T-cell Proliferation Research Articles

Effects of Cardiorespiratory Fitness on Immune Cell Mitochondrial Metabolism in Health and Disease

Problem: Cardiorespiratory fitness (CRF) and mitochondrial function are important factors in health and disease. Even in immune cells, an effective metabolism is crucial for most major cellular functions, such as T-cell proliferation. Little is known about the relationship between CRF and mitochondrial metabolism of immune cells. Methods: In this narrative review, we collected the most recent literature in the field of exercise immunology and mitochondrial immune cell metabolism. We included studies, which used Oxygraph- or Seahorse techniques for measurement of mitochondrial function in various immune cell populations and subtypes associated with topics in the field of exercise in health and disease. Results: 20 studies were included in our analysis. Cell populations were PBMCs (peripheral blood mononuclear **cells), lymphocytes, T cells, NK cells, platelets, and neutrophils. While mitochondrial function was inconstantly affected through acute bouts of exercise, most of the studies, which included training interventions, reported a higher mitochondrial function in immune cells, which were in some studies correlated with the CRF. In various disease conditions, mitochondrial function of immune cells was impaired compared to healthy controls. › Discussion: If a regular exercise training is followed by an improvement in immune cell mitochondrial respiration, regular exercise could become even more important in the context of optimizing immune function and immune regulation in prevention and therapy.

PLGA-LEC/F127 hybrid nanoparticles loaded with curcumin and their modulatory effect on monocytes.

Aim: To investigate the effect of surfactant type on curcumin-loaded (CUR) PLGA nanoparticles (NPs) to modulate monocyte functions. Materials & methods: The nanoprecipitation method was used, and PLGA NPs were designed using PluronicF127 (F127) and/or lecithin (LEC) as surfactants. Results: The Z-average of the NPs was <200nm, they had a spherical shape, Derjaguin-Muller-Toporov modulus >0.128MPa, they were stable during storage at 4°C, ζ-potential ∼-40mV, polydispersity index <0.26 and % EE of CUR >94%. PLGA-LEC/F127NPs showed favorable physicochemical and nanomechanical properties. These NPs were bound and internalized mainly by monocytes, suppressed monocyte-induced reactive oxygen species production,and decreased the ability of monocytes to modulate T-cell proliferation. Conclusion: These results demonstrate the potential of these NPs for targeted therapy.

Intermittent MEK Inhibition with GITR Costimulation Rescues T-cell Function for Increased Efficacy with CTLA-4 Blockade in Solid Tumor Models.

MEK inhibitors (MEKi) have shown limited success as a treatment for MAPK/ERK pathway-dependent cancers due to various resistance mechanisms tumor cells can employ. CH5126766 (CKI27) is an inhibitor that binds to MEK and prevents release of RAF, reducing the relief of negative feedback commonly observed with other MEKis. We observed that CKI27 increased MHC expression in tumor cells and improved T cell-mediated killing. Yet, CKI27 also decreased T-cell proliferation, activation, and cytolytic activity by inhibiting the MAPK/ERK pathway that is activated downstream of T-cell receptor signaling. Therefore, we aimed to balance the positive and negative immunomodulatory effects of MEKis for optimal combination with immunotherapy. Intermittent administration of CKI27 allowed T cells to partially recover and costimulation via GITR and OX-40 agonist antibodies completely alleviated inhibition of function. In Kras mutant lung and colon tumor mouse models, intermittent CKI27 and anti-GITR significantly decreased tumor growth and prolonged survival when further combined with CTLA-4 immune checkpoint blockade. Moreover, this triple combination increased CD8+ and CD4+ T-cell proliferation, activation, and effector/memory subsets in the tumor-draining lymph nodes and tumors and led to intratumoral regulatory T-cell destabilization. These data, collectively, will allow for more informed decisions when optimizing combination regimens by overcoming resistance, reducing toxicity, and generating long-term immune responses.

