Allogeneic Dendritic Cells Research Articles

Immunotherapeutic allogeneic dendritic cell and autologous tumor cell fusion vaccine alone or combined with radiotherapy in canine oral malignant melanoma is safe and potentially effective.

Immunotherapy represents a promising breakthrough in cancer management and is being explored in canine melanomas. Dendritic cells (DCs) play a crucial role in priming T-cell-mediated immune reactions through the antigen-presenting function. Combining immunotherapy and radiation therapy may generate more substantial anti-cancer efficacy through immunomodulation. Our research reported a preliminary result of the safety and outcome of a kind of immunotherapy, the allogeneic dendritic cell and autologous tumor cell fusion vaccine, alone or in combination with hypofractionated radiation therapy, in canine oral malignant melanoma. Two groups of dogs with histopathological diagnoses of oral malignant melanoma were recruited. In group 1 (DCRT), dogs received a combination of DC fusion vaccine and radiotherapy. In group 2 (DC), dogs received DC fusion vaccine alone. DC vaccination was given once every 2 weeks for four doses. Radiotherapy was performed weekly for five fractions. Dogs that received carboplatin were retrospectively collected as a control group (group 3). Five dogs were included in group 1 (two stage II, three stage III), 11 in group 2 (three stage I/II, eight stage III/IV), and eight (two stage I/II, six stage III/IV) in the control group. Both DC and DCRT were well-tolerated, with only mild adverse events reported, including mucositis, gastrointestinal discomfort, and injection site reactions. The median progression-free intervals in groups 1, 2, and 3 were 214 (95% CI, NA, due to insufficient data), 100 (95% CI, 27-237), and 42 days (95% CI, NA-170), respectively, which were not significantly different. The 1-year survival rates were 20, 54.5, and 12.5% in groups 1, 2, and 3. Dogs in the DCRT group exhibited significantly higher TGF-β signals than the DC group throughout the treatment course, indicating a possible higher degree of immunosuppression. The manuscript demonstrated the safety of dendritic cell/tumor cell fusion vaccine immunotherapy, alone or in combination with radiotherapy. The results support further expansion of this immunotherapy, modification of combination treatment and protocols, and investigation of combining DC vaccine with other treatment modalities. Preclinical Trials, PCTE0000475.

Immunological profiling of glioblastoma cellular composition to predict response to dendritic cell vaccination.

e14037 Background: Glioblastomas are the most common primary malignant brain tumors, comprising 2% of all adult cancers. Despite recent advancements in genetic classification and targeted therapies for specific mutations, there has been no significant improvement in overall life expectancy for the majority of patients. Last year, our research group reported promising results from a phase II prospective trial involving allogeneic dendritic cell vaccination. To date, six out of 37 reported cases remain alive without tumor recurrence, exhibiting a performance status of 90 to 100% according to the Karnofsky Performance Scale (KPS) three years after the initiation of vaccination. Methods: In this study, we focused on the immune cell infiltration observed at the time of tumor excision in enrolled patients. We utilized flow cytometry to quantify cells expressing membrane CD86, CD45, HLA-DR, PD-L1, CD11b, and CD14, in addition to assessing IDH-1 status, age, gender, KPS, gender, ECOG, symptomatology, and the number of vaccines administered during the trial. Our goal was to predict responsiveness in terms of overall survival using a neural network prediction algorithm with three hidden nodes and five seeds for improved reproducibility. We employed the random holdback method for algorithm validation. Results: From the 37 enrolled patients, we successfully obtained viable tumor cells from 19 frozen samples. The model achieved an RSquare value of 0.71 after training and 0.66 after validation (where 1 indicates a perfect model and 0 implies no improvement over a constant model). Similar RSquare values in both training and validation sets indicate strong predictive power. Positive predictors of overall survival included HLA-DR positivity, PD-L1 positivity, IDH-1 mutation status, the presence of focal deficits, and higher KPS scores, while negative predictors were CD86 expression, older age, symptoms of intracranial hypertension (ICH), and higher ECOG performance status. Conclusions: In conclusion, the neural network algorithm described herein enables the prediction of responsiveness to dendritic cell vaccination in terms of overall survival, based on clinical features and the profile of immune cell infiltration observed at the time of tumor excision.

Vaccination Using an Allogeneic Leukemia-Derived Dendritic Cell Vaccine, Maintains and Improves Frequencies of Circulating Antigen Presenting Dendritic Cells Correlating with Relapse Free and Overall Survival in AML Patients

