Murine Lymphoma Model Research Articles

CD38-specific immunoPET imaging for multiple myeloma diagnosis and therapeutic monitoring: preclinical and first-in-human studies.

CD38 is a glycoprotein highly specific to multiple myeloma (MM). Therapeutics using antibodies targeting CD38 have shown promising efficacy. However, the efficient stratification of patients who may benefit from daratumumab (Dara) therapy and timely monitoring of therapeutic responses remain significant clinical challenges. To address these issues, we developed a novel nanobody-based PET tracer, [68Ga]Ga-TOHP-CD3813, which exhibits rapid clearance from the blood and rapid accumulation in targeted tumor lesions, facilitating the detection of CD38 expression in murine models of MM and lymphoma. Furthermore, we conducted the world's first-in-human trials using CD38-targeted nanobodies to validate and assess the clinical imaging effectiveness of [68Ga]Ga-TOHP-CD3813 in guiding cancer immunotherapy. We prepared a new PET imaging probe based on a CD38-targeted nanobody CD3813, [68Ga]Ga-TOHP-CD3813, via the site-specific radiolabeling for noninvasive PET imaging of CD38 expression. [68Ga]Ga-TOHP-CD3813 was assessed for its affinity and specificity to CD38 and its ability to image CD38 expression in MM and lymphoma xenograft models. Biodistribution and the relationship between tumor uptake and CD38 expression were evaluated. Subsequently, we conducted a translational PET imaging of 2 MM patients using [68Ga]Ga-TOHP-CD3813, while compared with [18F]FDG PET/CT head-to-head. Dosimetry was also calculated based on the animal data. TOHP-CD3813 retained a high affinity for CD38 with a KD of 0.0826 nmol/L. [68Ga]Ga-TOHP-CD3813 was successfully synthesized at room temperature within 10min, exhibiting optimal radiochemical properties. Preclinical assessments revealed rapid blood clearance, high CD38 affinity, and significant uptake in CD38-positive xenograft mouse models (6.50 ± 2.69%ID/g). [68Ga]Ga-TOHP-CD3813 showed pronounced accumulation in the kidneys and bladder, with moderate liver uptake, indicating its potential as a viable clinical PET radiotracer for diagnosing MM. Additionally, in first-in-human trials, [68Ga]Ga-TOHP-CD3813 PET/CT provides a substantial improvement over [18F]FDG PET/CT for the visualization of MM. [68Ga]Ga-TOHP-CD3813, with its high affinity, specificity, and robust imaging capabilities, rapidly and specifically accumulates in tumors with high CD38 expression, offering a significant advantage over [18F]FDG PET/CT for visualizing MM and enabling same-day PET imaging. Initial human trial results are promising, suggesting its potential as a companion diagnostic tool for optimizing CD38-targeted treatments in tumors. Ongoing larger trials aim to further confirm these findings.

A novel murine syngeneic CD8 peripheral T-cell lymphoma model with preclinical applications

Peripheral T-cell Lymphoma (PTCL) represents a heterogenous group of aggressive non-Hodgkin Lymphomas with poor prognostic outcomes and limited treatment options. The development and refinement of therapeutic strategies for PTCL are impeded by a paucity of reliable preclinical models that accurately mimic the disease’s pathophysiology. There is a dire need for more physiologically relevant models for PTCL. Here we describe a spontaneousCD8+ peripheral T-cell lymphoma cell line (LM-23) derived from a 12-week-old female Balb/cJ mouse. Both intravenous and subcutaneous administration of this cell line to syngeneic Balb/cJ mice resulted in rapid establishment of tumor growth. CHOP and anti-PD1 treatment both displayed no benefit to mice in regulating tumor growth. Such results along with its phenotypic characteristics, rapid growth, and metastatic behavior in syngeneic mice highlight its value in studying the elusive disease and discovery of novel therapeutics.

Synthesis and Anti-Cancer Activity of the Novel Selective Glucocorticoid Receptor Agonists of the Phenylethanolamine Series.

Glucocorticoids (GCs) are widely used for treating hematological malignancies despite their multiple adverse effects. The biological response to GCs relies on glucocorticoid receptor (GR) transrepression (TR) that mediates the anticancer effects and transactivation (TA) associated with the side effects. Selective GR agonists (SEGRAs) preferentially activating GR TR could offer greater benefits in cancer treatment. One of the well-characterized SEGRAs, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium-chloride (CpdA), exhibited anticancer activity; however, its translational potential is limited due to chemical instability. To overcome this limitation, we obtained CpdA derivatives, CpdA-01-CpdA-08, employing two synthetic strategies and studied their anti-tumor activity: 4-(1-hydroxy-2-(piperidin-1-yl)ethyl)phenol or CpdA-03 demonstrated superior GR affinity and stability compared to CpdA. In lymphoma Granta and leukemia CEM cell lines, CpdA-03 ligand exhibited typical SEGRA properties, inducing GR TR without triggering GR TA. CpdA-03 effects on cell viability, growth, and apoptosis were similar to the reference GR ligand, dexamethasone (Dex), and the source compound CpdA. In vivo testing of CpdA-03 activity against lymphoma on the transplantable P388 murine lymphoma model showed that CpdA-03 reduced tumor volume threefold, outperforming Dex and CpdA. In conclusion, in this work, we introduce a novel SEGRA CpdA-03 as a promising agent for lymphoma treatment with fewer side effects.