Co-stimulation of CD28/CD40 signaling molecule potentiates CAR-T cell efficacy and stemness

CD19 chimeric antigen receptor T (CD19CAR-T) cells have achieved promising outcomes in relapsed/refractory B-cell malignancies. However, recurrences occur due to the loss of CAR-T cell persistence. We developed dual T/B-cell co-stimulatory molecules (CD28 and CD40) in CAR-T cells to enhance intense tumoricidal activity and persistence. CD19.28.40z CAR-T cells promoted pNF-κB and pRelB downstream signaling while diminishing NFAT signaling upon antigen exposure. CD19.28.40z CAR-T cells demonstrated greater proliferation which translated into effective anti-tumor cytotoxicity in long-term co-culture assay. Repetitive weekly antigen stimulation unveiled continuous CAR-T cell expansion while preserving central memory T-cell subset and lower expression of exhaustion phenotypes. The intrinsic genes underlying CD19.28.40z CAR-T cell responses were compared to conventional CARs and demonstrated the up-regulated genes associated with T-cell proliferation and memory as well as down-regulated genes related to apoptosis, exhaustion, and glycolysis pathway. Enrichment of genes toward T-cell stemness, particularly SELL, IL-7r, TCF7, and KLF2, was observed. Effective and continuing anti-tumor cytotoxicity in vivo was exhibited in both B-ALL and B-NHL xenograft models while demonstrating persistent T-cell memory signatures. The functional enhancement of CD37.28.40z CAR-T cell activities against CD37+ tumor cells was further validated. The modification of dual T/B-cell signaling molecules remarkably maximized the efficacy of CAR-T cell therapy.

MDB-97. INVESTIGATING THE IMMUNOMODULATORY ROLE OF V-DOMAIN IG SUPPRESSOR OF T-CELL ACTIVATION IN AN AGGRESSIVE MURINE MODEL OF PEDIATRIC MEDULLOBLASTOMA

Abstract BACKGROUND Efforts to treat relapsed medulloblastoma patients with immune checkpoint inhibitors aim to enhance T-cell recognition and elimination of tumor cells. However, significant barriers to treatment efficacy include low numbers of infiltrating effector T-cells and paucity of PD-L1 expression in the tumor microenvironment. Thus, identifying alternative immune checkpoints on the predominant immune cells that infiltrate tumors represents an attractive therapeutic alternative. METHODS Medulloblastoma tumor cells (mCB MYC DNp53) are orthotopically implanted into the right cerebellum of C57/BL6J mice. Once moribund, tumors are harvested and analyzed by flow cytometry. For in vitro studies, tumor cells are co-cultured at increasing concentrations with naïve splenic CD4+ T-cells, CD3/CD28 beads, and rIL-2 for 5 days and then analyzed by flow cytometry. RESULTS Flow cytometric analysis of tumors reveals an abundance of CD45highCD11bhigh tumor-associated macrophages (TAMs), CD45intCD11bint tumor-associated microglia, and CD4+FoxP3+Treg cells. Across all three immune populations, we observe high expression of the inhibitory immune checkpoint, V-domain Ig Suppressor of T-cell Activation (VISTA), with the highest MFI observed on Mø-like TAMs (n=9 tumors; p&amp;lt;0.05). Compared to sham controls, infiltration of Mø-like TAMs is specific to the tumor and express higher levels of MHCII than Mg-like cells (n=14 tumors, 6 sham; p &amp;lt;0.05). Additionally, we find that medulloblastoma tumor cells (mCB MYC DNp53) express VISTA’s binding partner, VSIG3. Tumor cells co-cultured with CD4+ T-cells can inhibit T-cell proliferation in vitro. Preliminary results suggest that addition of 2ug/mL of anti-VISTA blocking antibody (13F3) can rescue proliferation. CONCLUSIONS Tumors are highly infiltrated by VISTA+ T-regs and TAMs. These TAMs upregulate MHCII, suggesting that they are presenting antigen in the context of an inhibitory molecule. Furthermore, our in vitro results warrant further investigation into the role of the VISTA/VSIG3 axis in inhibiting T-cells and supports blocking VISTA as a potential strategy to promote T-cell activation and an anti-tumor response.

