Human Lymphoma Research Articles

A Cell-Based Assay to Measure the Activity of the Complement Convertases

IntroductionThe complement system serves as a crucial defense mechanism against invading pathogens, but dysregulation of this system can result in harmful consequences. Central to the complement cascade are the classical/lectin and the alternative pathway convertases. Aberrant regulation of the convertases is often implicated in the development of rare complement-related diseases. However, analyzing convertase activity poses a significant challenge due to their labile nature and intricate interactions with serum proteins. MethodsIn this study, we propose a novel assay for the functional evaluation of these complexes. Our approach leverages a widely available human lymphoma cell line, which, when sensitized with antibodies, triggers activation of the classical pathway with a substantial amplification by the alternative pathway. The combined action of two C5 blockers eculizumab and crovalimab let the cascade proceed up to the level of convertases but not further. In the next step, C5 inhibitors are washed away and guinea pig serum in EDTA buffer supports the development of lytic sites on the platform of pre-existing convertases. ResultsThe assay detects recombinant gain-of-function components of both convertase types within human serum or plasma. Furthermore, we demonstrate the assay's practical utility in analyzing nephrological patients harboring C3 genetic variants and illustrate its capacity to distinguish between patients and asymptomatic relatives carrying the same pathogenic C3 variant. ConclusionsWe provided a proof of concept of a new assay that detects convertase overactivity in individuals carrying variants of both pathogenic character or these of unknown significance in ubiquitous complement proteins like C3.

Germinal center cytokine driven epigenetic control of Epstein-Barr virus latency gene expression.

Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide and is associated with multiple human lymphomas that express characteristic EBV latency programs used by the virus to navigate the B-cell compartment. Upon primary infection, the EBV latency III program, comprised of six Epstein-Barr Nuclear Antigens (EBNA) and two Latent Membrane Protein (LMP) antigens, drives infected B-cells into germinal center (GC). By incompletely understood mechanisms, GC microenvironmental cues trigger the EBV genome to switch to the latency II program, comprised of EBNA1, LMP1 and LMP2A and observed in GC-derived Hodgkin lymphoma. To gain insights into pathways and epigenetic mechanisms that control EBV latency reprogramming as EBV-infected B-cells encounter microenvironmental cues, we characterized GC cytokine effects on EBV latency protein expression and on the EBV epigenome. We confirmed and extended prior studies highlighting GC cytokine effects in support of the latency II transition. The T-follicular helper cytokine interleukin 21 (IL-21), which is a major regulator of GC responses, and to a lesser extent IL-4 and IL-10, hyper-induced LMP1 expression, while repressing EBNA expression. However, follicular dendritic cell cytokines including IL-15 and IL-27 downmodulate EBNA but not LMP1 expression. CRISPR editing highlighted that STAT3 and STAT5 were necessary for cytokine mediated EBNA silencing via epigenetic effects at the EBV genomic C promoter. By contrast, STAT3 was instead necessary for LMP1 promoter epigenetic remodeling, including gain of activating histone chromatin marks and loss of repressive polycomb repressive complex silencing marks. Thus, EBV has evolved to coopt STAT signaling to oppositely regulate the epigenetic status of key viral genomic promoters in response to GC cytokine cues.

TYM-3-98, a novel selective inhibitor of PI3Kδ, demonstrates promising preclinical antitumor activity in B-cell lymphomas

AimsPI3Kδ is expressed predominately in leukocytes and is commonly found to be aberrantly activated in human B-cell lymphomas. Although PI3Kδ has been intensively targeted for discovering anti-lymphoma drugs, the application of currently approved PI3Kδ inhibitors has been limited due to unwanted systemic toxicities, thus warranting the development of novel PI3Kδ inhibitors with new scaffolds. Main methodsWe designed TYM-3-98, an indazole derivative, and evaluated its selectivity for all four PI3K isoforms, as well as its efficacy against various B-cell lymphomas both in vitro and in vivo. Key findingsWe identified TYM-3-98 as a highly selective PI3Kδ inhibitor over other PI3K isoforms at both molecular and cellular levels. It showed superior antiproliferative activity in several B-lymphoma cell lines compared with the approved first-generation PI3Kδ inhibitor idelalisib. TYM-3-98 demonstrated a concentration-dependent PI3K/AKT/mTOR signaling blockage followed by apoptosis induction. In vivo, TYM-3-98 showed good pharmaceutical properties and remarkably reduced tumor growth in a human lymphoma xenograft model and a mouse lymphoma model. SignificanceOur findings establish TYM-3-98 as a promising PI3Kδ inhibitor for the treatment of B-cell lymphoma.

Anlotinib Inhibiting Mantle Cell Lymphoma Proliferation and Inducing Apoptosis through PI3K/AKT/mTOR Pathway.

