and cellular assays, blocking adenosine production from AMP. Expression of CD73 on immune cells is sufficient to drive immunosuppression in vitro when AMP is present. We have previously shown that ORIC-533 can rescue activation of CD8+ T- cells exposed to AMP and restore proliferation, cytokine secretion and CD25 expression at low nanomolar concentrations. To understand the role of CD73 in dendritic cell (DC) priming of helper CD4+ T-cells, we utilized co-culture of allogeneic immature DC and CD4+ T-cells, where AMP to adenosine conversion severely suppresses activation of helper CD4+ T-cells. Treatment with ORIC-533 completely restored activation of CD4+ T-cells in this setting. To better define the biologically relevant AMP concentrations likely to be encountered in tumors, we determined the concentration of AMP in a panel of primary patient tumors. We discovered that intratumoral AMP levels can reach up to 500 micromoles, emphasizing the requirement for CD73 inhibitors to be efficacious in a high AMP environment. We previously demonstrated that nanomolar concentrations of ORIC-533, unlike other CD73 inhibitors tested, can efficiently rescue cytotoxic T-cell function in the presence of millimolar AMP concentrations. Surprisingly, in these studies we found that inhibitors of adenosine receptors, A2A or A2A/B, can only rescue CD8+ T-cell function in the context of low micromolar AMP or adenosine and thus are likely to be ineffective in patient tumors with high AMP. Taken together, these preclinical results indicate the CD73 inhibitor ORIC-533 has best-in-class properties in reversing immunosuppression in tumors. ORIC-533 is currently undergoing GLP studies to support an IND filing in the first half of 2021. Citation Format: Dena Sutimantanapi, Chelsea Chen, Bob Warne, Alex Pankov, Jae Chang, Lori Friedman, Melissa Junttila, Tatiana Zavorotinskaya. Blocking adenosine production with ORIC-533, a CD73 inhibitor with best-in-class properties, reverses immunosuppression in high-AMP environments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB163.

Novel Variant Ig Domain (vIgD) Proteins Generated Via Directed Evolution of Igsf Domains Have Therapeutic Efficacy in Animal Models of Graft Versus Host Disease