MDB-89. DETERMINING THE IMMUNOMODULATORY POTENTIAL OF PI3KΓ IN AGGRESSIVE MEDULLOBLASTOMA AND THE TUMOR MICROENVIRONMENT

Abstract BACKGROUND Medulloblastoma (MB) is the most common pediatric embryonal brain tumor arising in the cerebellum. It has a “cold” tumor microenvironment (TME) characterized by an abundance of immunosuppressive Tumor Associated Macrophages (TAMs) and low T-cell infiltration. TAMs include tissue resident microglia and migrating bone marrow derived macrophages. While microglia play important roles in the central nervous system through their immune surveillance and ability to activate other immune cells, they can be polarized by tumor cells toward an anti-inflammatory state which allows for maintained tumor growth and proliferation. A potential new target to combat the immunosuppressed TME of MB is the class IB isoform PI3Kγ. Inhibiting this isoform has been attributed to controlling a critical switch between immune suppression and immune stimulation in TAMs but has yet to be examined in MB and its TME. We hypothesize that inhibiting PI3Kγ in TAMs and MB tumor cells will induce an anti-tumorigenic immune response within the MB TME and promote tumor clearance. METHODS Engineered murine MB cells, mCB DNp53 MYC, are treated with a pan-PI3K or specific PI3Kγ inhibitor for 48 hours before being co-cultured with naïve splenic CD4 T-cells from C57BL/6J mice for 5 days. Proliferation of the T cells is measured using Cell Trace Violet through flow cytometry on a Cytek Aurora. RESULTS Pan-PI3K inhibition revealed that tumor cells treated with the inhibitor prior to co-culturing did not suppress T-cell proliferation as drastically when compared to untreated tumor cells. Treatment with a selective inhibitor is on-going. CONCLUSIONS These results suggest that inhibition of the PI3K pathway in our MB tumor cells dampens their ability to suppress T cell proliferation. The PI3K pathway may play an active role in tumor cell’s ability to induce immunosuppression. Inhibiting this pathway may be a new avenue for therapeutic intervention for relapsed/refractory MB.

IL-15/IL-15Rα-Fc-Fusion Protein XmAb24306 Potentiates Activity of CD3 Bispecific Antibodies through Enhancing T-Cell Expansion.

An insufficient quantity of functional T cells is a likely factor limiting the clinical activity of T-cell bispecific antibodies, especially in solid tumor indications. We hypothesized that XmAb24306 (efbalropendekin alfa), a lymphoproliferative interleukin (IL)-15/IL-15 receptor α (IL-15Rα) Fc-fusion protein, may potentiate the activity of T-cell dependent (TDB) antibodies. The activation of human peripheral T cells by cevostamab, an anti-FcRH5/CD3 TDB, or anti-HER2/CD3 TDB resulted in the upregulation of the IL-2/15Rβ (CD122) receptor subunit in nearly all CD8+ and majority of CD4+ T cells, suggesting that TDB treatment may sensitize T cells to IL-15. XmAb24306 enhanced T-cell bispecific antibody-induced CD8+ and CD4+ T-cell proliferation and expansion. In vitro combination of XmAb24306 with cevostamab or anti-HER2/CD3 TDB resulted in significant enhancement of tumor cell killing, which was reversed when T-cell numbers were normalized, suggesting that T-cell expansion is the main mechanism of the observed benefit. Pretreatment of immunocompetent mice with a mouse-reactive surrogate of XmAb24306 (mIL-15-Fc) resulted in a significant increase of T cells in the blood, spleen, and tumors and converted transient anti-HER2/CD3 TDB responses to complete durable responses. In summary, our results support the hypothesis that the number of tumor-infiltrating T cells is rate limiting for the activity of solid tumor-targeting TDBs. Upregulation of CD122 by TDB treatment and the observed synergy with XmAb24306 and T-cell bispecific antibodies support clinical evaluation of this novel immunotherapy combination.

Nivolumab Reaches Brain Lesions in Patients with Recurrent Glioblastoma and Induces T-cell Activity and Upregulation of Checkpoint Pathways.

Glioblastoma (GBM) is an aggressive brain tumor with poor prognosis. Although immunotherapy is being explored as a potential treatment option for patients with GBM, it is unclear whether systemic immunotherapy can reach and modify the tumor microenvironment in the brain. We evaluated immune characteristics in patients receiving the anti-PD-1 immune checkpoint inhibitor nivolumab 1 week prior to surgery, compared with control patients receiving salvage resection without prior nivolumab treatment. We observed saturating levels of nivolumab bound to intratumorally and tissue-resident T cells in the brain, implicating saturating levels of nivolumab reaching brain tumors. Following nivolumab treatment, significant changes in T-cell activation and proliferation were observed in the tumor-resident T-cell population, and peripheral T cells upregulated chemokine receptors related to brain homing. A strong nivolumab-driven upregulation in compensatory checkpoint inhibition molecules, i.e., TIGIT, LAG-3, TIM-3, and CTLA-4, was observed, potentially counteracting the treatment effect. Finally, tumor-reactive tumor-infiltrating lymphocytes (TIL) were found in a subset of nivolumab-treated patients with prolonged survival, and neoantigen-reactive T cells were identified in both TILs and blood. This indicates a systemic response toward GBM in a subset of patients, which was further boosted by nivolumab, with T-cell responses toward tumor-derived neoantigens. Our study demonstrates that nivolumab does reach the GBM tumor lesion and enhances antitumor T-cell responses both intratumorally and systemically. However, various anti-inflammatory mechanisms mitigate the clinical efficacy of the anti-PD-1 treatment.

