Syngeneic Lymphoma Model Research Articles

Development of Syngeneic Murine Cell Lines of Germinal Center-Derived B-Cell Lymphomas

Background. Germinal center (GC)-derived B-cell lymphomas are clinically and biologically heterogeneous. Next generation sequencing has enabled identification of genetic aberrations involved in lymphoma pathogenesis, which may provide prognostic predictions and potential therapeutic targets. However, the mechanisms driving pathogenesis are still not fully understood, especially in the context of an intact tumor microenvironment, in part because there are no available syngeneic models of lymphoma faithfully representing the pathological features of human disease. Therefore, there is a critical need for lymphoma models which can be studied in immunocompetent mice. Methods. We established eight murine cell lines derived from spontaneous B-cell lymphomas with GC origin arising in genetically engineered mouse models (GEMM). These cell lines were developed by passing an original tumor from a GEMM through several passages of Rag1-/- and wild type mice, which enabled selection of more fit/aggressive clones that grow in vitro. We also tested multiple culture conditions and characterized the lines via flow cytometry, VDJ and targeted sequencing, karyotyping, and evaluation of tumor development and its microenvironment in vivo. Results. This resource describes eight cell lines driven by recurrent genetic mutations found in diffuse large B-cell lymphoma and follicular lymphoma patients. The first cell line is defined by (1) BCL2 overexpression (hereafter B2). Four cell lines couple BCL2 overexpression with epigenetic dysregulation, defined by (2) BCL2 and gain-of-function (GOF) mutation of EZH2 Y641F (hereafter EZB), (3) EZB and MYC overexpression (hereafter EBM), (4) BCL2 with knockout of KMT2D (hereafter KB), and (5) BCL2 with knockout of SETD2 (hereafter S2B). Finally, three cell lines are defined by mutations to the Toll-like and B-cell receptors pathways, defined by (6) BCL2 and GOF mutations in Myd88 L252P and CD79b Y195H (hereafter MBC79), (7) BCL2, Myd88 L252P and GOF mutation in Card11 L251P (hereafter MBC11), and (8) BCL2 and Card11 L251P (hereafter BC11). Doubling time of these lines ranges from 30-72 hours, with EZB and EBM on the fast end and S2B on the slow end. By flow cytometry, we show that B2, MBC79, and MBC11 lose B-cell surface markers and are very plasmacytic (>95% B220- CD19- CD138+). In contrast, the EZB, EBM, KB, BC11, and S2B are mostly characterized by GC B-cell markers (B220+ CD19+ CD138- FAS+ CD38- GL7+). By VDJ sequencing, we found that EZB, BC11, and S2B are very monoclonal, with one rearrangement arising from the original tumor and taking over to represent >95% of clones. In contrast, the MCB11 and MBC79 lines are very polyclonal, with the top 10 rearrangements being equally represented, and tending to shift between passages. Thus far, S2B and EZB have been shown to grow in immunocompetent mice, representing novel syngeneic lymphoma models. While mice injected with S2B can survive for up to 30 days, mice injected with EZB die significantly earlier. We established two EZB lines from early (P1) and late (P6) passages of a tumor arising from the same original mouse. Notably, we show that mice injected with P1 survive 15-20 days, whereas mice injected with P6 survive 10-15 days. Although both P1 and P6 grow at similar rates in vitro, P1 develops high grade follicular lymphomas in C57BL6 recipients, whereas P6 develops DLBCL, suggesting additional passaging drives selection for more aggressive disease. Imaging of the tumors reveals a lack of follicular pattern. Notably, P6 tumors display less infiltration of CD4 and CD8 T-cells and low abundance or absence of FDC meshwork, as compared to P1. Imaging of mice engrafted with luciferase transduced P1 and P6 shows dissemination into spleen, lymph nodes, liver, and kidney, with more advanced disease in P6 injected mice. Conclusion. While conventional preclinical tumor mouse models are often used for drug sensitivity and treatment prediction studies, they are time-consuming and expensive. These cell lines serve as a preclinical tool to test novel therapeutic strategies and develop a deeper understanding of the molecular mechanisms driving lymphomagenesis, therapeutic response, and resistance in genetically defined subtypes of GC-derived B-cell lymphomas. Most importantly, this resource meets a critical need in the field for syngeneic models of lymphoma to study disease progression and precision therapy in immunocompetent mice.