Preclinical Development of a Romidepsin Nanoparticle Demonstrates Superior Tolerability and Efficacy in Models of Human T-Cell Lymphoma and Large Granular Lymphocyte Leukemia.

Histone deacetylase (HDAC) inhibitors are a widely recognized and valued treatment option for patients with relapsed or refractory peripheral T cell lymphomas (PTCL). Romidepsin is a relatively selective Class I HDAC inhibitor originally approved for patients with relapsed or refractory (R/R) cutaneous T cell lymphoma (CTCL) and subsequently R/R PTCL. Unfortunately, the FDA approval of romidepsin for R/R PTCL was withdrawn due to a negative Phase 4 post-marketing requirement (PMR), diminishing further the treatment options for patients with PTCL. Herein we describe the development of a first-in-class polymer nanoparticle of romidepsin (Nanoromidepsin) using an innovative amphiphilic di-block copolymer-based nanochemistry platform. Nanoromidepsin exhibited superior pharmacologic disposition, with improved tolerability and safety in murine models of T-cell lymphoma. Nanoromidepsin also exhibited superior anti-tumor efficacy in multiple models including in vitro T cell lymphoma (TCL) cell lines, ex vivo LGL leukemia primary patient samples, and murine TCL xenografts. Nanoromidepsin demonstrated greater accumulation in tumors and a statistically significant improvement in overall survival (OS) compared to romidepsin in murine xenograft models. These findings collectively justify the clinical development of Nanoromidepsin in patients with T-cell malignancies.

Comparative analysis of Bcl-2 family protein overexpression in CAR T cells alone and in combination with BH3 mimetics.

Approximately 50% of patients with hematologic malignancies relapse after chimeric antigen receptor (CAR) T cell treatment; mechanisms of failure include loss of CAR T persistence and tumor resistance to apoptosis. We hypothesized that both of these challenges could potentially be overcome by overexpressing one or more of the Bcl-2 family proteins in CAR T cells to reduce their susceptibility to apoptosis, both alone and in the presence of BH3 mimetics, which can be used to activate apoptotic machinery in malignant cells. We comprehensively investigated overexpression of different Bcl-2 family proteins in CAR T cells with different signaling domains as well as in different tumor types. We found that Bcl-xL and Bcl-2 overexpression in CAR T cells bearing a 4-1BB costimulatory domain resulted in increased expansion and antitumor activity, reduced exhaustion, and decreased apoptotic priming. In addition, CAR T cells expressing either Bcl-xL or a venetoclax-resistant Bcl-2 variant led to enhanced antitumor efficacy and survival in murine xenograft models of lymphoma and leukemia in the presence or absence of the BH3 mimetic venetoclax, a clinically approved BH3 mimetic. In this setting, Bcl-xL overexpression had stronger effects than overexpression of Bcl-2 or the Bcl-2(G101V) variant. These findings suggest that CAR T cells could be optimally engineered by overexpressing Bcl-xL to enhance their persistence while opening a therapeutic window for combination with BH3 mimetics to prime tumors for apoptosis.

Abstract 4690: SC-2882, a novel QPCTL inhibitor, with anti-tumor activity in solid tumor models

Abstract Background: SC-2882 is an orally administered first-in-class small molecule inhibitor of glutaminyl-peptide cyclotransferase like (QPCTL). QPCTL is an intracellular enzyme that modifies several proteins containing an N-terminal glutamine or glutamate, resulting in pyroglutamylated amino acids. Pyroglutamylation can alter protein function in different ways and dependent on context. Key substrate proteins include the “don’t eat me” signal protein CD47 and several chemokines (CCL2 and CCL7) (Nat Med 2019, Nat Imm. 2022). Previously, we have shown activity of SC-2882 in a murine model of diffuse large B-cell lymphoma (Di Siervi, 2023). Here we report for the first time, the evaluation of SC-2882 in several preclinical mouse models for solid tumors. Methods: Potency of SC-2882 was evaluated in a cell-based assay in several human and mouse tumor cell lines: SIRPα binding to CD47 in response to increasing concentrations of SC-2882 was measured in a flowcytometry-based assay. Several syngeneic mouse tumor models were evaluated for their response towards SC-2882 as a single agent and in combination with either a murine anti-PD-L1 antibody or cisplatin. Animals were dosed orally with SC-2882 once daily for the duration of the experiment. Results: In all human and mouse cancer cell lines tested, SC-2882 was able to reduce SIRPα binding to CD47 significantly. This reduction was equivalent to the reduction observed in QPCTL genetic knockouts created in the same cell line. In the same experiments, no differences in CD47 expression were observed. From anti-tumor efficacy studies, we learned that SC-2882 was well tolerated in mice as a single agent and in combination with anti-PD-L1 or cisplatin for at least 4 weeks (this was the maximum period tested). No clinical signs or changes in body weight were observed, even over prolonged periods of treatment. Tumor growth inhibition and tumor shrinkage by SC-2882 was observed in 5 different syngeneic mouse tumor models. In two out of these five models, SC-2882 was able to reduce tumor growth as a single agent. In the other three models, tumor growth inhibition was observed in combination with either anti-PD-L1 or cisplatin. Conclusions: SC-2882 is an orally administered small molecule inhibitor with a favorable PK profile. Preclinical studies show that SC-2882 was well tolerated in mice. In several mouse tumor models, SC-2882 reduced tumor growth in either single agent treated animals or in combination with anti-PD-L1 or cisplatin. SC-2882 is currently in IND enabling studies and scheduled to enter the clinic in 2024. Citation Format: Jasper Mullenders, Marcel Scheepstra, Michel R. Faas, Annette B. Galler, Bastiaan Evers, Jens U. Wuerthner. SC-2882, a novel QPCTL inhibitor, with anti-tumor activity in solid tumor 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 4690.

