Addition Of Ibrutinib Research Articles

Ibrutinib combined with low-dose histone deacetylases inhibitor chidamide synergistically enhances the anti-tumor effect in B-cell lymphoma.

Aberrant activity of histone deacetylases (HDACs) is frequently detected in B-cell lymphomas, which indicated the therapeutic implications of HDAC inhibitors for B-cell malignancies. We have discovered that lymphoma cells treated with HDAC inhibitor presented with activation of Bruton tyrosine kinase (BTK) which played an important role in the development of B-cell malignancies. Therefore, our study intended to explore whether the addition of ibrutinib (BTK inhibitor) to chidamide (HDAC inhibitor) could generate combined anti-tumor effects in B-cell lymphomas. Using cell viability assay, cell cycle and apoptosis kit, we demonstrated an evident synergistic action of ibrutinib and chidamide in inhibiting tumor cell proliferation and motility. Consistent with in vitro data, the synergistic anti-tumor effects were also observed in multiple tumor-bearing mice models. By performing RNA-seq and flow cytometry of tumor tissue, the enhancement of anti-tumor immunity was observed with the co-treatment of chidamide and ibrutinib. Together, these mechanistic insights indicated that simultaneously targeting BTK and HDAC could be a promising clinical therapy for B-cell lymphomas.

Ibrutinib with R-CHOP Differentially Affects Genetic Subtypes of DLBCL.

The genetic classification of DLBCL identified subgroups that benefit from addition of ibrutinib and its underlying mechanism.

Ibrutinib- and bortezomib-extended R-CHOP induction in elderly higher-risk patients newly diagnosed with diffuse large B-cell lymphoma – first analysis of toxicity and efficacy signals

Diffuse large-cell B-cell lymphoma (DLBCL) is the most common lymphoid malignancy. About 30–40% of the patients will not be cured by standard Rituximab (R)-CHOP-like immune-chemotherapy, and many of them experience relapse and eventually succumb to their disease. Enhancing first-line efficacy in patients at higher risk, among them many elderly, is key to improve long-term outcomes. Numerous attempts to combine R-CHOP with targeted agents failed in large randomized phase III trials. The addition of Ibrutinib enhanced survival in younger patients, but increased toxicity across all age groups, especially in the elderly. Older DLBCL patients impose particular challenges, since they often present with more advanced disease, and exhibit treatment-relevant comorbidities. ImbruVeRCHOP trial aims at identifying patients who need that benefit from rationally augmented first-line regimens without experiencing overt toxicity and detecting their molecular signatures of response. This first analysis presents encouraging feasibility, safety, and preliminary response data in elderly high-risk DLBCL patients.

Ibrutinib, Fludarabine, Cyclophosphamide, and Obinutuzumab (GA101) (iFCG) for First-Line Treatment of Patients with CLL with Mutated IGHV and without TP53 Aberrations

Ibrutinib, Fludarabine, Cyclophosphamide, and Obinutuzumab (GA101) (iFCG) for First-Line Treatment of Patients with CLL with Mutated IGHV and without TP53 Aberrations

Intratumoral CpG, Local Radiation, and Oral Ibrutinib Combine to Produce Effective in Situ Vaccination in Patients with Low-Grade B-Cell Lymphoma

