Increased T-cell Proliferation Research Articles

Incorporating IL7 receptor alpha signaling in the endodomain of B7H3-targeting chimeric antigen receptor T cells mediates antitumor activity in glioblastoma.

CAR-T-cell therapy has shown promise in treating hematological malignancies but faces challenges in treating solid tumors due to impaired T-cell function in the tumor microenvironment. To provide optimal T-cell activation, we developed a B7 homolog 3 protein (B7H3)-targeting CAR construct consisting of three activation signals: CD3ζ (signal 1), 41BB (signal 2), and the interleukin 7 receptor alpha (IL7Rα) cytoplasmic domain (signal 3). We generated B7H3 CAR-T cells with different lengths of the IL7Rα cytoplasmic domain, including the full length (IL7R-L), intermediate length (IL7R-M), and short length (IL7R-S) domains, and evaluated their functionality in vitro and in vivo. All the B7H3-IL7Rα CAR-T cells exhibited a less differentiated phenotype and effectively eliminated B7H3-positive glioblastoma in vitro. Superiority was found in B7H3 CAR-T cells contained the short length of the IL7Rα cytoplasmic domain. Integration of the IL7R-S cytoplasmic domain maintained pSTAT5 activation and increased T-cell proliferation while reducing activation-induced cell death. Moreover, RNA-sequencing analysis of B7H3-IL7R-S CAR-T cells after coculture with a glioblastoma cell line revealed downregulation of proapoptotic genes and upregulation of genes associated with T-cell proliferation compared with those in 2nd generation B7H3 CAR-T cells. In animal models, compared with conventional CAR-T cells, B7H3-IL7R-S CAR-T cells suppressed tumor growth and prolonged overall survival. Our study demonstrated the therapeutic potential of IL7Rα-incorporating CAR-T cells for glioblastoma treatment, suggesting a promising strategy for augmenting the effectiveness of CAR-T cell therapy.

Machine Learning Links T-cell Function and Spatial Localization to Neoadjuvant Immunotherapy and Clinical Outcome in Pancreatic Cancer.

Tumor molecular data sets are becoming increasingly complex, making it nearly impossible for humans alone to effectively analyze them. Here, we demonstrate the power of using machine learning (ML) to analyze a single-cell, spatial, and highly multiplexed proteomic data set from human pancreatic cancer and reveal underlying biological mechanisms that may contribute to clinical outcomes. We designed a multiplex immunohistochemistry antibody panel to compare T-cell functionality and spatial localization in resected tumors from treatment-naïve patients with localized pancreatic ductal adenocarcinoma (PDAC) with resected tumors from a second cohort of patients treated with neoadjuvant agonistic CD40 (anti-CD40) monoclonal antibody therapy. In total, nearly 2.5 million cells from 306 tissue regions collected from 29 patients across both cohorts were assayed, and over 1,000 tumor microenvironment (TME) features were quantified. We then trained ML models to accurately predict anti-CD40 treatment status and disease-free survival (DFS) following anti-CD40 therapy based on TME features. Through downstream interpretation of the ML models' predictions, we found anti-CD40 therapy reduced canonical aspects of T-cell exhaustion within the TME, as compared with treatment-naïve TMEs. Using automated clustering approaches, we found improved DFS following anti-CD40 therapy correlated with an increased presence of CD44+CD4+ Th1 cells located specifically within cellular neighborhoods characterized by increased T-cell proliferation, antigen experience, and cytotoxicity in immune aggregates. Overall, our results demonstrate the utility of ML in molecular cancer immunology applications, highlight the impact of anti-CD40 therapy on T cells within the TME, and identify potential candidate biomarkers of DFS for anti-CD40-treated patients with PDAC.

Janus kinase inhibitors modify the fatty acid profile of extracellular vesicles and modulate the immune response

BackgroundJanus kinase inhibitors (jakinibs) are immunomodulators used for treating malignancies, autoimmune diseases, and immunodeficiencies. However, they induce adverse effects such as thrombosis, lymphocytosis, and neutropenia that could be mediated by extracellular vesicles (EVs). These particles are cell membrane-derived structures that transport cellular and environmental molecules and participate in intercellular communication. Jakinibs can modify the content of EVs and enable them to modulate the activity of different components of the immune response. Objectiveto evaluate the interactions between immune system components of healthy individuals and EVs derived from monocytic and lymphoid lineage cells generated in the presence of baricitinib (BARI) and itacitinib (ITA) and their possible effects. MethodsEVs were isolated from monocytes (M) and lymphocytes (L) of healthy individuals, as well as from U937 (U) and Jurkat (J) cells exposed to non-cytotoxic concentrations of BARI, ITA, and dimethyl sulfoxide (DMSO; vehicle control). The binding to and engulfment of EVs by peripheral blood leukocytes of healthy individuals were analyzed by flow cytometry using CFSE-stained EVs and anti-CD45-PeCy7 mAb-labeled whole blood. The effect of EVs on respiratory burst, T-cell activation and proliferation, cytokine synthesis, and platelet aggregation was evaluated. Respiratory burst was assessed in PMA-stimulated neutrophils by the dihydrorhodamine (DHR) test and flow cytometry. T-cell activation and proliferation and cytokine production were assessed in CFSE-stained PBMC cultures stimulated with PHA; expression of the T-cell activation markers CD25 and CD69 and T-cell proliferation were analyzed by flow cytometry, and the cytokine levels were quantified in culture supernatants by Luminex assays. Platelet aggregation was analyzed in platelet-rich plasma (PRP) samples by light transmission aggregometry. The EVs’ fatty acid (FA) profile was analyzed using methyl ester derivatization followed by gas chromatography. ResultsITA exposure during the generation of EVs modified the size of the EVs released; however, treatment with DMSO and BARI did not alter the size of EVs generated from U937 and Jurkat cells. Circulating neutrophils, lymphocytes, and monocytes showed a 2-fold greater tendency to internalize ITA-U-EVs than their respective DMSO control. The neutrophil respiratory burst was attenuated in greater extent by M-EVs than by L-EVs. Autologous ITA-M-EVs reduced T-cell proliferation by decreasing IL-2 levels and CD25 expression independently of CD69. A higher accumulation of pro-inflammatory cytokines was observed in PHA-stimulated PBMC cultures exposed to M-EVs than to L-EVs; this difference may be related to the higher myristate content of M-EVs. Platelet aggregation increased in the presence of ITA-L/M-EVs by a mechanism presumably dependent on the high arachidonic acid content of the vesicles. ConclusionsCellular origin and jakinib exposure modify the FA profile of EVs, enabling them, in turn, to modulate neutrophil respiratory burst, T-cell proliferation, and platelet aggregation. The increased T-cell proliferation induced by BARI-L/M-EVs could explain the lymphocytosis observed in patients treated with BARI. The higher proportion of arachidonic acid in the FA content of ITA-L/M-EVs could be related to the thrombosis described in patients treated with ITA. EVs also induced a decrease in the respiratory burst of neutrophils.

