Cells In Preclinical Models Research Articles

Depletion of conventional CD4+ T cells is required for robust priming and dissemination of tumor antigen-specific CD8+ T cells in the setting of anti-CD4 therapy

BackgroundOvercoming immune suppression is a major barrier to eliciting potent CD8+ T cell responses against cancer. Treatment with anti-CD4 monoclonal antibody is an effective means for eliminating CD4+Foxp3+ regulatory (Treg)...

Trastuzumab-Mediated Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Enhances Natural Killer Cell Cytotoxicity in HER2-Overexpressing Ovarian Cancer.

Ovarian cancer is the deadliest gynecologic cancer. Although human epidermal growth factor receptor-2 (HER2) overexpression, a poor prognostic molecular marker in ovarian cancer, is found in almost 30% of ovarian cancer cases, there are no established therapies for HER2-overexpressing ovarian cancer. In this study, we investigated the efficacy of combined samfenet, a biosimilar compound of trastuzumab, and natural killer (NK) cells in preclinical model of HER2-overexpressing ovarian cancer. Firstly, we screened the HER2 expression in three ovarian cancer cell lines and eight ovarian cancer patient-derived tumor xenograft (PDTX) samples. Then, immunohistochemistry and silver in situ hybridization (SISH) were performed following clinical criteria. HER2-overexpressing cells exhibited the highest sensitivity to samfenet compared with low-HER2-expressing cells. In addition, the combination of samfenet with natural killer (NK) cells resulted in significantly enhanced sensitivity to HER2-overexpressing cells and showed a significant antitumor effect on PDTX mice compared with monotherapy. It is known that anti-HER2-humanized IgG1 monoclonal antibodies, including trastuzumab, induce antibody-dependent cellular cytotoxicity (ADCC). Consequently, the combination of samfenet with NK cells demonstrated NK cell-mediated ADCC, as confirmed using an in vitro NK cytotoxicity assay and in vivo antitumor efficacy. A transferase dUTP nick end labeling (TUNEL) assay using xenografted tumors further supported the ADCC effects based on the increase in the number of apoptotic cells in the combination group. Furthermore, high HER2 expression was associated with shorter progression-free survival and overall survival based on public mRNA expression data. In this study, we demonstrated that the combination of samfenet and NK cell therapy could be a promising treatment strategy for patients with HER2-overexpressing ovarian cancer, through ADCC effects. Therefore, this study supports a rationale for further clinical studies of the combination of samfenet and NK cells as a therapy for patients with HER2-overexpressing ovarian cancer.

TRAIL-induced cytokine production via NFKB2 pathway promotes neutrophil chemotaxis and immune suppression in triple negative breast cancers.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapeutic that induces apoptosis in cancer cells while sparing the non-malignant cells in preclinical models. However, its efficacy in clinical trials has been limited, suggesting unknown modulatory mechanisms responsible for the lack of TRAIL activity in patients. Here, we hypothesized that TRAIL treatment elicits transcriptional changes in triple negative breast cancer (TNBC) cells that alter the immune milieu. To test this, we performed an RNAseq analysis of MDA-MB-231 cells treated with TRAIL, followed by validation in additional TNBC cell lines. TRAIL significantly induces expression of multiple cytokines such as CXCLs 1, 2, 3, 8,11 and IL-6, which are known to modify neutrophil function. Mechanistically, the induction of these cytokines was predominantly mediated by death receptor 5, caspase 8 (but not caspase 8 enzymatic activity), and the non-canonical NFKB2 pathway. The cytokines produced by the TRAIL-treated TNBC cells enhanced chemotaxis of healthy human donor isolated neutrophils. In vivo , TRAIL treated TNBC murine xenograft tumors showed activation of the NFKB2 pathway, elevated production of CXCLs and IL-6, and increased neutrophil recruitment into the tumors. Moreover, donor isolated neutrophils preincubated in supernatants from TRAIL-treated TNBC cells exhibited impaired cytotoxic effect against TNBC cells. Transcriptomic analysis of neutrophils incubated with either TRAIL alone or supernatant of TRAIL-treated TNBC cells revealed increased expression of inflammatory cytokines, immune modulatory genes, immune checkpoint genes, and genes implicated in delayed neutrophil apoptosis. Functional studies with these neutrophils confirmed their suppressive effect on T cell proliferation and an increase in Treg suppressive phenotype. Collectively, our study demonstrates a novel role of TRAIL-induced NFKB2-dependent cytokine production that promotes neutrophil chemotaxis and immune suppression.

Age-related macular degeneration: suitability of optogenetic therapy for geographic atrophy.

Age-related macular degeneration (AMD) is a growing public health concern given the aging population and it is the leading cause of blindness in developed countries, affecting individuals over the age of 55 years. AMD affects the retinal pigment epithelium (RPE) and Bruch's membrane in the macula, leading to secondary photoreceptor degeneration and eventual loss of central vision. Late AMD is divided into two forms: neovascular AMD and geographic atrophy (GA). GA accounts for around 60% of late AMD and has been the most challenging subtype to treat. Recent advances include approval of new intravitreally administered therapeutics, pegcetacoplan (Syfovre) and avacincaptad pegol (Iveric Bio), which target complement factors C3 and C5, respectively, which slow down the rate of enlargement of the area of atrophy. However, there is currently no treatment to reverse the central vision loss associated with GA. Optogenetics may provide a strategy for rescuing visual function in GA by imparting light-sensitivity to the surviving inner retina (i.e., retinal ganglion cells or bipolar cells). It takes advantage of residual inner retinal architecture to transmit visual stimuli along the visual pathway, while a wide range of photosensitive proteins are available for consideration. Herein, we review the anatomical changes in GA, discuss the suitability of optogenetic therapeutic sensors in different target cells in pre-clinical models, and consider the advantages and disadvantages of different routes of administration of therapeutic vectors.