Introduction: Local treatment with intratumoral CpG (a toll-like receptor 9 agonist, SD-101) and low-dose radiation can elicit antitumor immune responses and global tumor reduction in patients with low-grade lymphoma (Frank, Cancer Discov, 2018). Ibrutinib compromises B-cell survival by inhibiting Bruton's tyrosine kinase, but also modulates T-cells by inhibiting interleukin-2-inducible T-cell kinase. In a mouse model of lymphoma, ibrutinib plus intratumoral CpG was curative of systemic disease, an effect that was T-cell dependent (Sagiv-Barfi, Blood, 2015). Thus, we initiated a phase I/II clinical trial combining oral ibrutinib, intratumoral CpG and local low-dose radiation in adults with recurrent low-grade lymphoma (NCT02927964). Methods: Enrolled patients received intratumoral injections of CpG (SD-101, 3mg) weekly for 5 doses, starting on the second day of a 2-day course of local radiation (4Gy total) to the same site. Daily oral ibrutinib (560mg) began on day 9. Treatment-emergent adverse events (AEs), ibrutinib dose modifications and adherence were recorded at every visit. Revised Lugano criteria (Cheson et al., JCO, 2014) were used to assess response to therapy, based on CT scans at 3, 6, 12, 18, and 24 months. Fine needle aspirates (FNAs) were obtained from CpG-injected and non-injected nodal tumor sites pre- and post-treatment and analyzed by flow cytometry and single-cell RNA sequencing (scRNAseq). When available, viably preserved tumor and peripheral blood cells were used for in vitro immune response assays. Results: As of July 16, 2020, 18 patients had been treated, with a median follow-up of 12 months. Ten were male and 8 were female. All but one had a diagnosis of follicular lymphoma; one patient had marginal zone lymphoma. All were previously treated with an average of 2 prior lines of therapy. AEs were consistent with known effects of ibrutinib (including diarrhea and rash) and of CpG (including fever and flu-like reaction) with no unexpected AEs to suggest synergistic toxicity. There were no grade 4 or 5 events. AEs led to ibrutinib dose reduction or discontinuation in 2 patients and dose interruption in 6 patients. At the time of analysis, 9 of 18 evaluable patients had achieved a partial response (50% ORR) and 12 of 18 patients experienced at least a 30% reduction in the distant uninjected lesions (Figure 1A). Most responses have been maintained for at least 6 months, many longer (Figure 1B). Flow cytometry revealed decreased T follicular helper cells and increased CD4 and/or CD8 effector T-cells, CD137+ activated T-cells, and NK cells in CpG-injected tumors. Abscopal immune effects in distant non-injected lesions included an increase in Granzyme B+ CD8 T-cells, most prominent after the addition of ibrutinib. scRNAseq data showed significant transcriptional shifts in tumor cells and in tumor-infiltrating T-cells, including signatures of interferon response and T cell activation and cytotoxicity. Finally, in vitro assays showed tumor-specific immune responses in peripheral blood T-cells of all 6 evaluable patients tested thus far. Conclusion: Early data suggest that the combination of oral ibrutinib, intratumoral CpG, and local low-dose radiation is safe and can generate systemic antitumor immune responses and systemic tumor shrinkage in low-grade lymphoma. Disclosures Khodadoust: Seattle Genetics: Consultancy; Kyowa Kirin: Consultancy. Levy:Quadriga: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; GigaGen: Membership on an entity's Board of Directors or advisory committees; Teneobio: Membership on an entity's Board of Directors or advisory committees; Sutro: Membership on an entity's Board of Directors or advisory committees; Checkmate: Membership on an entity's Board of Directors or advisory committees; Nurix: Membership on an entity's Board of Directors or advisory committees; Dragonfly: Membership on an entity's Board of Directors or advisory committees; Abpro: Membership on an entity's Board of Directors or advisory committees; Apexigen: Membership on an entity's Board of Directors or advisory committees; Spotlight: Membership on an entity's Board of Directors or advisory committees; 47 Inc.: Membership on an entity's Board of Directors or advisory committees; XCella: Membership on an entity's Board of Directors or advisory committees; Immunocore: Membership on an entity's Board of Directors or advisory committees; Walking Fish: Membership on an entity's Board of Directors or advisory committees; Viracta: Membership on an entity's Board of Directors or advisory committees.

S2516 Rare Cause of Diarrhea in a Liver Transplant Recipient

INTRODUCTION: We present a case of a 63-year-old male who underwent a liver transplant that was found to have diffuse mantle cell lymphoma (MCL) after work-up for diarrhea. CASE DESCRIPTION/METHODS: A 63-year old orthotopic liver transplant (OLT) patient with a past medical history of Hepatitis C cirrhosis complicated by hepatocellular carcinoma pre-transplant presented to the clinic with progressive non-bloody diarrhea and weight loss of 15 pounds. The patient denied night sweats and fatigue. There were no concerning findings on abdominal exam. Abdominal Magnetic Resonance Imaging was completed 3 months before he visited our clinic for abnormal liver function tests and showed stable prominent mesenteric lymph nodes. Colonoscopy showed mild erythema in the rectum and sigmoid colon up to 30 cm proximal to the anal verge (Figure 1). Biopsies were taken from the duodenum, right colon, and left colon. Histologic sections of colonic mucosa revealed prominent infiltrate of small atypical lymphocytes that have round to irregular nuclei, mature chromatin, and scant to moderate clear cytoplasm (Figure 2). The biopsies were suspicious for lymphoma, which prompted a bone marrow biopsy. Pathology revealed 10% of the marrow was mantle cell; Fluorescence In Situ Hybridization revealed 11 ;14 translocations. The patient was later diagnosed with stage 4 MCL with diffuse gastrointestinal involvement; all biopsies tested positive for MCL. DISCUSSION: Often MCL presents in men in their 60's with lymphadenopathy, blood and bone marrow involvement, and splenomegaly. Our patient’s lack of classic symptoms made the diagnosis of MCL quite challenging. To our knowledge, MCL is a rare cause of diarrhea in a post-OLT patient. It is plausible that his atypical presentation may be related to partial masking of symptoms related to immunosuppression. Post-transplant patients are at an increased risk for Hodgkin's Lymphoma in the setting of immunosuppression. However, an increased risk of MCL has not been shown as a complication of immunosuppression. The challenge in this patient is balancing his chemotherapeutic and anti-rejection medications. Cyclosporine was continued with the addition of Ibrutinib. While Ibrutinib has shown promising results in patients with MCL, there have been cases reporting hepatotoxic reactions. Thus far, our patient has not had any drug-induced liver injury from Ibrutinib. In conclusion, it is important to maintain MCL along with a broad differential when working up diarrhea in post-transplant patients.Figure 1Figure 2