Sting Activation Improves T-Cell Engaging Immunotherapy of Acute Myeloid Leukemia

Background: Despite emerging therapeutic options for Acute myeloid leukemia (AML) patients, for the majority of relapsed/refractory patients the outcome remains poor. Allogeneic stem cell transplantation has proven the power of T cells in eradicating residual leukemic cells, but alternative strategies based on T-cell recruiting bispecifics have so far failed to induce sustained responses. Resistance is mediated by the immunosuppressive tumor-microenvironment and secretion of immune dampening metabolites by AML cells. We hypothesized that combining a CD33-directed T cell engager (CD33 BiTE ® molecule or AMG 330) with a cGAS-STING agonist has the potential to augment anti-leukemic activity. Methods: In vitro co-culture assays of human T cells and AML cell lines for up to 72 h were performed to study the effect of AMG 330 in combination with the physiological STING-agonist 2‘-3‘ cGAMP (cGAMP). The cytotoxicity against the AML cell lines HL-60, MOLM-13 and OCI-AML-3 was assessed by flow cytometry. T-cell proliferation, T-cell degranulation (CD107a), as well as Granzyme B and TRAIL expression were characterized by flow cytometry. The secretion of effector cytokines (IFNy, TNF, IL-2, IFNa2a, CXCL10) was analyzed via CBA and ELISA assays. To better understand how AMG 330 and cGAMP work together, immunoblotting, transcriptomic analysis (bulk RNA-sequencing) and comprehensive CRISPRCas9 knockout-studies were carried out. Results: We observed a significant increase in AMG 330-mediated cytotoxicity of AML target cells upon co-application of cGAMP (Fig1A). The latter observation was enhanced upon prolongation of the culture and was also notable at low effector-to-target ratios. Monotherapy or addition of cGAMP by itself exerted only minor cytotoxicity against AML cells. The increase in AMG 330 mediated cytotoxicity through cGAMP was accompanied by an increase in T-cell proliferation, degranulation, Granzyme B secretion and TRAIL expression. Moreover, we observed a strong increase in secretion of proinflammatory cytokines (IFNy, IL-2 and IFNa2a). Bulk RNA-sequencing of AMG 330 and cGAMP stimulated co-cultures revealed type-I-IFN signature in the target (HL-60) and the effector cell (T cells) cell population. In addition, distinct phenotypes were found in each cell population: T cells demonstrated a marked increase in TNFa, IFNy and GZMB expression through the co-treatment, whereas HL-60 cells displayed an IFNy signaling signature (Fig1B). Importantly, the responsiveness of AML target cells to STING activation in terms of interferon stimulated gene (ISG)-induction was increased by the co-treatment, evidenced by further upregulation of STAT1, IRF7, WARS, CXCL11, IFI16, RSAD2 and CXCL10. An increase in type-I-IFN production and subsequent ISG-induction could also be confirmed on the protein level by measuring IFNα2a and CXCL-10. Knockout-studies revealed that the cGAMP enhanced AMG 330-mediated cytotoxicity was fully dependent on target cell intrinsic STING and IRF3 signaling, but also dependent on functional IFNy and TNFa signaling. Notably, the increased IFNy production by T cells upon cGAMP addition was dependent on functional target cell STING and IFNy signaling. These observations imply a crosstalk between effector and target cells and an intricate interdependence between target cell intrinsic IFNγ- and STING-signaling. Of note, the deletion of the IFNy-receptor in AML target cells also reduced AMG 330-dependent cytotoxicity under steady state conditions. Finally, we demonstrated that the combination of IFNy, TNFa and cGAMP sufficed to induce an apoptotic cell death of AML cells, similar to the cGAMP containing T-cell-HL-60 cocultures. Conclusion: We propose a novel mechanism by which AMG 330-activated T cells prime and sensitize AML target cells in a forward feedback loop towards STING activation, leading to increased type-I-IFN production and induction of ISGs. The beneficial effect of physiological cGAMP in enhancing AMG 330-mediated cytotoxicity was accompanied by the pronounced expression of effector cytokines and an overall cytotoxic T-cell phenotype. We established a key role for interferon gamma in AMG 330-mediated cytotoxicity of AML cells and in rendering AML cells responsive to STING agonism. We are currently conducting in vivoexperiments to further validate our findings.

A synthetic biology approach to engineering circuits in immune cells.

A synthetic circuit in a biological system involves the designed assembly of genetic elements, biomolecules, or cells to create a defined function. These circuits are central in synthetic biology, enabling the reprogramming of cellular behavior and the engineering of cells with customized responses. In cancer therapeutics, engineering T cells with circuits have the potential to overcome the challenges of current approaches, for example, by allowing specific recognition and killing of cancer cells. Recent advances also facilitate engineering integrated circuits for the controlled release of therapeutic molecules at specified locations, for example, in a solid tumor. In this review, we discuss recent strategies and applications of synthetic receptor circuits aimed at enhancing immune cell functions for cancer immunotherapy. We begin by introducing the concept of circuits in networks at the molecular and cellular scales and provide an analysis of the development and implementation of several synthetic circuits in T cells that have the goal to overcome current challenges in cancer immunotherapy. These include specific targeting of cancer cells, increased T-cell proliferation, and persistence in the tumor microenvironment. By harnessing the power of synthetic biology, and the characteristics of certain circuit architectures, it is now possible to engineer a new generation of immune cells that recognize cancer cells, while minimizing off-target toxicities. We specifically discuss T-cell circuits for antigen density sensing. These circuits allow targeting of solid tumors that share antigens with normal tissues. Additionally, we explore designs for synthetic circuits that could control T-cell differentiation or T-cell fate as well as the concept of synthetic multicellular circuits that leverage cellular communication and division of labor to achieve improved therapeutic efficacy. As our understanding of cell biology expands and novel tools for genome, protein, and cell engineering are developed, we anticipate further innovative approaches to emerge in the design and engineering of circuits in immune cells.

