Genetic Inhibition of PD-L1 on Myeloma Cells Improves Survival in Contrast to the Treatment with Checkpoint Inihibitors

Checkpoint inhibition (CPI) has shown dramatic improvements in overall survival in many malignant diseases. However, in multiple myeloma (MM) the results were disappointing resulting in an early termination of clinical trials. Despite the advantages in therapy the disease remains incurable. Methods: We analyzed the efficiency and immunological mechanisms of PD-1/PD-L1 blockade using KaLwRij mice that develop MM upon injection of 5T33 myeloma cells. Treatment of mice started d19 post inoculation. Bone marrow (BM) and spleen cells were analyzed by flow cytometry for the phenotype of immune cells. Results: Comprehensive immunophenotyping including the analysis of T and NK cell subpopulations revealed no differences of early MM disease stage compared to healthy control groups. Treatment of mice with mAbs blocking PD-1 or PD-L1 had no effect on tumor growth and survival. It was demonstrated that HDAC inhibitors beside their direct effect on malignant cells may increase the immunogenicity of malignant cells by improving the presentation of tumor antigens and modulate the immunological composition of the tumor microenvironment (TME). The pan-HDAC inhibitor panobinostat that is approved for the treatment of myeloma patients inhibited the development of myeloma in treated mice. Surprisingly, the combined application of the anti-PD-1 blocking antibody with panobinostat reduced the anti-myeloma effect of the compound and resulted in decreased survival. By analyzing the phenotype of immune cells in the different populations, we found in the panobinostat treated animal group an increase in the CXCR4 expressing CD4+ NKT cells. Additionally, the CD8+ T cells expressing CD1d and CXCR4 decreased compared to the other groups in the spleen. CD1d is a MHC like receptor for glycolipids activating NKT cells, whereas CXCR4 is a BM homing receptor and linked to metastasis and tumor aggressiveness. We found a gradually increase of CXCR4+ NKT cells in the BM corresponding to MM disease progression. Interestingly, we observed a shift in the CD4+/CD4- NKT cell ratio during disease progression, whereby the CXCR4+ CD4- NKT cells seem to be associated with advanced tumor growth, while the increase of CXCR4+ CD4+ NKT are associated with prolonged survival as observed in in the panobinostat treated group. To further analyze the role of PD-L1 expression on myeloma cells we generated a PD-L1 KO of the 5T33 cell line using the CRISPR/Cas9 technology. We found no differences in the expression of surface molecules such as MHC class I and II, co-stimulatory or adhesion molecules, proliferation and migration of the genetically engineered cells in comparison to the mock control. Interestingly, mice inoculated with the 5T33 PD-L1 KO cells showed a significant longer survival compared with the 5T33 mock injected, indicating that blocking of the PD-L1 molecule on myeloma cells plays an important role in the pathogenesis of MM and its direct blocking on malignant cells rather than in the TME might have an impact on the clinical efficiency. When analyzing the spleen of the mock vs PD-L1 KO myeloma inoculated mice, we found the same downregulation of CXCR4 and CD1d on CD8+ T cells in the PD-L1 KO myeloma group as observed in the panobinostat treated group with extended survival. In addition, we used NOD. scid. Il2Rγc null (NSG) mice to proof that the survival prolongation is a result of the immunological response to PD-L1 and that the myeloma cells are not otherwise effected in their tumor cell properties in vivo. NSG mice experience the same tumor burden post 5T33 mock and PD-L1 KO challenge, assuming that the previous observed survival prolongation is exclusively dependent on the PD-L1 tumor- immune cell interaction. Conclusion: We found that PD-1 blockade might negatively affect and inhibit the therapeutic efficacy of HDAC inhibitors such as panobinostat. Genetic down regulation of PD-L1 on the myeloma cells enables a significant improvement and longer survival. These results give new insights into the complexity of the action of CPI in the treatment of malignant diseases which might help to develop combinatorial approaches of checkpoint inhibitors in clinical trials. Furthermore, the increase of CD4- CXCR4 expressing NKT cells in the BM might be used as biomarker to monitor MM disease progression, whereas the increase of the CD4+/CD4- NKT cell ratio in the BM might be associated with the shrinkage of MM tumor burden. Disclosures Brossart: BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; MSD: Honoraria.