Abstract 5244: SIRPα knockout iPSC-derived macrophages (iMACs) are resistant to CD47-dependent inhibition of phagocytosis and efficiently kill tumor cells in pre-clinical models

Abstract The CD47-SIRPα axis is a critical checkpoint that prevents SIRPα-positive macrophages from phagocytosing CD47-expressing solid tumors. Several agents aiming to block this axis have recently entered early clinical trials including anti-CD47 and anti-SIRPα monoclonal antibodies (mAb). These checkpoint inhibitors (CPI) aim to modulate the phagocytotic activity of endogenous tumor associated macrophages (TAMs). However, the adoptive transfer of macrophages resistant to CD47-based inhibition in the tumor microenvironment (TME) could also increase clinical efficacy while avoiding side effects linked to the use of anti-CD47 mAb. Cell therapy based on autologous macrophages have gained increasing attention for cancer treatment due to their ability to infiltrate into the immunosuppressive TME and their unique immunomodulatory characteristics. However, due to required intricate genetic manipulation of autologous macrophage cell product for each patient, optimization and consistency of the cell product remains challenging. In contrast, the use of induced pluripotent stem cell (iPSC)-derived macrophages (iMACs) facilitates the introduction of genetic modifications to further optimize the iMAC cell product and limits the need for combination therapies. The knockout (KO) of the SIRPα gene in iMACs is a promising genetic modification that results in a potent iMAC cell therapy product resistant to phagocytosis inhibition by CD47-expressing tumor cells. A SIRPα KO was introduced in a fully characterized GMP iPSC line that was then differentiated to iMACs using Evotec’s 3D differentiation protocol. The SIRPα KO iMACs were then evaluated for their antibody-dependent cellular phagocytosis (ADCP) capacity in comparison to antibody-loaded wildtype (WT) iMACs when co-cultured with CD47-expressing tumor cells. SIRPα KO iPSCs showed differentiation potential comparable to WT iPSC and resulted in iMACs expressing typical markers of fully differentiated macrophages. SIRPα KO iMACs exhibited increased phagocytic potency and killing capacity compared to WT iMACs when both cell types were exposed to CD47-positive tumor cells and loaded with the same tumor-targeting mAb. This increased phagocytosis of tumor cells by antibody-loaded SIRPα KO iMACs was also comparable to ADCP observed for WT iMACs in the presence of an anti-CD47 blocking antibody. Using Evotec’s gene editing platform we were able to efficiently generate SIRPα-deficient iPSCs that serve as the starting material to manufacture highly pure, genetically modified iMACs that lack SIRPα expression rendering them resistant to CD47-dependent inhibition of phagocytosis. This novel allogeneic off-the-shelf iMAC cell product overcomes the need to combine this cell therapeutic with CD47-SIRPα axis CPIs and provides the basis to develop innovative treatments for solid tumors. Citation Format: Kathrin Haake, Michela Mirenda, Quentin Bernard, Philip Hublitz, Lucie Gouxette, Martin Briscadieu, Philine Scheinpflug, Garima Singh, Alica Hinkelmann, Tanja Schneider, Michael Esquerré, Audrey Holtzinger, Michael Epstein, Daniel Sommermeyer, Michael Paillasse, Andreas Scheel, Markus Dangl, Monika Braun, Nadja Wagner. SIRPα knockout iPSC-derived macrophages (iMACs) are resistant to CD47-dependent inhibition of phagocytosis and efficiently kill tumor cells in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5244.

Abstract 2006: Deciphering the interplay of β-catenin/YAP signaling and alveolar lineage plasticity during targeted therapy in non-small cell lung cancer

Abstract Molecularly targeted therapies have significantly enhanced clinical outcomes by directly addressing oncogenic driver mutations in genes such as EGFR. However, despite the success of treatments targeting these mutations, long-term patient survival remains limited. This study investigates the molecular mechanisms underlying drug-tolerance in response to targeted therapies in non-small cell lung cancer (NSCLC). Using single-cell RNA sequencing of oncogene-driven human NSCLCs at the residual disease (RD) state during active targeted therapy, our investigation reveals an increase of an alveolar type I/II (AT1/2) mixed lineage program in surviving cancer cells, resembling an alveolar cell-like state. We observed increased WNT3A/4 ligands, FZD2 receptors, β-catenin signaling, and AT1/2 signatures at the RD stage. Consistent with clinical results, AT1/2 signatures and target genes of β-catenin and YAP are increased in drug-tolerant persister (DTP) cells in preclinical models. Additionally, we discovered that β-catenin and YAP form a stable nuclear protein complex in DTP cells. Disrupting β-catenin/YAP protein expression and stability in DTP cells by knocking down FZD2 or interrupting the destruction complex of β-catenin/YAP leads to re-sensitization to the EGFR inhibitor Osimertinib, suggesting a potential therapeutic approach. Furthermore, this therapeutic strategy decreased AT1/2 gene signatures. Our results highlight an interdependence between β-catenin and YAP, with both proteins playing crucial roles in sustaining DTP cells and promoting the alveolar cell like lineage plasticity. These findings shed light on the complex interplay between β-catenin/YAP signaling and alveolar signatures in the residual cancer state, offering mechanistic insights and potential therapeutic strategies to address drug tolerance and improve clinical outcomes. Citation Format: Yu-Ting Chou, Wei Wu, Daniel Kerr, Macey Slota, Trever Bivona. Deciphering the interplay of β-catenin/YAP signaling and alveolar lineage plasticity during targeted therapy in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2006.