Bruton tyrosine kinase inhibitors preserve anti-CD19 chimeric antigen receptor T-cell functionality and reprogram tumor micro-environment in B-cell lymphoma

Background aimsCombination therapy is being actively explored to improve the efficacy and safety of anti-CD19 chimeric antigen receptor T-cell (CART19) therapy, among which Bruton tyrosine kinase inhibitors (BTKIs) are highly expected. BTKIs may modulate T-cell function and remodel the tumor micro-environment (TME), but the exact mechanisms involved and the steps required to transform different BTKIs into clinical applications need further investigation. MethodsWe examined the impacts of BTKIs on T-cell and CART19 phenotype and functionality in vitro and further explored the mechanisms. We evaluated the efficacy and safety of CART19 concurrent with BTKIs in vitro and in vivo. Moreover, we investigated the effects of BTKIs on TME in a syngeneic lymphoma model. ResultsHere we identified that the three BTKIs, ibrutinib, zanubrutinib and orelabrutinib, attenuated CART19 exhaustion mediated by tonic signaling, T-cell receptor (TCR) activation and antigen stimulation. Mechanistically, BTKIs markedly suppressed CD3-ζ phosphorylation of both chimeric antigen receptor and TCR and downregulated the expression of genes associated with T-cell activation signaling pathways. Moreover, BTKIs decreased interleukin 6 and tumor necrosis factor alpha release in vitro and in vivo. In a syngeneic lymphoma model, BTKIs reprogrammed macrophages to the M1 subtype and polarized T helper (Th) cells toward the Th1 subtype. ConclusionsOur data revealed that BTKIs preserved T-cell and CART19 functionality under persistent antigen exposure and further demonstrated that BTKI administration was a potential strategy for mitigating cytokine release syndrome after CART19 treatment. Our study lays the experimental foundation for the rational application of BTKIs combined with CART19 in clinical practice.

Pharmacologic targeting of Nedd8-activating enzyme reinvigorates T-cell responses in lymphoid neoplasia

Neddylation is a sequential enzyme-based process which regulates the function of E3 Cullin-RING ligase (CRL) and thus degradation of substrate proteins. Here we show that CD8+ T cells are a direct target for therapeutically relevant anti-lymphoma activity of pevonedistat, a Nedd8-activating enzyme (NAE) inhibitor. Pevonedistat-treated patient-derived CD8+ T cells upregulated TNFα and IFNγ and exhibited enhanced cytotoxicity. Pevonedistat induced CD8+ T-cell inflamed microenvironment and delayed tumor progression in A20 syngeneic lymphoma model. This anti-tumor effect lessened when CD8+ T cells lost the ability to engage tumors through MHC class I interactions, achieved either through CD8+ T-cell depletion or genetic knockout of B2M. Meanwhile, loss of UBE2M in tumor did not alter efficacy of pevonedistat. Concurrent blockade of NAE and PD-1 led to enhanced tumor immune infiltration, T-cell activation and chemokine expression and synergistically restricted tumor growth. shRNA-mediated knockdown of HIF-1α, a CRL substrate, abrogated the in vitro effects of pevonedistat, suggesting that NAE inhibition modulates T-cell function in HIF-1α-dependent manner. scRNA-Seq-based clinical analyses in lymphoma patients receiving pevonedistat therapy demonstrated upregulation of interferon response signatures in immune cells. Thus, targeting NAE enhances the inflammatory T-cell state, providing rationale for checkpoint blockade-based combination therapy.

Preclinical study of 212Pb alpha-radioimmunotherapy targeting CD20 in non-Hodgkin lymphoma.

Despite therapeutic advances, Non-Hodgkin lymphoma (NHL) relapses can occur. The development of radioimmunotherapy (RIT) with α-emitters is an attractive alternative. In this study, we investigated the potential of α-RIT in conjunction with 212Pb-rituximab for the treatment of NHL. EL4-hCD20-Luc cells (mouse lymphoma cell line) were used for in vitro and in vivo studies. Biodistribution and efficacy studies were performed on C57BL/6 mice injected intravenously with 25 × 103 cells. 212Pb-rituximab (0.925-7.4 kBq/mL) inhibit proliferation of EL4-hCD20-Luc cells in vitro. Biodistribution of 203/212Pb-rituximab in mice showed a significant tumour uptake and suggested that the liver, spleen, and kidneys were the organs at risk. For efficacy studies, mice were treated at either 11 days (early stage) or 20-30 days after injection of tumour cells (late stage). Treatment with 277.5 kBq 212Pb-rituximab significantly prolonged survival. Even at an advanced tumour stage, significant tumour regression occurred, with an increase in the median survival time to 28 days, compared with 9 days in the controls. These results show the efficacy of 212Pb-rituximab in a murine syngeneic lymphoma model, in terms of significant tumour regression and increased survival, thereby highlighting the potency of α-RIT for the treatment of NHL.

Dual Targeting Oncoproteins MYC and HIF1α Regresses Tumor Growth of Lung Cancer and Lymphoma.