This study investigates the inhibitory mechanism of anlotinib on human Mantle Cell Lymphoma (MCL) cells through in vitro and in vivo experiments. In vitro cellular experiments validate the effects of anlotinib on MCL cell proliferation and apoptosis. Moreover, a subcutaneous xenograft nude mice model of Mino MCL cells was established to assess the anti-tumour effect and tumour microenvironment regulation of anlotinib in vivo. The results indicate that MCL cell proliferation was significantly inhibited upon anlotinib exposure. The alterations in the expression of apoptosis-related proteins further confirm that anlotinib can induce apoptosis in MCL cells. Additionally, anlotinib significantly reduced the PI3K/Akt/mTOR phosphorylation level in MCL cells. The administration of a PI3K phosphorylation agonist, 740YP, could reverse the inhibitory effect of anlotinib on MCL. In the xenograft mouse model using Mino MCL cells, anlotinib treatment led to a gradual reduction in body weight and a significant increase in survival time compared to the control group. Additionally, anlotinib attenuated PD-1 expression and elevated inflammatory factors, CD4, and CD8 levels in tumour tissues. Anlotinib effectively inhibits proliferation and induces apoptosis in MCL both in vitro and in vivo. This inhibition is likely linked to suppressing phosphorylation in the PI3K/Akt/mTOR pathway.

Pegylated-liposomes increase the efficacy of Idelalisib in lymphoma B-cells

New drugs and technologies are continuously developed to improve the efficacy and minimize the critical side effects of cancer treatments. The present investigation focuses on the development of a liposomal formulation for Idelalisib, a small-molecule kinase inhibitor approved for the treatment of lymphoid malignancies. Idelalisib is a potent and selective antitumor agent, but it is not indicated nor recommended for first-line treatment due to fatal and serious toxicities. Herein, liposomes are proposed as a delivery tool to improve the therapeutic profile of Idelalisib. Specifically, PEGylated liposomes were prepared, and their physicochemical and technological features were investigated. Light-scattering spectroscopy and cryo-transmission electron microscopy revealed nanosized unilamellar vesicles, which were proved to be stable in storage and in simulated biological fluids.The cytotoxicity of the liposome formulation was investigated in a human non-Hodgkin's lymphoma B cell line. Idelalisib was able to induce death of tumor cells if delivered by the nanocarrier system at increased efficacy. These findings suggest that combining Idelalisib and nanotechnologies may be a powerful strategy to increase the antitumor efficacy of the drug.

Effects of Cannabidiol, ∆9-Tetrahydrocannabinol, and WIN 55-212-22 on the Viability of Canine and Human Non-Hodgkin Lymphoma Cell Lines.

In our previous study, we demonstrated the impact of overexpression of CB1 and CB2 cannabinoid receptors and the inhibitory effect of endocannabinoids (2-arachidonoylglycerol (2-AG) and Anandamide (AEA)) on canine (Canis lupus familiaris) and human (Homo sapiens) non-Hodgkin lymphoma (NHL) cell lines' viability compared to cells treated with a vehicle. The purpose of this study was to demonstrate the anti-cancer effects of the phytocannabinoids, cannabidiol (CBD) and ∆9-tetrahydrocannabinol (THC), and the synthetic cannabinoid WIN 55-212-22 (WIN) in canine and human lymphoma cell lines and to compare their inhibitory effect to that of endocannabinoids. We used malignant canine B-cell lymphoma (BCL) (1771 and CLB-L1) and T-cell lymphoma (TCL) (CL-1) cell lines, and human BCL cell line (RAMOS). Our cell viability assay results demonstrated, compared to the controls, a biphasic effect (concentration range from 0.5 μM to 50 μM) with a significant reduction in cancer viability for both phytocannabinoids and the synthetic cannabinoid. However, the decrease in cell viability in the TCL CL-1 line was limited to CBD. The results of the biochemical analysis using the 1771 BCL cell line revealed a significant increase in markers of oxidative stress, inflammation, and apoptosis, and a decrease in markers of mitochondrial function in cells treated with the exogenous cannabinoids compared to the control. Based on the IC50 values, CBD was the most potent phytocannabinoid in reducing lymphoma cell viability in 1771, Ramos, and CL-1. Previously, we demonstrated the endocannabinoid AEA to be more potent than 2-AG. Our study suggests that future studies should use CBD and AEA for further cannabinoid testing as they might reduce tumor burden in malignant NHL of canines and humans.

TIM-3+ CD8 T cells with a terminally exhausted phenotype retain functional capacity in hematological malignancies.

Chronic antigen stimulation is thought to generate dysfunctional CD8 T cells. Here, we identify a CD8 T cell subset in the bone marrow tumor microenvironment that, despite an apparent terminally exhausted phenotype (TPHEX), expressed granzymes, perforin, and IFN-γ. Concurrent gene expression and DNA accessibility revealed that genes encoding these functional proteins correlated with BATF expression and motif accessibility. IFN-γ+ TPHEX effectively killed myeloma with comparable efficacy to transitory effectors, and disease progression correlated with numerical deficits in IFN-γ+ TPHEX. We also observed IFN-γ+ TPHEX within CD19-targeted chimeric antigen receptor T cells, which killed CD19+ leukemia cells. An IFN-γ+ TPHEX gene signature was recapitulated in TEX cells from human cancers, including myeloma and lymphoma. Here, we characterize a TEX subset in hematological malignancies that paradoxically retains function and is distinct from dysfunctional TEX found in chronic viral infections. Thus, IFN-γ+ TPHEX represent a potential target for immunotherapy of blood cancers.