A novel strategy to elicit enduring anti-morphine immunity and relief from addiction by targeting Acr1 protein nano vaccine through TLR-2 to dendritic cells

Morphine addiction poses a significant challenge to global healthcare. Current opioid substitution therapies, such as buprenorphine, naloxone and methadone are effective but often lead to dependence. Thus, exploring alternative treatments for opioid addiction is crucial. We have developed a novel vaccine that presents morphine and Pam3Cys (a TLR-2 agonist) on the surface of Acr1 nanoparticles. This vaccine has self-adjuvant properties and targets TLR-2 receptors on antigen-presenting cells, particularly dendritic cells. Our vaccination strategy promotes the proliferation and differentiation of morphine-specific B-cells and Acr1-reactive CD4 T-cells. Additionally, the vaccine elicited the production of high-affinity anti-morphine antibodies, effectively eliminating morphine from the bloodstream and brain in mice. It also reduced the expression of addiction-associated μ-opioid receptor and dopamine genes. The significant increase in memory CD4 T-cells and B-cells indicates the vaccine's ability to induce long-lasting immunity against morphine. This vaccine holds promise as a prophylactic measure against morphine addiction.

Elevated memory T-cell conversion in a preclinical mouse model of hemophilia A.

One of the major challenges in the choice of the best therapeutic approach for the treatment of patients affected by hemophilia A (HA) is the definition of criteria predicting the formation of factor VIII (FVIII) neutralizing antibodies, called inhibitors. Both genetic and environmental elements influencing the immune response toward FVIII have been identified but still not all the factors causing the pathological rejection of FVIII have been identified. Since there is a connection between coagulation and inflammation, here we assessed the role played by the FVIII deficiency in shaping the humoral and cellular response toward an antigen other than FVIII itself. To this aim, we challenged both HA and wild-type (WT) mice with either FVIII or ovalbumin (OVA) and followed antigen-specific antibody level, immune cell population frequency and phenotype up to 9 weeks after the last antigen booster. The activation threshold was evaluated in vitro by stimulating the murine T cells with a decreasing dose of α-CD3. The humoral response to FVIII was similar between the two groups while both the in vivo and in vitro experiments highlighted an antigen-independent sensitivity of HA compared with WT T cells causing an increase in memory T-cell conversion and proliferation capability.

Interpretation of T-cell and NK-cell lymphoid proliferations and lymphomas in the 5th edition of the WHO classification of haematolymphoid tumours

The 5th edition of the World Health Organization (WHO) classification of haematolymphoid tumours used the hierarchical system to classify T-cell and NK-cell lymphoid proliferations and lymphomas (T/NK-LPD/LYM) based on research advances and clinicopathological characteristics of the diseases. In this edition of classification, tumour-like lesions were included, some tumors were added/deleted, the names or terms of certain diseases were refined, and the diagnostic criteria or subtypes of some diseases were revised. This group of diseases was reintegrated from non-clonal hyperplasia to highly aggressive lymphoma, which would further reflect the nature of T/NK-LPD/LYM and benefit to clinical application.

The role of OX40 ligand/OX40 axis signalling in atopic dermatitis.

Atopic dermatitis (AD) is a heterogeneous inflammatory condition involving multiple immune pathways mediated by pathogenic T cells. OX40 ligand (OX40L) and OX40 are costimulatory immune checkpoint molecules that regulate effector and memory T-cell activity and promote sustained immune responses in multiple immunological pathways, including T helper (Th)2, Th1, Th17 and Th22. As such, OX40L/OX40 signalling between antigen-presenting cells (APCs) and activated T cells postantigen recognition promotes pathogenic T-cell proliferation and survival. Under inflammatory conditions, OX40L is upregulated on APCs, enhancing the magnitude of antigen-specific T-cell responses and secretion of proinflammatory cytokines. In AD, OX40L/OX40 signalling contributes to the amplification and chronic persistence of T-cell-mediated inflammation. Recent therapeutic success in clinical trials has highlighted the importance of the OX40L/OX40 axis as a promising target for the treatment of AD. Here, we discuss the many factors that are involved in the expression of OX40L and OX40, including the cytokine milieu, antigen presentation, the inflammatory environment in AD, and the therapeutic direction influenced by this costimulatory pathway.