Preclinical Evidence for the Efficacy of CD79b Immunotherapy in B Cell Precursor Acute Lymphoblastic Leukemia

Patients with B cell precursor acute lymphoblastic leukemia (BCP-ALL) have a favorable prognosis, yet current treatment protocols are based on intensive cytotoxic chemotherapy and therapy options are limited when patients relapse. Novel immunotherapy approaches are therefore needed. We recently reported that the preB cell receptor signaling unit CD79a (known as Igα) is crucial for BCP-ALL engraftment in vivo, particularly in the central nervous system (CNS) employing BCR-ABL + and E2A-PBX1 + patient derived xenograft (PDX) models (Lenk et al., Communications Biology, 2021). CD79a forms a heterodimer with CD79b (known as Igß), which is also expressed on the surface of mature B cells as part of the B cell antigen receptor. Accordingly, the CD79b antibody drug conjugate (ADC) Polatuzumab Vedotin (PolVed) has shown therapeutic efficacy in the treatment of refractory/relapsed (r/r) diffuse large B cell lymphoma. Moreover, CD79a/CD79b may also be present on the cell surface at the pro/pre B cell stage. We therefore hypothesize that CD79b can serve as a therapeutic target in BCP-ALL. First, to substantiate that CD79b is important for BCP-ALL engraftment in vivo, a murine/murine transplantation model was applied. B cell precursors were isolated from mice harboring CD79b with a non-functional signaling domain (CD79b-ITAM-KO) or wildtype mice, malignantly transformed with a BCR-ABL fusion construct and transplanted into NSG-mice. Whereas control cells caused overt leukemia in all animals within 25 days, no animal injected with CD79b-ITAM-KO cells had developed leukemia by the time of sacrifice at 162 days (P<0.001). To investigate the frequency of surface (s)CD79b expression in BCP-ALL patients, we measured sCD79b levels via flow cytometry in diagnostic samples of pediatric BCP-ALL patients with different cytogenetic backgrounds. We detected sCD79b-positivity (defined as ≥10% sCD79b + BCP-ALL cells) in 23/94 patients including BCR-ABL +, E2A-PBX1 +, MLL-rearranged (MLLr), TEL-AML1 + and B-other BCP-ALL patients indicating a population of sCD79b + patients within different cytogenetic BCP-ALL subgroups (Figure 1a). To validate CD79b as a therapeutic target, we applied an unconjugated monoclonal CD79b antibody (CD79b-mAB) to PDX mice bearing either pediatric BCR-ABL + or E2A-PBX1 + PDX samples with high sCD79b expression (49.6% sCD79b + cells and 37.7% sCD79b + cells, respectively). Treatment was initiated one day after BCP-ALL injection, modelling a minimal residual disease (MRD) situation. Therapy with CD79b-mAB (1mg/kg) resulted in a mild reduction of leukemia burden in the spleen (SP) and bone marrow (BM) of PDX mice and a significant reduction of CNS-involvement in both PDX-models as compared to control treated mice (P<0.05 and P<0.01, respectively). To test the hypothesis that treatment of BCP-ALL with a CD79b-ADC outperforms CD79b-mAB, we applied PolVed (1mg/kg) in NSG mice bearing the same E2A-PBX1 + and BCR-ABL + PDX cells. PolVed therapy resulted in a significant reduction of BCP-ALL engraftment in SP, BM and CNS (P<0.01, respectively) and a significant survival prolongation compared to control treated mice in both models (P<0.01, respectively). Of note, 4/5 PolVed-treated E2A-PBX1-PDX animals were free of leukemia by the time of sacrifice (236 days) (Figure 1B). To test the efficacy of PolVed on different BCP-ALL subgroups, we conducted a preclinical phase II-like PDX study, using sCD79b high and sCD79b low PDX samples (defined by sCD79b-expression above or below the median) (5E2A-PBX1 +, 3 BCR-ABL +, 2 MLLr, 1 E2A-HLF + and 1 ETV6-NTRK3 +). Two NSG mice per patient were injected with PDX cells, randomly assigned into treatment groups and PolVed therapy was initiated when 1% PDX-cells were detected in the peripheral blood, modelling an overt leukemia situation. sCD79b low PDX mice did not respond to PolVed treatment (Figure 1c), but we detected a response to therapy and a significant survival prolongation in 5/6 sCD79b high PDX samples irrespective of the cytogenetic background (P<0.05) (Figure 1d). Taken together, our data indicate that a subgroup of BCP-ALL patients is sCD79 + positive and may respond to PolVed treatment. Therefore, we suggest CD79b as a novel therapeutic target in BCP-ALL and propose PolVed as a potential therapeutic agent in r/r disease. *LL and DW contributed equally to this work Figure 1 Figure 1. Disclosures Lenk: OSE Immunotherapeutics: Research Funding. Richter: HTG Molecular Diagnostics, Inc.: Current Employment, Research Funding. Schrappe: SHIRE: Other: research support; JazzPharma: Honoraria, Other: research support; Servier: Honoraria, Other: research support; SigmaTau: Other: research support; Novartis: Honoraria; JazzPharma: Honoraria; Novartis: Honoraria, Other: research support; Servier: Honoraria; Amgen: Other: research support. Cario: Novartis: Other: Lecture Fee. Brüggemann: Incyte: Other: Advisory Board; Amgen: Other: Advisory Board, Travel support, Research Funding, Speakers Bureau; Janssen: Speakers Bureau. Schewe: Jazz Pharmaceuticals: Other: Advisory Board; SOBI: Other: Advisory Board; Bayer: Other: Advisory Board; OSE Immunotherapeutics: Research Funding.