Experimental model of primary intraocular lymphoma based on BALB/CaNn strain and A20 cells is optimal for investigational research.

The purpose of this project was to compare the characteristics of two experimental murine models of primary intraocular lymphoma (PIOL) and determine which experimental model is most suitable for further investigational research to elucidate the pathophysiology of PIOL and to find new therapeutical strategies. In both experimental models PIOL was induced in immunocompetent mice with intravitreal injection of syngeneic B-cell lymphoma cell lines. Murine strain C3H/HeN and cell line 38C13 were used in the first model and BALB/CaNn mice and cell line A20 in the second model. During the experiments, thorough clinical evaluation (using photo documentation, ultrasonography, and MRI) and histological evaluation were performed. In both models, the percentage of PIOL development was high, reaching nearly 80%. Disease progression was faster in C3H/HeN with exophthalmos occurring on average on day 10. Vitreous involvement was a predominant sign in the clinical presentation of this group. In BALB/CaNn mice exophthalmos occurred on average on day 22. The predominant clinical sign in the BALB/CaNn group was tumorous infiltration of the retina, optic disc, and tumorous retinal detachment. Slower progression of the disease in BALB/CaNn mice, greater possibility to examine the retina due to mild vitreous involvement, and later occurrence of exophthalmos makes this strain more suitable for further investigational research.

Promising preclinical efficacy of intravitreally administered tafasitamab and anti‐Hu CD199 on immunocompetent murine model of primary intraocular lymphoma

Aims/Purpose: This project aimed to evaluate the efficacy of anti‐CD19 monoclonal antibody tafasitamab and experimental chemokine receptor blocker anti‐Hu CD199 and to study the impact of the microbiome on immunotherapy in an experimental immunocompetent murine model of primary intraocular lymphoma (PIOL).Methods: PIOL was induced in immunocompetent mice BALB/CaNn by intravitreal injection of syngeneic lymphoma A20‐CD19 cell suspension. To eliminate intestinal microflora, 50% of the mice were pre‐treated with broad‐spectrum antibiotics (metronidazole and ciprofloxacin). On days 3 and 7, mice were treated intravitreally either with tafasitamab (20 μg) or anti‐Hu CD199 (1 μg). Following the injection mice were monitored clinically 2 times per week. The experiment was terminated at the first signs of exophthalmos or on day 30. Eyes were collected post‐mortem and histologically processed.Results: In both treated groups the signs of PIOL were delayed in comparison to the control group. The untreated group started to show signs of PIOL on day 7. In the group treated with tafasitamab first signs of PIOL started to appear on days 10–14. In the group which received experimental anti‐Hu CD199, signs of PIOL first appeared on day 17. Development of exophthalmos was delayed as well, most significantly in the group treated with anti‐Hu CD199. So far, we did not observe statistically significant differences in PIOL behaviour and the effect of the treatment between the group with the intact microbiome and the group treated with antibiotics.Conclusions: Results of in vivo experiments confirm the efficacy of both tafasitamab and experimental anti‐Hu CD199 administered intravitreally in immunocompetent mice with PIOL. However, experimental chemokine receptor blocker anti‐Hu CD199 shows greater potential in the intravitreal treatment of PIOL.Financial support of this project: AZV MZ CR NU20‐03‐00253, SVV UK 260631.

Photoablation with the Aurolase System Reduces T Cell Exhaustion and Synergizes with Immunotherapies in Lymphoma