Background. Active immunotherapy relies on the patient's immune system for induction of an effective immune response. Not only the frequency, number and exhaustion or activation status of CD8 T cells is important for an efficacious tumor cell lysis, but the quality, number and distribution of different antigen presenting cells is of equal importance. Vididencel is an allogeneic leukemia-derived dendritic cell vaccine used in AML patients in first complete remission (CR1/CRi), but with MRD positivity, to mount an immune response through intradermal vaccination (ADVANCE-II, Clintrials.gov: NCT03697707). Intradermal vaccination is known to induce a specific immune response, by recruitment of skin and blood derived dendritic cells, which process antigen and present them to the immune system (Zuo et al., 2021). In this study circulating dendritic cells and monocytes have been investigated before and during vaccination to investigate the effect of vididencel treatment on the composition of antigen presenting cells in relation to clinical responses. Methods. A total of 20 evaluable AML patients, in CR1/CRi, with measurable residual disease (MRD +) and ineligible for HSCT at inclusion, received 4 biweekly doses of vididencel, followed by 2 booster doses at week 14 and 18. Peripheral blood mononuclear cells were taken at baseline, week 6, 11, 14, 18, 20 and 32, ficoll isolated and cryopreserved in liquid nitrogen. Analysis of immune cell composition was performed by spectral flow cytometry, using a single 40-marker panel. Immune subset analysis of dendritic (live, single, CD45+, Lin-(CD3, 56, 19), CD16-, CD15-) cells was performed and correlated to clinical response, relapse free and overall survival, by high dimensional analysis using FlowSOM. Results. Dendritic cell subsets increased upon vaccination, with levels before and during vaccination being the highest in patients remaining in CR. Gating of the dendritic cell subsets by spectral flow cytometry analyses could be hampered by presence of leukemic blasts in the peripheral blood, expressing for example CD123, a marker required for gating of pDCs. Using gating on the lineage negative, CD15-, CD16- and HLA-DR+ cells, a deep immunophenotyping was performed on dendritic cells in peripheral blood. Baseline analysis showed a significantly higher frequency of CD45RA+, HLA-DR+ cells in patients who remain in CR. Using tSNE and UMAP plots generated to analyse changes in the Lin-, CD14-, CD15-, CD16- and CD11b-, HLA-DR+ population; changes and increases in several subsets were observed, most notably in subsets with cDC1, cDC2 and pDC characteristics, like expression of CD141, CLEC9A, CD1c and CD123. Higher frequencies at baseline of dendritic cells (cDC1 and cDC2) correlated positively with both relapse-free and overall survival. In patients remaining in CR, vaccination further increased or maintained dendritic cell subset frequencies, of which classical cDC1 and cDC2 correlated with longer overall survival (p<0.05). Conclusion: Patients who remain in CR after vididencel treatment had highest baseline levels of HLA-DR+ CD45RA+ cells, which could be myeloid cells able to differentiate into dendritic cell subsets, as in general CD45RA+ is lost during maturation from precursor to matured dendritic cells. Vaccination might improve and induce maturation of dendritic cell subsets, such as cDC1, cDC2 and pDC, which enhance antigen capturing, processing and presentation to tumor-reactive T cells, ultimately leading to improved survival.

Induction of Cellular and Humoral Immune Responses Is Associated with Durable Remissions in MRD+ AML-Patients after Maintenance Treatment with an Allogeneic Leukemia-Derived Dendritic Cell Vaccine

Background. Maintaining remission in AML after intensive chemotherapy continues to be a challenge, specifically in patients with measurable residual disease (MRD+). Immunotherapy could be an effective way to induce durable disease control for patients in remission. This phase 2 study used an allogenic leukemia-derived dendritic cell vaccine to induce an effective anti-leukemic response to control or eradicated MRD, leading to durable disease control. (ADVANCE-II, Clintrials.gov: NCT03697707). Methods. A total of 20 evaluable AML patients, in complete remission (CR1/CRi), MRD + and ineligible for allo-HSCT at inclusion, were scheduled to receive 4 biweekly doses of vididencel, followed by 2 booster doses at week 14 and 18. Patients aged 34-79 (median 60 years) had an ELN2017 risk score assessed of 13 favorable, 6 intermediate and 1 adverse, with mutations in NPM1 (n=10), CBFB-MYH11 (n=4), IDH2 (n=2), RUNX1-RUNX1T1 (n=2), TP53 (n=1) and CEBPA (n=1). Patients were vaccinated after a median of 8.9 months (range 3 to 60.8 Mo) after reaching CR1/CRi. MRD was assessed at baseline, week 14, 20 and 32 by flow cytometry and/or molecular analyses on bone marrow samples. Immune responses were evaluated on peripheral blood samples taken before and during treatment by a) IFNγ ELISPOT using three antigens (WT1, PRAME and RHAMM), known to be expressed by vididencel, by b) flow cytometry, using a single 40-marker panel, and c) skin biopsies taken 2 days after the first, fourth and sixth dose to assess immune responses in the skin. Evaluation of relapse-free and overall survival was done at data cut-off (3 rd July 2023). Results. All 20 patients have received 4 initial vaccinations, 17 patients received all booster vaccinations and 1 patient received only one booster vaccination. No serious adverse events (AE) or severe AE (grade 3 or higher) related to the treatment have been reported. Related AEs are mainly injection site reactions, such as redness, warmth and swelling, occurring within 48 hours after intradermal administration. To date, after a median follow-up of 24.8 Mo (range 6.6- 54.8) (N=20) 14 patients are still alive with 12 being relapse-free for 24.4 to 54.7 months. The median RFS and OS (not shown) have not yet been reached (Figure 1A). Estimated RFS and OS at 12 months is 73% (range 51-86%) and 87% (range 67-95%), respectively, whereas the 24 months RFS is 59% (range 35-77%) and OS is 73% (range 50-87%). Induction of at least one vaccine induced response (VIR), as measured by IFNγ ELISPOT, was observed in 17 out of 20 patients, with a sustained vaccine induced response (sVIR) in 9 patients. There is a clear correlation (LogRank p=0.03) between number of VIRs and overall survival (Figure 1B), with best survival in patients with at least 3 VIRs. Of these patients, 4 out of 7 had shown MRD conversion (MRD -). Analysis of the immune cell composition at baseline and after vaccination revealed that higher baseline levels of B-cells (p <0.05) , cDC1, cDC2 were observed in patients remaining in CR. Vaccination also improved immune cell frequencies of dendritic cells with highest levels correlating with longer RFS (p<0.05) and OS (p<0.05). Immune histochemistry on skin biopsies confirmed the large influx of CD4+ and CD8+ T-cells as well as antigen presenting cells, in all patients explaining the clinical observation of swelling, warmth and redness in almost all patients. Conclusion/discussion. Vaccination with vididencel resulted in durable disease control, reflected by 2-year RFS and OS of 59% and 73%, respectively and the emerging plateau in the corresponding Kaplan Meier curves. Immune response assays indicate the induction of a strong immune response, with induction of specific T-cell responses towards tumor associated antigens, increased frequencies of B-cells and dendritic cells, all correlating with longer RFS and OS. The locally skin induced immune response leading to influx of a plethora of immune cells, confirmed the mode of action of immune priming through intradermal administration of the cancer vaccine. These results warrant further studies combining vididencel with SOC such as hypomethylating agents in AML maintenance.