Latent Epstein-Barr virus infection collaborates with Myc over-expression in normal human B cells to induce Burkitt-like Lymphomas in mice.

Epstein-Barr virus (EBV) is an important cause of human lymphomas, including Burkitt lymphoma (BL). EBV+ BLs are driven by Myc translocation and have stringent forms of viral latency that do not express either of the two major EBV oncoproteins, EBNA2 (which mimics Notch signaling) and LMP1 (which activates NF-κB signaling). Suppression of Myc-induced apoptosis, often through mutation of the TP53 (p53) gene or inhibition of pro-apoptotic BCL2L11 (BIM) gene expression, is required for development of Myc-driven BLs. EBV+ BLs contain fewer cellular mutations in apoptotic pathways compared to EBV-negative BLs, suggesting that latent EBV infection inhibits Myc-induced apoptosis. Here we use an EBNA2-deleted EBV virus (ΔEBNA2 EBV) to create the first in vivo model for EBV+ BL-like lymphomas derived from primary human B cells. We show that cord blood B cells infected with both ΔEBNA2 EBV and a Myc-expressing vector proliferate indefinitely on a CD40L/IL21 expressing feeder layer in vitro and cause rapid onset EBV+ BL-like tumors in NSG mice. These LMP1/EBNA2-negative Myc-driven lymphomas have wild type p53 and very low BIM, and express numerous germinal center B cell proteins (including TCF3, BACH2, Myb, CD10, CCDN3, and GCSAM) in the absence of BCL6 expression. Myc-induced activation of Myb mediates expression of many of these BL-associated proteins. We demonstrate that Myc blocks LMP1 expression both by inhibiting expression of cellular factors (STAT3 and Src) that activate LMP1 transcription and by increasing expression of proteins (DNMT3B and UHRF1) known to enhance DNA methylation of the LMP1 promoters in human BLs. These results show that latent EBV infection collaborates with Myc over-expression to induce BL-like human B-cell lymphomas in mice. As NF-κB signaling retards the growth of EBV-negative BLs, Myc-mediated repression of LMP1 may be essential for latent EBV infection and Myc translocation to collaboratively induce human BLs.

Estrogen receptors alpha and beta expression in different canine cancer types with an emphasis on hematopoietic malignancies.

Estrogen receptors (ERs) are located in both healthy and neoplastic tissues. The type of estrogen receptor expressed varies depending on its location, tumor type, and species. Estrogen action is mediated by binding to ER and activating the transcriptional and signaling processes that result in the control of gene expression. There are two main types of estrogen receptors: ER alpha (ERα) and ER beta (ERβ). Both receptors are functionally different, they may act antagonistically and are distributed in different tissues but their structure is similar - as they are composed of 5 different domains: A/B, C, D, E, and F. The signaling pathway and hence regulation of the gene expression by ERs is a complex and multifactorial process that involves both genomic and nongenomic actions. In the human reproductive tract, both ERα and β are present, with predominant expression of ERβ, while there are no satisfactory data distinguishing the type of ERs expressed in the canine reproductive tract. In mammary gland neoplasia, a decreased or lacking ERα expression in humans is associated with a poorer prognosis. This is similar to dogs, where higher ERα expression intensity was noted in benign tumors than in carcinomas. In human hematopoietic malignancies, ERβ is a predominant receptor. Selective and non-selective ERβ agonists have an antiproliferative and pro-apoptotic effect on human lymphoma cell lines and may be effective in the therapy of ERβ positive lymphomas and leukemias. In canine lymphoma tissues, none or only marginal expression of ERs was detected over the decades. Considering available data, we conducted preliminary studies proving that, in contrast to humans, the dominant ER expressed in canine hematopoietic tumors is ERα.

Effect of Celastrus Orbiculatus Extract on proliferation and apoptosis of human Burkitt lymphoma cells.