The immunomodulatory role of withania somnifera (L.) dunal in inflammatory diseases.

Withania somnifera (L.) Dunal (Solanaceae) (also known as Ashwagandha) is a botanical drug that has been used for centuries to treat many chronic diseases like high blood pressure, arthritis, diabetes, Alzheimer's disease, and depression. As many botanical drugs, w. Somnifera possesses anti-inflammatory, antioxidant, anticarinogenic, anti-diabetic, and anti-asthmatic properties. W. somnifera is often compared to the ginseng plant due to its ability to reduce stress, improve cognitive functions (e.g., memory), and promote a healthy immune system. It promotes immunomodulatory effects whose function is to balance the humoral and cellular responses of the adaptive immune system. The therapeutic effect of w. Somnifera is attributed to active ingredients like alkaloids, steroidal lactones (such as withanolides, withaferins), and steroidal saponins. Although w. Somnifera is safe and highly recommended for treating various diseases, the current knowledge and understanding of its operational mechanisms are limited. One of the proposed mechanisms states that w. Somnifera promotes cellular-mediated immunity or initiates chemical interactions that contribute to therapeutic effects. Withania somnifera has been shown to play a significant role in immunological diseases by modulating several cytokines, increasing T-cell proliferation and enhancing macrophages functions. In this review, we will discuss the latest therapeutic effects of w. Somnifera on a number of diseases through modulating immunological markers and which specific components of w. Somnifera induce these therapeutic activities. We will also focus on the chemical properties in w. Somnifera components and their immunomodulatory role in type 2 allergic diseases where type 2 inflammation is highly imbalanced.

Abstract P3068: TNFR1 Regulates Cellular Proliferation And Not Pathogenicity Of Cd4 + T-lymphocytes During Ischemic Heart Failure

Ischemic heart failure (HF) is associated with elevated inflammatory responses including pro-inflammatory cytokines, such as Tumor necrosis factor (TNF) α [and its cognate receptors, TNF receptor (TNFR) 1 and TNFR2] and pathological switching of CD4 + T-cells. TNF-TNFR2 signaling is known to promote CD4 + T-cell activation and proliferation. However, it is unknown if CD4 + T-cells also express TNFR1, and if elevated TNFα-TNFR1 signaling during HF mediates pathological effects of CD4 + T-cells. Using flow cytometry, we show that TNFR1 + CD4 + T-cells are amplified in the hearts at 3 days (d) and 8 weeks (wks) post-MI (HF) in mice. As compared to sham, while TNFα expression (and %) is blunted in CD4 + T-cells at 3 d post-MI, it is increased during HF with significantly higher expression in TNFR1 + T-cells as compared to TNFR1 - T-cells. Splenic CD4 + T-cells activated using anti-CD3/CD28 antibodies and treated with neutralizing anti-TNFR1 antibody showed increased T-cell proliferation, and Bcl-xL (pro-survival factor) and TNFα expression without affecting activation marker (CD69) suggesting a role of TNFR1 in mediating pro-apoptotic signaling without affecting activation. To confirm these results, we adoptively transferred (AT) HF-activated splenic WT and TNFR1 -/- T-cells (CD45.2) into CD45.1 naïve mice, and measured changes in cardiac function of recipient mice by echocardiography (as an indicator of pathological activity), and donor T-cells (as a measure of cell survival) in the blood, heart, spleen and mediastinal lymph nodes (mLNs) at 14 wks post-AT. Compared to WT, we observed amplified levels of donor TNFR1 -/- T-cells in the blood, spleen, and mLNs which were not different in the hearts. Consistently, recipient mice injected either with WT and TNFR1 -/- T-cells showed similar degrees of cardiac dysfunction. Interestingly, at 14 wks post-AT the expression of ki67, a proliferation marker, was magnified in HF-activated TNFR1 -/- T-cells (as compared to WT) infiltrated into the myocardium suggesting that TNF-TNFR1 mediates pro-survival and proliferative signaling in T-cells during HF, and not their pathogenicity. Our results also provide deeper insights into the paradoxical outcomes observed in the clinical trials antagonizing TNFα in human HF patients.

S173: T-CELL ACTIVATION AND MYELOMA CELL KILLING CONFIRM THE MODE OF ACTION OF RG6234, A NOVEL GPRC5D T-CELL ENGAGING BISPECIFIC ANTIBODY, IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA

Background: RG6234 is a novel T-cell engaging bispecific antibody targeting G protein-coupled receptor 5D (GPRC5D) with a unique 2:1 format. GPRC5D is highly expressed on multiple myeloma (MM) cells and concurrent binding of RG6234 to GPRC5D and CD3 on T cells results in immunological synapse formation and potent T-cell directed tumor cell killing. An ongoing Phase 1 dose-escalation study (NCT04557150) is investigating the safety, clinical activity, pharmacodynamics (PD), and pharmacokinetics of RG6234 monotherapy in patients (pts) with relapsed/refractory MM (RRMM). Clinical activity was observed during dose escalation, and the safety profile was manageable (Riley et al. EHA 2022). Aims: Here, we present preliminary clinical biomarker data highlighting PD effects after intravenous (IV) administration that confirm the mechanism of action and high potency of RG6234. Methods: Exploratory biomarker analyses included data from pts treated with RG6234 doses ranging from 0.006mg to 4.8mg in dose escalation. RG6234 was administered as an IV infusion under a step-up dosing regimen, reaching the target dose not later than 2 weeks after the priming dose. Peripheral biomarkers were evaluated using whole blood flow cytometry (n=28), plasma cytokine Protein Simple ELLA (n=33), and plasma sBCMA Protein Simple ELLA (n=26). MM cells were assessed at baseline and on-treatment by bone marrow (BM) aspirate flow cytometry and by BM biopsy CD138/CD8 immunohistochemistry. Informed consent was obtained from participating pts. The clinical cut-off date for the current analysis was January 31, 2022. Results: PD changes were observed in peripheral blood at all tested doses. Cytokines (IFNg, TNFa, CXCL10, IL6, IL10, IL2, IL8) and sCD25 peaked at 4 to 24 hours (h) following the first administration, while cytokine peak magnitudes decreased at subsequent administrations. Cytokine release was followed by transient reduction of circulating T cells at 4 h after infusion, with partial recovery of peripheral T-cell counts by Day 8 after first administration. Elevation of sCD25 and IFNg in plasma (~3.2 and 33 median fold change from baseline, respectively) together with increase in T-cell proliferation (~4 median fold increase of Ki67+CD8+ T-cells) within 72 h post first infusion indicated T-cell activation. Analysis of a limited number of paired baseline and on-treatment BM biopsies (n=13) revealed that the density of CD8+ tumor-infiltrating T cells increased upon treatment in responders, indicating T-cell recruitment towards the tumor. RG6234 induced rapid depletion of MM cells, as demonstrated by a decrease of sBCMA in the plasma of responding pts already 8 days after the first administration (median 33.5% reduction from baseline in responding pts; n=15). Moreover, at the end of Cycle 1, the majority of pts (14/15) had <1% of MM cells in BM based on flow cytometry readout. GPRC5D expression was detected at baseline in all pts with evaluable bone marrow aspirate and >20 detectable MM cells (n=16). Updated exploratory biomarker data will be presented. Summary/Conclusion: Cytokine release, T-cell activation, BM infiltration, and MM cell depletion are early PD changes seen after treatment with RG6234 and precede clinical responses. These PD changes indicate that RG6234 leads to T-cell engagement in the BM of pts with RRMM and clearly demonstrate rapid and effective T-cell mediated anti-MM activity.

A phase 1/2 dose escalation/expansion study of OR2805 alone or in combination in subjects with advanced solid tumors.

TPS2693 Background: M2-like macrophages are thought to promote an immunoinhibitory environment and prevent activation and proliferation of T cells, leading to resistance to checkpoint inhibitor (CPI) therapy. OR2805 is a fully human monoclonal antibody identified in a patient who had an exceptional response to CPI. OR2805 recognizes CD163, an immunosuppressive scavenger receptor highly expressed on M2 tumor-associated macrophages (TAMs). OR2805 treatment reprograms M2 macrophages to take on an immunostimulatory, M1-like phenotype. In macrophage/T cell coculture assays, OR2805 treatment relieved the suppressive effect of M2c macrophages as demonstrated by increased T-cell proliferation and stimulation of IFN-γ and perforin release. OR2805 restored the IFN-γ production of exhausted T cells and showed a synergistic effect on cocultures treated in combination with CPI. OR2805 monotherapy demonstrated significant anti-tumor activity in lung cancer xenograft models in NSG-SGM3 mice. Relieving the immune suppression of CD163-expressing TAMs by OR2805 to improve anti-tumor T-cell responses will be evaluated alone and in combination with CPI therapy. Methods: This phase 1/2, open-label, multicenter, dose-escalation/expansion study is assessing the safety, tolerability and preliminary activity of OR2805 alone and in combination with anti-PD-1 or chemotherapy in up to 325 adults (≥18 years) with histologically or cytologically confirmed metastatic or locally advanced solid tumors. The study is divided into 3 parts. Part A will enroll subjects with advanced solid tumors of any histology to describe the preliminary safety profile of OR2805 and determine the recommended phase 2 dose (RP2D) of OR2805 alone and in combination with an anti-PD-1 monoclonal antibody. Part B will further characterize the safety and preliminary antitumor activity of OR2805 either alone or in combination with an anti-PD-1 or chemotherapy in previously treated non-small cell lung cancer (NSCLC), melanoma and liposarcoma, as well as in previously untreated NSCLC in combination with an anti-PD-1 antibody. Part C will evaluate OR2805 monotherapy in relapsed squamous cell carcinoma of the head and neck and other tumor types with an emphasis on correlative biomarker analysis. OR2805 will be infused on Day 1 of 21-day cycles. Primary endpoints are adverse events, laboratory abnormalities, dose-limiting toxicities, and dose-level safety and activity. Secondary endpoints are objective response rates, duration of response, complete response, progression-free survival, overall survival, PK, and antidrug antibodies. Exploratory biomarkers of OR2805-mediated pharmacodynamic effects and immune stimulation will be explored. The study was opened September 2021 and is enrolling at sites in the United States with planned expansion within the US and to Europe. Clinical trial information: Clinical trial information: NCT05094804.

Design and Evaluation of TIM-3-CD28 Checkpoint Fusion Proteins to Improve Anti-CD19 CAR T-Cell Function.

Therapeutic targeting of inhibitory checkpoint molecules in combination with chimeric antigen receptor (CAR) T cells is currently investigated in a variety of clinical studies for treatment of hematologic and solid malignancies. However, the impact of co-inhibitory axes and their therapeutic implication remains understudied for the majority of acute leukemias due to their low immunogenicity/mutational load. The inhibitory exhaustion molecule TIM-3 is an important marker for the interaction of T cells with leukemic cells. Moreover, inhibitory signals from malignant cells could be transformed into stimulatory signals by synthetic fusion molecules with extracellular inhibitory receptors fused to an intracellular stimulatory domain. Here, we designed a variety of different TIM-3-CD28 fusion proteins to turn inhibitory signals derived by TIM-3 engagement into T-cell activation through CD28. In the absence of anti-CD19 CAR, two TIM-3-CD28 fusion receptors with large parts of CD28 showed strongest responses in terms of cytokine secretion and proliferation upon stimulation with anti-CD3 antibodies compared to controls. We then combined these two novel TIM-3-CD28 fusion proteins with first- and second-generation anti-CD19 CAR T cells and found that the fusion receptor can increase proliferation, activation, and cytotoxic capacity of conventional anti-CD19 CAR T cells. These additionally armed CAR T cells showed excellent effector function. In terms of safety considerations, the fusion receptors showed exclusively increased cytokine release, when the CAR target CD19 was present. We conclude that combining checkpoint fusion proteins with anti-CD19 CARs has the potential to increase T-cell proliferation capacity with the intention to overcome inhibitory signals during the response against malignant cells.