Abstract 5228: Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile

Abstract Acute myeloid leukemia (AML) is an aggressive hematological malignancy that, despite advances in treatment, has an overall 5-year survival rate of less than 30%. There is an urgent unmet need for new and effective therapies for AML. Chimeric antigen receptor (CAR)-modified cell therapies are emerging as promising options. However, the selection of suitable antigens is challenging, due to the heterogeneous expression of potential antigen targets in AML. Most antigens, such as CD123, are expressed on both leukemic cells and normal hematopoietic stem and progenitor cells. In addition, conventional autologous or allogeneic T cell therapies present many limitations, such as cytokine release syndrome (CRS) and graft-vs-host disease (GvHD). We therefore evaluated CD123-targeting CAR designs in vδ1-enriched γδT cells, an innate immune cell platform that is inherently non-MHC restricted, has low risks of CRS and GvHD, and is capable of discriminating between malignant and healthy cells in preclinical models. We engineered vδ1-enriched γδT cells from peripheral blood of healthy individuals to express various CD123-targeting CAR constructs. The CD123-targeting CAR-engineered vδ1-enriched γδT cells expressed high levels of natural cytotoxicity receptors, suggesting capability to target innate receptor ligands on AML cell surface. CD123-targeting CAR-expressing vδ1-enriched γδT cells demonstrated enhanced cytotoxicity in vitro against multiple AML cell lines, in both short-term co-culture and repeated antigen stimulation assays. CD123-targeting CAR-expressing vδ1-enriched γδT cells also showed enhanced efficacy and survival in vivo against mouse models of human AML. Interestingly, attenuation of CAR activity via novel designs preserved the inherent selectivity of vδ1-enriched γδT cells against AML cells relative to normal hematopoietic stem or progenitor cells, which also express CD123. Together, our results demonstrated through rational CAR designing and engineering in vδ1-enriched γδT cells, an allogeneic cell therapy targeting CD123 can be generated with selective cytotoxicity against AML cells while sparing normal hematopoietic stem cells. Such product designs have the potential to be a transformative therapeutic option for a broad population of AML patients. Citation Format: Mei Rosa Ng, Sarah L. Hayward, Di Zhang, Gary Gu, Bri Paulhus, Donjeta Gjuka, Collin Crowley, Sophie McLaughlin, Anya Lublinsky, Angel Mathew, Evans Berreondo Giron, Doanh Mai, Madisen Lee, Megha Wal, Rebecca Alade, Andre Simoes, Robin Lytle, James Gavin, Shira Cramer, Zhong-hua Yan, Taylor Hickman, Rashmi Choudhary, Cori Abikoff, Yana Wang, Istvan Kovacs, Nandhu M. Sobhana, Lan Cao. Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5228.

Early induction of cytokine release syndrome by rapidly generated CAR T cells in preclinical models

Cytokine release syndrome (CRS) is a significant side-effect of conventional chimeric antigen receptor (CAR) T-cell therapy. To facilitate patient accessibility, short-term (st) CAR T cells, which are administered to patients only 24 h after vector exposure, are in focus of current investigations. Their impact on the incidence and severity of CRS has been poorly explored. Here, we evaluated CD19-specific stCAR T cells in preclinical models. In co-culture with tumor cells and monocytes, stCAR T cells exhibited anti-tumoral activity and potent release of CRS-related cytokines (IL-6, IFN-γ, TNF-α, GM-CSF, IL-2, IL-10). When administered to NSG-SGM3 mice, stCAR T cells, but not conventional CAR T cells, induced severe acute adverse events within 24 h, including hypothermia and weight loss, as well as high body scores, independent of the presence of tumor target cells. Human (IFN-γ, TNF-α, IL-2, IL-10) and murine (MCP-1, IL-6, G-CSF) cytokines, typical for severe CRS, were systemically elevated. Our data highlight potential safety risks of rapidly manufactured CAR T cells and suggest NSG-SGM3 mice as sensitive model for their preclinical safety evaluation.