Introduction: Immunotherapy is less active in non-Hodgkin lymphoma due to immune evasion from the tumor microenvironment. For diffuse large B cell lymphoma (DLBCL), the overall response rates (ORR) for immune checkpoint inhibitors (ICIs) were low (4-18%). Bispecific antibodies (BiAb), such as FDA-approved Epcoritamab and Glofitamab, had a complete response (CR) rate of 39% for DLBCL. Methods that alter local and systemic immune microenvironments are needed to improve the response to immunotherapy. Unlike radiation, photothermal therapy (PTT) can generate a strong T-cell activation signal. PTT is created by irradiating specially designed gold nanoparticles with near-infrared (NIR) light. The nanoparticles absorb the light and transfer the energy into heat, leading to very localized ablations. The local high heat stimulates a cascade of pro-inflammatory cytokines, including IL-6, IL-1β, TNF-α, G-CSF, GM-CSF, and CCL2. We previously found that PTT with toll-like receptor 9 agonists had a significantly higher CD8 to CD4 ratio, cytotoxic T cell to regulatory T cell ratio, and effector memory T cells in a murine lymphoma model. The AuroLase system, which uses silica gold nanoshells and an FDA-cleared laser, has been undergoing clinical trials for various solid tumors. Here, we explore the systemic immune effects of the PTT device in a lymphoma murine model and evaluate its synergistic effects with ICIs and BiAbs. Methods: The AuroLase system and AuroShells were provided by Nanospectra. The lymphoma model uses A20 cells implanted in Balb/c mice. Single flank tumors were used for immunophenotyping, and a dual tumor model was used to evaluate the systemic anti-lymphoma effects of PTT and combination therapies. PTT (day 0) was performed by injecting 20ul of nanoshells intratumorally and then irradiating the tumor with the laser at 4W for 30sec-1min. Spleens were collected on days (d) 1, 4, and 8 for immunophenotype by flow cytometry. For systemic immune response, we used a dual tumor model and treated the left side with PTT on d0. Anti-PD1 (aPD1) antibodies and the CD20xCD3 BiAbs were injected intraperitoneally on d1, 3, and 5. Results: In a single tumor lymphoma model, PTT did not change the size of the spleen or the total T cell count, including CD4, CD8, and regulatory T cell (Treg), compared to tumor naïve or tumor-bearing mice. However, the treatment reduced splenocyte T cell PD1 levels on d1 & 4 post-ablation, down to the level of tumor naive mice, and returned to elevated levels by d8, similar to untreated mice. CD4 and CD8 T cell PD1 levels dropped from 3.49% to 0.78% and 28% to 0.45%, respectively, post-ablation (p<0.001). Treg PD1 levels also dropped from 11.5% to 2.4%. There was a reduction of the central memory T cells (CD44 hi CD62 hi) population on d1&4 post-ablation but started to increase by d8. The PD1 expression pattern was similar to the prior, with a reduction on d1&4 and recovery by d8. The effector memory T cells (CD44 hi CD62 lo) had an increased population by d8 with the same PD1 reduction pattern. The B cell population didn't change, but there was an increase in B cell maturation (CD80 +) on d1&4. As for the myeloid cells, there is a sharp increase in dendritic cell population on d1 and increased maturation/activation on d1&4. There was an increased population of macrophages on d4. There was a reduction of myeloid-derived suppressor cells on d1. In the dual tumor model, the treated tumor had a dramatic loss of tumor volume due to PTT. The untreated tumor did not have a tumor reduction with PTT alone. However, when combined with anti PD1 (aPD1) therapy with just three doses, the untreated side had significant tumor growth reduction. By day 20, the PTT+aPD1 group had a tumor size of 378 mm 3, while the PTT alone and aPD1 alone groups were 1207 and 985 mm 3, respectively. There was also a survival advantage with PTT+aPD1. Similarly, we found that PTT+BiAb significantly delayed tumor growth compared to PTT only. Survival data is still ongoing. Conclusion: PTT in lymphoma is understudied and can effectively activate an anti-lymphoma response. PTT resulted in a marked reduction in PD1 expression on all T cells and activated several myeloid immune cells. We found that PTT combined with aPD1 therapy or BiAbs had improved tumor suppression in a lymphoma model. This system provides a platform and opportunity to translate PTT into the clinics.

The Immune Checkpoint Siglec-15 in Promoting Immune Dysregulation in Non-Hodgkin's Lymphomas

Outcomes for patients with relapsed or refractory (r/r) hematologic malignancies remain dismal despite therapeutic advances over the last several years. This is particularly striking in patients with r/r aggressive mature B-cell lymphomas such as Diffuse Large B-cell Lymphoma (DLBCL) and Burkitt's Lymphoma (BL). With use of intensive multiagent chemo-immunotherapy, DLBCL, which accounts for approximately 10-20% of pediatric Non-Hodgkin's Lymphoma (NHL) cases and is the most common subtype in adults, is highly curable with 5-year event free survival >75%. However, approximately 30-40% of adults diagnosed with DLBCL will become refractory to or relapse after first-line treatment. Yet even with intensive salvage regimens, r/r DLBCL remains a clinical challenge. There remains an unmet need to identify specific defects in the immune response, among other pathways, that drive lymphomagenesis that will in turn inform the development of novel therapeutics to improve survival in this population of patients. Siglec-15 (Sig-15), a member of the sialic acid binding immunoglobulin-like lectin family of proteins, has recently been identified as a critical immune suppressor that is highly expressed in human cancers and intra-tumoral myeloid cells. Importantly, recent reports have demonstrated inhibition of Sig-15, has a restorative effect on local anti-tumor immune responses and abrogated tumor progression. While reported in solid malignancies and acute leukemias, a role for Siglec-15 in promoting disease progression in aggressive B-cell lymphomas has not yet been described. We have found higher expression of SIGLEC15 in DLBCL cell lines compared to normal B cells at the RNA level, through analyses of the publicly available ONCOMINE database. This was confirmed at the protein level by western blot demonstrating higher Sig-15 expression in various lymphoma cell lines (RAJI, SUDHL6, OCI-LY10, HBL-1) compared to healthy donor PBMCs. Further, immunohistochemistry evaluating Sig-15 expression was performed on a tumor microarray consisting of 139 cases of DLBCL from adult patients along with validation samples from 15 primary pediatric lymphoma cases (DLBCL, BL and Anaplastic Large Cell Lymphoma (ALCL)). Sig-15 was found to be highly expressed, with histologic scores ranging from weak (1+) to strong (3+) in 124 out of the 139 adult cases (Figure 1A) and in all pediatric lymphoma samples but with distinct staining patterns observed in the aggressive B-cell lymphomas. Specifically, Sig-15 appears to be highly expressed and associated with the cell membrane in most DLBCL and Burkitt's lymphoma. Expression is more variable in ALCL, primarily elevated in the cytoplasm at low levels and/or in macrophages (Figure 1A). There is heterogeneity in Sig-15 expression with preliminary analysis of RNA-seq data from a cohort of 693 DLBCL patient samples demonstrates higher levels in the germinal center B-cell (GCB) versus activated B-cell molecular (ABC) subtype of DLBCL when compared to healthy controls (Kruskal-Wallis, *p = 0.041). Finally, using a well-established murine lymphoma model, A20 cells were stably transduced with control, non-silencing shRNA (shNS) or shRNA against Sig-15 and injected into un-irradiated immune competent (wild type, WT) or immune deficient ( Rag1 -/-) BALB/C mice and monitored for signs of lymphoma development. Knockdown of Sig-15 in A20 cells abrogates disease progression in WT but not immune deficient mice (Log-rank, **p <0.0001, n=10 mice/group), consistent with a role for Sig-15 in immune evasion in lymphoma (Figure 1B). Together, these data implicate Sig-15 as an immune checkpoint that may be inhibited therapeutically to promote an immune response to lymphoma cells.