BIOM-08. DNA METHYLATION-BASED SUBGROUPING PREDICTS SURVIVAL BENEFIT FROM LOMUSTINE/TEMOZOLOMID COMBINATION THERAPY IN MGMT PROMOTOR-METHYLATED GLIOBLASTOMA

BACKGROUND The CeTeG/NOA-09 trial showed that lomustine/temozolomide chemotherapy prolongs survival for newly diagnosed MGMT-methylated glioblastoma patients. Previous reports on temozolomide monotherapy suggested, that the survival benefit of temozolomide in MGMT-methylated tumors may be restricted to the RTK II methylation subgroup and absent in RTK I and MES subgroups. To identify patients with a particularly strong benefit from CCNU/TMZ, we explored the association of methylation subgroups with outcome after lomustine/temozolomide therapy. METHODS All patients from the CeTeG/NOA-09 trial with sufficiently available tumor tissue (n = 98) underwent 850K methylation array analysis of their tumor and methylation subgroup annotation (Heidelberg brain tumor methylation classifier v11b4; calibrated score > 0.5 required). Overall survival (OS) was compared between a pooled cohort of tumors of the RTK I/MES subgroups and RTK II tumors. RESULTS In the CCNU/TMZ arm of CeTeG/NOA-09, OS was prolonged in RTK I/MES (n = 16; median not reached, 4-year OS 69%) as compared to RTK II patients (n = 14; median 20.6 months, 4-year OS 23%; p = 0.004 logrank test). In the standard temozolomide arm of CeTeG/NOA-09, OS tended to be shorter in RTK I/MES (n = 7; median 23.7 months, 4-year OS 17%) as compared to RTK II patients (n = 17; median 35.2 months; 4-year OS 38%, p = 0.15). CONCLUSION The CCNU/TMZ-dependent survival prolongation in patients with RTK I/MES tumors as opposed to RTK II seen in CeTeG/NOA-09 suggests that methylation-based subgrouping could be predictive for CCNU/TMZ efficacy in newly diagnosed MGMT-methylated glioblastoma.