Clinical and immunological relevance of SLAMF6 expression in the tumor microenvironment of breast cancer and melanoma

Compelling evidence shows that the frequency of T cells in the tumor microenvironment correlates with prognosis as well as response to immunotherapy. However, considerable heterogeneity exists within tumor-infiltrating T cells, and significance of their genomic and transcriptomic landscape on clinical outcomes remains to be elucidated. Signaling lymphocyte activation molecule 6 (SLAMF6) is expressed on intra-tumoral progenitor-exhausted T cells, which exhibit the capacity to proliferate, self-renew and produce terminally-exhausted T cells in pre-clinical models and patients. Here, we investigated the impact of SLAMF6 expression on prognosis in two immunologically different tumor types using publicly available databases. Our findings demonstrate that high SLAMF6 expression is associated with better prognosis, expression of TCF7 (encoding T-cell factor 1), and increased gene signatures associated with conventional type 1 dendritic cells and effector function of T cells in melanoma and breast cancer. Single-cell profiling of breast cancer tumor microenvironment reveals SLAMF6 expression overlaps CD8 T cells with a T-effector signature, which includes subsets expressing TCF7, memory and effector-related genes, analogous to progenitor-exhausted T cells. These findings illustrate the significance of SLAMF6 in the tumor as a marker for better effector responses, and provide insights into the predictive and prognostic determinants for cancer patients.

Aging and Metabolic Reprogramming of Adipose-Derived Stem Cells Affect Molecular Mechanisms Related to Cardiovascular Diseases.

We performed a systematic search of the PubMed database for English-language articles related to the function of adipose-derived stem cells in the pathogenesis of cardiovascular diseases. In preclinical models, adipose-derived stem cells protected arteries and the heart from oxidative stress and inflammation and preserved angiogenesis. However, clinical trials did not reiterate successful treatments with these cells in preclinical models. The low success in patients may be due to aging and metabolic reprogramming associated with the loss of proliferation capacity and increased senescence of stem cells, loss of mitochondrial function, increased oxidative stress and inflammation, and adipogenesis with increased lipid deposition associated with the low potential to induce endothelial cell function and angiogenesis, cardiomyocyte survival, and restore heart function. Then, we identify noncoding RNAs that may be mechanistically related to these dysfunctions of human adipose-derived stem cells. In particular, a decrease in let-7, miR-17-92, miR-21, miR-145, and miR-221 led to the loss of their function with obesity, type 2 diabetes, oxidative stress, and inflammation. An increase in miR-34a, miR-486-5p, and mir-24-3p contributed to the loss of function, with a noteworthy increase in miR-34a with age. In contrast, miR-146a and miR-210 may protect stem cells. However, a systematic analysis of other noncoding RNAs in human adipose-derived stem cells is warranted. Overall, this review gives insight into modes to improve the functionality of human adipose-derived stem cells.

Abstract B047: Phase I trial of αvβ3 integrin cytotoxin ProAgio in patients with previously treated advanced pancreatic cancer and other solid tumor malignancies

Abstract Background: ProAgio is a pegylated peptide drug that targets and kills cells that express integrin αvβ3 including cancer associated fibroblasts (CAFs) and endothelial cells within the tumor microenvironment. ProAgio utilizes a novel binding site to directly trigger apoptosis of target cells in pre-clinical models. Here we describe the interim results of a first-in-human phase 1 trial of ProAgio in patients with advanced solid malignancies, including pancreatic cancer. Methods: The trial is enrolling adults with previously treated and histologically confirmed advanced solid malignancy for which no curative therapy exists. The dose escalation cohort uses a modified 3+3 design to determine the recommended phase 2 dose (RP2D) of ProAgio. This will be followed by a dose expansion cohort which will enroll only participants with non-neuroendocrine advanced pancreatic cancer. The primary objective of the trial is to determine the RP2D of ProAgio. Secondary objectives assess the safety (CTCAE v. 4.03), pharmacokinetics and objective response rate (ORR) of Proagio. Ascending doses of ProAgio were administered as an intravenous infusion every 1-2 weeks depending on dose level. Response is determined by serial imaging (CT or MRI) and tumor marker assessment (CA 19-9 or appropriate tumor-specific marker). Exploratory objectives include assessing biologic effect of ProAgio on pre- and on-therapy tumor biopsy specimens (apoptosis of tumor cells, integrin αvβ3 expression, CAF population, tumor vasculature and architecture), as well as identifying putative surrogate circulating biomarkers (circulating αvβ3 expressing cells among others). Results: Sixteen participants (14 with pancreatic adenocarcinoma and 2 with other gastrointestinal malignancies) have been treated on the dose escalation as of June 30, 2023. Preliminary pharmacokinetic measurements demonstrate a plasma half-life of 17.35 hours, distribution volume of 17.62 L, and clearance of 0.522 L/hr. ProAgio has been exceptionally well tolerated with only transient grade 1 or 2 treatment-related adverse events observed in most participants. Two participants had grade 2 infusion related reaction but were able to continue treatment with increased dose of diphenhydramine pre-medication. A serious adverse event of grade 3 hyperglycemia was observed in 1 participant with insulin-dependent diabetes within 24 hours of ProAgio infusion. No other treatment-associated hyperglycemia has been observed. Two participants with pancreatic cancer receiving weekly dosing of ProAgio had stable disease for >4 months and interval stabilization in CA 19-9 tumor marker. Conclusions: ProAgio is safe and well-tolerated. Upon completion of the dose escalation, a dose expansion phase of 15 participants treated at the RP2D will begin enrolling. NCT05085548 Citation Format: Nebojsa Skorupan, Cody J. Peer, Eve Koehler, Seth Steinberg, Jane B Trepel, William D Figg, Zhi-Ren Liu, Christine Alewine. Phase I trial of αvβ3 integrin cytotoxin ProAgio in patients with previously treated advanced pancreatic cancer and other solid tumor malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B047.