The lymphoma incidence rate is on the rise, with invasive forms particularly prone to relapse following conventional treatment, posing a significant threat to human life and wellbeing. Numerous studies have shown that traditional Chinese botanical drug medicine offers promising therapeutic benefits for various malignancies, with previous experimental findings indicating that Celastrus orbiculatus extract effectively combats digestive tract tumors. However, its impact on lymphoma remains unexplored. This study aims to investigate the impact and underlying mechanisms of COE on the proliferation and apoptosis of Burkitt lymphoma cells. We diluted COE in RPMI-1640 medium to create various working concentrations and introduced it to human Burkitt lymphoma Raji and Ramos cells. To evaluate cell viability, we used the CCK-8 assay, and we observed morphological changes using HE staining. We also conducted Annexin V-PI and JC-1 staining experiments to assess apoptosis. By combining the cell cycle experiment with the EDU assay, we gained insights into the effects of COE on DNA replication in lymphoma cells. Using Western blotting, we detected alterations in apoptosis-related proteins. In vivo experiments revealed that following COE intervention, tumor volume decreased, survival time was prolonged, spleen size reduced, and the expression of tumor apoptosis-related proteins changed. Our findings indicate that COE effectively inhibits lymphoma cell proliferation and promotes apoptosis by regulating these apoptosis-related proteins.

338 Non-occupational herbicide and VOC exposures detected in dogs with multicentric lymphoma: a model for human non-Hodgkin lymphoma

OBJECTIVES/GOALS: The objective of this study was to determine whether pet dogs with multicentric lymphoma (ML), a spontaneous, immunocompetent model for human non-Hodgkin lymphoma (NHL), are exposed to higher concentrations of herbicides and volatile organic compounds (VOCs) compared to matched unaffected control dogs. METHODS/STUDY POPULATION: We are prospectively enrolling dogs with ML within a single high-risk breed, the boxer dog, along with age-matched control boxers sampled within the same season. We are measuring urinary concentrations of the herbicides glyphosate (in Roundup®) and 2,4-D, as well as stable urinary metabolites of the VOCs benzene and 1,3-butadiene. To assess the genotoxic potential of herbicide and VOC exposures, we are using reverse dosimetry to estimate plasma exposures, and exposing healthy canine PBMC’s to these concentrations of herbicides and VOCs in vitroto assess double stranded DNA damage using the Comet Chip assay. RESULTS/ANTICIPATED RESULTS: Preliminary data show significantly higher benzene exposures, measured by the stable benzene metabolite PHMA, and significantly higher 2,4-D exposures at the time of diagnosis in cases versus controls. All dogs had measurable exposures to 1,3-butadiene (measured as its stable metabolite DHBM) and glyphosate.In vitro results show significant genotoxicity thresholds of 0.1 uM for both glyphosate and 2,4-D in dog lymphoid cells. To date, these predicted plasma exposures have not been reached in vivo in boxer dogs with ML or unaffected control boxers. DISCUSSION/SIGNIFICANCE: Canine multicentric lymphoma resembles human NHL and is a potentially useful model of non-occupational chemical risk for NHL in people. The goal of this research is to identify potentially preventable non-occupational chemical risk for lymphoma and support evidence-based remediation strategies to decrease lymphoma risk in both humans and dogs.

Next Generation Sequencing and Insilico Identification of Human Anaplastic Lymphoma Kinase in Human Cancer with Biopython

Abstract: In this comprehensive molecular investigation, the focus centers on the anaplastic lymphoma kinase (ALK) within the context of lung cancer. Specifically, utilizing the sample with the PDB code 3AOX, the study employs a multifaceted approach combining advanced bioinformatics tools and structural biology methodologies.Commencing with RasMol, the analysis of the protein sample reveals a complex network of 196 hydrogen bonds within the ALK structure. This foundational information sets the stage for subsequent investigations. Visual representation is meticulously addressed, with RasMol utilized for color-coding alpha helices in magenta, beta helices in yellow, and the remaining residues in white. The presentation mode emphasizes helices in red, sheets in yellow, and loops in green, providing a vivid depiction of the structural elements within the ALK protein.Moving beyond visualization, PyMOL is engaged to represent the surface form, providing insights into the interactions between ALK and its environment. This is particularly crucial in understanding the structural dynamics of ALK in the context of lung cancer, where the protein sample serves as a representative model.Active site identification in Chain A is pursued using tools such as PyMol, offering a glimpse into potential functional regions crucial for ALK's role in lung cancer pathogenesis. The study extends to biophysical aspects, incorporating protein-ligand docking studies with Mektovi and Almita through CB DOCK. This approach sheds light on the intricate molecular interactions between ALK and specific ligands, providing valuable information for targeted therapies.Structural validation becomes paramount in ensuring the reliability of the 3AOX sample. The ERRAT structure validation server evaluates the overall quality factor, while Procheck on the SAVES server scrutinizes the Ramachandran plot, ensuring the conformational consistency of ALK's backbone dihedral angles.Biopython scripts play a pivotal role in extracting and analyzing data related to the alpha-beta class representation within the CATH database. This bioinformatics analysis adds another layer of understanding to ALK's structural classification, offering insights into its role in lung cancer.