Elranatamab (PF-06863135), a B-Cell Maturation Antigen (BCMA) Targeted CD3-Engaging Bispecific Molecule, for Patients with Relapsed or Refractory Multiple Myeloma: Results from Magnetismm-1

Elranatamab (PF-06863135), a B-Cell Maturation Antigen (BCMA) Targeted CD3-Engaging Bispecific Molecule, for Patients with Relapsed or Refractory Multiple Myeloma: Results from Magnetismm-1

Mapping the High-Risk Multiple Myeloma Cell Surface Proteome Identifies T-Cell Inhibitory Receptors for Immune Targeting

Multiple myeloma (MM) is an incurable malignancy of mature plasma cells. Despite major advances in the therapeutic armamentarium of MM, only 50% of patients survive more than 5 years after diagnosis, with significantly lower rates (21%) for high-risk patients.Chimeric Antigen Receptor (CAR) T-cell therapy targeting BCMA (B-cell maturation antigen) shows high response rates in relapsed/refractory patients. However, most patients have disease remission that lasts less than 18 months, prompting the search for additional and synergistic therapeutic approaches.We unbiasedly mapped the cell surface proteome of MM by integrating Mass-Spectrometry (MS) and RNA-seq analyses from 7 MM cell lines and 904 primary MM patient samples bearing high-risk cytogenetics. To identify cell surface proteins, we ran a pool of 4,761 proteins and 16,000+ transcripts through five repositories. An integrated scoring database was developed by scoring each ID based on the number of databases (0-5) it was identified in, with 0 if the molecule was not found in any and 5 if the protein was found in all five.We identified 402 proteins with a surface score of 3 or higher in MM cell lines and patient samples by transcriptomics and proteomics. We prioritized the 326 candidates that were more highly expressed in patients. Based on functional enrichment analyses, we found the proteins formed three main networks with immune mechanisms representing the largest cluster (227 out of 326 cell surface proteins) followed by transporters and adhesion proteins.Based on a pipeline we previously established (1), we further selected 97 candidates minimally expressed in normal tissues. This list included current therapeutic targets such as BCMA, SLAMF7, ITGB7 and LY9. Validation in primary patient samples by western blot and flow-cytometric analyses, enabled the identification of 10 top candidates (CCR1, CD320, FCRL3, IL12RB1, ITGA4, LAX1, LILRB4, LRRC8D, SEMA4A, SLAMF6) that resulted most frequently and highly expressed. We found that LAX1, LILRB4 and SEMA4A significantly impact myeloma patient overall survival based on Kaplan-Meier analysis in the MM Research Foundation (MMRF) cohort (2). CCR1, IL12RB1, LILRB4 and SEMA4A were upregulated by the treatment with Bortezomib or Venetoclax that conversely, decreased BCMA expression in MM U266 cells. By stratifying the patient population, we found that the SEMA4A and LAX1 were up-regulated in patients with t(4;14) compared to patients with no cytogenetic abnormality; LILRB4 in patients with t(14;16) and CCR1 patients with t(14;16) and t(14;20). By calculating co-expression levels CCR1-LILRB4 and CCR1-FCRL3 resulted co-expressed in 100% of patients. For safety purposes (3), we excluded candidates with high (>55%) protein abundance in highly-purified normal hematopoietic stem cells and activated T-cells, narrowing down the list to 6 top candidates (CCR1, FCRL3, IL12RB1, LILRB4, LRRC8D, SEMA4A).To define the function of this group of promising cell surface targets, we used a CRISPR/Cas9 inducible system in KMS11 MM cells. We found that knock-out of CCR1, LRRC8D and SEMA4A individually reduces the MM cell growth by ~60%, 50% and 50% respectively, and almost completely abrogates MM cell migration through porous chambers by >80%. By co-culturing irradiated KO and control MM cells with healthy donor T-cells we also found that lack of CCR1 increased T-cell proliferation by 50% compared to controls and enhanced killing of MM cells, suggesting that CCR1 may suppress T-cell mediated immune responses in addition to play a role in MM cell survival and migration.This study suggests the contribution of an altered MM surfaceome to disease development and may lead to potential novel immunotherapeutic approaches for high-risk MM.

EFFICACY AND SAFETY OF ELRANATAMAB (PF-06863135), A B-CELL MATURATION ANTIGEN (BCMA)-CD3 BISPECIFIC ANTIBODY, IN PATIENTS WITH RELAPSED OR REFRACTORY MULTIPLE MYELOMA