Oral Decitabine/Cedazuridine Is a Tolerable and Effective Ambulatory Regimen for Patients with Advanced Myeloproliferative Neoplasms

Background: Treatment options are limited for patients (pts) with advanced myeloproliferative neoplasms (MPNs). Most pts become refractory to JAK2 inhibitor (JAKi) therapy by 3 years with subsequent survival of only 11-16 months. Sequential treatment with chromatin-modifying agents including decitabine (D) can upregulate CXCR4 expression, correct abnormal trafficking of myelofibrosis (MF) stem cells, and eliminate JAK2V617F+ MF stem and progenitor cells in preclinical models ( Wang et al, Cancer Res 2019, Blood 2010). Combination therapy with intravenous D and ruxolitinib (Rux) had a favorable effect on overall survival (OS) in a phase 2 study of MPN-accelerated/blast phase pts ( Mascarenhas et al, Blood Adv 2020) and prolonged survival in a cohort of DIPSS-plus high-risk MF pts ( Bader et al, Leuk Res 2015). Oral decitabine/cedazuridine (DC), therefore, represents an attractive ambulatory treatment option for pts with advanced MPNs. Methods: We retrospectively reviewed the electronic health records of pts with advanced high-risk MF (AHR-MF) or MPN- accelerated phase (MPN-AP) seen at our institution that were treated with DC with or without a JAKi from 1/2021-7/2023. MPN-AP was defined by the presence of 10-19% blasts in peripheral blood/ bone marrow. AHR-MF was defined by the presence of 4-9% circulating blasts or 5-9% blasts in the bone marrow, and/or refractoriness to a JAKi. Results: A total of 14 pts, 7 each with MPN-AP and AHR-MF, respectively received DC therapy (Table 1). Most pts (8/14 or 57%) were older than 75 years. All pts were classified as high risk/very high-risk (MIPPS70 plus v.2). Ten (71%) pts had one or more high molecular risk mutations ( ASXL1, EZH2, SRSF2, IDH1/2, U2AF1), with the most prevalent being ASXL1 mutations in 8/14 (57%) pts. Other somatic mutations included NF1, KRAS, NRAS, BRAF, GATA2, STAG2, DNMT3A and TET2 genes. Eight of the 14 pts (57%) had either an unfavorable or very high-risk karyotype (MIPSS70 plus v.2). Thirteen pts had previously failed JAKi therapy. The median number of cycles of DC therapy was 2 (range,1-6) and 4 (range,1-26) for MPN-AP and AHR-MF pts, respectively and 4/7 pts received a concurrent JAKi in each group. Two pts in AHR-MF cohort were successfully bridged to transplant. Two pts in the MPN-AP group discontinued treatment after cycle 1 due to neutropenic sepsis and progression to blast phase, respectively. Among MPN-AP pts who received ≥ 2 cycles of DC, circulating blasts were reduced to ≤ 5% in 4/5 pts, normalization of thrombocytopenia and improvement in hemoglobin by ≥ 1.5 g/dl each occurred in 3/5 pts (Fig.1). In the AHR-MF cohort, 6/7 pts received ≥ 2 cycles of DC with elimination of circulating blasts in one pt. WBC and platelet count were effectively reduced in 4/4 and 2/2 pts with baseline WBC of >50x 10 3/uL and platelet count >1000 x 10 3/uL, respectively. Spleen size reduction was achieved in two pts with combination of DC + Rux who were previously refractory to a JAKi. One pt achieved complete resolution of palpable splenomegaly after 2 cycles of combination therapy. The predominant treatment-related adverse event was neutropenic fever in three pts requiring hospitalization. DC cycle length was reduced from 5 to 3/28 days in 7/14 pts, which reduced the degree of therapy related neutropenia and anemia. Pts remained ambulatory during treatment except for hospitalizations related to neutropenic fever. With a median follow-up of 109 days (range, 28 days - 29 months), median OS has not been reached. Three pts in the MPN-AP cohort progressed to MPN-blast phase (BP), including two pts who only received one cycle of DC. Two pts with MPN-BP died after 28 and 68 days of DC therapy, respectively. None of the AHR-MF pts progressed to MPN-AP/BP. The most durable response was observed in a pt who has received a total of 26 cycles of DC + Rux with stable disease for >2 years and loss of BRAF and KRAS mutations after 9 months of treatment. Conclusions: DC therapy appears to be a well-tolerated and effective oral ambulatory regimen with clinically meaningful responses in elderly, transplant ineligible pts as well as a bridge to transplant in high-risk pts with advanced MPNs. The ease of outpatient administration significantly reduces pt treatment burdens and positively impacts their quality of life. These data provide a rationale for studying oral decitabine/cedazuridine +/- JAKi in a prospective manner, especially in pts with AHR-MF refractory to JAKi prior to evolution MPN-AP/BP.