A valproátterápia túlélésre gyakorolt hatása gliomás betegekben

Összefoglaló. Bevezetés: A gliomák, ezen belül a glioblastoma kezelése továbbra is megoldatlan onkológiai problémát jelent. A szekunder szimptómás epilepsziabetegség megjelenése pozitív prognosztikai faktornak tekinthető a korai diagnosztizálás és az antiepileptikumok potenciális tumorellenes hatásának köszönhetően. A valproát túlélést hosszabbító hatása már több mint 20 éve az alap- és klinikai kutatások tárgyát képezi. Napjainkban ismert citotoxikus, proapoptotikus, antiangiogenetikus és hiszton-deacetiláz-gátló hatásmechanizmusa. Célkitűzés: Kutatásunk célja a valproát túlélést hosszabbító hatásának vizsgálata egy hazai gliomás betegcsoportban. Módszer: Egycentrumos, retrospektív klinikai vizsgálatot végeztünk. A vizsgálatba 122 felnőtt beteget vontunk be, akiknél 2000 januárja és 2018 januárja között supratentorialis glioma miatt műtét történt, és rohamtevékenység miatt antiepileptikumot (valproát, levetiracetám, karbamazepin) szedtek. Egyúttal gyógyszert nem szedő kontrollcsoportot is kialakítottunk. A populációt vizsgálati és kontrollcsoportokra osztottuk 28 : 52 arányban. Leíró statisztikai, Kaplan–Meier- és log-rank analízist végeztünk. Eredmények: A vizsgált szövettani kategóriák túlélési analízise az irodalmi adatokkal megegyező értékeket mutatott. A progressziómentes (PFS: p = 0,031) és a teljes (OS: p = 0,027) túlélés tekintetében is szignifikáns eltérés mutatkozott a különböző antiepileptikumot szedő betegcsoportok között, amely még kifejezettebbé vált a valproátot és az egyéb antiepileptikumot szedő betegek túlélési idejének összehasonlítása során (PFS: p = 0,006; OS: p = 0,015). Következtetés: Vizsgálatunkban a valproát betegeink PFS- és OS-idejének meghosszabbodását eredményezte. Az irodalmi adatok és kutatásunk alapján megfontolandónak tartjuk a valproát első vonalban történő alkalmazását onkoterápiában részesülő, epilepsziás, agyi gliomás betegekben. Orv Hetil. 2021; 162(24): 960–967. Summary. Introduction: Gliomas still prove to be a serious oncological problem. The presence of epilepsy may present a favorable prognosis due to early diagnosis and the potential antitumor effects of antiepileptic drugs. The survival prolongation effect of valproate has been studied for more than 20 years, nowadays its proapoptotic, anti-angiogenetic, cytotoxic and histone deacetylase inhibitory effects are well known. Objective: Our goal was to investigate the survival-enhancing effects of valproate in a Hungarian patient cohort of primary brain tumors. Method: A single-center based retrospective clinical trial was designed. In our study, we included 122 patients harboring supratentorial glioma who underwent surgery and experienced seizures between 2000 January and 2018 January. The patients were grouped by the antiepileptic therapies and survival analysis was performed. Results: The Kaplan–Meier curves of the histological categories showed the survival values consistent with the data of the literature. The progression-free (PFS: p = 0.031) and the overall (OS: p = 0.027) survival of the antiepileptic drug categories were significantly different. It was performed by comparing the valproate group and the population formed by the other groups which also showed a significant increase in the survival values (PFS: p = 0.006; OS: p = 0.015). Conclusion: Our results show that valproate increases the PFS and OS period of glioma patients in comparison to other antiepileptic drugs. Our data suggest that the use of valproic acid should be considered as a first-line antiepileptic agent in certain well-selected epileptic patients with glioma as a supplement to the oncotherapy. Orv Hetil. 2021; 162(24): 960–967.

Antitumor Effects of Combining Docetaxel (Taxotere) with the Antivascular Action of Ultrasound Stimulated Microbubbles

Ultrasound stimulated microbubbles (USMB) are being investigated for their potential to promote the uptake of anticancer agents into tumor tissue by exploiting their ability to enhance microvascular permeability. At sufficiently high ultrasound transmit amplitudes it has also recently been shown that USMB treatments can, on their own, induce vascular damage, shutdown blood flow, and inhibit tumor growth. The objective of this study is to examine the antitumor effects of ‘antivascular’ USMB treatments in conjunction with chemotherapy, which differs from previous work which has sought to enhance drug uptake with USMBs by increasing vascular permeability. Conceptually this is a strategy similar to combining vascular disrupting agents with a chemotherapy, and we have selected the taxane docetaxel (Taxotere) for evaluating this approach as it has previously been shown to have potent antitumor effects when combined with small molecule vascular disrupting agents. Experiments were conducted on PC3 tumors implanted in athymic mice. USMB treatments were performed at a frequency of 1 MHz employing sequences of 50 ms bursts (0.00024 duty cycle) at 1.65 MPa. USMB treatments were administered on a weekly basis for 4 weeks with docetaxel (DTX) being given intravenously at a dose level of 5 mg/kg. The USMB treatments, either alone or in combination with DTX, induced an acute reduction in tumor perfusion which was accompanied at the 24 hour point by significantly enhanced necrosis and apoptosis. Longitudinal experiments showed a modest prolongation in survival but no significant growth inhibition occurred in DTX–only and USMB-only treatment groups relative to control tumors. The combined USMB-DTX treatment group produced tumor shrinkage in weeks 4–6, and significant growth inhibition and survival prolongation relative to the control (p<0.001), USMB-only (p<0.01) and DTX-only treatment groups (p<0.01). These results suggest the potential of enhancing the antitumor activity of docetaxel by combining it with antivascular USMB effects.