Blocking Orai1 constitutive activity inhibits B-cell cancer migration and synergistically acts with drugs to reduce B-CLL cell survival

Orai1 channel capacity to control store-operated Ca2+ entry (SOCE) and B-cell functions is poorly understood and more specifically in B-cell cancers, including human lymphoma and leukemia. As compared to normal B-cells, Orai1 is overexpressed in B-chronic lymphocytic leukemia (B-CLL) and contributes in resting B-CLL to mediate an elevated basal Ca2+ level through a constitutive Ca2+ entry, and in BCR-activated B-cell to regulate the Ca2+ signaling response. Such observations were confirmed in human B-cell lymphoma and leukemia lines, including RAMOS, JOK-1, MEC-1 and JVM-3 cells. Next, the use of pharmacological Orai1 inhibitors (GSK-7975 A and Synta66) blocks constitutive Ca2+ entry and in turn affects B-cell cancer (primary and cell lines) survival and migration, controls cell cycle, and induces apoptosis through a mitochondrial and caspase-3 independent pathway. Finally, the added value of Orai1 inhibitors in combination with B-CLL drugs (ibrutinib, idelalisib, rituximab, and venetoclax) on B-CLL survival was tested, showing an additive/synergistic effect including in the B-cell cancer lines. To conclude, this study highlights the pathophysiological role of the Ca2+ channel Orai1 in B-cell cancers, and pave the way for the use of ORAI1 modulators as a plausible therapeutic strategy.

The aryl hydrocarbon receptor differentially modulates the expression profile of antibody isotypes in a human B-cell line.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant and high affinity ligand for the aryl hydrocarbon receptor (AhR). In animal models, AhR activation by TCDD generally inhibits antibody secretion. However, it is less clear if this translates to human antibody production. Using a human Burkitt lymphoma B-cell line (CL-01) that can be stimulated to secrete Ig and undergo class switch recombination to other Ig isotypes, the current study evaluated the effects of AhR activation or antagonism on the human Ig isotypic expression profile with CD40L+IL-4 stimulation. Our results suggest that AhR agonists (TCDD and indirubin) have little to no effect on IgM or IgA secretion, which were also not induced with stimulation. However, AhR activation significantly inhibited stimulation-induced IgG secretion, an effect reversed by the AhR antagonist CH223191. Evaluation of Ig heavy chain (IgH) constant region gene expression (ie Cμ, Cγ1-4, Cα1-2, and Cε that encode for IgM, IgG1-4, IgA1-2, and IgE, respectively) demonstrated differential effects. While Cμ and Cα2 transcripts were unaffected by stimulation or AhR agonists, AhR activation significantly inhibited stimulation-induced Cγ2-4 and Cε mRNA transcripts, which was reversed by AhR antagonism. Notably, AhR antagonism in the absence of exogenous AhR ligands significantly increased IgG and IgA secretion as well as the expression of Cγ2-4 and Cε. These results suggest that modulation of AhR activity differentially alters the IgH isotypic expression profile and antibody secretion that may be partly dependent on cellular stimulation. Since a variety of chemicals from anthropogenic, industrial, pharmaceutical, dietary, and bacterial sources bind the AhR, the ability of environmental exposures to alter AhR activity (i.e. activate or inhibit) may have a direct influence on immune function and antibody-relevant disease conditions.

Abstract 4018: An innovative in vivo model for CAR-T cell therapy development: Tolerability and efficacy evaluation of CD19-targeting CAR-T cells on human lymphoma using the chicken CAM assay

Abstract BACKGROUND Chimeric antigen receptor (CAR) T cell therapy is a revolutionary approach in cellular immunotherapy, where a patient’s T cells are genetically engineered to recognize and attack cancer cells. In the last decade, several CAR-T cell therapies have been approved by the FDA. However, this encouraging outcome was obtained only with certain blood cancers, like B cell leukemias, lymphomas, etc. Many research/clinical trials are now underway worldwide to extend the CAR T-cell therapy benefits to a large spectrum of cancers, using rodents. This model presents several drawbacks, such as the immunodeficiency of humanized models, ethical constraints, time, and cost. An alternative, pertinent, 3Rs-compliant in vivo model for developing CAR-T therapy is urgently needed. Xenografts on the chicken embryo’s ChorioAllantoic Membrane (CAM) have proven extremely valuable for in vivo cancerology studies. In this work, we evaluated in vivo the anti-tumor efficacy of CD19-targeting CAR-T cells on human lymphoma using Inovotion’s CAM assay. METHODS Human lymphoma Raji cells (ATCC) were grafted on the CAM on embryonic development day (EDD) 9. A 2nd generation of CD19-targeting CAR-T cells (CAR-T-(CD3/4-1BB)) developed by Allogenica (France) were co-injected at EDD9 at E/T (effector CAR-T cell vs target Raji cell) ratio 1:1. One group was injected with CAR-T (CD3/4-1BB) only once at EDD9 and a second group received a first injection of CAR-T (CD3/4-1BB) at EDD9 and a second injection at EDD13. Embryonic viability was checked daily. On EDD18. The efficacy of CAR-T therapy was evaluated basing on tumor weight, metastatic invasion in the lower CAM and CAR-T infiltration in tumors detected by qPCR. Activation of the chicken embryo immune system was detected as well. RESULTS The treatment with CAR-T-(CD3/4-1BB) cells was well tolerated in ovo, no significant embryo mortality/abnormality was observed. CAR-T-(CD3/4-1BB) cells prevented tumor formation (tumor weight was 88.67% to 90.62% lower than control, p < 0.001); and prevented metastasis formation in the lower CAM (86.88% to 81.43% lower than control, p < 0.001). CONCLUSION Different to in vitro models and immunodeficient murine models, chicken embryo possesses a progressively mature immune system. Our results show that CAR-T-(CD3/4-1BB) cells can prevent tumor formation and metastatic invasion when co-injected with tumor cells. Inovotion’s CAM assay is a viable alternative in vivo model for developing CAR-T cell therapy on a large spectrum of cancers. It is fully 3Rs-compliant. Citation Format: Yan Wang, Xavier Rousset, Chloé Prunier, Emilien Dosda, Alejandra Gutierrez-Guerrero, Pauline Abrial, Inna Menkova, Jean Viallet. An innovative in vivo model for CAR-T cell therapy development: Tolerability and efficacy evaluation of CD19-targeting CAR-T cells on human lymphoma using the chicken CAM assay [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 4018.