Elranatamab (PF-06863135) is a humanized bispecific monoclonal antibody (IgG2a) that targets BCMA, a member of the tumor necrosis factor receptor superfamily expressed in multiple myeloma, and CD3 on T cells. We reported results for intravenous (IV) dosing (Raje et al. Blood. 2019;134(S1):1869) and now update for subcutaneous (SC) dosing from the ongoing Phase 1 study (MagnetisMM-1; NCT03269136). Patients received elranatamab at 80, 130, 215, 360, 600, and 1000 μg/kg SC weekly. A modified toxicity probability interval method was used for escalation, with monitoring for dose-limiting toxicity (DLT) to end of the first cycle. Treatment-emergent adverse events (TEAEs) were graded by Common Terminology Criteria for Adverse Events (v4.03), and cytokine release syndrome (CRS) by American Society for Transplantation and Cellular Therapy criteria (Lee et al. Biol Blood Marrow Transplant. 2019;25:625). Response was assessed by International Myeloma Working Group criteria. Pharmacokinetics, cytokine profiling, and T cell immunophenotyping were performed. 30 patients had received elranatamab as of 4-Feb-2021 at 80 (n = 6), 130 (n = 4), 215 (n = 4), 360 (n = 4), 600 (n = 6), or 1000 (n = 6) μg/kg SC weekly. Patients had a median of 8 prior treatments; 87% had triple refractory disease, 97% had prior anti-CD38 therapy, and 23% (7 patients) had prior BCMA-directed antibody drug conjugate (6 of 7 patients) or chimeric antigen receptor T cell therapy (3 of 7 patients). The most common all causality TEAEs included lymphopenia (n = 25, 83%; 20% G3, 63% G4), CRS (n = 22, 73%; 57% G1, 17% G2, none ≥G3), anemia (n = 18, 60%; 50% G3, 0% G4), thrombocytopenia (n = 16, 53%; 17% G3, 20% G4), neutropenia (n = 16, 53%; 23% G3, 30% G4), and injection site reaction (n = 15, 50%; 43% G1, 7% G2, none ≥G3). Both CRS and immune effector cell-associated neurotoxicity syndrome (n = 6, 20%) were limited to ≤G2 with median durations of 3 and 2.5 days, respectively. No DLT was observed. Exposure increased with dose, and T max ranged from 3–7 days. Cytokine increases occurred with the first dose, and increased T-cell proliferation was observed in peripheral blood. The overall response rate (ORR) for doses ≥215 μg/kg was 70% (n = 14/20) including partial response (PR; n = 1), very good PR (VGPR; n = 7), complete response (CR; n = 1), and stringent CR (sCR; n = 5). Median time to response was 22 days, and 3 of 4 patients (75%) with prior BCMA-directed therapy achieved response (VGPR, n = 2 and sCR, n = 1). Updated data, including duration of response, will be presented. Elranatamab demonstrated a manageable safety profile and, across the efficacious dose range (≥215 μg/kg), achieved ORR of 70% with CR/sCR rate of 30%, including responses after prior BCMA-directed therapy. These results demonstrate the safety and efficacy of elranatamab in this relapsed/refractory population and support ongoing development of elranatamab for patients with multiple myeloma, both as monotherapy and in combination with standard or novel therapies.

The effect of Curcumin on multi-level immune checkpoint blockade and T cell dysfunction in head and neck cancer

BackgroundDespite recent advances in understanding the complex immunologic dysfunction in the tumor microenvironment (TME), fewer than 20% of patients with head and neck squamous cell carcinoma (HNSCC) respond to immune checkpoint blockade (ICB). Thus, it is important to understand how inhibitory IC receptors maintain the suppressed dysfunctional TME, and to develop more effective combination immunotherapy. This study evaluated the immune-modulating effects of Curcumin, which has well-established anti-cancer and chemopreventive properties, and its long-term safety as a phytochemical drug. MethodsWe carried out the western blot and small interfering RNA (siRNA) transfection assay to evaluate the effects of Curcumin on IC ligands and IC ligands function in HNSCC. Through T-cell cytotoxicity assay and measurements of cytokine secretion, we assessed the effects of combination of Curcumin with programmed death-ligand 1 (PD-L1) Ab on cancer cell killing. Flow cytometry were used to analyze the effects of Curcumin on the expression of programmed cell death protein 1 (PD-1) and T-cell immunoglobulin and mucin-domain3 (TIM-3) on CD4, CD8 and Treg. Immunofluorescence, immunohistochemistry and western blot were used to detecte the cytokine (IFN-γ, Granzyme B), IC receptors (PD-1 and TIM-3) and its ligands (PD-L1, PD-L2, Galectin-9) in xenograft mouse model and 4-nitroquinoline-1-oxide (4-NQO) oral cancer model. ResultsWe found that Curcumin decreased the expression of IC ligands such as PD-L1, PD-L2, and Galectin-9 in HNSCC, leading to regulation of epithelial-to-mesenchymal transition-associated tumor invasion. Curcumin also effectively restored the ability of CD8+ cytotoxic T cells to lyse cancer cells. To evaluate the effect of Curcumin on the TME further, the 4-NQO oral cancer model was used. Curcumin increased T-cell proliferation, tumor-infiltrating lymphocytes (TILs), and effector cytokines, and decreased the expression of PD-1, TIM-3, suppressive IC receptors and their ligands (PD-L1, PD-L2, and Galectin-9) in the TME, implying reinvigoration of the exhausted CD8+ T cells. In addition, Curcumin inhibited expression of CD4+CD25+FoxP3+ Treg cells as well as PD-1 and TIM-3. ConclusionsThese results show that Curcumin reinvigorates defective T cells via multiple (PD-1 and TIM-3) and multi-level (IC receptors and its ligands) IC axis suppression, thus providing a rationale to combine Curcumin with conventional targeted therapy or ICB as a multi-faceted approach for treating patients with HNSCC.

MM-379: MagnetisMM-1: A Study of Elranatamab (PF-06863135), a B-Cell Maturation Antigen (BCMA)-Targeted, CD3-Engaging Bispecific Antibody, for Patients with Relapsed or Refractory Multiple Myeloma (MM)

<h3>Context</h3> Elranatamab (PF-06863135), a humanized bispecific monoclonal antibody (IgG2a), targets BCMA expressed in MM and CD3 on T-cells. <h3>Objective</h3> Characterize efficacy, safety, pharmacokinetics, and pharmacodynamics of elranatamab. <h3>Design</h3> Phase 1 study (MagnetismMM-1; NCT03269136). Data cut-off: 4 February 2021. <h3>Patients</h3> Relapsed or refractory MM. <h3>Intervention</h3> 80, 130, 215, 360, 600, or 1000 µg/kg/week subcutaneously. <h3>Main Outcome Measures</h3> 1) Treatment-emergent adverse events (TEAEs) graded by CTCAE v4.03 and cytokine release syndrome (CRS) by ASTCT criteria. 2) International Myeloma Working Group criteria-assessed response. 3) Pharmacokinetics, cytokine profiling, and T-cell immunophenotyping. <h3>Results</h3> Thirty patients received elranatamab at 80 (n=6), 130 (n=4), 215 (n=4), 360 (n=4), 600 (n=6), or 1000 (n=6) µg/kg. Patients had a median of 8 prior treatments; 87% had triple refractory disease, 97% prior anti-CD38 therapy, and 23% prior BCMA-directed antibody drug conjugate or chimeric antigen receptor T-cell therapy. Common all causality TEAEs included lymphopenia (n=25, 83%; 20% G3, 63% G4), CRS (n=22, 73%; none >G2), anemia (n=18, 60%; 50% G3, 0% G4), neutropenia (n=16, 53%; 23% G3, 30% G4), thrombocytopenia (n=16, 53%; 17% G3, 20% G4), and injection site reaction (n=15, 50%; none >G2). CRS and immune effector cell-associated neurotoxicity syndrome (n=6, 20%) were limited to ≤G2; median durations were 3 and 2.5 days, respectively. No dose-limiting toxicity was observed. Exposure increased with dose and Tmax ranged from 3–7 days. Cytokine increases occurred with the first dose, and increased T-cell proliferation was observed in peripheral blood. For doses ≥215 µg/kg, the confirmed overall response rate (ORR) was 70% (n=14/20); partial response (PR; n=1), very good PR (VGPR; n=7), complete response (CR; n=1), and stringent CR (n=5). Median time to response was 22 days. In this dosing group, 3 of 4 pts (75%) with prior BCMA-directed therapy achieved response. Confirmed ORR at the recommended phase 2 dose (RP2D) of 1000 µg/kg was 83% (n=5/6). <h3>Conclusions</h3> Elranatamab achieved confirmed ORR of 83% at RP2D with a manageable safety profile for patients with relapsed or refractory MM. These results support further development of elranatamab for patients with MM. <i>This study was sponsored by Pfizer.</i>