Antitumor Effects of Combining Docetaxel (Taxotere) with the Antivascular Action of Ultrasound Stimulated Microbubbles

Ultrasound stimulated microbubbles (USMB) are being investigated for their potential to promote the uptake of anticancer agents into tumor tissue by exploiting their ability to enhance microvascular permeability. At sufficiently high ultrasound transmit amplitudes it has also recently been shown that USMB treatments can, on their own, induce vascular damage, shutdown blood flow, and inhibit tumor growth. The objective of this study is to examine the antitumor effects of ‘antivascular’ USMB treatments in conjunction with chemotherapy, which differs from previous work which has sought to enhance drug uptake with USMBs by increasing vascular permeability. Conceptually this is a strategy similar to combining vascular disrupting agents with a chemotherapy, and we have selected the taxane docetaxel (Taxotere) for evaluating this approach as it has previously been shown to have potent antitumor effects when combined with small molecule vascular disrupting agents. Experiments were conducted on PC3 tumors implanted in athymic mice. USMB treatments were performed at a frequency of 1 MHz employing sequences of 50 ms bursts (0.00024 duty cycle) at 1.65 MPa. USMB treatments were administered on a weekly basis for 4 weeks with docetaxel (DTX) being given intravenously at a dose level of 5 mg/kg. The USMB treatments, either alone or in combination with DTX, induced an acute reduction in tumor perfusion which was accompanied at the 24 hour point by significantly enhanced necrosis and apoptosis. Longitudinal experiments showed a modest prolongation in survival but no significant growth inhibition occurred in DTX–only and USMB-only treatment groups relative to control tumors. The combined USMB-DTX treatment group produced tumor shrinkage in weeks 4–6, and significant growth inhibition and survival prolongation relative to the control (p<0.001), USMB-only (p<0.01) and DTX-only treatment groups (p<0.01). These results suggest the potential of enhancing the antitumor activity of docetaxel by combining it with antivascular USMB effects.

A Simple Self-Adjuvanting Biomimetic Nanovaccine Self-Assembled with the Conjugate of Phospholipids and Nucleotides Can Induce Strong Cancer Immunotherapeutic Effect

Background: Biomimetic nanoparticles have potential applications in many fields for their favorable properties. Results: Here, we developed a self-adjuvanting biomimetic anti-tumor nanovaccine, which was self-assembled with an amphiphilic conjugate synthetized with phospholipids of 1, 2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and hydrophilic Toll-like receptor (TLR9) agonists CpG ODN. The nanovaccine could not only provide effective initial antigens stimulation and sustained long-term antigen supply with a controlled release, but also induce antigens cross-presentation via MHC-I pathway initiating CD8+ T-cell responses. Moreover, the dense nucleotides shell around the nanovaccine could promote antigens endocytosis via various receptor-mediated pathways into dendritic cells. And CpG ODN interacted with TLR9 triggering the cytokines secretion of TNF-α and IL-10 further boosted the anti-tumor humoral and cellular immune responses, which led to significant tumor suppressive effect and remarkable survival prolongation. Conclusions: So, this nanovaccine self-assembled with phospholipid-nucleotide amphiphiles can serve as a safe, simple and efficient approach for anti-tumor immunotherapy.

PET, image-guided HDAC inhibition of pediatric diffuse midline glioma improves survival in murine models

Efforts at altering the dismal prognosis of pediatric midline gliomas focus on direct delivery strategies like convection-enhanced delivery (CED), where a cannula is implanted into tumor. Successful CED treatments require confirmation of tumor coverage, dosimetry, and longitudinal in vivo pharmacokinetic monitoring. These properties would be best determined clinically with image-guided dosimetry using theranostic agents. In this study, we combine CED with novel, molecular-grade positron emission tomography (PET) imaging and show how PETobinostat, a novel PET-imageable HDAC inhibitor, is effective against DIPG models. PET data reveal that CED has significant mouse-to-mouse variability; imaging is used to modulate CED infusions to maximize tumor saturation. The use of PET-guided CED results in survival prolongation in mouse models; imaging shows the need of CED to achieve high brain concentrations. This work demonstrates how personalized image-guided drug delivery may be useful in potentiating CED-based treatment algorithms and supports a foundation for clinical translation of PETobinostat.