Ibrutinib added to 10-day decitabine for older patients with AML and higher risk MDS

AbstractThe treatment of older, unfit patients with acute myeloid leukemia (AML) is challenging. Based on preclinical data of Bruton tyrosine kinase expression/phosphorylation and ibrutinib cytotoxicity in AML blasts, we conducted a randomized phase 2 multicenter study to assess the tolerability and efficacy of the addition of ibrutinib to 10-day decitabine in unfit (ie, Hematopoietic Cell Transplantation Comorbidity Index ≥3) AML patients and higher risk myelodysplasia patients (HOVON135/SAKK30/15 trial). In total, 144 eligible patients were randomly (1:1) assigned to either 10-day decitabine combined with ibrutinib (560 mg; sequentially given, starting the day after the last dose of decitabine) (n = 72) or to 10-day decitabine (n = 72). The addition of ibrutinib was well tolerated, and the number of adverse events was comparable for both arms. In the decitabine plus ibrutinib arm, 41% reached complete remission/complete remission with incomplete hematologic recovery (CR/CRi), the median overall survival (OS) was 11 months, and 2-year OS was 27%; these findings compared with 50% CR/CRi, median OS of 11.5 months, and 2-year OS of 21% for the decitabine group (not significant). Extensive molecular profiling at diagnosis revealed that patients with STAG2, IDH2, and ASXL1 mutations had significantly lower CR/CRi rates, whereas patients with mutations in TP53 had significantly higher CR/CRi rates. Furthermore, multicolor flow cytometry revealed that after 3 cycles of treatment, 28 (49%) of 57 patients with available bone marrow samples had no measurable residual disease. In this limited number of cases, measurable residual disease revealed no apparent impact on event-free survival and OS. In conclusion, the addition of ibrutinib does not improve the therapeutic efficacy of decitabine. This trial was registered at the Netherlands Trial Register (NL5751 [NTR6017]) and has EudraCT number 2015-002855-85.

Targeting CD38 Enhances the Antileukemic Activity of Ibrutinib in Chronic Lymphocytic Leukemia.

CD38 has emerged as a high-impact therapeutic target in multiple myeloma, with the approval of daratumumab (anti-CD38 mAb). The clinical importance of CD38 in patients with chronic lymphocytic leukemia (CLL) has been known for over 2 decades, although it's relevance as a therapeutic target in CLL remains understudied. We investigated the biological effects and antitumor mechanisms engaged by daratumumab in primary CLL cells. Besides its known immune-effector mechanisms (antibody-dependent cell-mediated cytotoxicity, complement-dependent death, and antibody-dependent cellular phagocytosis), we also measured direct apoptotic effects of daratumumab alone or in combination with ibrutinib. In vivo antileukemic activity was assessed in a partially humanized xenograft model. The influence of CD38 on B-cell receptor (BCR) signaling was measured via immunoblotting of Lyn, Syk, BTK, PLCγ2, ERK1/2, and AKT. In addition to immune-effector mechanisms; daratumumab also induced direct apoptosis of primary CLL cells, which was partially dependent on FcγR cross-linking. For the first time, we demonstrated the influence of CD38 on BCR signaling where interference of CD38 downregulated Syk, BTK, PLCγ2, ERK1/2, and AKT; effects that were further enhanced by addition of ibrutinib. In comparison to single-agent treatment, the combination of ibrutinib and daratumumab resulted in significantly enhanced anti-CLL activity in vitro and significantly decreased tumor growth and prolonged survival in the in vivo CLL xenograft model. Overall, our data demonstrate the antitumor mechanisms of daratumumab in CLL; furthermore, we show how cotargeting BTK and CD38 lead to a robust anti-CLL effect, which has clinical implications.