Identification of cross reactive T cell responses in adenovirus based COVID 19 vaccines

Vaccination has proven to be a valuable tool to combat SARS-CoV-2. However, reports of rare adverse reactions such as thrombosis/thrombocytopenia syndrome after ChAdOx1 nCoV-19 vaccination have caused scientific, public and media concern. ChAdOx1 was vectorised from the Y25 chimpanzee adenovirus, which was selected due to low human seroprevalence to circumvent pre-existing immunity. In this study, we aimed to explore patterns of T-cell activation after SARS-CoV-2 COVID-19 vaccine exposure in vitro using PBMCs collected from pre-pandemic ChAdOx1 nCoV-19 naïve healthy donors (HDs), and ChAdOx1 nCoV-19 and Pfizer vaccinated controls. PBMCs were assessed for T-cell proliferation using the lymphocyte transformation test (LTT) following exposure to SARS-CoV-2 COVID-19 vaccines. Cytokine analysis was performed via intracellular cytokine staining, ELISpot assay and LEGENDplex immunoassays. T-cell assays performed in pre-pandemic vaccine naïve HDs, revealed widespread lymphocyte stimulation after exposure to ChAdOx1 nCoV-19 (95%), ChAdOx-spike (90%) and the Ad26.COV2. S vaccine, but not on exposure to the BNT162b2 vaccine. ICS analysis demonstrated that CD4+ CD45RO+ memory T-cells are activated by ChAdOx1 nCoV-19 in vaccine naïve HDs. Cytometric immunoassays showed ChAdOx1 nCoV-19 exposure was associated with the release of proinflammatory and cytotoxic molecules, such as IFN-γ, IL-6, perforin, granzyme B and FasL. These studies demonstrate a ubiquitous T-cell response to ChAdOx1 nCoV-19 and Ad26.COV2. S in HDs recruited prior to the SARS-CoV-2 pandemic, with T-cell stimulation also identified in vaccinated controls. This may be due to underlying T-cell cross-reactivity with prevalent human adenoviruses and further study will be needed to identify T-cell epitopes involved.

Vascular endothelial growth factor secretion and immunosuppression are distinct potency mechanisms of human bone marrow mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are investigated as cellular therapeutics for inflammatory bowel diseases and associated perianal fistula, although consistent efficacy remains a concern. Determining host factors that modulate MSCs' potency including their secretion of angiogenic and wound-healing factors, immunosuppression, and anti-inflammatory properties are important determinants of their functionality. We investigated the mechanisms that regulate the secretion of angiogenic and wound-healing factors and immune suppression of human bone marrow MSCs. Secretory analysis of MSCs focusing on 18 angiogenic and wound-healing secretory molecules identified the most abundancy of vascular endothelial growth factor A (VEGF-A). MSC viability and secretion of other angiogenic factors are not dependent on VEGF-A secretion which exclude the autocrine role of VEGF-A on MSC's fitness. However, the combination of inflammatory cytokines IFNγ and TNFα reduces MSC's VEGF-A secretion. To identify the effect of intestinal microvasculature on MSCs' potency, coculture analysis was performed between human large intestine microvascular endothelial cells (HLMVECs) and human bone marrow-derived MSCs. HLMVECs do not attenuate MSCs' viability despite blocking their VEGF-A secretion. In addition, HLMVECs neither attenuate MSC's IFNγ mediated upregulation of immunosuppressive enzyme indoleamine 2,3-dioxygenase nor abrogate suppression of T-cell proliferation despite the attenuation of VEGF-A secretion. We found that HLMVECs express copious amounts of endothelial nitric oxide synthase and mechanistic analysis showed that pharmacological blocking reverses HLMVEC-mediated attenuation of MSC's VEGF-A secretion. Together these results suggest that secretion of VEGF-A and immunosuppression are separable functions of MSCs which are regulated by distinct mechanisms in the host.

Immune responsiveness in stable kidney transplantation patients: Complete inhibition of T-cell proliferation but residual T-cell activity during maintenance immunosuppressive treatment.