Abstract 2840: Development, characterization, and validation of a systemic human mantle cell lymphoma model utilizing luciferase expressing Jeko-1 tumor cells

Abstract Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphoma that is clinically and biologically heterogenous. MCL murine models are scarce and reliable in vivo models are necessary for pre-clinical testing and identification of improved novel therapeutics. To address this need, we established and characterized a human MCL in vivo model by systemic implantation of the luciferase-expressing Jeko-1 cell line (Jeko-1-luc) into immunodeficient NCG mice. Tumor growth kinetics was evaluated by testing multiple cell inoculums, and disease progression was monitored by in vivo serial bioimaging, three-dimensional optical imaging (3D), and ex-vivo endpoint analyses. Jeko-1-luc tumor growth profile revealed moderate latency with a median survival of 40 days post implant. Subsequently, we utilized 3D optical imaging and ex-vivo imaging that confirmed the localization of tumor growth predominantly in lungs, spleen, and skeletal regions. Additionally, we determined the surface expression of selected B-cell lineage markers including CD19, CD20, and CD22. Next, we evaluated the efficacy and effect on survival in response to various standard of care agents including Vincristine, Rituximab, Palbociclib, Temsirolimus, and Cyclophosphamide. The quantification of tumor burden by in vivo and ex-vivo bioluminescent imaging demonstrated that the highest therapeutic activity and overall survival was achieved by Palbociclib and Vincristine. Flow cytometric analyses further demonstrated that tumor cells obtained from blood and selected tissues including spleen and bone marrow showed reduced tumor burden in treatment groups relative to the control group. Taken together, these results illustrate the translational value of the Jeko-1-luc model and provides a platform for evaluating novel treatment modalities for mantle cell lymphoma. Citation Format: David Harris, Andrew Wong, Elizabeth Rainbolt, Nicole Westrick, Justin Avery, Joseph Kolb, Aswani Vunnava, Zachary Ward, Chassidy Hall, Bincy John. Development, characterization, and validation of a systemic human mantle cell lymphoma model utilizing luciferase expressing Jeko-1 tumor cells [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 2840.

Abstract 2356: NAV-006: A next-generation rituximab targeting CD20 for the treatment of B-cell lymphomas immunosuppressed by MUC16/CA125

Abstract Rituximab is a CD20-targeting antibody that is the standard-of-care for patients with Non-Hodgkin Lymphoma (NHL) cases. Rituximab’s mechanism of action includes complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), herein referred to as Humoral Immuno-Oncology (HIO) mechanisms. Recent clinical evidence suggest that high serum levels of the MUC16 (CA125) protein inversely correlate with the effectiveness of rituximab clinical activity in 40-50% of follicular lymphoma (FL) as well as diffuse large B-cell lymphoma patients with newly diagnosed or relapsed/refractory (R/R) disease. In this study we demonstrated that CA125 binds to and suppresses the activity of rituximab, tafasitamab as well as the bispecific mosunetuzumab and glofitamab antibodies, which are approved for treating cases with indolent disease. These therapies have an overall response rate around 50-60%, suggesting the presence of a mechanism that allows lymphoma cells to escape these agents’ immune-mediated killing effects. CA125 may facilitate this immunosuppressive mechanism, and in fact, upon binding rituximab, CA125 reduces its affinity for the Fc receptor, CD16a, thus suppressing ADCC. Using a proprietary technology called Block-Removed Immunoglobulin Technology (BRITE) that employs randomized amino acid substitution and HIO effector function screening, we have identified a rituximab variant named NAV-006. NAV-006 is refractory to the immunosuppressive effects mediated by CA125 via its reduced CA125 interaction and increased HIO activity as compared to parent rituximab as well as the other antibodies used to treat R/R FL. In an in vivo model of human B-cell lymphoma, NAV-006 showed superior efficacy compared to parent rituximab. These data warrant further investigation of NAV-006 as a next generation anti-CD20 antibody that could improve upon the efficacy of antibody-mediated therapies used to treat NHL patients with high levels of CA125. Citation Format: Luigi Grasso, J. Bradford Kline, Nicholas C. Nicolaides. NAV-006: A next-generation rituximab targeting CD20 for the treatment of B-cell lymphomas immunosuppressed by MUC16/CA125 [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 2356.