Simple SummaryBoth MYC and HIF1α are critical transcriptional factors involved in the initiation, transformation, progression and maintenance in a variety of human tumors, but till now no study indicates whether it is possible to simultaneously targeting both MYC and HIF1α for therapeutics. Here we report that echinomycin simultaneously inhibited MYC and HIF1α through proteasomal degradation. Treatment of echinomycin regressed tumor cell growth both in vitro cultured cells and in vivo mouse models of lung cancer and lymphoma. β-TrCP and VHL are involved in the degradation of MYC and HIF1α induced by echinomycin, respectively. Our data provided a new approach to target these and potentially other oncogenic proteins for cancer therapy.MYC and HIF1α are among the most important oncoproteins whose pharmacologic inhibition has been challenging for the diverse mechanisms driving their abnormal expression and because of the challenge in blocking protein-DNA interactions. Surprisingly, we found that MYC and HIF1α proteins in echinomycin-treated cells were degraded through proteasome dependent pathways, respectively by the β-TrCP- or VHL-dependent mechanisms. The degradation is induced in a variety of cancer types, including those with mutations in the p53 tumor and LKB tumor suppressors and the KRAS oncogene. Consistent with inhibition of MYC and HIF1α, administration of echinomycin inhibited growth of lung adenocarcinoma xenograft and a syngeneic lymphoma model in mice. Furthermore, echinomycin efficiently induced regression of syngeneic mouse lymphoma driven by MYC over-expression. Our data demonstrated a new mechanism by which echinomycin simultaneously targets MYC and HIF1α for degradation to inhibit growth of lung cancer and lymphoma. Given the broad impact of β-TrCP or VHL in stability of oncogenic proteins, echinomycin may emerge as a non-PROTAC (proteolysis targeting chimera) degrader of oncogenic proteins.

Regulation of PD-L1 expression is a novel facet of cyclic-AMP-mediated immunosuppression.

Cyclic-AMP (cAMP) exerts suppressive effects in the innate and adaptive immune system. The PD-1/PD-L1 immune checkpoint down-regulates T-cell activity. Here, we examined if these two immunosuppressive nodes intersect. Using normal and malignant lymphocytes from humans, and the phosphodiesterase 4b (Pde4b) knockout mouse, we found that cAMP induces PD-L1 transcription and protein expression. Mechanistically, we discovered that the cAMP effectors PKA and CREB induce the transcription/secretion of IL-10, IL-8 and IL-6, which initiate an autocrine loop that activates the JAK/STAT pathway and ultimately increase PD-L1 expression in the cell surface. This signaling axis is disarmed at two specific nodes in subsets of diffuse large B cell lymphoma, which may help explain the variable PD-L1 expression in these tumors. In vivo, we found that despite its immunosuppressive attributes, the PDE4 inhibitor roflumilast did not decrease the clinical activity of checkpoint inhibitors, an important clinical observation given the approved use of these agents in multiple diseases. In summary, we discovered that PD-L1 induction is a part of the repertoire of immunosuppressive actions mediated by cAMP, defined a cytokine-mediated autocrine loop that executes this action and, reassuringly, showed that PDE4 inhibition does not antagonize immune checkpoint blockade in an in vivo syngeneic lymphoma model.

The Inhibitor of Apoptosis Protein Livin Confers Resistance to Fas-Mediated Immune Cytotoxicity in Refractory Lymphoma.

Death receptor Fas-mediated apoptosis not only eliminates nonspecific and autoreactive B cells but also plays a major role in antitumor immunity. However, the possible mechanisms underlying impairment of Fas-mediated induction of apoptosis during lymphomagenesis remain unknown. In this study, we employed our developed syngeneic lymphoma model to demonstrate that downregulation of Fas is required for both lymphoma development and lymphoma cell survival to evade immune cytotoxicity. CD40 signal activation significantly restored Fas expression and thereby induced apoptosis after Fas ligand treatment in both mouse and human lymphoma cells. Nevertheless, certain human lymphoma cell lines were found to be resistant to Fas-mediated apoptosis, with Livin (melanoma inhibitor of apoptosis protein; ML-IAP) identified as a driver of such resistance. High expression of Livin and low expression of Fas were associated with poor prognosis in patients with aggressive non-Hodgkin's lymphoma. Livin expression was tightly driven by bromodomain and extraterminal (BET) proteins BRD4 and BRD2, suggesting that Livin expression is epigenetically regulated in refractory lymphoma cells to protect them from Fas-mediated apoptosis. Accordingly, the combination of CD40-mediated Fas restoration with targeting of the BET proteins-Livin axis may serve as a promising immunotherapeutic strategy for refractory B-cell lymphoma. SIGNIFICANCE: These findings yield insights into identifying risk factors in refractory lymphoma and provide a promising therapy for tumors resistant to Fas-mediated antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/20/4439/F1.large.jpg.