The recommended immunosuppressive treatment after kidney transplantation consists of tacrolimus, mycophenolate mofetil, and low-dose corticosteroids. Drug concentrations are monitored using therapeutic drug monitoring (TDM), which does not necessarily correlate with pharmacodynamic activity. To find the balance between optimal efficacy and minimal toxicity, it might be more informative to monitor patients' immunological status rather than drug concentrations. We selected a panel of T-cell-based immune assays, which were used for immunomonitoring of 14 stable kidney transplantation patients. Whole blood was incubated with a T-cell stimulus, after which T-cell proliferation, T-cell activation marker expression and cytokine production were measured to study residual immune activity invitro (before drug intake; drug added to the incubation) and exvivo (after drug intake). T-cell proliferation was completely suppressed in all patients over the full day, while IL-2, IFN-γ, CD71, and CD154 showed fluctuations over the day with a strong inhibition (75%-25%) at 2 h post-dose. The level of inhibition was variable between patients and could not be related to pharmacokinetic parameters or the presence of regulatory or senescence immune cells. Moreover, the level of inhibition did not correlate with the invitro tacrolimus drug effect as studied by incubating pre-dose blood samples with additional tacrolimus. Overall, IL-2, IFN-γ, CD71, and CD154 seem to be good markers to monitor residual immune activity of transplantation patients. To evaluate the correlation between these pharmacodynamic biomarkers and clinical outcome, prospective observational studies are needed.

Correlative biomarker analyses for optimal therapeutic dose determination of ABBV-383, a B-cell maturation antigen (BCMA) x CD3 bispecific antibody, in patients with relapsed/refractory multiple myeloma (RRMM).

7532 Background: ABBV-383 is a BCMA x CD3 bispecific antibody with 2 high-affinity BCMA-binding domains and a low-affinity CD3 engaging arm that has been shown to decrease the negative impact of soluble BCMA (sBCMA) and drive sustained T-cell activation with reduced cytokine release in preclinical models of MM. Here, we describe correlative biomarker results of baseline and longitudinal sBCMA levels and immune profiles in the ongoing first-in-human trial that supports 60 mg Q4W as the optimal therapeutic dose of ABBV-383 monotherapy. Methods: Peripheral blood and serum samples from patients with RRMM enrolled in the phase 1 open-label, dose-expansion study (NCT03933735) who received ABBV-383 at 20, 40, or 60 mg Q3W or 60 mg Q4W were collected at baseline (post-dexamethasone, pre–ABBV-383), on treatment, and at disease progression. Samples were analyzed by flow cytometry for immune cell populations, Luminex for cytokines, and electrochemiluminescence ligand binding for sBCMA. Results: Median sBCMA levels at baseline were highly variable among patients, but did not associate with clinical response (≥ partial response [PR]) to ABBV-383 at the selected optimal dose level of 60 mg Q4W (&lt;PR: 244.7 ng/mL; ≥PR: 52.2 ng/mL; P=0.13). Reduction in sBCMA levels over time associated with clinical response (Cmin at cycle 5: &lt;PR, 244.7 ng/mL; ≥PR, 4.7 ng/mL; P=0.04). ABBV-383 treatment led to a rapid and transient increase of proinflammatory cytokines, including IL-6, IL-8, IL-10, TNF-α (Cmax within cycle 1: 29, 349.5, 1375, 456.5 pg/mL, respectively), and promoted T-cell redistribution (89% reduction of CD8+ T cells from periphery), activation (1.9-fold increase in CD69+CD8+ T cells), and proliferation (2-fold increase in Ki67+CD8+ T cells) within cycle 1 in 60-mg Q4W-treated patients. The frequency of baseline CD8+ T-cell exhaustion (PD-1/TIM3) did not impact clinical response at the optimal dose level of 60 mg Q4W ABBV-383 (&lt;PR: 3.5%; ≥PR: 3.9%; P=0.9). Peak induction levels of proinflammatory cytokines during cycle 1 correlated with occurrence of cytokine release syndrome (≥G1) as well as clinical response across dose levels. Conclusions: High-avidity BCMA binding coupled to low-affinity T-cell engagement distinguishes the resulting mechanism of action for ABBV-383. Responses to 60 mg Q4W ABBV-383 were independent of baseline sBCMA levels and resulted in rapid and transient proinflammatory cytokine production that promoted robust T-cell redistribution, activation, and proliferation, indicating that the optimal dose of ABBV-383 maximizes its clinical potential as a convenient, safe, and effective therapy for MM. Clinical trial information: NCT03933735 .