Intradermal Vaccination with Vididencel in MRD+ AML-Patients Leads to Increase in Antigen Presenting Cells and T-Cells to the Injection Site, Visualized Using Imaging Mass Cytometry, Showing Local Immune Cell Interactions Leading to Systemic Immune Responses

Background. Vididencel is an allogeneic leukemia-derived dendritic cell vaccine being developed as maintenance treatment in AML patients. Intradermal vaccination using vididencel is known to induce a local skin reaction, with local antigen processing by skin dendritic cells and direct presentation to T cells as well as dendritic cells, crawling out of the skin and presentation of the antigen in the draining lymph nodes for a full-blown immune response. It was previously shown that vididencel is able to induce an influx of immune cells in the skin using standard immunohistochemistry (van de Loosdrecht, 2018). In a phase 2 study vididencel was investigated as AML maintenance treatment after induction of CR1 with chemotherapy (ADVANCE-II, Clintrials.gov: NCT03697707). Using imaging mass cytometry up to 43 markers can be assessed in the same tissue section, to allow in depth analysis of the immune cells on a single cell level infiltrating in the dermis at the injection site. Methods. AML patients, in first complete remission with measurable residual disease (MRD +) and not planned for HSCT at inclusion, received 4 biweekly doses of vididencel followed by 2 booster doses at week 14 and 18. Skin biopsies of the injection site were taken 2 days after the 1 st, 4 th and 6 th vaccination. Control samples were collected from unvaccinated/unaffected skin in two patients and from healthy controls. Samples were fixed and embedded in paraffin, and stained by standard immunohistochemistry for immune cell markers like CD4, CD8 and CD68 in the central lab. For patients treated at Haukeland University Hospital (N=6), tissue sections were prepared, mounted and stained with the panel of 29 antibodies labelled with heavy metal isotopes for use in the Hyperion mass cytometry imaging. For each sample a region of interest (ROI) of 2.8mm 2 was scanned based on the localization of the immune infiltrates, guided by standard H&E staining from neighboring slides. Results In 88.5% of patients injection site reactions were reported in this phase 2 study, characterized by redness, swelling and warmth at the site of intradermal injection, which were mild (maximum grade 1 or 2), and were of short duration (generally resolved within 12 days). Skin biopsies of a subset of 6 patients were analyzed using Hyperion. In standard single stain immunohistochemistry a large influx of CD4, CD8 and CD68 cells was observed in contrast to normal skin. Imaging mass cytometry confirmed these dense immune infiltrations. Using additional markers to characterize monocytes, dendritic cells and T-cells revealed that these cells are in close proximity and suggestive for cell-to-cell interaction (Figure 1). At sites of dense immune infiltration, influx of GranzymeB expressing CD8 T-cells and CD38 positive CD4 T-cells were observed as well as CD1c+ or CD141+ dendritic cells with high HLA-DR expression. These T-cells and dendritic cells are in close contact suggestive for patients' dendritic cells migrating from blood to skin, inducing a local immune response. Conclusion/discussion. Vaccination with vididencel triggered an inflammatory skin reaction in the majority of patients, with redness, warmth and swelling, of short duration, indicative for an active immune response at the site of injection. Skin biopsies using standard immunohistochemistry showed influx of activated CD4 and CD8 T cells as well as antigen presenting cell, including dendritic cells.Extended analysis with multiparameter imaging mass cytometry showed specific immune cell subsets infiltrating in these site of injection sites. The observed co-localization of the different immune subsets following vaccination supports the hypothesis of local antigen capturing by blood derived and local antigen presenting cells (Zuo et al, 2021). The local immune response may ultimately leads to a systemic immune response and disease control.

Durable Responses and Survival in High-risk Myelodysplastic Syndrome and Acute Myeloid Leukemia Patients Receiving the Allogeneic Leukemia-derived Dendritic Cell Vaccine DCP-001.

Durable Responses and Survival in High-risk Myelodysplastic Syndrome and Acute Myeloid Leukemia Patients Receiving the Allogeneic Leukemia-derived Dendritic Cell Vaccine DCP-001.

Proinflammatory allogeneic dendritic cells enhance the therapeutic efficacy of systemic anti-4-1BB treatment.