Abstract 2721: FF01: a novel humanized heavy chain antibody targeting endogenous retroviruses with anti-lymphoma activity

Abstract Background: Human endogenous retroviruses (HERVs) are transposable elements (TE) derived from retrovirus infections that occurred millions of years ago. Despite constituting 8% of our genome, their biological role still needs to be explored. HERV-K (HML-2), a subgroup of HERVs, represents the most recent retroviruses integrated into the human germline DNA. Members of the HML-2 subgroup retain complete ORF with coding potential and have garnered the most attention in disease-related studies. HERVs exhibit physiological expression in embryonic stem cells but are heavily methylated and remain almost silent in normal adult tissues. Although the mechanisms underlying HERV-K contribution to tumor cell growth and survival remain poorly understood, aberrant expression of HERVs has been demonstrated in various human tumor tissues and lymphoma. These findings led us to explore HERV-K as a potential therapeutic target in B cell lymphomas .Methods: RNA data were analyzed with Telescope. Validations were performed by qPCR, Western blot, flow cytometry, and ELISA. Env structure was obtained with AlphaFold and peptides were purchased from GeneScript. Antibody-dependent cellular cytotoxicity (ADCC) was performed with the ADCC Reporter Bioassay (Promega). Internalization was assessed using the Incucyte Fabfluor-pH Antibody labeling reagents. Results: We assessed the TE expression in RNA-seq datasets of diffuse large B cell lymphoma (DLBCL) and B cells from healthy individuals. An MDS plot showed that TEs could separate neoplastic from healthy samples, demonstrating that tumoral and normal specimens have distinct TE and HERV expression profiles. Focusing on tumor samples, DLBCLs were divided into four clusters with different prognoses. Similar results were obtained in a 47 B-cell lymphoma cell line panel. Focusing on the HML-2 family in lymphoma patients, we identified many of them upregulated in tumors. We validated RNA and protein expression of the HML-2 envelope (env) in seventeen lymphoma cell lines, and in two of them, we also proved the protein surface expression. Env-specific siRNA reduced env expression determined a decrease in cell proliferation, suggesting a contribution to lymphoma cell growth. In silico examination of the env identified four linear regions with potential for antibody binding, and peptides covering them were synthesized. We screened 150 lymphoma patients' serum samples for IgG antibodies binding to the peptides, and all peptides showed positive hits. We used a phage display screen to obtain one nanobody, FF01, binding one peptide. FF01 induced ADCC in the cell line with high surface env expression and not in a negative one. FF01 was internalized in an expressing cell, proving the principle for using it as a component of an antibody-drug conjugate. Conclusions: Our data demonstrate that HERVs are in DLBCL patients and propose FF01 as a new candidate to target HERV Env+ lymphomas. Citation Format: Filippo Spriano, Luciano Cascione, Jacopo Sgrignani, Nikolai Bendik, Sara Napoli, Giulio Sartori, Eleonora Cannas, Tao Gongo, Alberto J. Arribas, Davide Rossi, Davide F. Robbiani, Andrea Cavalli, Francesco Bertoni. FF01: a novel humanized heavy chain antibody targeting endogenous retroviruses with anti-lymphoma activity [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 2721.

Abstract 1772: A naturally occurring canine model of peripheral T-cell lymphoma, not otherwise specified