Development of CDX-527: a bispecific antibody combining PD-1 blockade and CD27 costimulation for cancer immunotherapy

CD27 is a costimulatory molecule that provides a complementary target to the PD-1/PD-L1 checkpoint axis on T cells. Combining a CD27 agonist antibody with PD-1/PD-L1 blockade has shown synergistic antitumor activity in preclinical models, which led to clinical studies of the combination in cancer patients. We theorized that coupling CD27 costimulation with PD-1/PD-L1 blockade in a bispecific antibody (BsAb) may provide greater immune activating properties than combining the individual mAbs due to enhanced CD27 activation by cross-linking through PD-L1 and Fc receptors. To test this approach, we developed CDX-527, a tetravalent PD-L1xCD27 IgG1-scFv BsAb. CDX-527 potently inhibits PD-1 signaling and induces CD27-mediated T cell costimulation through PD-L1 cross-linking. In mixed lymphocyte reaction assays, CDX-527 is more potent than the combination of the parental antibodies, suggesting that cross-linking through both Fc receptors and PD-L1 results in enhanced CD27 agonist activity. CDX-527 was shown to mediate effector function against tumor cells overexpressing either CD27 or PD-L1. In human CD27 transgenic mice, we observed that antigen-specific T cell responses to a vaccine are greatly enhanced with a surrogate PD-L1xCD27 BsAb. Furthermore, the BsAb exhibits greater antitumor activity than the combination of the parental antibodies in a syngeneic lymphoma model. A pilot study of CDX-527 in cynomolgus macaques confirmed a mAb-like pharmacokinetic profile without noted toxicities. These studies demonstrate that CDX-527 effectively combines PD-1 blockade and CD27 costimulation into one molecule that is more potent than combination of the parental antibodies providing the rationale to advance this BsAb toward clinical studies in cancer patients.

Parsaclisib Is a Next-Generation Phosphoinositide 3-Kinase δ Inhibitor with Reduced Hepatotoxicity and Potent Antitumor and Immunomodulatory Activities in Models of B-Cell Malignancy.

The clinical use of first-generation phosphoinositide 3-kinase (PI3K)δ inhibitors in B-cell malignancies is hampered by hepatotoxicity, requiring dose reduction, treatment interruption, and/or discontinuation of therapy. In addition, potential molecular mechanisms by which resistance to this class of drugs occurs have not been investigated. Parsaclisib (INCB050465) is a potent and selective next-generation PI3Kδ inhibitor that differs in structure from first-generation PI3Kδ inhibitors and has shown encouraging anti-B-cell tumor activity and reduced hepatotoxicity in phase 1/2 clinical studies. Here, we present preclinical data demonstrating parsaclisib as a potent inhibitor of PI3Kδ with over 1000-fold selectivity against other class 1 PI3K isozymes. Parsaclisib directly blocks PI3K signaling-mediated cell proliferation in B-cell lines in vitro and in vivo and indirectly controls tumor growth by lessening immunosuppression through regulatory T-cell inhibition in a syngeneic lymphoma model. Diffuse large B-cell lymphoma cell lines overexpressing MYC were insensitive to proliferation blockade via PI3Kδ signaling inhibition by parsaclisib, but their proliferative activities were reduced by suppression of MYC gene transcription. Molecular structure analysis of the first- and next-generation PI3Kδ inhibitors combined with clinical observation suggests that hepatotoxicity seen with the first-generation inhibitors could result from a structure-related off-target effect. Parsaclisib is currently being evaluated in multiple phase 2 clinical trials as a therapy against various hematologic malignancies of B-cell origin (NCT03126019, NCT02998476, NCT03235544, NCT03144674, and NCT02018861). SIGNIFICANCE STATEMENT: The preclinical properties described here provide the mechanism of action and support clinical investigations of parsaclisib as a therapy for B-cell malignancies. MYC overexpression was identified as a resistance mechanism to parsaclisib in DLBCL cells, which may be useful in guiding further translational studies for the selection of patients with DLBCL who might benefit from PI3Kδ inhibitor treatment in future trials. Hepatotoxicity associated with first-generation PI3Kδ inhibitors may be an off-target effect of that class of compounds.

Abstract 2392: CDX-527: A novel bispecific immune-modulating antibody targeting CD27 and PD-L1

Abstract CD27 is a costimulatory molecule that provides a complementary target to the PD-1/PD-L1 axis on T cells. Combining a CD27 agonist monoclonal antibody (mAb) with PD-1/PD-L1 blockade has been shown to promote synergistic antitumor activity in preclinical models. In advanced cancer patients, the combination of the CD27 agonist mAb, varlilumab, with the PD-1 mAb, nivolumab, was generally well tolerated, induced strong biological effects, and led to sustained clinical responses in some patients that generally have low response rates to checkpoint inhibitors. We theorized that combining CD27 costimulation with PD-1/PD-L1 blockade in a bispecific antibody (bsAb) may provide greater immune activating properties than combining the individual mAbs due to enhanced CD27 activation by crosslinking through PD-L1 in addition to Fc receptors. To test this approach, we developed CDX-527, a tetravalent human anti-CD27/PD-L1 IgG1 bsAb. CDX-527 inhibits PD-1 signaling in vitro in a manner similar to clinically approved PD-1/PD-L1 antibodies. CDX-527 also elicits potent T cell costimulation through PD-L1 crosslinking as measured by cytokine induction and cell proliferation, whereas the PD-L1 or CD27 parental antibodies show no significant activity. In a mixed lymphocyte reaction assay, CDX-527 is more potent than the combination of the parental antibodies, suggesting that cross-linking through both Fc receptors and PD-L1 results in enhanced CD27 agonist activity. In human CD27 transgenic mice, we observed that antigen-specific T cell responses to a vaccine in vivo are significantly more enhanced with a surrogate CD27/PD-L1 bsAb than with either individual antibody. Furthermore, the surrogate bsAb exhibits greater antitumor activity than the combination of the parental antibodies in a syngeneic lymphoma model. Taken together, these results suggest that the enhanced activity of CDX-527 can be attributed to more efficient cross-linking of the bispecific antibody acting on the CD27 receptor, resulting in stronger T cell activation, combined with efficient PD-1/PD-L1 blockade, providing a novel approach to immunotherapy of cancers. We have initiated development activities for CDX-527 including a pilot study in cynomolgus macaques to investigate the PK and PD properties and provide guidance for the design of a GLP toxicology study. Citation Format: Laura A. Vitale, Lawrence J. Thomas, Thomas O'Neill, Jenifer Widger, Laura Mills-Chen, Andrea Crocker, Colleen Patterson, Anna Wasiuk, Eric Forsberg, James Boyer, Crystal Sisson, Jeffrey Weidlick, Shannon Renn-Bingham, Ioannis Papayannopoulos, Russ Hammond, Joel Goldstein, Henry C. Marsh, Tibor Keler, Li-Zhen He. CDX-527: A novel bispecific immune-modulating antibody targeting CD27 and PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2392.