As an immune adjuvant, proinflammatory allogeneic dendritic cells (AlloDCs) have demonstrated promising immune-priming effects in several preclinical and clinical studies. The effector cells, including NK cells and T cells are widely acknowledged as pivotal factors in the effectiveness of cancer immunotherapy due to their ability to selectively identify and eradicate malignant cells. 4-1BB, as a costimulatory receptor, plays a significant role in the stimulation of effector cell activation. This study evaluated the anti-tumor effects when combining intratumoral administration of the immune-adjuvant AlloDCs with systemic α4-1BB treatment directly acting on effector cells. In both the CT-26 murine colon carcinoma model and B16 murine melanoma model, AlloDCs demonstrated a significant enhancement in the therapeutic efficacy of α4-1BB antibody. This enhancement was observed through the delayed growth of tumors and prolonged survival. Analysis of the tumor microenvironment (TME) in the combined-treatment group revealed an immune-inflamed TME characterized by increased infiltration of activated endogenous DCs and IFNγ+ CD8+ T cells, showing reduced signs of exhaustion. Furthermore, there was an augmented presence of tissue-resident memory (TRM) CD8+ T cells (CD103+CD49a+CD69+). The combination treatment also led to increased infiltration of CD39+CD103+ tumor-specific CD8+ T cells and neoantigen-specific T cells into the tumor. Additionally, the combined treatment resulted in a less immunosuppressive TME, indicated by decreased infiltration of myeloid-derived suppressor cells and Tregs. These findings suggest that the combination of intratumoral AlloDCs administration with systemic agonistic α4-1BB treatment can generate a synergistic anti-tumor response, thereby warranting further investigation through clinical studies.

Estimation of Sensitization Status in Renal Transplant Recipients by Assessing Indirect Pathway CD4 + T Cell Response to Donor Cell-pulsed Dendritic Cell.

Generation of donor-specific human leukocyte antigen antibody (DSA) via indirect allorecognition is detrimental to long-term survival of transplant organs. The detection of such immune responses would make it possible to define patients with high risk of sensitization. In this study, we established a novel method for evaluating indirect allorecognition to assess sensitization in kidney transplant recipients. Recipient CD14 + monocytes were mixed with donor peripheral blood mononuclear cells; cultured in the presence of IL-4, GM-CSF, IL-1β, and TNFα; and used as pulsed dendritic cells (DCs). Cell proliferation and cytokine production were evaluated by carboxyfluorescein diacetate succinimidyl ester-based T cell proliferation assay and Enzyme-Linked ImmunoSpot assay, respectively. CD4 + T cell proliferation was strongly observed in following coculture with allogeneic antigen-pulsed DC leading to interferon-γ and IL-21 production. About 1% of CD4 + T cells exhibited Tfh-like phenotype (PD-1 high CXCR5 + ICOS + CD40L + ). Recipient DC pulsed with donor peripheral blood mononuclear cells was cocultured with recipient CD45RA+CD4+ and CD45RA-CD4+ (generally defined as naive and memory in humans, respectively) T cells. Irrespective of preformed or de novo DSA status, CD45RA + CD4 + T cells constantly produced IL-21. In contrast, IL-21-produced CD45RA - CD4 + T cells were significantly higher in preformed DSA-positive patients than those in negative patients (80.8 ± 51.2 versus 14.8 ± 20.4, P < 0.001). In de novo DSA-positive patients, IL-21-produced CD45RA - CD4 + T cells were significantly increased after transplantation compared with before transplantation (9.23 ± 9.08 versus 43.9 ± 29.1, P < 0.001). Assessment of indirect pathway CD4 + T cell response could provide new insights into the underlying mechanism of de novo DSA production, leading to the development of effective strategies against antibody-mediated rejection.

Adjuvant Vaccination with Allogenic Dendritic Cells Significantly Prolongs Overall Survival in High-Grade Gliomas: Results of a Phase II Trial

Immunotherapy for cancer treatment has gained increased attention in recent years. Recently, our group reported the case of a patient with glioblastoma who underwent vaccination based on dendritic cells and experienced a strong Th1 immune response together with near-complete tumor remission. Here we report the results of a phase I/II prospective, non-controlled clinical trial with 37 patients harboring glioblastoma or grade 4 astrocytomas. At the time of first recurrence after surgery, patients began receiving monthly intradermal injections of allogenic DC-autologous tumor cell hybridomas. Overall survival, quality of life, and immunological profiles were assessed prospectively. Compared with patients in the Genomic Data Commons data bank, overall survival for vaccinated patients with glioblastoma was 27.6 ± 2.4 months (vs. 16.3 ± 0.7, log-rank p < 0.001, hazard ratio 0.53, 95%CI 0.36-0.78, p < 0.01), and it was 59.5 ± 15.9 for vaccinated astrocytoma grade 4 patients (vs. 19.8 ± 2.5, log-rank p < 0.05, hazard ratio 0.18, 95%CI 0.05-0.62, p < 0.01). Furthermore, seven vaccinated patients (two IDH-1-mutated and five wild type) remain alive at the time of this report (overall survival 47.9 months, SD 21.1, range: 25.4-78.6 months since diagnosis; and 34.2 months since recurrence, range: 17.8 to 40.7, SD 21.3). We believe that the data reported here can foster the improvement of treatment protocols for high-grade gliomas based on cellular immunotherapy.

Leveraging a powerful allogeneic dendritic cell line towards neoantigen-based cancer vaccines.