Clinico-Genomic Characterization of AML Patients Based on IL2RA (CD25) Expression Uncovers an Association with Stem Cell Signatures and FLT3-ITD Status and Informs Drug Combinations

Background Acute myeloid leukemia (AML) is a heterogeneous disease with poor outcomes, thus there remains a need to integrate molecular information to identify patients most likely to respond to drugs and combinations. Disease segmentation based on transcriptomics has provided valuable insight into disease risk and the likelihood of response to targeted compounds. IL2RA(CD25) is a receptor expressed on both AML leukemic and immune cells, and has shown initial promise as a potential dual target of leukemic blasts and regulatory T (Treg) cells in pre-clinical models (Pousse et al. Front. Oncol. 2023). We used a systems approach based on a recently described transcriptomic classifier for AML (Hamidi et al. ASH 2021) and other molecular tools to characterize the association between IL2RA expression levels and AML genomic markers, clinical features and patient outcomes. In addition, we evaluated IL2RA in relation to ex vivo drug sensitivity, to identify patient segments who are most likely to benefit from CD25-targeting drugs. Methods BEAT-AML (NCT01728402) RNAseq data from patient samples, associated ex vivo drug sensitivity data (N=283), was VOOM normalized. Patients were binned into quartiles based on IL2RA expression. Gene signature scoring was performed using xCell cell type enrichment algorithm and GSVA for Hallmark pathways and scRNAseq signature based on Van Galen et al. Cell 2019. Associations were performed with clinical outcome (log-rank test), gene signatures (Spearman correlation), mutations (Wilcoxon test) and ex vivo drug sensitivity (Kruskal Wallis test). Results We characterized the BeatAML dataset and found that elevated IL2RA expression associates with inferior overall survival (p=0.014) and high-risk features. In addition, we identified a strong association between IL2RA expression and FLT3-ITD status (Figure), as well as other genetic alterations. Using correlation analyses, we established an association between IL2RA expression levels and “primitive” AML signatures (leukemic stem cell (LSC), R=0.4, p=6.8x10-12, hematopoietic stem cell (HSC)-like, R=0.43, p=6.8x10-14) and Tregs (R=0.47, p=1.7x10-15), and an anti-correlation with promonocytic signatures (R=-0.23, p=1.1x10-4). Interestingly, HSC-like and Treg signatures were also correlated (R=0.19, p=0.00087), consistent with an association between stem cell abundance and a repressive immune microenvironment. Finally, using a multivariate model adjusting for the effect of Tregs and LSCs, the prognostic value of IL2RA remained significant (p=0.029). We previously used unsupervised machine learning clustering based on consensus non-negative factorization (cNMF) to discover novel transcription-based classification (Hamidi ASH 2021). Using this methodology, we identified a strong correlation between IL2RA expression in cNMF subtypes (p=1.6x10-10). Moreover, this method uncovered patient subtypes in which IL2RA correlates with LSC (cNMF 6.3), Treg (6.4, 6.6) or both signatures (6.1, 6.2). This differential association was independent of prognostic category, maturation state or venetoclax sensitivity (Table). Transcription-based classifiers have been shown to be highly predictive of ex vivo drug sensitivity. Consistent with an association between IL2RA expression and FLT3-ITD status, we identified strong correlations between IL2RA and sensitivity to FLT-3 and other tyrosine kinase inhibitors, providing a rationale for combining these compounds with CD25-targeting therapeutics. In contrast, there was no correlation between IL2RA levels and venetoclax AUC (p=0.25), while the cNMF classification system similarly revealed that patient segments with increased IL2RA expression had heterogeneous venetoclax sensitivity (Table), supporting CD25 targeting in patients who may not respond to venetoclax. Conclusion AML patients with elevated IL2RA expression have inferior prognosis, enrichment of stem-like and Treg signatures and FLT3-ITD alterations. Transcriptomics-based clustering could be used to guide combination therapies based on potential impact of CD25 targeting on Tregs, leukemic cells, or both populations. Ex vivo drug sensitivity analyses support combinations of CD25-targeting agents with FLT3 inhibitors, as well as venetoclax. Additional work to evaluate these and other combinations using functional assays is warranted.

Genetic Engineering of Therapeutic Cells with the Sodium Iodide Symporter (NIS) to Enable Noninvasive In Vivo Therapy Tracking.

Noninvasive long-term imaging of therapeutic cells in preclinical models can be achieved through introducing a reporter gene into the cells of interest. Despite important recent developments such as gene editing, cell engineering based on lentiviruses remains a mainstream tool for gene transfer applicable to a variety of different cell types.In this chapter, we describe how to use lentivirus-based genetic engineering to render different candidate cell therapies in vivo traceable by radionuclide imaging. We illustrate this reporter gene technology using the sodium iodide symporter (NIS), which is compatible with both positron emission tomography (PET) and single-photon emission computed tomography (SPECT). For preclinical experimentation, we fused NIS with a suitable fluorescent protein such as monomeric GFP or RFP to streamline cell line generation and downstream analyses of ex vivo tissue samples. We present protocols for reporter gene engineering of human cardiac progenitor cells, regulatory T cells, and effector T cells as well as for the characterization experiments required to validate NIS-fluorescent protein reporter function in these candidate therapeutic cells.