Takeaim Lymphoma: An Open-Label, Dose Escalation and Expansion Trial of Emavusertib (CA-4948) in Combination with Ibrutinib in Patients with Relapsed or Refractory Hematologic Malignancies

Background: Emavusertib (CA-4948) is a novel oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4), which is essential for toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling in B cell proliferation. Additional inhibitory activity against FMS-like tyrosine kinase 3 (FLT3) and CDC-like kinases (CLK1/2/4) is also present in emavusertib. IRAK4 forms a Myddosome complex with MYD88 adaptor protein and drives overactivation ofnuclear factor-kappa B (NF-κB), causing inflammation and tumor growth. Emavusertib has been reported to be well tolerated and active as monotherapy in heavily pretreated patients with relapsed/refractory (R/R) non-Hodgkin lymphoma (NHL) (Nowakowski 2020). Preclinical studies demonstrated that tumor resistance and survival via IRAK4 activation could be delayed or reversed. Emavusertib crossed the blood-brain barrier in a murine PDX model of PCNS lymphoma, blocked the activated IRAK4 pathway, and induced tumor response and prolonged survival (von Roemeling 2022). In combination with Bruton tyrosine kinase (BTK) inhibitors, emavusertib showed in vitro synergy and re-sensitization of resistant cell lines, overcoming BTK resistance (Guidetti 2023a and 2023b). In vivo synergy in B-cell NHL was also demonstrated. Adaptive chemotherapy treatment resistance through IRAK4 upregulation and activation was also described in other disease models (Melgar 2019, Li 2019). This abstract presents preliminary efficacy and safety data of emavusertib + ibrutinib in R/R NHL patients. Aim:Assessment of safety and clinical activity of emavusertib in combination with ibrutinib. Methods: This is an ongoing open-label trial (NCT03328078) of emavusertib as monotherapy and in combination with ibrutinib. Part A1 - completed; dose escalation of emavusertib as monotherapy; the recommended phase 2 dose (RP2D) is 300 mg BID with continuous oral dosing (Nowakowski 2020). Part A2 - completed; dose escalation of emavusertib in combination with ibrutinib at 420 and 560 mg QD (Joffe 2022). Part B - ongoing; an expansion cohort of PCNSL patients with resistance to prior BTK inhibitors for additional safety and efficacy related to exposure: emavusertib at 100 or 200 mg BID + ibrutinib at 560 mg QD in 28-Day cycles. Results:As of 19 July 2023, 18 heavily pretreated NHL (including five PCNSL) patients received emavusertib at 100, 200, or 300 mg BID in combination with full ibrutinib doses per label. Median age was 63.5 years (range 50-92). Median number of prior lines of anti-cancer therapies was 3 (range 1-10). Eleven of 18 patients failed prior BTK inhibitors. Median treatment duration was 95.5 days (21-587 days), suggesting acceptable safety and tolerability. No DLTs were observed at the 200 mg BID dose level, and two reversible DLTs (stomatitis and syncope) were observed at the 300 mg BID dose level. The preliminary efficacy data of 16 evaluable patients in combination with ibrutinib showed 5 CRs (2 MCL, 3 PCNSL) and 1 PR (1 CLL). All 3 PCNSL patients with CR were previously treated with BTK inhibitors. Conclusion: The combination ofemavusertib plus ibrutinib (ema+ibr) is well tolerated with an acceptable long-term safety profile and promising efficacy, showing several objective responses in heavily pretreated and/or BTK inhibitor resistant patients. Emavusertib may have the potential to overcome BTK inhibitor resistance and the combination of ema+ibr has the potential to show increased anti-cancer activity compared to ibrutinib monotherapy.

242 Inhibition of STAT3/5 pathway by multi-kinase inhibitor IQDMA reduces tumor growth and outperforms conventional phototherapeutic treatment regime in cutaneous T cell lymphoma murine model

242 Inhibition of STAT3/5 pathway by multi-kinase inhibitor IQDMA reduces tumor growth and outperforms conventional phototherapeutic treatment regime in cutaneous T cell lymphoma murine model

Melatonin modulates L-arginine metabolism in tumor-associated macrophages by targeting arginase 1 in lymphoma.