PS1091 TREATMENT OF ACTIVATED B‐CELL LYMPHOMA WITH RITUXIMAB,DOXORUBICIN,ETOPOSIDE,PREDNISOLONE/IFOSFAMIDE+CISPLATIN PLUS LENALIDOMIDE (R‐ACEP/ICL), CONFERS LONG PROGRESSION FREE AND OVERALL SURVIVAL RATES

Background:gene profiling (GEP) revealed the main two types of DLBCL [the germinal center B‐cell (GCB) and the non‐germinal center or Activated B‐cell lymphoma(ABC)]. Immuno‐histochemistry plays an important role in differentiating between the two types using several algorithms, however, Hans algorithm and the improved Hans are considered the more reliable. Patients with GCB are still doing well in the era of type I and II anti‐CD20, however, ACB patients are experiencing bad prognosis with shorter progression free and overall survival rates, therefore, we have developed a high‐dense high intense protocol to treat those population with ABC.Aims:the study aims at evaluating a new dose intense chemotherapy in patients with ABC lymphoma with end points including progression free and overall survivalMethods:this is a multicenter study Al Bairouni university cancer center in Damascus, Syria between 2013 and 2015 where we recruited 122 patients having ABC DLBCL confirmed by immuno‐histochemistry using the improved Hans algorithm. Patients received a dose dense protocol as follows: (Rituximab 375 mg/m2 with Doxorubicin 75 mg/m2, Cyclophosphamide 1200 mg/m2, Etopdside 300 mg/m2 and Prednisolone 1 mg/kg for 5 days) repeated every 21 days for 6 cycles with 5 days G‐csf support. Then partial and complete responders were given (Ifosfamide 4 gr/m2 day 1–3 + Cisplatine 40 mg/m2 day 1–3) for 4 cycles repeated every 15 days with 4 days G‐csf support. Lenalidomide 10 mg/d was given upfront lasting for 1 year. End‐points were progression free survival and overall survival at 5 years.Results:overall response rate (ORR) was documented in 118 patients (96.7%) (92 complete responders and 26 partial responders). 10 patients progressed at different stages of follow up with a progression free survival (PFS) of 88.6% (95% CI 83–94). However, 113 patients were found to still alive at 5 years with an overall survival (OS) rate of 92.6 (95% CI 86–97). In spite of G‐csf support, neutropenia of different grades was documented in 23 patients (18.8%). Acute and transient renal failure was observed and managed in 15 patients (12.2%) mainly in the first 6 cycles. Other side effects were manageable.Summary/Conclusion:ABC forms a special entity of DLBCL carrying a bad prognosis compared with GCB subtype. Progress was made using the R‐CHOP, however, results were still poor until the addition of Ibrutinib, Lenalidomide or Bortezomib to R‐CHOP in different trials which improved OS to exceed 90%. In this study, we present a dose intense protocol conferring a good PFS and OS rates with acceptable toxicity profile. However, a big randomized trials including both ABC and GCB patients in order to identify factors having bad impact on progression.

PS1092 TANDEM AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR TREATMENT OF ADULT T-CELL LYMPHOBLASTIC LYMPHOMA: A MULTIPLE CENTER PROSPECTIVE STUDY IN SOUTHWESTERN CHINA

Background: gene profiling (GEP) revealed the main two types of DLBCL [the germinal center B-cell (GCB) and the non-germinal center or Activated B-cell lymphoma(ABC)]. Immuno-histochemistry plays an important role in differentiating between the two types using several algorithms, however, Hans algorithm and the improved Hans are considered the more reliable. Patients with GCB are still doing well in the era of type I and II anti-CD20, however, ACB patients are experiencing bad prognosis with shorter progression free and overall survival rates, therefore, we have developed a high-dense high intense protocol to treat those population with ABC. Aims: the study aims at evaluating a new dose intense chemotherapy in patients with ABC lymphoma with end points including progression free and overall survival Methods: this is a multicenter study Al Bairouni university cancer center in Damascus, Syria between 2013 and 2015 where we recruited 122 patients having ABC DLBCL confirmed by immuno-histochemistry using the improved Hans algorithm. Patients received a dose dense protocol as follows: (Rituximab 375 mg/m2 with Doxorubicin 75 mg/m2, Cyclophosphamide 1200 mg/m2, Etopdside 300 mg/m2 and Prednisolone 1 mg/kg for 5 days) repeated every 21 days for 6 cycles with 5 days G-csf support. Then partial and complete responders were given (Ifosfamide 4 gr/m2 day 1–3 + Cisplatine 40 mg/m2 day 1–3) for 4 cycles repeated every 15 days with 4 days G-csf support. Lenalidomide 10 mg/d was given upfront lasting for 1 year. End-points were progression free survival and overall survival at 5 years. Results: overall response rate (ORR) was documented in 118 patients (96.7%) (92 complete responders and 26 partial responders). 10 patients progressed at different stages of follow up with a progression free survival (PFS) of 88.6% (95% CI 83–94). However, 113 patients were found to still alive at 5 years with an overall survival (OS) rate of 92.6 (95% CI 86–97). In spite of G-csf support, neutropenia of different grades was documented in 23 patients (18.8%). Acute and transient renal failure was observed and managed in 15 patients (12.2%) mainly in the first 6 cycles. Other side effects were manageable. Summary/Conclusion: ABC forms a special entity of DLBCL carrying a bad prognosis compared with GCB subtype. Progress was made using the R-CHOP, however, results were still poor until the addition of Ibrutinib, Lenalidomide or Bortezomib to R-CHOP in different trials which improved OS to exceed 90%. In this study, we present a dose intense protocol conferring a good PFS and OS rates with acceptable toxicity profile. However, a big randomized trials including both ABC and GCB patients in order to identify factors having bad impact on progression.