In recent years, immunotherapy has finally found its place in the anti-cancer therapeutic arsenal, even becoming standard of care as first line treatment for metastatic forms. The clinical benefit provided by checkpoint blockers such as anti-PD-1/PD-L1 in many cancers revolutionized the field. However, too many patients remain refractory to these treatments due to weak baseline anti-cancer immunity. There is therefore a need to boost the frequency and function of patients' cytotoxic CD8+ cellular effectors by targeting immunogenic and tumor-restricted antigens, such as neoantigens using an efficient vaccination platform. Dendritic cells (DC) are the most powerful immune cell subset for triggering cellular immune response. However, autologous DC-based vaccines display several limitations, such as the lack of reproducibility and the limited number of cells that can be manufactured. Here we discuss the advantages of a new therapeutic vaccine based on an allogeneic Plasmacytoid DC cell line, which is easy to produce and represents a powerful platform for priming and expanding anti-neoantigen cytotoxic CD8+ T-cells.

A New Plasmacytoid Dendritic Cell-Based Vaccine in Combination with Anti-PD-1 Expands the Tumor-Specific CD8+ T Cells of Lung Cancer Patients.

The purpose of immune checkpoint inhibitor (ICI)-based therapies is to help the patient's immune system to combat tumors by restoring the immune response mediated by CD8+ cytotoxic T cells. Despite impressive clinical responses, most patients do not respond to ICIs. Therapeutic vaccines with autologous professional antigen-presenting cells, including dendritic cells, do not show yet significant clinical benefit. To improve these approaches, we have developed a new therapeutic vaccine based on an allogeneic plasmacytoid dendritic cell line (PDC*line), which efficiently activates the CD8+ T-cell response in the context of melanoma. The goal of the study is to demonstrate the potential of this platform to activate circulating tumor-specific CD8+ T cells in patients with lung cancer, specifically non-small-cell lung cancer (NSCLC). PDC*line cells loaded with peptides derived from tumor antigens are used to stimulate the peripheral blood mononuclear cells of NSCLC patients. Very interestingly, we demonstrate an efficient activation of specific T cells for at least two tumor antigens in 69% of patients irrespective of tumor antigen mRNA overexpression and NSCLC subtype. We also show, for the first time, that the antitumor CD8+ T-cell expansion is considerably improved by clinical-grade anti-PD-1 antibodies. Using PDC*line cells as an antigen presentation platform, we show that circulating antitumor CD8+ T cells from lung cancer patients can be activated, and we demonstrate the synergistic effect of anti-PD-1 on this expansion. These results are encouraging for the development of a PDC*line-based vaccine in NSCLC patients, especially in combination with ICIs.

Membrane-coated nanoparticles for direct recognition by T cells.

The direct modulation of T cell responses is an emerging therapeutic strategy with the potential to modulate undesired immune responses including, autoimmune disease, and allogeneic cells transplantation. We have previously demonstrated that poly(lactide-co-glycolide) particles were able to modulate T cell responses indirectly through antigen-presenting cells (APCs). In this report, we investigated the design of nanoparticles that can directly interact and modulate T cells by coating the membranes from APCs onto nanoparticles to form membrane-coated nanoparticles (MCNPs). Proteins within the membranes of the APCs, such as Major Histocompatibility Complexclass II and co-stimulatory factors, were effectively transferred to the MCNP. Using alloreactive T cell models, MCNP derived from allogeneic dendritic cells were able to stimulate proliferation, which was not observed with membranes from syngeneic dendritic cells and influenced cytokine secretion. Furthermore, we investigated the engineering of the membranes either on the dendritic cells or postfabrication of MCNP. Engineered membranes could be to promote antigen-specific responses, to differentially activate T cells, or to directly induce apoptosis. Collectively, MCNPs represent a tunable platform that can directly interact with and modulate T cell responses.

Use of an Allogeneic Leukemia-Derived Dendritic Cell Vaccine in MRD+ AML-Patients Results in MRD Conversion, Improved Relapse-Free Survival and Vaccine Induced Immune Responses to Tumor Antigens