125I seed combined with DCS loaded with tumor antigen to convert irradiated liver cancer into effective in situ vaccine.

e14573 Background: With unique feature of self-immune tolerance and high radio-sensitivity, hepatocellular carcinoma (HCC) can gradually develop undesired effects. Iodine-125 (125I) seed, as a source of internal radiation therapy, may have a favorable adaptability and immunostimulation. In this study, we investigated whether 125I seeds can stimulate local tumor infiltrating lymphocytes (TILs) and whether a combination of 125I seeds and dendritic cells (DCs) loaded with tumor antigen (Ag+DCs) can enhance both local tumor control and abscopal effect in murine subcutaneous tumor models. The related mechanism of anti-tumor immune response was also explored. Methods: H22/Hepa1-6 HCC cells were examined for radiosensitivity and immunosensitivity. Cell apoptosis and expression of MHC-I (major histocompatibility complex class I) and PD-L1 (programmed death-ligand 1) were detected before and after irradiation. The tumor cells were injected subcutaneously as primary and abscopal tumor. Bone marrow-derived dendritic cells (BM-DCs) were induced from mouse bone marrow cells and incubated with tumor cell lysate. 125I seeds were implanted into the primary tumors, followed by intratumoral injection of Ag+DCs and intraperitoneal injection of αPD1 antibody (αPD1-ab). DC migration was detected by transwell chambers in vitro and CFSE (carboxyfluorescein succinimidyl ester)-labeled DCs in vivo. DC maturation was tested by flow cytometry. Analyses of tumor growth and survival rates were performed in vitro, and that of, TILs, T-cell proliferation, cytotoxicity test, immunostimulatory cytokines release, and immunological memory were performed in vivo. In situ vaccine and abscopal effect were verified in orthotopic mouse models of liver cancer and lung metastatic tumor. Results: 125I seeds alone could induce TILs and activate cytotoxic T cells (CTLs), but these effects were limited. The combination treatment of 125I seeds with Ag+DC administration inhibited primary tumor growth and significantly prolonged survival time in association with a significant increase in T-cell proliferation and interferon-γ release. In addition, triple-combination treatment with αPD1-ab amplified these responses, leading to significant regression of primary and metastatic tumors and successful stimulation of immunological memory. Conclusions: 125I seeds can safely activate anti-tumor immune response.125I seeds combined with Ag+DC administration is able to convert irradiated liver cancer into effective in situ vaccine. Furthermore, triple-combination therapy of 125I seeds, Ag+DC, and αPD1-ab can be a promising approach to activate systemic anti-tumor immunity, which is a potential novel individualized therapy for patients with solid cancers.

Efficacy and safety of elranatamab (PF-06863135), a B-cell maturation antigen (BCMA)-CD3 bispecific antibody, in patients with relapsed or refractory multiple myeloma (MM).

8006 Background: Elranatamab (PF-06863135) is a humanized bispecific monoclonal antibody (IgG2a) that targets BCMA, a member of the tumor necrosis factor receptor superfamily expressed in MM, and CD3 on T cells. We reported results for intravenous (IV) dosing (Raje et al. Blood. 2019;134(S1):1869) and now update for subcutaneous (SC) dosing from the ongoing Phase 1 study (MagnetisMM-1). Methods: Patients (pts) received elranatamab at 80, 130, 215, 360, 600, and 1000μg/kg SC weekly. A modified toxicity probability interval method was used for escalation, with monitoring for dose-limiting toxicity (DLT) to end of the first cycle. Treatment-emergent adverse events (TEAEs) were graded by Common Terminology Criteria for Adverse Events (v4.03), and cytokine release syndrome (CRS) by American Society for Transplantation and Cellular Therapy criteria (Lee et al. Biol Blood Marrow Transplant. 2019;25:625). Response was assessed by International Myeloma Working Group criteria. Pharmacokinetics, cytokine profiling, and T cell immunophenotyping were performed. Results: 30 pts had received elranatamab as of 4-Aug-2020 at 80 (n = 6), 130 (n = 4), 215 (n = 4), 360 (n = 4), 600 (n = 6), or 1000 (n = 6) μg/kg SC weekly. Pts had a median of 8 prior treatments; 87% had triple refractory disease, 97% had prior anti-CD38 therapy, and 23% had prior BCMA-directed antibody drug conjugate or chimeric antigen receptor T cell therapy. The most common all causality TEAEs included lymphopenia (n = 24, 80%; 20% G3, 60% G4), CRS (n = 22, 73%; none &gt; G2), anemia (n = 17, 57%; 43% G3, 3% G4), injection site reaction (n = 16, 53%; none &gt; G2), thrombocytopenia (n = 16, 53%; 23% G3, 17% G4), and neutropenia (n = 12, 40%; 17% G3, 17% G4). Both CRS and immune effector cell-associated neurotoxicity syndrome (n = 6, 20%) were limited to ≤G2 with median durations of 2 and 1.5 days, respectively. No DLT was observed. Exposure increased with dose, and Tmax ranged from 3–7 days. Cytokine increases occurred with the first dose, and increased T-cell proliferation was observed in peripheral blood. The overall response rate (ORR) for doses ≥215μg/kg was 75% (n = 15/20) including partial response (PR; n = 6), very good PR (VGPR; n = 3), complete response (CR; n = 1), and stringent CR (sCR; n = 5). Median time to response was 22 days, and 3 of 4 pts (75%) with prior BCMA-directed therapy achieved response (VGPR, n = 2 and sCR, n = 1). Updated data, including duration of response, will be presented. Conclusions: Elranatamab demonstrated a manageable safety profile, and SC doses ≥215μg/kg achieved ORR of 75% with CR/sCR rate of 30%. These results demonstrate the safety and efficacy of SC elranatamab in this relapsed/refractory population and support ongoing development of elranatamab for pts with MM, both as monotherapy and in combination with standard or novel therapies. Clinical trial information: NCT03269136.