TMIC-61. INVESTIGATING REPROGRAMING OF THE GBM’S TUMOR MICROENVIRONMENT AS A RESULT OF CO-TRANSFER OF HEMATOPOIETIC STEM CELLS (HSCS) WITH ADOPTIVE CELLULAR THERAPY

Abstract Glioblastoma (GBM) is an incredibly aggressive and prevalent primary CNS tumor with dismal survival outcomes. GBM’s intra-tumor heterogeneity and the powerful regulatory mechanisms within the tumor microenvironment (TME) that hamper immune activation have proved to be formidable challenges to the development of effective therapies. Fortunately, immunotherapy has emerged as a potentially powerful approach to achieve of immune activation within the TME through adoptive immunotherapy and checkpoint inhibition Our previous work has demonstrated that the survival benefit of adoptive cellular therapy (ACT) is significantly enhanced by concomitant transfer of bone marrow derived hematopoietic stem cells (HSC) with tumor-reactive T cells in preclinical models. Using a novel organotypic brain slice culture (BSC) model study using glioma-bearing C57BL/6J mice, we interrogate tumor microenvironment and directly demonstrate that the presence of HSC-derived cells in the microenvironment increases T cell migration to tumor, and increased tumor cell death. In addition, we can investigate reprogramming of the TME as a result of treatment with this ACT+HSC platform. These studies are unique in that through this model system, we are capable of studying the cell-cell interactions occurring within the tumor microenvironment TME longitudinally while accounting for the complexity and the spatial orientation of the components within the TME that promote immunosuppression and tumor escape.

Development of potent antibody drug conjugates against ICAM1+ cancer cells in preclinical models of cholangiocarcinoma

As a highly lethal adenocarcinoma of the hepatobiliary system, outcomes for cholangiocarcinoma (CCA) patients remain prominently poor with a 5-year survival of <10% due to the lack of effective treatment modalities. Targeted therapeutics for CCA are limited and surgical resection of CCA frequently suffers from a high recurrence rate. Here we report two effective targeted therapeutics in this preclinical study for CCA. We first performed a quantitative and unbiased screening of cancer-related antigens using comparative flow cytometry in a panel of human CCA cell lines, and identified intercellular adhesion molecule-1 (ICAM1) as a therapeutic target for CCA. After determining that ICAM1 has the ability to efficiently mediate antibody internalization, we constructed two ICAM1 antibody-drug conjugates (ADCs) by conjugating ICAM1 antibodies to different cytotoxic payloads through cleavable chemical linkers. The efficacies of two ICAM1 ADCs have been evaluated in comparison with the first-line chemodrug Gemcitabine in vitro and in vivo, and ICAM1 antibodies coupled with warhead DX-8951 derivative (DXd) or monomethyl auristatin E (MMAE) elicit a potent and consistent tumor attenuation. In summary, this study paves the road for developing a promising targeted therapeutic candidate for clinical treatment of CCA.

Remibrutinib (LOU064) inhibits neuroinflammation driven by B cells and myeloid cells in preclinical models of multiple sclerosis

BackgroundBruton’s tyrosine kinase (BTK) is a key signaling node in B cell receptor (BCR) and Fc receptor (FcR) signaling. BTK inhibitors (BTKi) are an emerging oral treatment option for patients suffering from multiple sclerosis (MS). Remibrutinib (LOU064) is a potent, highly selective covalent BTKi with a promising preclinical and clinical profile for MS and other autoimmune or autoallergic indications.MethodsThe efficacy and mechanism of action of remibrutinib was assessed in two different experimental autoimmune encephalomyelitis (EAE) mouse models for MS. The impact of remibrutinib on B cell-driven EAE pathology was determined after immunization with human myelin oligodendrocyte glycoprotein (HuMOG). The efficacy on myeloid cell and microglia driven neuroinflammation was determined in the RatMOG EAE. In addition, we assessed the relationship of efficacy to BTK occupancy in tissue, ex vivo T cell response, as well as single cell RNA-sequencing (scRNA-seq) in brain and spinal cord tissue.ResultsRemibrutinib inhibited B cell-dependent HuMOG EAE in dose-dependent manner and strongly reduced neurological symptoms. At the efficacious oral dose of 30 mg/kg, remibrutinib showed strong BTK occupancy in the peripheral immune organs and in the brain of EAE mice. Ex vivo MOG-specific T cell recall response was reduced, but not polyclonal T cell response, indicating absence of non-specific T cell inhibition. Remibrutinib also inhibited RatMOG EAE, suggesting that myeloid cell and microglia inhibition contribute to its efficacy in EAE. Remibrutinib did not reduce B cells, total Ig levels nor MOG-specific antibody response. In brain and spinal cord tissue a clear anti-inflammatory effect in microglia was detected by scRNA-seq. Finally, remibrutinib showed potent inhibition of in vitro immune complex-driven inflammatory response in human microglia.ConclusionRemibrutinib inhibited EAE models by a two-pronged mechanism based on inhibition of pathogenic B cell autoreactivity, as well as direct anti-inflammatory effects in microglia. Remibrutinib showed efficacy in both models in absence of direct B cell depletion, broad T cell inhibition or reduction of total Ig levels. These findings support the view that remibrutinib may represent a novel treatment option for patients with MS.