Background. Maintenance therapy in AML patients with measurable residual disease (MRD) remains challenging. Immunotherapy could be an effective way to induce durable disease control. This phase II study aims to show efficacy of priming the immune system, using an allogenic leukemia-derived dendritic cell vaccine (DCP-001), to induce an effective anti-leukemic response to control or kill remaining malignant cells (ADVANCE-II, Clintrials.gov: NCT03697707). Methods. A total of 20 evaluable AML patients in first complete remission (CR1) with MRD positivity and ineligible for HSCT received 4 biweekly doses of 25e6 or 50e6 cells per intradermal vaccination (cells/vc) and 2 doses as boosts at week 14 and 18 of 10e6 cells/vc. MRD, as primary endpoint, was assessed at baseline, week 14, 20 and 32 by flow cytometry and/or molecular analyses. Evaluation of secondary endpoints relapse-free (RFS) and overall survival (OS) was done at data cut-off (18th July 2022). Peripheral blood samples were taken before and during treatment for analysis of vaccine induced responses as measured by IFNγ ELISPOT and general immune profiles by flow cytometry using a 40-marker panel. Results. Patients aged 34-79 (median 60y) had a cytogenetic risk status assessed of 4 better, 15 intermediate and 1 poor-risk, with mutations in NPM1 (n=10), CBFB-MYH11 (n=4), IDH2 (n=2), RUNX1-RUNX1T1 (n=2), TP53 (n=1) and CEBPA (n=1). All 20 patients have received 4 initial vaccinations, 17 patients received all booster vaccinations and 1 patient received one booster. No serious adverse events (AE) or severe AE (grade 3 or higher) related to the treatment have been reported. Related AEs are mainly injection site reactions, such as redness, warmth and swelling, occurring within 48 hours after intradermal administration. To date, median RFS and OS have not yet been reached (Figure 1), with a median follow up period of 15.4 (range 6.6- 16.8) months for the entire study population. Estimated RFS and OS at 6 months is 83.9% (range 64-94%) and 97% (range 79-99%), respectively. Estimated RFS and OS at 12 months is 70% (range 48-85%) and 87% (67-95%), respectively. MRD response (conversion or >10-fold decrease of MRD) was observed in 7 patients: 5 patients converted to negative and 2 showed a MRD decrease. Responses were observed within 14-20 weeks and durable in 4 out of 5 patients, remaining MRD negative up to week 32. Seven patients remained in CR with stable MRD levels and 6 patients relapsed before week 32. Three patients with stable MRD became eligible for HSCT during study and were transplanted successfully. Immune responses were observed to tumor associated antigens expressed by DCP-001; WT1, RHAMM and PRAME, with specific vaccine induced responses (VIR) in the majority of patients (17/20) and highest number of VIR per antigen observed in patients with MRD responses (Figure 2). Differences in immune profiles at baseline were observed; relapsed patients had a high number of CD8RO+ cells, in particular CD8 CM. On the contrary, MRD responders had the highest level of CD8RA+ cells at baseline, with CD8 EMRA cells at the highest level. Patients with stable MRD had comparable, but slightly lower CD8RO+ levels than MRD responders. Conclusion/discussion. Vaccination with DCP-001 resulted in durable MRD responses with five patients converting to MRD negativity, remaining relapse-free and alive during follow up. This translates in promising survival data with the median RFS for the full patient population not reached to date, at a median FU time of 15.4 months. Highest level of vaccine-induced T-cell responses to leukemia specific antigens were detected in MRD responders. Baseline levels of CD8 memory profiles were markedly different in MRD responders, which might be indicative that lower number of memory cells, and thus especially higher number of CD8 EMRA could reflect the ability to induce a tumor specific cytotoxic T-cell response after vaccination. Relapse on study was limited, with an estimated RFS at 6 months of 83.7%, which is higher than expected for this MRD+ AML patient population, which would be around 45% as shown for placebo-treated patients from a recent phase III study in a similar patient population (Quazar AML001). Immunotherapy with DCP-001 as AML maintenance therapy is safe and effective in triggering immune responses and converting patients from MRD positive to MRD negative, leading to promising relapse-free survival. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Intratumoral administration of pro-inflammatory allogeneic dendritic cells improved the anti-tumor response of systemic anti-CTLA-4 treatment via unleashing a T cell-dependent response

ABSTRACT Immune checkpoint inhibitors (ICIs) have revolutionized the oncology field. However, a significant number of patients do not respond, at least partly due to the lack of preexisting anti-tumor T-cell immunity. Therefore, it is emergent to add an immune-priming step to improve efficacy. Here, we report a combined approach consisting of intratumoral administration of pro-inflammatory allogeneic dendritic cells (AlloDCs) and systemic treatment with αCTLA-4 that can drastically improve the anti-tumor efficacy compared to αCTLA-4 monotherapy. When evaluated in mice with large established CT-26 tumors, monotherapy with αCTLA-4 neither delayed tumor progression nor improved mice survival. However, combination treatment of AlloDCs and αCTLA-4 drastically improved the effectiveness, with 70% of mice being cured. This effect was T cell-dependent, and all survived mice rejected a subsequent tumor re-challenge. Further investigation revealed an immune-inflamed tumor microenvironment (TME) in the combination treatment group characterized by enhanced infiltration of activated antigen-presenting endogenous DCs and CD8+ T cells with a tissue-resident memory (TRM) phenotype (CD49a+CD103+). This correlated with elevated levels of tumor-specific CD39+CD103+CD8+ T cells in the tumor and “tumor-matching” NKG2D+CD39+CX3CR1+CD8+ T cells in peripheral blood. Moreover, splenocytes from mice in the combination treatment group secreted significantly higher IFN-γ upon stimulation with the peptide from the endogenous CT-26 retroviral gp70 (model neoantigen), confirming the induction of a tumor-specific CD8+ T-cell response. Taken together, these data indicate a strong anti-tumor synergy between AlloDCs and αCTLA-4 that warrant further clinical investigation with the corresponding human AlloDC product (ilixadencel) for patients receiving αCTLA-4 therapy.

CD96 Downregulation Promotes the Immune Response of CD4 T Cells and Associates with Ankylosing Spondylitis.

Inhibitory receptors (IRs) play an indispensable role in regulating T cell activation and expansion. This study is aimed at exploring the correlation between IRs and ankylosing spondylitis (AS). Bioinformatics analysis of two datasets (GSE25101 and GSE73754), including 68 AS cases and 36 healthy controls, demonstrated that “T cell receptor signaling pathway” was significantly enriched, and two IRs (CD112R and CD96) were downregulated in AS cases. Real-time Quantitative PCR Detecting System (qPCR) analysis confirmed the decreased expression of CD112R and CD96 in the peripheral blood of AS patients. Flow cytometry demonstrated that the frequency of CD96-positive cells among CD4 T cells in AS patients was significantly reduced and that expressed on the cells was also significantly lower than the healthy controls. In addition, the expression of CD96 was altered on human primary CD4 T cells extracted from 3 healthy volunteers and cocultured with allogeneic dendritic cells (DCs). Also, low expression of CD96 elevated the phosphorylation of ERK in CD4 T cells and increased the level of TNF-α, IL-23, IL-17A, IL-6, and IFN-γ in the cell culture supernatant. These results suggested that CD96 is crucial for the pathogenesis of AS and may be a potential target in the treatment of the disease.