Abstract Despite being the most common subtype of human peripheral T-cell lymphoma (PTCL), PTCL-not otherwise specified (PTCL-NOS) remains a poorly understood diagnosis of exclusion with poor survival times and treatment responses. Canine PTCL clinically and immunophenotypically resembles human PTCL-NOS, and PTCL is more common in dogs than humans, leading to their interest as a naturally occurring preclinical model. Canine models of cancer offer several unique advantages: in contrast to traditional rodent models, these are spontaneous tumors in genetically diverse and immunocompetent animals living in a shared environment with humans, but relative to their human counterparts, they offer accelerated lifespans with shorter clinical courses of disease. Here, we analyzed the gene expression profile of 96 canine CD4+ PTCLs to determine similarities to human PTCL-NOS and investigate a possible cell of origin. Bulk RNA-seq was performed on lymph node aspirates of 96 cases of canine CD4+ PTCL diagnosed by flow cytometry and sorted CD4+ nodal lymphocytes and CD4+ thymocytes from healthy control dogs. Raw reads were aligned to the Ensembl canine reference genome (ROS_Cfam_1.0) and tabulated. Data normalization and differential expression analysis was conducted with DESeq2, and differentially expressed genes were compared to human PTCL-NOS and various stages of T-cell development via gene set enrichment analyses (GSEA). Global gene expression in canine CD4+ PTCL was analogous to that of human PTCL-NOS (NES = 2.3, p < 0.001). Additionally, canine CD4+ PTCL had increased expression of GATA3 (log2fc = 1.9, padj = 6.5e-07) and was enriched for gene signatures associated with downregulation of PTEN (NES = 1.7, p = 0.0001) and upregulation of PI3K/AKT/mTOR signaling (NES = 1.5, padj = 0.0007), resembling the more aggressive human GATA3-PTCL subtype. Canine CD4+ PTCL was also enriched for human and murine gene signatures associated with early thymocyte progenitor cells and had increased expression of genes of immaturity, including CD34 (log2fc = 7.8, padj = 2.6e-14), KIT (log2fc = 3.3, padj = 7.7e-05), and CCR9 (log2fc = 4.8, padj = 3.9e-11), although surface CD34 expression is not detected by flow cytometry. These findings were confirmed when we compared the canine PTCL gene expression programs to a canine single cell transcriptomic atlas of hematopoietic precursors and T cells across canine bone marrow, thymic, and lymph node tissues. Canine CD4+ PTCL cells were significantly (padj < 0.05) enriched for gene signatures associated with early thymic and bone marrow precursors (NES 1.3-2.1), and negatively enriched for gene signatures associated with naïve nodal CD4+ T cells (NES = -3.2, padj = 0.006). In conclusion, the gene expression profile of canine CD4+ PTCL resembles human PTCL-NOS, supporting its potential as a naturally occurring preclinical model, and a subset of these neoplasms may arise from a thymic precursor cell of origin. Citation Format: Eileen Owens, Adam Harris, Anne Avery. A naturally occurring canine model of peripheral T-cell lymphoma, not otherwise specified [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 1772.

Abstract 3999: Augmentation of antitumor effects by CAR-T cells secreting anti-PD-1 single-chain antibody fused with IL-21

Abstract Background: Chimeric antigen receptor (CAR) T cell therapy has shown remarkable responses in hematologic malignancies. However, many patients suffer from limited response and tumor relapse due to lack of persisting CAR-T cells and immune escape. Delivery of immunomodulatory cytokines is one of the strategies to improve immune function; however, the systemic effect of cytokines can be detrimental. Methods: To prevent the toxic effects of cytokines, we proposed the concept of targeting cytokines to tumor-specific T cells in vivo. To this end, we developed CAR-T cells secreting a fusion protein (PD-1Ab21) composed of anti-PD-1 single chain antibody and IL-21, which target IL-21 to CAR-T cells to promote their proliferation and differentiation into memory cells while blocking the PD-1 signal of CAR-T cells. The secretion and functions of PD-1Ab21 were identified. The proliferation and differentiation of CAR-T cells secreting PD-1Ab21 (PD-1Ab21-CAR-T) were compared in vitro with that of the conventional CAR-T cells. The therapeutic effects of CAR-T cells secreting PD-1Ab21 were studied in immunodeficient mice engrafted with human Burkitt lymphoma Daudi, multiple myeloma cell U266 and Her2/neu+ breast cancer cell BT474. Results: The results showed that the culture supernatant of PD-1Ab21-CAR-T cells, like anti-PD-1 antibody, blocked the binding of PD-1 to PD-L1 and stimulated STAT3 phosphorylation in human T lymphocytic leukemia cell Hut78 with the same activity as IL-21. In the conventional culture milieu with high dose of IL-2, the proliferation of the PD-1Ab21-CAR-T cells was not different from that of conventional CAR-T cells. While cultured with low dose of IL-2 that could not support the proliferation of conventional CAR-T cells, the PD-1Ab21-CAR-T cells still showed significant proliferation. A major fraction of the PD-1Ab21-CAR-T products was shown to co-express CCR7, CD62L and CD45RA, and express high level of TCF-1, indicating a memory stem-like phenotype. While the most of conventional CAR-T cells showed effector phenotype defined as CD45RA+CCR7-. Furthermore, adoptive transfer of PD-1Ab21-CAR-T cells exhibited significantly improved CAR-T cell expansion and resulted in superior tumor eradication in the xenograft models of human B-cell malignancy, multiple myeloma and Her2/neu+ breast cancer. The enhanced cellular function by secreting PD-1Ab21 was mediated through an autocrine and/or paracrine manners. Conclusion: We developed an enhanced CAR-T cells. Our data provide preclinical evidence to support the translation of this design for an improved CAR-T cell-mediated anti-tumor response. Citation Format: Ying Li, Jitao Zhao, Xiangrui Cheng, Hang Li, Ran Tian, Qianqian He, Shengdian Wang. Augmentation of antitumor effects by CAR-T cells secreting anti-PD-1 single-chain antibody fused with IL-21 [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 3999.