Ibrutinib, Fludarabine, Cyclophosphamide, and Obinutuzumab (iFCG) for Firstline Treatment of Patients with CLL with Mutated IGHV and without TP53 Aberrations

Ibrutinib, Fludarabine, Cyclophosphamide, and Obinutuzumab (iFCG) for Firstline Treatment of Patients with CLL with Mutated IGHV and without TP53 Aberrations

Ibrutinib inhibits mesenchymal stem cells-mediated drug resistance in diffuse large B-cell lymphoma

目的探讨依布替尼克服弥漫大B细胞淋巴瘤（DLBCL）细胞耐药的机制。方法①体外实验：以DLBCL细胞系SUDHL10细胞（GCB亚型）、HBL-1（ABC亚型）以及8例DLBCL患者原代细胞为研究对象，与正常人骨髓基质细胞（MSC）共培养后，显微镜下计数向MSC趋化迁移及与MSC黏附的DLBCL细胞数，ELISA法检测MSC的CXCL12表达水平，流式细胞术检测DLBCL细胞的CXCR4表达水平；以携带有CXCR4的慢病毒转染HBL-1细胞，米托蒽醌、依布替尼处理后与MSC共培养，流式细胞术检测细胞凋亡水平；倒置显微镜下观察HBL-1细胞集落形成情况。②体内实验：以HBL-1细胞构建的NOD/SCID肿瘤模型小鼠为研究对象，观察依布替尼治疗后肿瘤体积变化。结果①依布替尼处理后，DLBCL细胞向MSC的迁移数和与MSC的黏附比例明显降低（P值均<0.05），并呈剂量依赖性。②与依布替尼处理前比较，处理后MSC的CXCL12表达水平降低（SUDHL10细胞：660 pg/ml对1 400 pg/ml，P=0.004；HBL-1细胞：720 pg/ml对1 490 pg/ml，P=0.018；DLBCL原代细胞：850 pg/ml对1 450 pg/ml，P=0.004），DLBCL细胞的CXCR4表达水平降低（P值均<0.05）。③共培养时，对照组、米托蒽醌组、依布替尼组、米托蒽醌组+依布替尼组的HBL-1细胞凋亡比例分别为15.1%、17.5%、23.5%、58.7%，转染过表达CXCR4后，HBL-1细胞凋亡比例分别为14.2%、16.1%、22.5%、38.3%，共培养联合用药组HBL-1细胞凋亡比例高于单药培养组，差异均有统计学意义（P值均<0.05）。④对照组、MSC组、依布替尼组、MSC组+依布替尼组集落数分别为113±5、205±4、62±9、123±3（每孔2.5×103），模型小鼠皮下肿瘤体积分别为6 500、17 000、4 000、10 000 mm3，依布替尼处理后较处理前集落数和肿瘤体积明显减少，差异均有统计学意义（P值均<0.05）。结论依布替尼靶向作用于CXCL12/CXCR4轴，抑制CXCR4表达从而克服MSC介导的耐药作用，并且能够在体内外抑制MSC促淋巴瘤细胞集落形成的作用。为依布替尼治疗复发耐药DLBCL提供了理论依据。

Ibrutinib, fludarabine, cyclophosphamide, and obinutuzumab (GA101) (iFCG) for previously untreated patients with chronic lymphocytic leukemia (CLL) with mutated IGHV and non-del (17p).

7522 Background: Pts with mutated IGHV ( IGHV-M) have favorable long-term outcomes after FCR. Methods: We designed an investigator-initiated phase II trial with ibrutinib, fludarabine, cyclophosphamide, and obinutuzumab (iFCG) for previously untreated pts with IGHV-M CLL (NCT02629809). The intent was to limit FC to 3 courses, potentially reducing short- and long-term toxicity, while maintaining efficacy through addition of ibrutinib and obinutuzumab. Key eligibility included age ≥18, IGHV-M, no del17p. Pts received 3 courses of iFCG. G-CSF was not mandated. Primary endpoint: CR/CRi with bone marrow (BM) MRD-neg (4-color flow-cytometry) after 3 courses of iFCG. Pts meeting primary endpoint received ibrutinib with obinutuzumab (iG) for C3-6, then ibrutinib C7-12. Pts not achieving primary endpoint received iG (C4-12). All pts who are MRD neg at 1 year will stop all therapy, including ibrutinib. Pts MRD+ at 1 year may continue ibrutinib. Historic C3 BM MRD-neg with FCR in IGHV-M 26% (Strati, Blood 2014). Target BM MRD-neg after iFCG x3 is 45%. Sample size 45. Results: 23 pts started treatment. Median age 59 yrs (25-71). Prognostic markers [del13q (n=17), negative (n=3); trisomy 12 (n=3)]. 18 pts completed 3 courses of iFCG and had initial response assessment (the remaining 5 pts too early). All 18 pts had a response; 14/18 (78%) achieved MRD-neg in BM at 3 month with 7/18 achieving CR/CRi (all MRD neg). No pt has progressed, and all but one continue to receive treatment. Of the 23 pts, 11 pts had G3-4 neutropenia and 5 pts had G3-4 thrombocytopenia. 4 pt had neutropenic fever. 1 pt who achieved MRD-neg CR developed pulmonary MAC infection, and declined further therapy. 1 pt had atrial fibrillation. G3 ALT developed in 3 pts. FC was dose reduced in 10 pts; ibrutinib dose-reduced in 2 pts. Conclusions: iFCG achieves high rate of MRD-neg remission after 3 courses. Pt enrollment continues, and updated results will be presented at the ASCO meeting. Clinical trial information: NCT02629809. [Table: see text]