The immunoglobulin superfamily (IgSF) is a large, diverse family of proteins extensively targeted for treatment of cancers and autoimmune diseases. Most of the therapeutic strategies targeting this family have focused on high affinity antibodies binding a single receptor. Wild-type IgSF receptors typically exhibit low affinities for their counter-structures, limiting their utility in therapeutic modulation of immune responses. We have developed a novel variant Ig domain™ (vIgD™) directed evolution platform to affinity mature human IgSF extracellular domains. In this platform, libraries of mutagenized IgSF domains are selected for altered affinity to specific recombinant protein counterstructures. Fc fusion proteins incorporating the resulting engineered IgSF domains are then tested in vitro for their ability to either agonize or antagonize T cell responses.We successfully engineered the IgSF protein ICOSL, which binds to the costimulatory molecule ICOS on T cells and triggers T cell activation. We used our directed evolution platform to engineer a first-in-class single vIgD domain with higher affinity for ICOS and high affinity binding to the costimulatory molecule CD28, which ICOSL does not normally bind. Following the creation of dual ICOS/CD28 antagonism, the resulting domains were used to generate individual constructs encoding Fc-fusion proteins. These enhanced ICOS/CD28 dual antagonist Fc-fusion proteins were transfected into HEK-293 cells and protein produced by these cells was purified by protein A affinity chromatography. The resultant purified proteins were then tested in a FACS based binding assay on cells transfected with each costimulatory receptor showing up to a 2-3-fold increase in ICOS binding and high affinity binding to CD28. Various ICOS/CD28 dual antagonist Fc-fusion proteins were then tested in soluble format over a range of concentrations for functional activity in mixed lymphocyte reaction cultures (MLR) where allogeneic dendritic cells (DC) were used to stimulate purified human T cells. Most Variants showed significant ability to inhibit the MLR response as read out measuring proliferation and cytokine production. The potency of the Fc-fusion proteins varied somewhat depending on their relative affinities for CD28 and ICOS, with all being superior to wild type ICOSL protein and many showing superiority to the CD28 blocking reagent belatacept (CTLA4-Fc). The level of inhibition correlated well with binding of the Fc-fusion proteins to the T cells in culture.To assess the function of the ICOS/CD28 dual antagonist Fc-fusion proteins in vivo, a subset were tested in the human PBMC-NSG™ GvHD mouse model. This model monitors graft versus host disease generated by xenogeneic responses of T cells in human PBMCs transferred into NSG™. Administration of the dual ICOS/CD28 antagonist Fc-fusion proteins significantly protected mice from the effects of xenogeneic T cell activation in vivo with treated animals showing dramatically enhanced survival and greatly reduced disease scores. Interestingly, the level of protection correlated with the potency of the molecules in the in vitro MLR assay with less potent molecules in MLR assays failing to protect while more potent molecules protected well. Belatacept was also effective in protecting animals from GvHD, but not as well as the ICOS/CD28 dual antagonists produced by our vIgD platform. Collectively, these data demonstrate the vIgD directed evolution platform can generate IgSF proteins with altered affinities and improved binding properties. Variants engineered for desired immunological properties can effectively perturb T cell costimulation and affect T cell responses both in vitro and in vivo with the in vitro potency of the proteins correlating to in vivo potency. The dual ICOS/CD28 antagonists produced with the vIgD platform can be used therapeutically in disease settings where attenuation of T cell responses is beneficial, including GvHD and potentially other autoimmune diseases. Phase I enabling studies are in progress. [Display omitted] DisclosuresLevin:Alpine Immune Sciences: Employment, Equity Ownership. Evans:Alpine Immune Sciences: Employment, Equity Ownership. Rickel:Alpine Immune Sciences: Employment, Equity Ownership. Wolfson:Alpine Immune Sciences: Employment, Equity Ownership. Dillon:Alpine Immune Sciences: Employment, Equity Ownership. Kornacker:Alpine Immune Sciences: Employment, Equity Ownership, Patents & Royalties. Swanson:Alpine Immune Sciences: Employment, Equity Ownership, Patents & Royalties. Peng:Alpine Immune Sciences: Employment, Equity Ownership.

Reduced CXCL1 production by endogenous IL-37 expressing dendritic cells does not affect T cell activation.

The dendritic cell (DC)-derived cytokine profile contributes to naive T cell differentiation, thereby directing the immune response. IL-37 is a cytokine with anti-inflammatory characteristics that has been demonstrated to induce tolerogenic properties in DC. In this study we aimed to evaluate the influence of IL-37 on DC-T cell interaction, with a special focus on the role of the chemokine CXCL1. DC were cultured from bone marrow of human IL-37 transgenic (hIL-37Tg) or WT mice. The phenotype of unstimulated and LPS-stimulated DC was analyzed (co-stimulatory molecules and MHCII by flow cytometry, cytokine profile by RT-PCR and ELISA), and T cell stimulatory capacity was assessed in mixed lymphocyte reaction. The role of CXCL1 in T cell activation was analyzed in T cell stimulation assays with anti-CD3 or allogeneic DC. The expression of the co-stimulatory molecules CD40, CD80 and CD86, and of MHCII in LPS-stimulated DC was not affected by endogenous expression of IL-37, whereas LPS-stimulated hIL-37Tg DC produced less CXCL1 compared to LPS-stimulated WT DC. T cell stimulatory capacity of LPS-matured hIL-37Tg DC was comparable to that of WT DC. Recombinant mouse CXCL1 did not increase T cell proliferation either alone or in combination with anti-CD3 or allogeneic DC, nor did CXCL1 affect the T cell production of interferon-γ and IL-17. Endogenous IL-37 expression does not affect mouse DC phenotype or subsequent T cell stimulatory capacity, despite a reduced CXCL1 production. In addition, we did not observe an effect of CXCL1 in T cell proliferation or differentiation.

Preclinical Characterization of Linrodostat Mesylate, a Novel, Potent, and Selective Oral Indoleamine 2,3-Dioxygenase 1 Inhibitor.