Tuning Antibody Presentation to Enhance T-Cell Activation for Downstream Cytotoxicity

Cytotoxic effector cells are an integral component of the immune response against pathogens and diseases such as cancer and thus of great interest to researchers who wish to enhance the native immune response. Although researchers routinely use particles to stimulate cytotoxic T cells, few studies have comprehensively investigated: (1) beyond initial activation responses (i.e., proliferation and CD25/CD69 expression) to downstream cancer-killing effects and (2) how to drive cytotoxic T-cell responses by adjusting biomolecular and physical properties of particles. In this study, we designed particles displaying an anti-CD3 antibody to activate cytotoxic T cells and study their downstream cytotoxic effects. We evaluated the effect of antibody immobilization, particle size, molecular surface density of an anti-CD3 antibody, and the inclusion of an anti-CD28 antibody on cytolytic granule release by T cells. We found that immobilizing the anti-CD3 antibody onto smaller nanoparticles elicited increased T-cell activation products for an equivalent delivery of the anti-CD3 antibody. We further established that the mechanism behind increased cancer cell death was associated with the proximity of T cells to cancer cells. Functionalizing particles additionally with the anti-CD28 antibody at an optimized antibody density caused increased T-cell proliferation and T-cell binding but we observed no effective increase in cytotoxicity. Meaningfully, our results are discussed within the context of commercially available and widely used anti-CD3/28 Dynabeads. These results showed that T-cell activation and cytotoxicity can be optimized with a molecular presentation on smaller particles and thus, offer exciting new possibilities to engineer T-cell activation responses for effective outcomes.

Immune monitoring in mesothelioma patients identifies novel immune-modulatory functions of gemcitabine associating with clinical response.

BackgroundGemcitabine is a frequently used chemotherapeutic agent but its effects on the immune system are incompletely understood. Recently, the randomized NVALT19-trial revealed that maintenance gemcitabine after first-line chemotherapy significantly prolonged progression-free survival (PFS) compared to best supportive care (BSC) in malignant mesothelioma. Whether these effects are paralleled by changes in circulating immune cell subsets is currently unknown. These analyses could offer improved mechanistic insights into the effects of gemcitabine on the host and guide development of effective combination therapies in mesothelioma.MethodsWe stained peripheral blood mononuclear cells (PBMCs) and myeloid-derived suppressor cells (MDSCs) at baseline and 3 weeks following start of gemcitabine or BSC treatment in a subgroup of mesothelioma patients included in the NVALT19-trial. In total, 24 paired samples including both MDSCs and PBMCs were included. We performed multicolour flow-cytometry to assess co-inhibitory and-stimulatory receptor- and cytokine expression and matched these parameters with PFS and OS.FindingsGemcitabine treatment was significantly associated with an increased NK-cell- and decreased T-regulatory cell proliferation whereas the opposite occurred in control patients. Furthermore, myeloid-derived suppressor cells (MDSCs) frequencies were lower in gemcitabine-treated patients and this correlated with increased T-cell proliferation following treatment. Whereas gemcitabine variably altered co-inhibitory receptor expression, co-stimulatory molecules including ICOS, CD28 and HLA-DR were uniformly increased across CD4+ T-helper, CD8+ T- and NK-cells. Although preliminary in nature, the increase in NK-cell proliferation and PD-1 expression in T cells following gemcitabine treatment was associated with improved PFS and OS.InterpretationGemcitabine treatment was associated with widespread effects on circulating immune cells of mesothelioma patients with responding patients displaying increased NK-cell and PD-1 + T-cell proliferation. These exploratory data provide a platform for future on treatment-biomarker development and novel combination treatment strategies.

The mTOR-glycolytic pathway promotes T-cell immunobiology in oral lichen planus.

Oral lichen planus (OLP) is a T-cell-mediated inflammatory mucosal disease. T cells require rapid metabolic reprogramming for their effector functions after activation by immunologic stimuli. The mammalian target of rapamycin (mTOR) is a central player in the metabolic reprogramming and immune responses of T cells. The present study investigated the role of mTOR in the immunometabolism of OLP. mTOR and its direct target eukaryotic initiation factor 4E binding protein 1 (4E-BP1) were highly phosphorylated in peripheral T cells of OLP patients. Rapamycin-mediated blockage of mTOR activation restrained both T-cell proliferation and DNA synthesis, promoted apoptosis, and decreased Th1/Th2 and Th17/Treg ratios. Dual blockage of mTOR and phosphatidylinositol 3-kinase (PI3K) exerted stronger inhibition on T-cell immunobiology than selective repression of PI3K alone. Rapamycin also blocked the glycolytic pathway in T cells. Moreover, glucose-induced activation of mTOR-glycolytic pathway increased T-cell proliferation, DNA synthesis, and the Th17/Treg ratio, and decreased T-cell apoptosis. In contrast, inhibition of glycolysis by 2-Deoxy-d-glucose (2-DG) yielded the opposite effects on T-cell immunobiology by blocking the mTOR pathway. In conclusion, enhanced activation of the mTOR-glycolytic pathway promoted T-cell immunobiology, suggesting that dysregulation of immunometabolism might be associated with T-cell dysfunction in OLP.