Humanized mice (humice) carrying patient-derived xenograft (PDX) as a platform to develop immunotherapy in bladder cancer (BCa).

381 Background: Immunotherapy with anti-programmed cell death 1 (PD1) or PD ligand 1 (PD-L1) antibody has emerged as a promising therapeutic modality, but has a response rate of approximately 20% in BCa. There are various drawbacks associated with current animal models. The objective of this study is to establish and characterize humice carrying PDXs in which both the immune cells and BCa cells are derived from humans. Methods: NOD-scid IL2Rgammanull or NSG, mice received CD34+ hematopoietic progenitor cells (HPC) cells i.v. after whole body radiation. PDXs were established through direct implantation of human BCa clinical specimens into NSG mice. Immune cell subpopulations were analyzed through flow cytometry analysis. Humice carrying HLA-unmatched PDXs were treated with an anti-PD1 antibody pemborlizumab (pembro) or in combination with a BKT/ITK inhibitor ibrutinib to determine the anti-tumor efficacy and toxicity. Results: PDXs retained the morphology fidelity and 92-97% of genetic alterations of parental patient cancers. Of the first 8 PDXs tested, 3 had high PD-L1 ( &gt; 10 FPKM) as determined by RNA-seq which was further confirmed with flow cytometry analysis. Major human immune cell sub-populations were reconstituted in humice. No xenograft versus host disease was observed before pembro treatment. In humice with HPC donor 6466, pembro significantly inhibited tumor growth (p = 0.0016 at Day 29) of PDX BL293, but had no effect in another PDX BL440 with the same HPC donor 6466, or with the same PDX BL293 but with a different HPC donor 912. In another set of humice (HPC donor 710) carrying PDX BL293, pembro alone inhibited tumor growth. However, addition of ibrutinib did not potentiate the efficacy of pembro, but increased toxicity. Tumor regression with pembro treatment was associated with decrease of CD4+PD1+, CD8+PD1+ cells at peripheral blood and increased CD45+ and CD8+ cells in PDXs. Conclusions: Humice carrying PDXs reconstitute with human immune system, and can potentially be used to screen for effective immunotherapeutic agents or combinations, and to study resistant mechanisms.