Tumors can exploit the indoleamine 2,3-dioxygenase 1 (IDO1) pathway to create an immunosuppressive microenvironment. Activated IDO1 metabolizes tryptophan into immunosuppressive kynurenine, leading to suppressed effector T-cell (Teff) proliferation, allowing for tumor escape from host immune surveillance. IDO1 inhibition counteracts this immunosuppressive tumor microenvironment and may improve cancer outcomes, particularly when combined with other immunotherapies. Linrodostat mesylate (linrodostat) is a potent, selective oral IDO1 inhibitor that occupies the heme cofactor-binding site to prevent further IDO1 activation and is currently in multiple clinical trials for treatment of patients with advanced cancers. Here, we assess the in vitro potency, in vivo pharmacodynamic (PD) activity, and preclinical pharmacokinetics (PKs) of linrodostat. Linrodostat exhibited potent cellular activity, suppressing kynurenine production in HEK293 cells overexpressing human IDO1 and HeLa cells stimulated with IFNγ, with no activity against tryptophan 2,3-dioxygenase or murine indoleamine 2,3-dioxygenase 2 detected. Linrodostat restored T-cell proliferation in a mixed-lymphocyte reaction of T cells and allogeneic IDO1-expressing dendritic cells. In vivo, linrodostat reduced kynurenine levels in human tumor xenograft models, exhibiting significant PD activity. Linrodostat demonstrated a PK/PD relationship in the xenograft model, preclinical species, and samples from patients with advanced cancers, with high oral bioavailability in preclinical species and low to moderate systemic clearance. Our data demonstrate that linrodostat potently and specifically inhibits IDO1 to block an immunosuppressive mechanism that could be responsible for tumor escape from host immune surveillance with favorable PK/PD characteristics that support clinical development.

A New Pipeline to Predict and Confirm Tumor Neoantigens Predict Better Response to Immune Checkpoint Blockade.

Mutations that drive oncogenesis in cancer can generate neoantigens that may be recognized by the immune system. Identification of these neoantigens remains challenging due to the complexity of the MHC antigen and T-cell receptor interaction. Here, we describe the development of a systematic approach to efficiently identify and validate immunogenic neoantigens. Whole-exome sequencing of tissue from a patient with melanoma was used to identify nonsynonymous mutations, followed by MHC binding prediction and identification of tumor clonal architecture. The top 18 putative class I neoantigens were selected for immunogenicity testing via a novel in vitro pipeline in HLA-A201 healthy donor blood. Naïve CD8 T cells from donors were stimulated with allogeneic dendritic cells pulsed with peptide pools and then with individual peptides. The presence of antigen-specific T cells was determined via functional assays. We identified one putative neoantigen that expanded T cells specific to the mutant form of the peptide and validated this pipeline in a subset of patients with bladder tumors treated with durvalumab (n = 5). Within this cohort, the top predicted neoantigens from all patients were immunogenic in vitro. Finally, we looked at overall survival in the whole durvalumab-treated bladder cohort (N = 37) by stratifying patients by tertile measure of tumor mutation burden (TMB) or neoantigen load. Patients with higher neoantigen and TMB load tended to show better overall survival. IMPLICATIONS: This pipeline can enable accurate and rapid identification of personalized neoantigens that may help to identify patients who will survive longer on durvalumab.

Ex vivo modelling of PD-1/PD-L1 immune checkpoint blockade under acute, chronic, and exhaustion-like conditions of T-cell stimulation