Constitutive Turbodomains enhance expansion and antitumor activity of allogeneic BCMA CAR T cells in preclinical models.

The magnitude of CAR T cell expansion has been associated with clinical efficacy. Although cytokines can augment CAR T cell proliferation, systemically administered cytokines can result in toxicities. To gain the benefits of cytokine signaling while mitigating toxicities, we designed constitutively active synthetic cytokine receptor chimeras (constitutive Turbodomains) that signal in a CAR T cell-specific manner. The modular design of Turbodomains enables diverse cytokine signaling outputs from a single homodimeric receptor chimera and allows multiplexing of different cytokine signals. Turbodomains containing an IL-2/15Rβ-derived signaling domain closely mimicked IL-15 signaling and enhanced CAR T cell potency. Allogeneic TurboCAR T cells targeting BCMA showed no evidence of aberrant proliferation yet displayed enhanced expansion and antitumor activity, prolonging survival and preventing extramedullary relapses in mouse models. These results illustrate the potential of constitutive Turbodomains to achieve selective potentiation of CAR T cells and demonstrate the safety and efficacy of allogeneic BCMA TurboCAR T cells, supporting clinical evaluation in multiple myeloma.

γ-Secretase inhibitor in combination with BCMA chimeric antigen receptor T-cell immunotherapy for individuals with relapsed or refractory multiple myeloma: a phase 1, first-in-human trial

γ-Secretase inhibitors (GSIs) increase B cell maturation antigen (BCMA) density on malignant plasma cells and enhance antitumour activity of BCMA chimeric antigen receptor (CAR) T cells in preclinical models. We aimed to evaluate the safety and identify the recommended phase 2 dose of BCMA CAR T cells in combination with crenigacestat (LY3039478) for individuals with relapsed or refractory multiple myeloma. We conducted a phase 1, first-in-human trial combining crenigacestat with BCMA CAR T-cells at a single cancer centre in Seattle, WA, USA. We included individuals aged 21 years or older with relapsed or refractory multiple myeloma, previous autologous stem-cell transplant or persistent disease after more than four cycles of induction therapy, and Eastern Cooperative Oncology Group performance status of 0-2, regardless of previous BCMA-targeted therapy. To assess the effect of the GSI on BCMA surface density on bone marrow plasma cells, participants received GSI during a pretreatment run-in, consisting of three doses administered 48 h apart. BCMA CAR T cells were infused at doses of 50 × 106 CAR T cells, 150 × 106 CAR T cells, 300 × 106 CAR T cells, and 450 × 106 CAR T cells (total cell dose), in combination with the 25 mg crenigacestat dosed three times a week for up to nine doses. The primary endpoints were the safety and recommended phase 2 dose of BCMA CAR T cells in combination with crenigacestat, an oral GSI. This study is registered with ClinicalTrials.gov, NCT03502577, and has met accrual goals. 19 participants were enrolled between June 1, 2018, and March 1, 2021, and one participant did not proceed with BCMA CAR T-cell infusion. 18 participants (eight [44%] men and ten [56%] women) with multiple myeloma received treatment between July 11, 2018, and April 14, 2021, with a median follow up of 36 months (95% CI 26 to not reached). The most common non-haematological adverse events of grade 3 or higher were hypophosphataemia in 14 (78%) participants, fatigue in 11 (61%), hypocalcaemia in nine (50%), and hypertension in seven (39%). Two deaths reported outside of the 28-day adverse event collection window were related to treatment. Participants were treated at doses up to 450 × 106 CAR+ cells, and the recommended phase 2 dose was not reached. Combining a GSI with BCMA CAR T cells appears to be well tolerated, and crenigacestat increases target antigen density. Deep responses were observed among heavily pretreated participants with multiple myeloma who had previously received BCMA-targeted therapy and those who were naive to previous BCMA-targeted therapy. Further study of GSIs given with BCMA-targeted therapeutics is warranted in clinical trials. Juno Therapeutics-a Bristol Myers Squibb company and the National Institutes of Health.

The Importance of Stem Cells Isolated from Human Dental Pulp and Exfoliated Deciduous Teeth as Therapeutic Approach in Nervous System Pathologies.

Despite decades of research, no therapies are available to halt or slow down the course of neuro-degenerative disorders. Most of the drugs developed to fight neurodegeneration are aimed to alleviate symptoms, but none has proven adequate in altering the course of the pathologies. Cell therapy has emerged as an intriguing alternative to the classical pharmacological approach. Cell therapy consists of the transplantation of stem cells that can be obtained from various embryonal and adult tissues. Whereas the former holds notable ethical issue, adult somatic stem cells can be obtained without major concerns. However, most adult stem cells, such as those derived from the bone marrow, are committed toward the mesodermal lineage, and hence need to be reprogrammed to induce the differentiation into the neurons. The discovery of neural crest stem cells in the dental pulp, both in adults' molar and in baby teeth (dental pulp stem cells and stem cells from human exfoliated deciduous teeth, respectively) prompted researchers to investigate their utility as therapy in nervous system disorders. In this review, we recapitulate the advancements on the application of these stem cells in preclinical models of neurodegenerative diseases, highlighting differences and analogies in their maintenance, differentiation, and potential clinical application.