Preclinical Analyses Support Clinical Investigation of Combined Anti-CD19 CAR-T Cell, JCAR017 with Ibrutinib for the Treatment of Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) drives systemic immune suppression and T cell dysfunction in patients, highlighting an important consideration in this setting for the manufacturing and efficacy of adoptive cell therapies using autologous T cells. In clinical studies, anti-CD19 CAR-T cells produce durable and complete responses in leukemic and some lymphomatous B cell malignancies. While preconditioning with cyclophosphamide (Cy) and fludarabine (Flu) has improved CAR-T responses in CLL patients, reported complete response rates still have been below 50%; additional therapeutic strategies likely will be required.Ibrutinib, an irreversible inhibitor of BTK, has been approved as a frontline treatment option for patients with CLL. The potent off-BTK activity of ibrutinib on ITK and TEC family kinases could affect CAR T cell biology. Recent work highlighted the ability of ibrutinib to restore CLL patient T cell functionality, enhance CAR-T production and potentially improve clinical efficacy. Additional preclinical work demonstrated improved tumor clearance when anti-CD19 CAR T cells were combined with ibrutinib in several murine tumor models. A preclinical evaluation of the combination between the anti-CD19 CAR-T product, JCAR017, and ibrutinib was performed to determine feasibility for clinical use in CLL. JCAR017 is a second generation CAR-T cell product candidate that contains a 41BB costimulatory endo-domain and is currently in phase 1 trials for non-Hodgkin lymphoma (NHL).A series of in vitro studies assessed the functional activity of JCAR017 cells (derived from 3 healthy donors), in combination with ibrutinib (500-0.05nM), across a dose range covering the cMax and cMin. Cytolytic activity was monitored by co-culturing CAR-T cells with ibrutinib-resistant K562 CD19 tumor cells at an effector-to-target ratio of 2.5:1. Ibrutinib, at concentrations tested, did not inhibit the cytolytic function of JCAR017 cells. For cells derived from some donors, addition of ibrutinib appeared to increase % target killing. To address ibrutinib effects on JCAR017 activation, cell surface markers and cytokines were tracked over 4 days following stimulation with irradiated K562 CD19 cells. We observed no significant effect on JCAR017 surface expression of CD25, CD38, CD39, CD95, CD62L, CCR7, or CD45RO, or of EGFRt, a surrogate transduction marker. With addition of ibrutinib, we observed a modest decrease in the percentage of cells expressing CD69, CD107a and PD-1. With 5 and 50nM of ibrutinib, there was a 19.5% (p<0.01) average increase in IFNγ production. At supraphysiological concentrations (500nM) we observed a 20% (p<0.05) decrease in IL-2 production, suggesting ibrutinib at high concentrations may dampen T cell activation. CAR-T cell expansion after repeated antigen stimulation has been shown to be a predictor of in vivo efficacy. JCAR017 cells stimulated every 3-4 days with irradiated target cells in the presence of ibrutinib showed no inhibition of initial growth. However, after 5 rounds of stimulation, JCAR017 + ibrutinib cells from 1 donor had enhanced proliferation compared to control, untreated cells (p<0.05). Interestingly, after 5 rounds of serial stimulation, we observed an increased proportion of CD4+CXCR3+CRTh2- Th1 cells with 500nM ibrutinib treatment compared to control (p<0.01).We assessed the in vivo anti-tumor activity of JCAR017 in combination with ibrutinib using NSG mice injected with 5x105 Nalm6-luciferase cells. After tumor engraftment, a suboptimal dose (5x105) of JCAR017 cells was transferred to mice and ibrutinib (25 mg/kg qd) was administered for the duration of the study. Ibrutinib treatment alone had no effect on tumor burden compared to vehicle treatment. Mice treated with a suboptimal JCAR017 dose + ibrutinib showed decreased tumor burden (p<0.05) and increased median survival from 44 days to >80 days (p<0.001) compared to the group receiving the suboptimal JCAR017 dose + vehicle. Similar effects were seen in replicate studies using JCAR017 cells produced from multiple donors. Ex vivo evaluation for CAR-T quantitation and immunophenotyping was also performed.Taken together, the results suggest that ibrutinib enhances intrinsic JCAR017 activity and may improve outcomes in CLL patients treated with anti-CD19 CAR T therapy, irrespective of BTK mutational status. A Phase 1b study of JCAR017 in combination with ibrutinib for BTKi R/R CLL is planned. DisclosuresQin:Juno Therapeutics: Employment. Baturevych:Juno Therapeutics: Employment. Mudri:Juno Therapeutics: Employment, Equity Ownership. Salmon:Juno Therapeutics: Employment. Ports:Juno Therapeutics: Employment.

Abstract B193: Ibrutinib potentiates the effects of mTOR inhibitors and pazopanib in renal cell carcinoma in vitro and in vivo