PS1075 ANTITUMOR ACTIVITY OF PEMBROLIZUMAB PLUS DINACICLIB IN PATIENTS WITH DIFFUSE LARGE B CELL LYMPHOMA: THE PHASE 1B KEYNOTE‐155 STUDY

Background:The cyclin‐dependent kinase inhibitor dinaciclib induces apoptosis by CDK9 inhibition with antitumor activity in preclinical syngeneic lymphoma models and early phase trials of hematologic malignancies including diffuse large B‐cell lymphoma (DLBCL). Murine syngeneic tumor models (MC‐38) showed combined anti‐tumor activity of anti‐PD‐1 antibody and dinaciclib via enhanced dinaciclib‐induced immunogenic cell death. KEYNOTE‐155 (NCT02684617) evaluated the antitumor activity of pembrolizumab plus dinaciclib in patients (pts) with relapsed/refractory (rr) CLL, MM, and DLBCL.Aims:To present data from an interim analysis of safety and efficacy in pts with rrDLBCL.Methods:In this phase 1b, two‐part, non‐randomized, open‐label study, pts with rrDLBCL who received ≥2 prior therapies were enrolled into an initial dose‐evaluation phase (two 21‐day cycles of pembrolizumab 200 mg [day 1] and dinaciclib 7 mg/m2 [day 1], 10 mg/m2 [day 8] in cycle 1 and 14 mg/m2 [day 1, day 8] from cycle 2 onwards). The DLBCL expansion cohort was opened for the signal‐detection phase (≥30 patients) if ≤4 pts had dose‐limiting toxicities (DLTs) in the dose‐evaluation phase. Treatment continued for up to 35 cycles or until disease progression (PD) or unacceptable toxicity. Response was assessed every 3 cycles for DLBCL (Revised Response Criteria for Malignant Lymphoma 2007). Pts who discontinued before PD were followed Q12W until documented PD, new anticancer treatment, or death. The primary endpoint was safety and tolerability. Secondary endpoints included ORR by investigator, DOR, PFS, and OS. AEs were graded per National Cancer Institute Common Terminology Criteria for AEs. Efficacy and safety were assessed in all pts who received ≥1 dose of pembrolizumab or dinaciclib.Results:As of Apr 25, 2018, 38 pts with rrDLBCL were enrolled and treated. Median age was 64.5 years (range, 39–85), 22 (58%) pts had ECOG PS 1, and 22 (58%) had ≥3 prior therapies. After a median follow‐up of 2.79 mo (range, 1.5–6.8), 36 (95%) pts discontinued treatment: 21 (55%) PD, 12 (32%) physician decision (10 [26%] clinical progression, 1[3%] logistics, 1 [3%] new therapy), 2 (5%) pt decision, and 1 (3%) AE. Median time on treatment was 51 days (range, 1–471). Among the 12 pts in all disease cohorts enrolled in the dose‐evaluation phase, DLTs occurred in 1 of 6 (17%) evaluable pts (grade 4 thrombocytopenia) with rrDLBCL. Of 38 pts with rrDLBCL, 24 (63%) had a treatment‐related AE. The most common treatment‐related AEs included decreased platelets (21%), lymphopenia (16%), nausea (13%), anemia (13%), diarrhea (13%), and pyrexia (13%). 12 (32%) pts had a grade 3–4 treatment‐related AE. The most common grade 3–4 treatment‐related AEs included lymphopenia (13%), decreased neutrophils (11%), decreased platelets (8%), leukopenia (8%), and laboratory tumor lysis syndrome (5%). There was no clinical TLS and no treatment‐related deaths. ORR was 18% (95% CI, 8–34) (3 CR and 4 PR) and median DOR was 4.9 mo (range, 2.1–13.8); 2 pts remained on treatment at data cutoff. With 36 (95%) events, median PFS was 2.1 mo (95% CI, 2–3) with 6‐mo and 12‐mo PFS rates of 13% and 8%, respectively. 19 (50%) pts died, with median OS of 9.8 mo (95% CI, 3 to not reached), and 12‐mo OS rate of 43%.Summary/Conclusion:Despite limited overall response, the observed CRs and durability (up to 13 mo) provide proof of concept for activity of this combination in rrDLBCL and rationale for further studies to define optimal dosing of dinaciclib, given the brevity of CDK9 inhibition observed in preclinical models.