Blockade of PD-1/PD-L1 interactions is proving an exciting, durable therapeutic modality in a range of cancers whereby T cells are released from checkpoint inhibition to revive their inherent anti-tumour activity. Here we have studied various ways to model ex vivo T cell function in order to compare the impact of the clinically utilised anti-PD-1 antibody, pembrolizumab (Keytruda) on the activation of human T cells: focussing on the release of pro-inflammatory IFNγ and anti-inflammatory IL-10 to assess functionality. Firstly, we investigated the actions of pembrolizumab in an acute model of T-cell activation with either immature or mature allogeneic dendritic cells (DCs); pembrolizumab enhanced IFNγ and IL-10 release from purified CD4+ T-cells in the majority of donors with a bias towards pro-inflammatory cytokine release. Next, we modelled the impact of pembrolizumab in settings of more chronic T-cell activation. In a 7-day antigen-specific response to EBV peptides, the presence of pembrolizumab resulted in a relatively modest increase in both IFNγ and IL-10 release. Where pembrolizumab was assessed against long-term stimulated CD4+ cells that had up-regulated the exhaustion markers TIM-3 and PD-1, there was a highly effective enhancement of the otherwise exhausted response to allogeneic DCs with respect to IFNγ production. By contrast, the restoration of IL-10 production was considerably more limited. Finally, to assess a direct clinical relevance we investigated the consequence of PD-1/PD-L1 blockade in the disease setting of dissociated cells from lung and colon carcinomas responding to allogeneic DCs: here, pembrolizumab once more enhanced IFNγ production from the majority of tumour preparations whereas, again, the increase in IL-10 release was modest at best. In conclusion, we have shown that the contribution of PD-1—revealed by using a canonical blocking antibody to interrupt its interaction with PD-L1—to the production of an exemplar pro- and anti-inflammatory cytokine, respectively, depends in magnitude and ratio on the particular stimulation setting and activation status of the target T cell. We have identified a number of in vitro assays with response profiles that mimic features of dissociated cell populations from primary tumours thereby indicating these represent disease-relevant functional assays for the screening of immune checkpoint inhibitors in current and future development. Such in vitro assays may also support patient stratification of those likely to respond to immuno-oncology therapies in the wider population.

Mixed cultures of allogeneic dendritic cells are phenotypically and functionally stable - a potential for primary cell-based "off the shelf" product generation.

Vaccination against tumors using antigen-pulsed dendritic cell (DC) vaccines has greatly evolved over the last decade, with hundreds of active human clinical trials well on the way. The use of an autologous source for DC-based vaccine therapeutics remains the obvious choice in the majority of clinical studies; however, novel evidence suggests that an allogeneic source of DCs can yield success if administered in the right context. One of the challenges facing successful DC vaccination protocols is the generation of large enough numbers of DCs intended for vaccination and standardization of these procedures. In addition, variations in the quality of DC vaccines due to donor-to-donor variation represent an important therapeutic factor. To this day it has not been shown whether DCs from different donors can readily co-exist within the same co-culture for the extended periods required for vaccine manufacture. We demonstrate that generation of allogeneic DC co-cultures, generated from multiple unrelated donors, allows the preservation of their phenotypical and functional properties in vitro for up to 72 hours. Therefore, in the case of an allogeneic vaccination approach, one could ensure large numbers of DCs generated from a primary cell source intended for multiple vaccinations. By generating large amounts of ex vivo manufactured DCs from multiple donors, this would represent the possibility to ensure sufficient amounts of equipotent “off the shelf” product that could e.g. be used for an entire cohort of patients within a study.

Promoting Antigen Escape from Dendritic Cell Endosomes Potentiates Anti-Tumoral Immunity

Background: Dendritic cells (DCs) are usually very efficient at activating CD8 T cells. However, their capacity to cross-present soluble antigens can be limited by non-specific degradation of captured antigens during endosome maturation, which would lead to the destruction of immunogenic epitopes. To bypass this limitation, we tested a new antigen formulation using AccumTM, a technology originally designed to enhance accumulation of antibody-drug conjugate in target cells by promoting endosome-to-cytosol escape. Methods: Following chemical linking and characterization of the Accum-antigen complex, a series of in vitro antigen presentation assays were conducted to assess the T-cell activation potency of the antigen formulation. The generated cellular product was then tested in vivo for its capacity to elicit anti-tumoral responses in both prophylactic and therapeutic vaccination settings. Finally, allogeneic DCs pulsed with tumor lysate linked to AccumTM were characterized for their therapeutic potency. Results: Linking the Accum moiety to the experimental xenoantigen ovalbumin caused endosomal damages resulting in enhanced intracellular protein processing by primary DCs. As a result, production of pro-inflammatory cytokines and chemokines was enhanced by responding T cells. When evaluated for their anti-tumoral properties, Accum-ovalbumin-pulsed DCs elicited potent effector and central memory CD8 T cells, which explains the complete and long-lasting protection observed in vaccinated mice despite multiple EG.7 T-cell lymphoma challenges. When combined with the anti-PD-1 immune checkpoint inhibitor, therapeutic vaccination using both syngeneic and allogeneic DCs pulsed with Accum-linked ovalbumin triggered potent anti-tumoral responses; an observation further enhanced using Accum-linked lymphoma tumor lysate proteins. Conclusion: These highly favorable therapeutic effects highlight the promising potential of AccumTM as a distinct and potent technology platform suitable for the design of next generation cell cancer vaccines.