L-Arginine metabolism plays a crucial role in determining the M1/M2 polarization of macrophages. The M1 macrophages express inducible nitric oxide synthase (iNOS), while the M2 macrophages express arginase 1 and metabolize arginine into nitric oxide and urea, respectively. The tumor microenvironment promotes M2 macrophage polarization and consequently switches the metabolic fate of arginine from nitric oxide towards urea production. Importantly, infiltration of M2 macrophages or tumor-associated macrophages (TAMs) has been correlated with poor prognosis of various cancer types. Melatonin is well reported to have antitumor and immunomodulatory properties. However, whether and how it impacts the polarization of TAMs has not been elucidated. Considering the crucial role of arginine metabolism in macrophage polarization, we were interested to know the fate of L-arginine in TAMs and whether it can be reinstated by melatonin or not. We used a murine model of Dalton's lymphoma and established an in vitro model of TAMs. For TAMs, we used the ascitic fluid of tumor-bearing hosts to activate the macrophages in the presence and absence of lipopolysaccharide (LPS). In these groups, L-arginine metabolism was evaluated, and then the effect of melatonin was assessed in these groups, wherein the metabolic fate of arginine as well as the expression of iNOS and arginase 1 were checked. Furthermore, in the in vivo system of the tumor-bearing host, the effect of melatonin was assessed. The in vitro model of TAMs showed a Th2 cytokine profile, reduced phagocytic activity, and increased wound healing ability. Upon investigating arginine metabolism, we observed high urea levels with increased activity and expression of arginase 1 in TAMs. Furthermore, we observed reduced levels of LPS-induced nitric oxide in TAMs; however, their iNOS expression was comparable. With melatonin treatment, urea level decreased significantly, while the reduction in nitric oxide level was not as significant as observed in its absence in TAMs. Also, melatonin significantly reduced arginase activity and expression at the transcriptional and translational levels, while iNOS expression was affected only at the translational level. This effect was further investigated in the in vivo system, wherein melatonin treatment reversed the metabolic fate of arginine, from urea towards nitric oxide, within the tumor microenvironment. This effect was further correlated with pro-apoptotic tumor cell death in the in vivo system. Our results reinforced the immunomodulatory role of melatonin and offered a strong prospect for activating the anti-tumor immune response in cancer conditions.

The antagonist of β-adrenergic receptor propranolol inhibits T cell lymphoma growth and enhances antitumor efficacy of cisplatin in vivo: A role of modulated apoptosis, glucose metabolism, pH regulation, and antitumor immune response

Accumulating evidence has shown a vital role of stress-regulatory hormones, including epinephrine, in the progression of numerous cancers, including T cell lymphoma. Further, the antitumor and chemosensitizing potential of propranolol, an inexpensive β-adrenergic receptor antagonist has also been reported against breast, colon, ovarian, and pancreatic cancers. However, in vivo antitumor and chemopotentiating activity of propranolol have not yet been examined against malignancies of hematological origin, including T cell lymphoma. Therefore, the present study is designed to evaluate the antitumor and chemopotentiating action of propranolol in a T cell lymphoma murine model. In this study, T cell lymphoma-bearing mice were treated with vehicle alone (PBS) or containing propranolol followed by administration of with or without cisplatin. The progression of the tumor was assessed along with analysis of tumor cell apoptosis, glucose metabolism, pH regulation, and antitumor immune response. The apoptosis was estimated by cellular and nuclear morphology analysis through Wright-Giemsa, annexin-V, and DAPI staining. ELISA was used to detect the epinephrine level in serum. The glucose, lactate, and NO levels were measured in the tumor ascitic fluid by calorimetric methods. RT-PCR and Western blot were used to assess the levels of various crucial regulators at gene and protein levels, respectively. Our results showed that propranolol exerts antitumor as well as chemopotentiating ability in DL-bearing mice by altering apoptosis, glycolysis, acidification of TME, and immunosuppression.

CLN-978, a novel half-life extended CD19/CD3/HSA-specific T cell-engaging antibody construct with potent activity against B-cell malignancies with low CD19 expression

BackgroundDespite significant progress in the development of T cell-engaging therapies for various B-cell malignancies, a high medical need remains for the refractory disease setting, often characterized by suboptimal target levels.MethodsTo...

Degradation of CD47‐SIRPα axis by pomalidomide potentiates CD20 antibody‐dependent cellular phagocytosis against B‐cell lymphoma