Abstract LB-303: Novel peptide is synergistic with checkpoint inhibitor antiCTLA4

Abstract Specific peptides are known to modulate cellular immune responses. Isolates of leukocytes containing tyrosine and glycine peptide sequences have been previously tested in HIV-positive, colon cancer, and other patients, where they were found to be safe and to demonstrate pleiotropic effects on CD4+ T cells, CD8+ T cells, and natural killer cells, as well as increased production of interferon gamma and other cytokines. We hypothesized, therefore, that Tyr-Gly peptides may enhance anti-tumor responses in combination with checkpoint inhibitors through stimulation of the immune system. INT-151 (synthetic Tyr-Gly), originally identified as the active component of a leukocyte isolate, was tested alone and in combination with the checkpoint inhibitor anti-CTLA4 in a murine syngeneic lymphoma model (E.G7-OVA) for anti-tumor effect and survival. Immune profiles in control and treatment groups were assessed for tumors and blood using FACs analysis to detect CD8+ T cells, CD4+ T cells, regulatory T cells (Treg), macrophages, and myeloid derived suppressor cells (MDSC). E.G7-OVA tumor bearing mice with an average tumor volume of 43 mm3, were treated 3x weekly for 17 days with INT-151 or anti-CTLA4 alone or in combination. The treatment group receiving INT-151 plus anti-CTLA4 had the lowest tumor volume (351 ± 38.9 mm3, N=6) compared with those treated with vehicle (876.7 ± 142.9 mm3, p < 0.04, N=6), INT-151 alone (821 ± 52.2 mm3, p = 0.8, N=6), and anti-CTLA4 alone (616 ± 117.4 mm3, p = 0.1, N=6). Tumor growth inhibition was highest in the combination group (60 %) compared to single agents anti-CTLA4 (30 %) and INT-151 (7 %). Immune cell populations increased in tumors were CD8+ T cells, CD4+ T cells and M1 macrophages in the combination group. Regulatory T cells, mMDSC or gMDSC remained unchanged in any of the treatment groups compared to controls. The highest ratio of CD8+T/Treg cells of 8.5±2.1 was found in the combination group (INT-151+anti-CTLA4, p<0.001) compared to 4.4±0.9 for anti-CTLA4 and 2.5±0.5 for vehicle and INT-151 (3.2±1.05). The E.G7-OVA mouse model also demonstrated increased survival on day 40 in groups treated with INT-151 plus anti-CTLA4 (55%, N=9) compared to single agent INT-151 (30 %, N=10), anti-CTLA4 (0%, N=8) and vehicle (0 %, N=10). These data demonstrate synergistic activity of INT-151 in a syngeneic murine lymphoma model. Combining a checkpoint inhibitor with INT-151 could be a safe and promising therapeutic strategy for lymphoma and other malignancies. Citation Format: Carola Leuschner, Clifford H. Kern. Novel peptide is synergistic with checkpoint inhibitor antiCTLA4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-303.

A Flagellin-Derived Toll-Like Receptor 5 Agonist Stimulates Cytotoxic Lymphocyte-Mediated Tumor Immunity

Toll-like receptor (TLR) mediated recognition of pathogen associated molecular patterns allows the immune system to rapidly respond to a pathogenic insult. The “danger context” elicited by TLR agonists allows an initially non-immunogenic antigen to become immunogenic. This ability to alter environment is highly relevant in tumor immunity, since it is inherently difficult for the immune system to recognize host-derived tumors as immunogenic. However, immune cells may have encountered certain TLR ligands associated with tumor development, yet the endogenous stimulation is typically not sufficient to induce spontaneous tumor rejection. Of special interest are TLR5 agonists, because there are no endogenous ligands that bind TLR5. CBLB502 is a pharmacologically optimized TLR5 agonist derived from Salmonella enterica flagellin. We examined the effect of CBLB502 on tumor immunity using two syngeneic lymphoma models, both of which do not express TLR5, and thus do not directly respond to CBLB502. Upon challenge with the T-cell lymphoma RMAS, CBLB502 treatment after tumor inoculation protects C57BL/6 mice from death caused by tumor growth. This protective effect is both natural killer (NK) cell- and perforin-dependent. In addition, CBLB502 stimulates clearance of the B-cell lymphoma A20 in BALB/c mice in a CD8+ T cell-dependent fashion. Analysis on the cellular level via ImageStream flow cytometry reveals that CD11b+ and CD11c+ cells, but neither NK nor T cells, directly respond to CBLB502 as determined by NFκB nuclear translocation. Our findings demonstrate that CBLB502 stimulates a robust antitumor response by directly activating TLR5-expressing accessory immune cells, which in turn activate cytotoxic lymphocytes.