IL-7 During Antigenic Stimulation Using Allogeneic Dendritic Cells Promotes Expansion of CD45RA-CD62L+CD4+ Invariant NKT Cells With Th-2 Biased Cytokine Production Profile.

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes cells that recognize glycolipid antigens associated with CD1d, non-classical antigen presenting proteins. They can drive either pro-inflammatory (Th-1) or anti-inflammatory (Th-2) immune microenvironment through the production of both Th-1 and Th-2 type cytokines upon activation, thus play a vital role in cancer, infection, and autoimmune diseases. Adoptive cell therapy using ex vivo expanded iNKT cells is a promising approach to enhance anti-tumor immunity or immunosuppression. However, overcoming phenotypic and functional heterogeneity and promoting in vivo persistency of iNKT cells remains to be a challenge. Here, we compared various methods for ex vivo expansion of human iNKT cells and assessed the quality of expansion, phenotype, and cytokine production profile of expanded iNKT cells. While a direct stimulation of iNKT cells in peripheral blood mononuclear cells with agonist glycolipid led to the expansion of iNKT cells in varying degrees, stimulation of enriched iNKT cells by irradiated autologous peripheral blood mononuclear cells or allogeneic dendritic cells resulted in consistent expansion of highly pure iNKT cells. Interestingly, the mode of antigenic stimulation influenced the dominant subtype of expanded iNKT cells. Further, we evaluated whether additional IL-7 or IL-15 during antigenic stimulation with allogeneic dendritic cells can improve the phenotypic heterogeneity and modify cytokine production profile of iNKT cells expanded from 18 consecutive donors. The presence of IL-7 or IL-15 during antigenic stimulation did not affect the fold of expansion or purity of expanded iNKT cells. However, IL-7, but not IL-15, led to a better expansion of CD4+ iNKT cells, enhanced Th-2 type cytokine production of CD4+ iNKT cells, and maintained the expansion of central memory (CD45RA-CD62L+) CD4+ iNKT cells. Our results suggest the addition of IL-7 during antigenic stimulation with allogeneic dendritic cells can promote the expansion of CD62L+Th-2+CD4+ human iNKT cells that can be used as novel immunotherapeutic to control excessive inflammation to treat various autoimmune diseases.

Conversion from MRD Positive to Negative Status in AML Patients in CR1 after Treatment with an Allogeneic Leukemia-Derived Dendritic Cell Vaccine