Introduction: Anti-CD20 monoclonal antibodies (mAbs) have revolutionized the treatment of B-cell lymphomas (BCL). The chemo-free combination with lenalidomide (Len) and rituximab (R2) as a first-line therapy has shown promising activity in indolent lymphomas. However, the efficacy of R2 regimen is yet unsatisfactory in aggressive settings, thereby novel synergistic strategy with next-generation CD20 mAbs and/or newer immunomodulatory drugs (IMiD) are urgently needed. Pomalidomide (Pom) is more powerful than Len in immunomodulatory properties including redirection of tumor-associated macrophages (TAM), which may confer a therapeutic opportunity to potentiate antibody-dependent cellular phagocytosis (ADCP) in elimination of lymphoma cells. This study aimed to evaluate the macrophage-based anti-lymphoma activities of Pom plus CD20 mAbs. Methods: BCL cell lines (SU-DHL-4, Raji and Daudi) were co-cultured with human monocyte-derived macrophages and treated with various combinations with IMiDs (Len or Pom) plus CD20 mAbs (rituximab or obinutuzumab). Phagocytosis and cell viability of lymphoma cells were tested by flow cytometry. Protein mass spectrometry and co-immunoprecipitation (co-IP) were performed to identify Pom-redirected neosubstrates of cereblon (CRBN). IMiD-responsive CrbnI391V mice were bred to Eμ-Myc lymphoma mice to evaluate the in vivo immunomodulatory effects of Pom on macrophages. Results: In vitro co-culture systems showed that Pom plus obinutuzumab elicited the strongest ADCP effects, resulting in robust phagocytosis of tumor cells by macrophages and markedly decreased tumor cell number, when compared to other combinations including Len plus rituximab (R2), Len plus obinutuzumab (GALEN) or Pom plus rituximab. Mechanistically, we identified CD47, a well-known negative regulator of macrophage phagocytosis, as a novel CRBN neosubstrate in BCL cells using mass spectrometry screening and co-IP validation. Interestingly, we found that only Pom rather than Len or thalidomide could efficiently induce degradation of total and surface CD47 in SU-DHL-4 cells. We further confirmed that knockdown of CRBN abrogated Pom-induced CD47 degradation, indicating the on-target effect. More importantly, based on the murine spontaneous lymphoma models (Eμ-Myc CrbnI391V mice), we found administration of Pom elicited a shift from M2 (CD206+) to M1 (CD86+) phenotype of tumor-associated macrophages and significant decrease of SIRPα+ populations in both M1 and M2 subtypes. Conclusion: Our study provides evidence for the superior effects of Pom plus obinutuzumab by harnessing the anti-lymphoma activity of TAMs, and suggests degradation of CD47-SIRPα axis as a novel mechanism underlying the drug synergy. Further preclinical and clinical investigations are needed to determine the in vivo synergistic efficacy and potential toxicity of this combined treatment. The research was funded by: This study was supported by the National Natural Science Foundation of China (No. 81470336 to KZ). Keywords: Aggressive B-cell non-Hodgkin lymphoma, Combination Therapies, Immunotherapy No conflicts of interests pertinent to the abstract.

Adoptive Transfer of Photosensitizer-Loaded Cytotoxic T Cells for Combinational Photodynamic Therapy and Cancer Immuno-Therapy.

Adoptive cell transfer (ACT) has shown remarkable therapeutic efficacy against blood cancers such as leukemia and lymphomas, but its effect is still limited due to the lack of well-defined antigens expressed by aberrant cells within tumors, the insufficient trafficking of administered T cells to the tumor sites, as well as immunosuppression induced by the tumor microenvironment (TME). In this study, we propose the adoptive transfer of photosensitizer (PS)-loaded cytotoxic T cells for a combinational photodynamic and cancer immunotherapy. Temoporfin (Foscan®), a clinically applicable porphyrin derivative, was loaded into OT-1 cells (PS-OT-1 cells). The PS-OT-1 cells efficiently produced a large amount of reactive oxygen species (ROS) under visible light irradiation in a culture; importantly, the combinational photodynamic therapy (PDT) and ACT with PS-OT-1 cells induced significant cytotoxicity compared to ACT alone with unloaded OT-1 cells. In murine lymphoma models, intravenously injected PS-OT-1 cells significantly inhibited tumor growth compared to unloaded OT-1 cells when the tumor tissues were locally irradiated with visible light. Collectively, this study suggests that combinational PDT and ACT mediated by PS-OT-1 cells provides a new approach for effective cancer immunotherapy.

Multiagent Intratumoral Immunotherapy Can Be Effective in A20 Lymphoma Clearance and Generation of Systemic T Cell Immunity

The use of immunotherapies has shown promise against selective human cancers. Identifying novel combinations of innate and adaptive immune cell-activating agents that can work synergistically to suppress tumor growth and provide additional protection against resistance or recurrence is critical. The A20 murine lymphoma model was used to evaluate the effect of various combination immunotherapies administered intratumorally. We show that single-modality treatment with Poly(I:C) or GM-CSF-secreting allogeneic cells only modestly controls tumor growth, whereas when given together there is an improved benefit, with 50% of animals clearing tumors and surviving long-term. Neither heat nor irradiation of GM-CSF-secreting cells enhanced the response over use of live cells. The use of a TIM-3 inhibitory antibody and an OX40 agonist in combination with Poly(I:C) allowed for improved tumor control, with 90% of animals clearing tumors with or without a combination of GM-CSF-secreting cells. Across all treatment groups, mice rejecting their primary A20 tumors were immune to subsequent challenge with A20, and this longstanding immunity was T-cell dependent. The results herein support the use of combinations of innate and adaptive immune activating agents for immunotherapy against lymphoma and should be investigated in other cancer types.

The dual role of CD70 in B-cell lymphomagenesis.