Abstract 3433: Novel dual antagonist anti-LILRB1/LILRB2 antibodies reprogram myeloid cells and potentiate T cells

Abstract Background: Genome-wide association studies have identified HLA-G and LILRB1/2 loci associated with cancer risk. LILRB1 and LILRB2 are distinct inhibitory immune checkpoint receptors that recognize MHC-I ligands, particularly immunosuppressive HLA-G. LILRB1-mediated inhibition can lead to impairment of T-cell cytotoxicity and preventing the efficient engulfment of tumor cells by macrophages as a “don’t eat me” signal. LILRB2 is expressed primarily by myeloid cells, especially in the tumor microenvironment, as an important immunosuppressive signal to inhibit the stimulation of an immune response. A dual antagonist antibody targeting both LILRB1 and LILRB2 could be more effective for cancer immunotherapy than targeting either LILRB1 or LILRB2 individually. Methods: Mice were immunized with recombinant D1-D2 domain of LILRB1 and LILRB2 proteins. Resulted heavy chain and light chain variable regions of IgG were PCR amplified from splenocytes and bone marrow plasma cells. PCR amplicons were built into yeast libraries. Yeast displayed scFv were screened for LILRB1 and LILRB2 binding. The dual binders were converted to effector silent human IgG1 and evaluated for ability to block HLA-G binding to LILRB1/RB2. The dual binder and dual blocker were further assessed in series of assays using human T cells and various primary myeloid cell subsets, including human monocyte-derived macrophages (hMDMs), tumor-associated macrophages (TAMs), dendritic cells (DCs) and monocytes. Results: We have discovered a panel of anti-LILRB1/2 antibodies that bind to both LILRB1 and LILRB2 with high affinity and block the interactions to HLA-G efficiently. The anti-LILRB1/2 antibodies inhibit both LILRB1 and LILRB2 signaling and modulate the function of immune cells. Anti-LILRB1/LILRB2-mediatted LILRB1 blockade: 1) enhances phagocytosis of tumor cells by macrophages; 2) releases HLA-G mediated inhibition on TCR activation in Jurkat T cells over-expressing LILRB1; 3) promotes the activation of human primary T cells stimulated with allogenic DCs. Anti-LILRB1/LILRB2-mediatted LILRB2 blockad:1) enhances TNFα expression and inhibits IL10 expression in LPS-stimulated human PBMC; 2) reprograms both hMDMs and TAMs induced by tumor cells to polarize toward a more inflammatory phenotype, including enhanced T cell responses to stimulation by DCs and macrophages, increased expression of M1 markers and secreted proinflammatory cytokine TNFα, decreased expression of M2 markers and secreted anti-inflammatory cytokine IL10. Conclusions: We have generated dual antagonist anti-LILRB1/LILRB2 antibodies. The anti-LILRB1/LILRB2 can reprogram suppressive myeloid cells to a stimulatory state, enhance phagocytotic function of myeloid cells, and promote the activation of lymphoid cells. The data support further development of anti-LILRB1/LILRB2 as an immunotherapeutic for solid tumor malignancies. Citation Format: Guangan Hu, Quanju Zhao, Lai Shi, Nan Bing, Dong Zhang. Novel dual antagonist anti-LILRB1/LILRB2 antibodies reprogram myeloid cells and potentiate T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3433.

Abstract 3278: Identification of epigenetic therapy induced tumor antigens to promote proliferation and infiltration of antigen specific T cells in non-small cell lung cancer

Abstract Among the hallmarks of non-small cell lung cancer (NSCLC) is cancer immunoediting. Immunoediting results when cancer cells evade immune recognition via positive selection for cells that do not present immunogenic antigens recognized by CD8+ T cells. An epigenetic therapy-based approach has been developed in our lab to enhance expression of tumor antigens using combinatorial DNA methyltransferase inhibition (DNMTi) paired with histone deacetylase inhibition (HDACi). However, durable responses from the application of epigenetic therapy alone or in combination with other therapies have remained elusive in NSCLC. This may be because enhanced proliferation and infiltration of tumor-associated antigen specific CD8+ T cells are necessary to recognize epigenetically induced antigens to halt disease progression. In the current study, we identified epigenetic therapy inducible self-antigens that can potentially be used to activate and guide the differentiation of CD8+ T cells specific to NSCLC tumor antigens. The identification of these candidates was derived from analyses of paired RNAseq data from NSCLC patients treated with combination epigenetic therapy. From these analyses, differentially expressed transcripts selected from total transcriptome data were filtered to remove non-protein coding genes and genes with detectable expression in normal tissues as indicated by GTEx. HLA binding affinity (HLA-A*0201) of selected targets was defined by NetMHC 4.0 artificial neural network-based prediction to identify strong binding peptide candidates. The combined results of these data filtering facilitated the identification of 34 epigenetic therapy induced genes and associated peptides (9mers) with predicted high binding affinity for HLA-A*0201. The predicted peptides of target genes were selected for single peptide pulsing in allogenic dendritic cells to generate antigen specific CD8+ T cells for TCR sequencing (TCRseq). TCRseq libraries derived from peptide stimulation experiments revealed distinct V and J gene frequencies and emergence of antigen specific clonotypes, which suggest clonal expansion. To validate whether epigenetic therapy could induce target genes expression in an independent experimental model, in vitro human NSCLC cell lines were treated with DNMTi and HDACi to mimic the clinical drug paradigm and transcriptional augmentation was assessed by qRT-PCR. In this orthogonal system, we validated eight target genes that were induced by epigenetic therapy in vitro. Altogether, these data suggest that a conserved subset of genes, whose derived proteins are predicted to be presented in class I MHC and act as T cell epitopes, is induced by combination epigenetic treatment. Our studies provide a basis for the development of novel and personalized strategies to improve outcomes and quality of life for more patients with NSCLC. Citation Format: Julia An, Valsamo Anagnostou, Kristen A. Marrone, Victor E. Velculescu, Julie R. Brahmer, Stephen B. Baylin, Michael J. Topper. Identification of epigenetic therapy induced tumor antigens to promote proliferation and infiltration of antigen specific T cells in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3278.