Background. Persistence of measurable residual disease (MRD) in patients with acute myeloid leukemia (AML) remains a poor prognostic factor and unmet medical need. The allogeneic leukemia-derived dendritic cell vaccine, DCP-001, has shown in a phase I study to generate both humoral and cellular immune responses and is safe for clinical practice (van de Loosdrecht et al., Cancer Immunol. Immunother. 2018). In the current phase II study (Clintrials.gov: NCT03697707) we show the capability of DCP-001 to convert MRD positive patients to negative, leading to deeper remissions. Additionally, it is shown that DCP-001 induces immune responses, also specifically against tumor associated antigens known to be present in DCP-001 and relevant for AML. Methods. The current trial aims to enroll up to 20 AML -patients, ineligible for HSCT, who are in in first complete remission (CR1) but who are still MRD positive. MRD is assessed in the bone marrow through flow cytometry and/or qPCR (eg NPM1). Patients receive a primary vaccination regimen of 4 times 25.106or 50.106cells per vaccination, biweekly, followed by two booster vaccinations (10.106cells/vaccination) at week 14 and 18 after start of treatment. Primary endpoints of this trial are the safety and tolerability of the two vaccination schedules and the effect of vaccination on the MRD status. Additionally, cellular and humoral immune response induced by DCP-001 are evaluated in peripheral blood using several assay methods, including flow cytometry and IFN-ϒ ELISpot against known TAA's such as WT-1, RHAMM and PRAME. Results. Up to 15 July 2020, ten patients (age 41-76; 5 male, 5 female) have been enrolled and dosed within the study, completing the first dose cohort of 25.106cells/vaccination. All vaccinations were well tolerated and adverse events to the vaccine were limited to local injection site reactions such as redness, swelling and warmth (maximum grade 2). Three of the ten patient relapsed before the vaccination schedule was completed. Four patients could be evaluated for MRD; Two patients became MRD negative at the first timepoint after the initial vaccinations (week 14), and remained negative until end of active FU (week 32), two other patients remained in CR, but with MRD positivity. For the remaining three patients, to date, no MRD outcome is available yet but patients remain in CR (see swimmers plot for overview, Figure 1). Evaluation of the immune response before, during and after DCP-001 vaccination has only be performed in a small subset of three patients thus far. Of these patients one showed a clinical response becoming MRD negative after vaccination. In this patient we observed an increase in tumor specific functional T-cells assessed by IFNγ ELISPOT, either as a recall or primary response, as shown by the response observed to WT-1 (see for example Figure 2). These tumor associated antigens are known to be expressed by DCP-001 and included WT1, PRAME and RHAMM. Induction of specific memory CD8 and CD4 cells could be observed upon vaccination which might be related to the clinical response. Conclusion/discussion.Preliminary data from this ongoing study confirms that vaccination with DCP-001 is able to generate a tumor-specific immune response and may lead to potential tumor control. Two patients were actually converted from being MRD positive at start of vaccination to MRD negative during the study. These patients continue to be in complete remission for at least a year after vaccination. This study continues to enroll patients at a higher vaccine dose of 50.106cells per vaccination and additional data on induced immune responses and MRD status will be shown. Disclosures van de Loosdrecht: novartis: Honoraria; celgene: Honoraria. Wagner:Novartis: Membership on an entity's Board of Directors or advisory committees; Shire: Other: Travel grand; MSD: Membership on an entity's Board of Directors or advisory committees; Medac: Other: Travel grand; Kite/Gilead: Membership on an entity's Board of Directors or advisory committees. Platzbecker:Amgen: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Geron: Consultancy, Honoraria. Rovers:DCprime: Current Employment.

569P Intratumoral injection of allogeneic pro-inflammatory dendritic cells (ilixadencel) in combination with anti-CTLA-4 treatment induce complete tumour responses and anti-tumour immune memory in a mouse tumour model

569P Intratumoral injection of allogeneic pro-inflammatory dendritic cells (ilixadencel) in combination with anti-CTLA-4 treatment induce complete tumour responses and anti-tumour immune memory in a mouse tumour model

Abstract 3274: A robust enhancement of cytokine production in a human chronic activation model of T cell exhaustion in vitro through blockade of PD-1/PDL-1 interactions using pembrolizumab or nivolumab; correlation with dissociated tumor immune cell responses