Antitumor Activity of An RNA-Based Agonist of TLR7 and 8 In Preclinical Models of Hematological Malignancies

AbstractAbstract 421Treatment of hematological malignancies, non-Hodgkins lymphoma in particular, has evolved greatly in the past decade with the advent of rituximab, proteasome inhibitors and other new agents. Nonetheless, the need remains for effective agents with novel mechanisms of action, which lack cross-resistance to current standards of care. Agents that regulate host immune function may contribute to the control of hematological tumors by affecting innate and acquired immune responses targeting cancer cells. In this context, Toll-like receptors (TLR) are ideal targets for combination therapy as they play a key role in modulating immune responses and can influence the tumor microenvironment through induction of cytokines/chemokines and enhance antibody-dependent cell cytotoxicity (ADCC), a key mechanism involved in rituximab antitumor effects. We have shown that a novel class of synthetic oligoribonucleotide compounds, acting as TLR7 and 8 agonists, induce potent T helper 1-type immune responses in preclinical models (Proc Natl. Acad. Sci. USA, 104: 13750, 2007) and exert significant antitumor effects (Mol Cancer Ther. 9: 1788, 2010). In this study, we have evaluated a combination strategy involving a TLR7/8 dual agonist with rituximab, bortezomib or cyclophosphamide, in human xenograft and murine syngeneic lymphoma models. We performed ADCC assays using human peripheral blood mononuclear cells (PBMCs) stimulated with the agonist as effectors and rituximab-coated Raji, a B cell lymphoma, or Granta-519, a mantle cell lymphoma tumor cell line, as targets. Combining agonist-treated PBMCs with uncoated Raji cells or untreated PBMCs with rituximab-coated Raji cells resulted in about 35% cytotoxicity. When agonist-treated PBMCs were combined with rituximab-coated Raji cells, cytotoxicity was enhanced to 65%. Agonist treatment of PBMCs alone did not induce killing of uncoated Granta-519 cells whereas treatment with rituximab caused 25% cytotoxicity. Combining agonist treatment with rituximab coating of Granta-519 cells increased cytotoxicity to 51%. Thus, the agonist significantly enhances the ADCC activity of rituximab. NOD-SCID mice implanted i.v. with Raji cells were treated with 50 mg/kg agonist (s.c., twice weekly for 5 weeks), 10 mg/kg rituximab (i.p., twice weekly for 5 weeks), the combination of the two agents or PBS starting at day 3. The control group of mice that received PBS developed a disseminated systemic disease with a median survival 18.5 days and died within 24 days. Treatment with agonist or rituximab alone lead to moderate tumor growth inhibition with a median survival of 24 days (p = 0.043) or 36 days (p < 0.01), respectively. However, all mice in both groups developed lymphoma and died within 74 days. Treatment of mice with combination of agonist and rituximab resulted in long term tumor-free survival of 63% of the treated mice (p = 0.0005 compared to antibody alone group) with a median survival of 82 days. Similar antitumor effects were observed in HBL-2 mantle cell lymphoma xenografts in Rag2 mice. Flow cytometry analysis of human CD20+, CD19+ and CD20−CD19+ cells in the peripheral blood of mice bearing HBL-2 tumors showed that treatment with the agonist or rituximab lead to a 19% and 70% reduction of lymphoma cells, respectively. In contrast, treatment with agonist and rituximab increased clearance of total human cells to 97%. Interestingly, although rituximab was very effective in clearing CD20+ cells, it did not impact the number of CD20−CD19+ cells which accumulated over time and account for 8% of the total lymphoma cell population in the blood stream. Treatment with agonist and rituximab, significantly reduced the number of circulating CD20−CD19+ cells. The agonist also showed synergistic antitumor activity when combined with either cyclophosphamide or bortezomib in murine B-cell lymphoma xenografts from Em-myc transgenic mice and A20 lymphoma in BALB/c mice, respectively. Combination treatment in both models resulted in significant tumor growth inhibition compared to cyclophosphamide (p = 0.035) or bortezomib (p = 0.0001) alone. Our data suggest that this novel dual agonist of TLR7 and TLR8 could enhance the antitumor activity of rituximab as well as other standard agents in the treatment of non-Hodgkins lymphoma and other hematological malignancies. Based on these data we have selected the dual agonist IMO-4200 as lead candidate for clinical development. Disclosures:No relevant conflicts of interest to declare.