Safety and tumour-specific immunological responses of combined dendritic cell vaccination and anti-CD40 agonistic antibody treatment for patients with metastatic pancreatic cancer: protocol for a phase I, open-label, single-arm, dose-escalation study (REACtiVe-2 trial)

IntroductionThe prognosis of patients with advanced pancreatic ductal adenocarcinoma (PDAC) is dismal and conventional chemotherapy treatment delivers limited survival improvement. Immunotherapy may complement our current treatment strategies. We previously demonstrated...

Ilixadencel, a Cell-based Immune Primer, plus Sunitinib Versus Sunitinib Alone in Metastatic Renal Cell Carcinoma: A Randomized Phase 2 Study

BackgroundThe prognosis of patients with synchronous metastatic renal cell carcinoma (mRCC) is poor. Whereas single-agent tyrosine kinase inhibition (TKI) is clearly insufficient, the effects can be enhanced by combinations with immune checkpoint inhibitors. Innovative treatment options combining TKI and other immune-stimulating agents could prove beneficial. ObjectiveTo evaluate the clinical effects on metastatic disease when two doses of allogeneic monocyte-derived dendritic cells (ilixadencel) are administrated intratumorally followed by nephrectomy and treatment with sunitinib compared with nephrectomy and sunitinib monotherapy, in patients with synchronous mRCC. Design, setting, and participantsA randomized (2:1) phase 2 multicenter trial enrolled 88 patients with newly diagnosed mRCC to treatment with the combination ilixadencel/sunitinib (ILIXA/SUN; 58 patients) or sunitinib alone (SUN; 30 patients). Outcome measurements and statistical analysisThe primary endpoints were 18-mo survival rate and overall survival (OS). A secondary endpoint was objective response rate (ORR) assessed up to 18 mo after enrollment. Statistic evaluations included Kaplan-Meier estimates, log-rank tests, Cox regression, and stratified Cochran-Mantel-Haenszel tests. Results and limitationsThe median OS was 35.6 mo in the ILIXA/SUN arm versus 25.3 mo in the SUN arm (hazard ratio 0.73, 95% confidence interval 0.42–1.27; p = 0.25), while the 18-mo OS rates were 63% and 66% in the ILIXA/SUN and SUN arms, respectively. The confirmed ORR in the ILIXA/SUN arm were 42.2% (19/45), including three patients with complete response, versus 24.0% (six/25) in the SUN arm (p = 0.13) without complete responses. The study was not adequately powered to detect modest differences in survival. ConclusionsThe study failed to meet its primary endpoints. However, ilixadencel in combination with sunitinib was associated with a numerically higher, nonsignificant, confirmed response rate, including complete responses, compared with sunitinib monotherapy. Patient summaryWe studied the effects of intratumoral vaccination with ilixadencel followed by sunitinib versus sunitinib only in a randomized phase 2 study. The combination treatment showed numerically higher numbers of confirmed responses, suggesting an immunologic effect.

Harnessing dendritic cells for innovative therapeutic cancer vaccines.

The clinical activity of new immunotherapies in cancer, such as anti-Programmed cell death 1 (PD-1)/Programmed death-ligand 1, has revealed the importance of the patient's immune system in controlling tumor development. As in infectious diseases, dendritic cells (DCs) are critical for inducing immune responses in cancer. Unfortunately, autologous DC-based vaccines have not yet demonstrated their clinical benefit. Here, we review recent research using allogeneic DCs as alternatives to autologous DCs to develop innovative therapeutic cancer vaccines. A novel approach using an allogeneic plasmacytoid dendritic cell (PDC) line as an antigen presentation platform showed great potency when used to prime and expand antitumor-specific CD8+ T cells in vitro and in vivo in a humanized mouse model. This PDC platform, named PDC∗vac, was first evaluated in the treatment of melanoma with encouraging results and is currently being evaluated in the treatment of lung cancer in combination with anti-PD-1 immunotherapy. Therapeutic cancer vaccines are of particular interest because they aim to help patients, to mount effective antitumor responses, especially those who insufficiently respond to immune checkpoint inhibitors. The use of an allogeneic plasmacytoid DC-based platform such as PDC∗vac could greatly potentiate the efficacy of these new immunotherapies.