Abstract Prolonged exposure to antigens can lead to a state of reduced T cell responsiveness termed exhaustion. Restoring T cell functions from this state of reduced functionality by, for example, blockade of inhibitory immune checkpoints is now an established therapeutic modality with lasting impact on survival for some patients. Modelling human T cell exhaustion and its reversal in vitro can be challenging in medium or high-throughput assays. We have therefore sought to develop and characterize an in vitro model of T cell exhaustion using human T cells. Long-term (14-day) prolonged stimulation of human T cells in vitro led to both an up-regulation of established exhaustion markers PD-1, LAG-3 and TIM-3, and a lack of responsiveness to subsequent stimulation with human allogeneic monocyte-derived dendritic cells (DCs) as assessed by, for instance, IFNγ and TNFα production. Subsequent addition of blocking antibodies against PD-1 (pembrolizumab and nivolumab) led to a robust reversal of exhausted state by, for instance, restoration of cytokine secretion. The magnitude and nature of the response also correlated with the response of dissociated cells from lung and colon carcinomas. This response also showed greater sensitivity to PD-1 blockade compared to freshly isolated T cells or when using PBMCs stimulated with a pool of EBV peptides. By performing intracellular cytokine staining of human exhausted T cells stimulated with allogeneic dendritic cells, we further noted that PD-1 blockade using either pembrolizumab or nivolumab increased the percentage of cells producing IFNγ suggesting a greater responding pool of T cells. However, whilst the levels of the IFNγ in the supernatants of the exhausted T cell/allogeneic dendritic cell cultures was fully restored when compared to ‘fresh' T cell/allogeneic dendritic cell cultures, the percentage of IFNγ+ cells was not, nor was it fully restored by the inclusion of PMA/ionomycin. Taken together, this would suggest that PD-1 blockade induces responding cells to produce more IFNγ. We suggest that this chronic activation model of human T cell exhaustion with a robust assay window, and responsive to the clinically validated anti-PD-1 antibodies, nivolumab and pembrolizumab, may provide a platform for the discovery of new immuno-oncology therapeutics as well as the assessment of differences in mechanism of action between them. Citation Format: Omar S. Qureshi, Alexander Roberts, Lindsay Bentley, Tina Tang, Fay Stewart, Graham Wallace, Alison Cooper, Aaron Scott, David Thickett, Babu Naidu, Thomas Pinkney, Graham Taylor, Kristian Brock, Louise Healy, Zania Stamataki, Catherine Brady, S J. Curnow, John Gordon, Nicholas M. Barnes. A robust enhancement of cytokine production in a human chronic activation model of T cell exhaustion in vitro through blockade of PD-1/PDL-1 interactions using pembrolizumab or nivolumab; correlation with dissociated tumor immune cell responses [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 3274.

Phase I trial evaluating safety and efficacy of intratumorally administered inflammatory allogeneic dendritic cells (ilixadencel) in advanced gastrointestinal stromal tumors

BackgroundThe majority of patients with advanced gastrointestinal stromal tumor (GIST) develop resistance to imatinib, and subsequent treatments have limited efficacy. Ilixadencel (allogeneic inflammatory dendritic cells) is a cell-based immune primer injected intratumorally that previously has been clinically investigated in metastatic renal cell carcinoma and hepatocellular carcinoma.MethodsThe trial was a single arm phase I trial assessing safety and efficacy of ilixadencel in subjects with progressing advanced/metastatic GIST despite ongoing treatment with second or later lines of tyrosine kinase inhibitors (TKI). Three patients were progressing while on sunitinib (second line), one on regorafenib (third line), and two on pazopanib (fourth line). TKI treatment was maintained throughout, while two intratumoral injections of ilixadencel (10 × 106 viable and HLA-DR expressing cells per dose) were administered.ResultsNo severe adverse events were found to be related to ilixadencel administration. Four patients showed continued tumor progression at 3 months per RECIST 1.1 and Choi criteria. One patient (on third line regorafenib) had stable disease for 9 months and another patient (on second line sunitinib) had stable disease at end of study (12 months) as per RECIST 1.1. These two patients developed a partial response as per Choi criteria with a duration of 3 and 6 months, respectively. The median progression-free survival (PFS) was 4.0 months.ConclusionIlixadencel treatment presented an acceptable safety profile among advanced GIST patients who developed resistance to TKI. Encouraging radiological tumor responses were detected in 33% of treated patients, supporting further investigation.Clinical trial registrationwww.clinicaltrials.gov; NCT: 02432846; registration date: February 22, 2016.

Dendritic cell vaccine as an alternative to opioid analgesia in patients with advanced cervical cancer.

e18022 Background: Locally advanced and progressive cervical cancer is always accompanied by the pain syndrome. The common analgesics include non-steroidal anti-inflammatory drugs and opioids. The purpose of the study was to assess allogeneic dendritic cell vaccine (DCV) as an alternative analgesic in patients with advanced cervical cancer. Methods: The pain syndrome dynamics was assessed in 20 patients with advanced T3-4N1M0-1 cervical cancer receiving subcutaneous paravertebral injections of allogeneic DCV in a total alternating dose of 5-10 million cells every 2 weeks. Patients received DCV for one year, with a total of 24 vaccine therapy sessions and a total dosage of 180 million dendritic cells. The pain intensity was assessed on a verbal rating scale: 0 – no pain; 1 – mild pain; 2 – moderate pain; 3 – severe pain; 4 – extremely intense pain. Results: Prior to the therapy, 15 patients (75%) had severe pain; 2 (10%) - moderate pain; 3 (15%) - extremely intense pain. After 4-6 DCV injections, the pain intensity decreased, patients refused opioid analgesics. After 10-12 DCV sessions, 19 (95%) (p &lt; 0.05) women had no pain at all, patients denied additional pain relief with non-opioid analgesics. Unrelieved pain was registered only in one cervical cancer patient. Conclusions: DCV injections in patients with advanced cervical cancer provide pain relief thereby improving their quality of life.