Abstract 5627: Antitumor activity of soluble beta-1,3/1,6 glucans: Structure matters

Abstract Beta-glucan is a pathogen-associated molecular pattern recognized by a variety of innate immune cells. Beta-1,3/1,6 glucans possess numerous immune potentiating activities. In particular, antitumor activity has been demonstrated with the soluble beta glucan Imprime PGG used in combination with complement-activating antitumor monoclonal antibodies (MAb) in several tumor models. Structurally, beta-1,3/1,6 glucans can vary on the basis of chain length, types of linkages, branching, and tertiary structure. Previously, the antitumor activity of soluble beta-1,3/1,6 glucans in combination with antitumor MAb has only been reported using beta glucans derived from yeast. The primary objective of this study was to compare the antitumor activity of three structurally distinct soluble beta-1,3/1,6 glucans in vivo in a mouse syngeneic lymphoma model. Tumor-bearing mice were administered antitumor MAb plus beta glucan 2x/week for 4 weeks. Antitumor activity was based on inhibition of tumor growth at the tumor implantation site and enhanced long-term survival. In addition, critical innate immune cells responsible for antitumor activity were identified focusing on the role of neutrophils. Also, characterization of relevant human immune cells and their beta glucan receptors will be shown focusing on complement receptor 3 (CR3) and dectin-1. Beta glucan binding to immune cells and identification of relevant receptors were determined by flow cytometry using beta glucan-specific antibody and fluorescently-labeled beta glucans. Key findings are that soluble beta glucans, which differ structurally, do not induce similar antitumor activity, and antitumor activity is abrogated in granulocyte-deficient mice. Finally, soluble glucans bind to human neutrophils in a CR3-dependent manner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5627.

Role of complement activation component C5a on tumor progression (100.9)

Abstract Enhancement of anti-tumor mAb immunological mechanisms for tumor destruction may be obtained by promoting complement activation. However, complement activation components, such as C5a, trigger inflammation and chronic inflammation promotes tumor growth. Recently, it has been shown that C5a in the tumor could recruit and activate myeloid-derived suppressor cells to inhibit the anti-tumor CD8 T cell response. However, this effect was observed in only one tumor model. We chose to address the role of C5a on tumor growth by transfecting tumor cells with C5a. A human ovarian carcinoma, SKOV-3, and a murine lymphoma, RMA, were transfected which allows us to study C5a in an immunocompromised xenograft model and an immunocompetent syngeneic model. In vitro growth kinetics observed revealed no significant difference between C5a, control vector (CV), or wild-type cells. In both models, tumor-bearing mice with C5a-transfected tumor cells have significantly less tumor burden as compared to CV tumors. In the xenograft model, CD11b+DX5+ NK cells were significantly more and VEGF and arginase levels were significantly less in C5a expressing tumors. The syngeneic lymphoma model revealed C5a modulates tumor-infiltrating CD11b+ cells to produce more IL-12 and there is a greater influx of NK and CD4 T cells. Thus our studies demonstrate C5a chemoattracts innate NK cells and downregulates negative mediators in the xenograft model while in the syngeneic model C5a promotes shift to a Th1 response.

Chronic restraint stress impairs T-cell immunity and promotes tumor progression in mice

Long-term exposure to stressful situations can affect the immune system. The T-cell response is an important component of anti-tumoral immunity. Hence, impairment of the immune function induced by a chronic stressor has been postulated to alter the immunosurveillance of tumors, thus leading to a worse neoplastic prognosis. Here, we show that chronic restraint stress affects T-cell mediated immunity in mice. This was evidenced by a decrease of mitogen-induced T-cell proliferation, a reduction in CD4+T lymphocyte number and a decrease of tumor necrosis factor-alpha (TNF-α) and Interferon-gamma (IFN-γ) production in stressed mice. Additionally, mice subjected to chronic restraint stress displayed an enhancement of tumor growth in a syngeneic lymphoma model, i.e. an increase of tumor proliferation and a reduction of animal survival. Finally, stressed mice had a reduced specific cytotoxic response against these tumor cells. These results suggest that chronic exposure to stress promotes cancer establishment and subsequent progression, probably by depressing T-cell mediated immunity. The T-cell immunity impairment as well as the tumor progression enhancement emphasize the importance of the therapeutic management of stress to improve the prognosis of cancer patients.

NK Cells Negatively Regulate Antigen Presentation and Tumor-Specific CTLs in a Syngeneic Lymphoma Model

NK cells are known to kill tumor cells and produce proinflammatory cytokines that lead to the generation of tumor-specific CTLs. Many studies have used MHC class I-deficient tumor cells and/or adjuvants that induce NK cell responses. In this study, the focus was on less-immunogenic lymphoma cells that express MHC class I as a model to study NK cell responses to tumors that do not directly stimulate NK cell activation. When RMA tumor cells that expressed a truncated version of OVA, or RMA cells alone, were injected into mice that were depleted of NK cells, the mice developed an increased number of tumor-specific CTLs, increased IFN-gamma responses, and a higher amount of Ag presentation in draining LNs compared with mice with intact NK cells. These data suggest that NK cells can inhibit the development of effective adaptive immunity in the absence of signals that trigger NK cell activation.