CD70 is a costimulatory molecule that is transiently expressed on a small set of activated lymphocytes and is involved in T-cell-mediated immunity. However, the role of CD70 in B-cell malignancies remains controversial. We investigated the clinical relevance of CD70 genetic alterations and its protein expression in two diffuse large B-cell lymphoma (DLBCL) cohorts with different ethnic backgrounds. We also performed transcriptomic analysis to explore the role of CD70 alterations in tumour microenvironment. We further tested the blockade of CD70 in combination with PD-L1 inhibitor in a murine lymphoma model. We showed that CD70 genetic aberrations occurred more frequently in the Chinese DLBCL cohort (56/233, 24.0%) than in the Swedish cohort (9/84, 10.8%), especially in those with concomitant hepatitis B virus (HBV) infection. The CD70 genetic changes in DLBCL resulted in a reduction/loss of protein expression and/or CD27 binding, which might impair T cell priming and were independently associated with poor overall survival. Paradoxically, we observed that over-expression of CD70 protein was also associated with a poor treatment response, as well as an advanced disease stage and EBV infection. More exhausted CD8+ T cells were furthermore identified in CD70 high-expression DLBCLs. Finally, in a murine lymphoma model, we demonstrated that blocking the CD70/CD27 and/or PD1/PD-L1 interactions could reduce CD70+ lymphoma growth in vivo, by directly impairing the tumour cell proliferation and rescuing the exhausted T cells. Our findings suggest that CD70 can play a role in either tumour suppression or oncogenesis in DLBCL, likely via distinct immune evasion mechanisms, that is, impairing T cell priming or inducing T cell exhaustion. Characterisation of specific dysfunction of CD70 in DLBCL may thus provide opportunities for the development of novel targeted immuno-therapeutic strategies.

FGFR1 Is a Novel Therapeutic Target in Relapsed/Refractory Diffuse Large B Cell Lymphoma (RR-DLBCL)

More than 40% of pts DLBCL will not respond to first-line immunochemotherapy. The therapeutic response of DLBCL is rooted in its heterogeneous genetic landscape and its lymphoma microenvironment (LME). In this regard, the roles of cancer-associated fibroblasts (CAF) and tumor-associated macrophages (TAMs) in lymphoma progression and therapy response have been started to be elucidated (Kotlov ... Cerchietti. Cancer Discovery 2021). We analyzed (by transcriptomics and multiplex imaging) the LME cellular composition and heterogeneity in paired samples from diagnostic and relapsed (RR) DLBCLs (n = 10) and early and late murine lymphoma progression models (A20, Myc + Bcl2, Ezh2 mutant + Bcl6, and Setd2 mutant + Bcl2). We found that, in both human and mouse DLBCLs, the heterogeneity of the LME decreases as a function of lymphoma progression. RR-DLBCLs are characterized by a significant decrease in tumor CD3+ T-cell infiltration, lower T-cell diversity, and expansion of a subpopulation of CAFs upregulating the fibroblast growth factor receptor FGFR1 (CAFFGFR1). Thus, we investigated the role of CAFFGFR1 in pre-clinical models of RR-DLBCL biology and therapy. We first proved that lymphoma cells (murine and human, primary and relapsed, ABC- and GCB-DLBCLs) do not express FGFR1 and do not respond to its pharmacological inhibition or genetic knock-down in cell cultures. We then tested the anti-lymphoma effect of the novel selective oral FGFR1 inhibitor SSR128129E (FGFR1i) in two engrafted syngeneic mouse models DLBCL (A20 and Setd2+/-/Bcl2OE) implanted subcutaneously and intrasplenically, respectively, in immunocompetent mice. FGFR1i (oral, 10 mg/kg) significantly decreased tumor burden in both models (p<0.05). Analysis of the LME at the end of the treatment showed that the LME of FGFR1i treated mice had significantly increased TAMs (p<0.005). There were no differences in the proportions of other LME cell populations, including CAFs. To further investigate the role of TAMs concerning FGFR1i in RR-DLBCL harboring CAFFGFR1-rich LMEs, we established two RR-DLBCL patient-derived xenografts (PDX) models. Treatment of RR-DLBCL PDX-1 and PDX-2 with CHOP confirmed the chemorefractory nature of the models; however, both models were highly responsive to oral 10 mg/kg FGFR1i (p<0.05, both models vs. vehicle). To understand how the FGFR1i affected distinct CAF and TAM subpopulations, we conducted single-cell RNA-sequencing in LME from these PDXs. We found that FGFR1i-treated mice had a significant increase in inflammatory-like CAFs characterized by the expression of several monocyte/macrophage chemotactic factors, including CCL2, CCL7, CXCL3, and CXCL12, which agrees with the increase in the LM-TAM in the syngeneic models. In FGFR1i mice, we found a sub-set of TAMs that expressed high levels of classical and alternative complement receptors critical for phagocytosis, suggesting that this process can be relevant for the anti-lymphoma effect. To test this notion, we assessed the effect of FGFR1i in a murine lymphoma model depleted of macrophages using liposomal clodronate. We found a significant decrease in the anti-lymphoma effect of FGFR1i when TAMs were depleted (p<0.05). This suggests that phagocytic TAMs are important for the response to FGFR1i, a concept we are testing in additional DLBCL models. In conclusion, our results suggest that CAFFGFR1s could constitute a novel LME-resident therapeutic vulnerability in RR-DLBCL and that FGFR1i may require phagocytic TAMs for its anti-lymphoma effect. Given the FDA approval of several FGFR1/2 inhibitors for solid tumors, our results bare translational potential to benefit RR-DLBCL patients in the near term.