Phase 2 randomized study of rintatolimod following FOLFIRINOX in patients with locally advanced pancreatic adenocarcinoma.

TPS4214 Background: The immunosuppressive microenvironment of pancreatic ductal adenocarcinoma (PDAC) is a driver of its lethality and resistance to immunotherapy. Strategies which modulate the immunosuppressive milieu have the potential to improve the anti-tumor response and improve outcomes in patients with PDAC. Rintatolimod is a synthetic double-stranded RNA molecule that activates the toll-like receptor 3 (TLR3) present on dendritic cells, B-cells, natural killer cells and pancreatic epithelial cells. TLR3 stimulation may increase cytotoxic T-cell activity in PDAC through dendritic cell maturation, antigen priming and downstream proliferation of cytotoxic T-cells. Twenty-seven patients were treated with rintatolimod in a single center Named Patient Program at Erasmus University Medical Center in Rotterdam between January 2017 and February 2019. All had locally advanced or metastatic PDAC which had not progressed on FOLFIRINOX (median 8 cycles). Maintenance rintatolimod, administered intravenously twice weekly for up to 18 weeks, was generally well tolerated. The most common toxicities were grade 1-2 myalgia (30%), chills (52%) and fatigue (30%); no toxicities ≥ grade 3 were observed. Progression-free and overall survival (from cycle 1 FOLFIRINOX) were 13 and 19 months. Methods: This is a phase 2 randomized, open-label, multicenter clinical trial. The primary objective is to assess the efficacy of rintatolimod compared to observation in subjects with locally advanced PDAC which has not progressed after FOLFIRINOX. Eligible patients have completed at least 4 months of FOLFIRINOX, have no evidence of progressive disease, have measurable disease by RECIST v1.1, Karnofsky Performance Status ≥80 and have adequate organ function. Subjects with resected PDAC or metastatic disease are not eligible. Subjects are randomized 2:1 to rintatolimod vs. observation. Rintatolimod is administered intravenously twice weekly until disease progression in the experimental arm. Cross-sectional imaging is performed every 8 weeks in both arms. The primary endpoint is progression-free survival, as assessed by the investigator. Secondary endpoints include overall survival, objective response rate, duration of response and safety, as assessed by CTCAE v5.0. Exploratory endpoints include quality of life and changes in immune cell composition and T-cell response in the peripheral blood to characterize the immunomodulatory effects of rintatolimod. The planned sample size is 90 subjects. The final analysis will be performed after 64 events (disease progression or death) have been observed. The study is powered to detect a hazard ratio of 0.47 for median PFS with 80% power and significance level of 5%. Clinical trial information: NCT05494697 .

Glycoengineering-based anti-PD-1-iRGD peptide conjugate boosts antitumor efficacy through T cell engagement

Despite the important breakthroughs of immune checkpoint inhibitors in recent years, the objective response rates remain limited. Here, we synthesize programmed cell death protein-1 (PD-1) antibody-iRGD cyclic peptide conjugate (αPD-1-(iRGD)2) through glycoengineering methods. In addition to enhancing tissue penetration, αPD-1-(iRGD)2 simultaneously engages tumor cells and PD-1+ Tcells via dual targeting, thus mediating tumor-specific Tcell activation and proliferation with mild effects on non-specific Tcells. In multiple syngeneic mouse models, αPD-1-(iRGD)2 effectively reduces tumor growth with satisfactory biosafety. Moreover, results of flow cytometry and single-cell RNA-seq reveal that αPD-1-(iRGD)2 remodels the tumor microenvironment and expands a population of "better effector" CD8+ tumor infiltrating Tcells expressing stem- and memory-associated genes, including Tcf7, Il7r, Lef1, and Bach2. Conclusively, αPD-1-(iRGD)2 is a promising antibody conjugate therapeutic beyond antibody-drug conjugate for cancer immunotherapy.

Phase I dose escalation trial of the first-in-class TNFR2 agonist monoclonal antibody, HFB200301, in monotherapy and in combination with tislelizumab, an anti-PD-1 monoclonal antibody, in adult patients with advanced solid tumors.