Engineered Type-1 Regulatory T Cells as Cellular Therapy for Treatment of Immune Mediated Diseases

Abstract Type 1 regulatory cells (Tr1) are a promising cellular product for suppression of effector T cells in immune mediated diseases, including graft-versus-host-disease (GvHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) (Roncarolo et al. Immunity 2018). We have developed an in vitro protocol to produce Tr1 cells by lentiviral transduction of the human IL10 with a constitutive promoter into human CD4+ T cells (Locafaro et al. Molecular Therapy 2017). These engineered Tr1 cells, called LV-10, acquire a characteristic Tr1 cytokine profile (IL-10 and IFN-g high, IL-4 low and expression of intracellular perforin and granzyme B). In vitro, LV-10 cells suppress the proliferation of responder CD4+ T cells upon activation by allogeneic dendritic cells. LV-10 cells also degranulate in response to and kill myeloid cells, including myeloid blasts from patients with acute myeloid leukemia, through a granzyme B- and perforin-dependent mechanism. Interestingly, the ability to degranulate and kill myeloid cells is not present when LV-10 are activated and expanded with anti-CD3 and anti-CD28 coated beads, suggesting that signals beyond TCR, CD28, and IL-10 receptor pathway activation are necessary to reprogram LV-10 cells into cytotoxic cells. In vivo, LV-10 cells injected into NSG mice do not induce xeno-GvHD, in contrast to control CD4+ T cells. In addition, LV-10 cells suppress CD4-induced xeno-GvHD and prevent expansion of myeloid leukemic cells. Experiments are ongoing to compare the potency and in vivo survival of allogeneic vs autologous LV-10 cells. These findings demonstrate the promise of using LV-10 to treat immune mediated diseases, including GvHD in AML patients receiving allo-HSCT.

Immunological responses through miRNA signatures in GBM plasma extracellular vesicles from patients receiving experimental immunotherapy

Abstract Glioblastoma (GBM) patients have a median survival of 15 months despite aggressive treatment. Treatment-related pseudo-progression confounds outcome assessment by MRI, particularly in patients receiving immunotherapy. Extracellular vesicles (EVs) contain tumor-specific miRNA that could serve as a liquid biopsy to distinguish true progression from treatment-related pseudo-progression. Small plasma EVs were isolated by serial density gradient ultracentrifugation from 20 newly-diagnosed GBM patients enrolled in a clinical trial of allogeneic tumor lysate-pulsed autologous dendritic cell (DC) vaccination. Short non-coding RNA sequencing was performed for each patient at three time points (TP): pre-vaccine (TP1), post-initial vaccine (TP2), and at end of treatment (TP3). Ingenuity Pathway Analysis from 31 differentially expressed miRNAs across time points (p-value &amp;lt;0.05, |logFC|&amp;gt;1) revealed that glioma-related signaling pathways previously seen in small EVs from glioma patients compared to normal donors were replicated when comparing patients at the beginning of the treatment (TP1-vs-TP2) and between early to late time points (TP1-vs-TP3). In addition, pathways corresponding to immunological responses typically seen in GBM patients undergoing treatment, such as IL-10, IL-6, and Toll-like Receptor (TLR) signaling, were exclusively found after the initial vaccine towards the end of treatment (TP2-vs-TP3). In conclusion, signaling pathways derived from differentially expressed miRNAs across time points suggest that as patients progress through treatment, consistent differences in plasma small EVs miRNA expression profile and immunological responses could be utilized as predictors of treatment response.