Abstract Introduction: Ibrutinib is a first-in-class Bruton's tyrosine kinase (BTK) inhibitor approved for treatment of multiple B-cell malignancies that also inhibits EGFR and HER2 activity (Honigberg, 2010; Gao, 2014). Ibrutinib suppressed growth of EGFR-driven NSCLC or HER2-amplified breast cancer cell lines and reduced tumor growth in murine xenograft models demonstrating the potential of anti-tumor activity in solid tumors (Gao, 2014; Elias, 2013). Ibrutinib also modulated host tumor immunity and enhanced PD-L1 Mab activity in solid tumor models from tumor cells insensitive to BTK or HER kinase inhibition (Sagiv-Barfi, 2015). These results suggest the potential of ibrutinib to be clinically active across a variety of tumors via multiple mechanisms of action (MoA). Immunotherapeutic interventions have had varying success in renal cell carcinoma (RCC), the most common type of kidney cancer. In this study, we determined the therapeutic impact of ibrutinib alone and in combination with approved therapies in RCC in vitro and in vivo and further investigated its MoA. Methods: The effect of drugs on cell proliferation was determined with CellTiter-Glo (Promega), and apoptosis assayed with annexin-V/PI staining. Signaling pathways were evaluated with Western blotting. Single agent and combinations were studied in vivo by subcutaneous implantation of syngeneic Renca or human 786-0 RCC cell lines into mice. Results: Treating RCC cells with everolimus alone potently inhibited p-S6, a downstream mTOR target, and significantly inhibited proliferation. Pazopanib, a multi-kinase inhibitor, similarly inhibited pAkt. As reported, these inhibitors up-regulated pAkt and/or pErk, two key pro-survival molecules, after 1h and/or 24h treatment. Such compensatory changes to signaling pathways have been suggested to attenuate the anti-tumor activity of these inhibitors (Wang, 2008; Breuleux, 2009; Grabinski, 2012; Soares, 2013). Interestingly, addition of ibrutinib antagonized up-regulation of pAkt and pErk by everolimus or pazopanib, and enhanced their anti-proliferative activities. In contrast, ibrutinib did not enhance everolimus activity in Caki-1 cells where pAkt and pErk were unaffected by the combination. Ibrutinib alone did not or only weakly inhibited RCC proliferation in cell culture despite inhibition of EGF-induced pEGFR. In contrast, pAkt and pErk, were much less affected, suggesting that EGFR is not a primary driver of proliferation in RCC cells. In vivo, ibrutinib alone showed marginal or no anti-tumor activity in Renca or 786-0 mice models. However, ibrutinib significantly reduced tumor burden in combination with sirolimus (p&amp;lt;0.001) or everolimus (p&amp;lt;0.05) in the Renca and 786-0 RCC models, compared to mTOR inhibitor treatments alone. Conclusion: These results suggest that ibrutinib, a kinase inhibitor with immune modulatory properties, has anti-tumor activity against RCC when combined with mTOR inhibitors or pazopanib with different target spectrums. Although EGFR is not critical for proliferation in these untreated RCC lines, ibrutinib may prevent ErbB kinases from contributing to feedback up-regulation of Akt/Erk pathways by established drugs. These results provide a preclinical rationale for investigation of ibrutinib as a novel agent for RCC through positive interaction with mTOR inhibitors and/or pazopanib. Citation Format: Jun Chen, Jeff Hsu, Yujun Huang, Danelle F. James, Taisei Kinoshita, Betty Y. Chang. Ibrutinib potentiates the effects of mTOR inhibitors and pazopanib in renal cell carcinoma in vitro and in vivo. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B193.

Abstract 259: Ibrutinib enhances the anti-tumor efficacy of CTLA-4 blockade in lymphoma and colon cancer models

Abstract The anti-CTLA-4 antibody ipilimumab was the first immune checkpoint blockade antibody to show significant clinical efficacy. However, only a subset of the treated melanoma patients had long-term survival benefits (Schadendorf, D., et al. ECCO 2013). Combining ipilimumab with an agent that modulates immunity by a different mechanism may improve efficacy. Ibrutinib is a first-in-class, oral, covalent inhibitor of Bruton's tyrosine kinase (Btk), an essential enzyme in the B-cell receptor signaling pathway. Ibrutinib also inhibits the interleukin-2-inducible Tec kinase (Itk) that is required for the activation/survival of Th2 T cells. This activity is consistent with preclinical studies that demonstrated ibrutinib could potentiate Th1 immunity (Dubovsky, JA., et al. Blood 2013). We investigated whether ibrutinib could enhance the anti-tumor effect of CTLA-4 blockade using the A20 B-cell lymphoma and the CT26 colon carcinoma mouse models. BALB/c mice (9-11/group) were implanted with tumor cells subcutaneously on the hind flanks. Animals bearing 80-100 mm3 (D x d2 x 0.4) tumors were dosed daily with oral ibrutinib (24 mg/kg) with or without the anti-mouse CTLA-4 antibody 9D9 (100 μg/mouse) given every 2 days by intraperitoneal injections. Animals in the lymphoma and colon cancer models were treated for 13 and 37 days, respectively. Tumor volume was measured and survival recorded for up to 2 months after tumor inoculation. Neither the A20 tumors, which express Btk, nor the CT26 tumors were inhibited by ibrutinib. In the A20 model, although 9D9 alone resulted in complete tumor regression in 7 of 9 treated animals, tumors in 2 of the 7 mice relapsed after treatment had ended. In contrast, 9D9 in combination with ibrutinib led to rapid, complete, and durable responses in 7 of 9 treated animals that lasted until the end of the study. In the CT26 model, treatment with 9D9 alone had minimal effects on tumor growth, but the addition of ibrutinib led to complete tumor regression in 6 of 10 treated animals. Thus, ibrutinib enhanced the anti-tumor efficacy of an anti-CTLA-4 antibody in B-cell lymphoma and colon carcinoma mouse models. The mechanism of tumor eradication by the combination therapy is under investigation and will be discussed at presentation. Citation Format: Patrick Ng, Daniel Lu, Betty Chang. Ibrutinib enhances the anti-tumor efficacy of CTLA-4 blockade in lymphoma and colon cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 259. doi:10.1158/1538-7445.AM2015-259