2526 Background: Tumor necrosis factor receptor-2 (TNFR2) is expressed on effector CD8+, CD4+, and regulatory T (Treg) cells, natural killer (NK) cells, and myeloid cells. Activation of TNFR2 is anticipated to yield effective anti-tumor immunity by stimulating T-cell and NK-cell activation and proliferation in the tumor microenvironment. HFB200301 is a first-in-class anti-TNFR2 agonistic monoclonal antibody that triggers both innate and adaptive immune stimulation by binding to a specific epitope on TNFR2. We report initial results of an ongoing Phase I dose escalation, multicenter study (NCT05238883) of HFB200301 in monotherapy and in combination with tislelizumab (TIS) in patients (pts) with advanced refractory solid tumors. Methods: A Phase I trial designed to evaluate the safety, pharmacokinetics (PK), pharmacodynamics (PD), and initial efficacy of HFB200301 in monotherapy and in combination with TIS. Eligible pts, ≥18 years with an ECOG PS 0-1 and advanced solid tumors, will be enrolled in dose escalation at 5 different dose cohorts in monotherapy (Q4W), 3 different dose cohorts in combination with TIS (Q4W), or in dose expansion following determination of safety. Radiographic response is assessed every 8 weeks. Results: A total of 39 pts have been enrolled, 27 pts in monotherapy and 12 pts in combination. All pts received prior systemic therapy (median 2, range 1-4); 23 pts (59%) had prior checkpoint inhibitor treatment. No dose limiting toxicities were observed in either the monotherapy or combination arms. Treatment-related adverse events (TRAEs) occurred in 44% of monotherapy and in 50% of combination pts, with pruritis (11%), tremor (7%), fatigue (7%), asthenia (7%) and nausea (7%) being the most common TRAEs. No ≥ Grade 3 TRAEs were reported, and there were no drug discontinuations due to TRAEs. Preliminary HFB200301 PK analysis demonstrated non-linear clearance of HFB200301, with dose-proportional maximum concentration [Cmax], achieving sufficient exposures for peripheral target engagement in all tested doses. Emerging PD data demonstrates evidence of immune activation and expansion of CD8+ T cells, but not Tregs, in the tumor microenvironment and peripheral blood. Single cell RNAseq analysis of peripheral blood mononuclear cells demonstrated PD modulation in TNFR2 positive immune cell types post-HFB200301 treatment. Early response evaluation reveals ongoing durable clinical responses (&gt; 6 months) in a PD-L1+ pleural mesothelioma pt and an EBV+ gastric cancer pt from combination cohorts. Conclusions: HFB200301 demonstrates a favorable safety profile, dose-dependent PK, with PD and preliminary clinical activity in monotherapy and in combination with TIS in pts with heavily pre-treated refractory solid tumors. Clinical trial information: NCT05238883 .

Engineered HCMV-infected APCs enable the identification of new immunodominant HLA-restricted epitopes of anti-HCMV T-cell immunity.

Complications due to HCMV infection or reactivation remain a challenging clinical problem in immunocompromised patients, mainly due to insufficient or absent T-cell functionality. Knowledge of viral targets is crucial to improve monitoring of high-risk patients and optimise antiviral T-cell therapy. To expand the epitope spectrum, genetically-engineered dendritic cells (DCs) and fibroblasts were designed to secrete soluble (s)HLA-A*11:01 and infected with an HCMV mutant lacking immune evasion molecules (US2-6 + 11). More than 700 HLA-A*11:01-restricted epitopes, including more than 50 epitopes derived from a broad range of HCMV open-reading-frames (ORFs) were identified by mass spectrometry and screened for HLA-A*11:01-binding using established prediction tools. The immunogenicity of the 24 highest scoring new candidates was evaluated in vitro in healthy HLA-A*11:01+/HCMV+ donors. Thus, four subdominant epitopes and one immunodominant epitope, derived from the anti-apoptotic protein UL36 and ORFL101C (A11SAL), were identified. Their HLA-A*11:01 complex stability was verified in vitro. In depth analyses revealed highly proliferative and cytotoxic memory T-cell responses against A11SAL, with T-cell responses comparable to the immunodominant HLA-A*02:01-restricted HCMVpp65NLV epitope. A11SAL-specific T cells were also detectable in vivo in immunosuppressed transplant patients and shown to be effective in an in vitro HCMV-infection model, suggesting their crucial role in inhibiting viral replication and improvement of patient's outcome. The developed in vitro pipeline is the first to utilise genetically-engineered DCs to identify naturally presented immunodominant HCMV-derived epitopes. It therefore offers advantages over in silico predictions, is transferable to other HLA alleles, and will significantly expand the repertoire of viral targets to improve therapeutic options.