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In this paper, we have focused on the investigation of the expression level of the enhancer of zeste homolog 2 (EZH2) gene in bone marrow mononuclear cells of acute myeloid leukemia (AML) patients and analyze the relationship between EZH2 gene expression and EMI. The expression of EZH2mRNA in bone marrow mononuclear cells of 26 patients with incipient AML was detected by qRT-PCR, and the relationship between EZH2mRNA expression and clinical characteristics was analyzed. EZH2 mRNA expression was increased in 26 AML patients. EZH2 gene expression in male patients was significantly higher than that in female patients. The expression of EZH2 in the group with extramedullary infiltration (EMI) was significantly higher than that in the group without EMI. The patients were divided into different groups according to the chromosomal karyotype and prognosis. Statistical analysis showed that the expression level of the medium-risk group was significantly higher than that of the low-risk group, while there was no statistical difference in other groups ( P > 0.05 ). The expression of EZH2 gene in AML patients was closely related to EMI, and the expression of EZH2 in AML cells was closely related to cell migration ability. EZH2 is expected to be one of the indicators of disease recurrence.

Background We compared the efficacy, safety, and immunogenicity of MIL60 with reference bevacizumab as first-line treatment in patients with advanced or recurrent non-squamous non-small cell lung cancer (NSCLC) in this phase 3, randomized, double-blind study. Methods Patients with untreated advanced or recurrent NSCLC were randomized (1:1 ratio) to receive either MIL60 or bevacizumab in combination with paclitaxel/carboplatin. Patients with non-progressive disease continued maintenance single-agent MIL60 until disease progression, or intolerable toxicity. The primary endpoint was the 12-week objective response rates (ORR12) by independent review committee (IRC) using RECIST 1.1. Bioequivalence was established if the ORR ratio located between 0.75 and 1/0.75. The trial was registered with clinicaltrials.gov (NCT03196986). Findings Between Aug 23, 2017, and May 8, 2019, 517 patients were randomly assigned to MIL60 group (n=257) and bevacizumab group (n=260). In the full analysis set (FAS) population including all randomized and evaluable patients who received at least one dose of MIL60 or bevacizumab, the ORR12 in MIL60 group and bevacizumab group were 48.6% and 43.1%, respectively. The ORR ratio of these two groups were 1.14 (90% CI 0.97-1.33), which fell within the pre-specified equivalence boundaries (0.75-1/0.75). The median DOR was 5.7 months (95% CI 4.5-6.2) for MIL60 and 5.6 months (95% CI 4.3-6.4) for bevacizumab. No significant difference was noted in median PFS (7.2 vs. 8.1 months; HR 1.01, 95% CI 0.78-1.30, p=0.9606) and OS (19.3 vs. 16.3 months; HR 0.81, 95% CI 0.64-1.02, p=0.0755). Safety and tolerability profiles were similar between the two groups. No patient detected positive for Anti-drug antibody (ADA). Interpretation The efficacy, safety and immunogenicity of MIL60 were similar with bevacizumab, providing an alternative treatment option for advanced or recurrent non-squamous NSCLC. Funding This study was sponsored by Betta Pharmaceutical Co., Ltd.

Richter syndrome (RS) indicates the transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma (mostly DLBCL). Richter syndrome is a rare complication with an aggressive clinical course, bearing an unfavorable prognosis. Currently, there is no effective treatment for it. As a novel cellular‐based immune therapy, chimeric antigen receptor‐modified T (CART) cells treatment is gradually used in treating hematological malignancies, especially in CD19+ B‐cell malignancy. Therefore, CD19‐directed chimeric antigen receptor‐modified T cells (CART‐19) treatment is promising to be used as a new method for RS patients. In our study, one RS patient expressing high level of CD19 molecule was enrolled in clinical trial; he has received a series of treatments but did not achieve a satisfactory therapeutic effect. The patient totally received 3.55 × 108 autologous CART‐19 cells infusion. After CART‐19 infusion, the mainly clinical side effect was repeated fever. The maximal duration period was 24 days and the highest temperature was 40.1°C. Pancytopenia and significantly serum cytokines level rise were observed, including IFN‐γ, IL‐6, and IL‐10. Before discharge, the level of cytokines reduced to normal levels. In addition, we detected the serum biochemical indices as like K+, Ca2+, creatinine, and glutamic‐pyruvic transaminase, all of these indices were normal. This showed that there was no tumor necrosis syndrome after treatment. The proportion of B cells in patient's peripheral blood decreased from 72% to 40.2% after infusion, co‐occurring with reduction in lymph nodes and hematopoietic reconstitution. Based on the recent revolution in the therapeutic landscape for hematological malignancies including B‐cell lymphomas, CART‐CD19 cell therapy as a new therapeutic option for RS might be available in the coming years. It aims to reduce patient's tumor burden, prolong their survival time, and provide opportunities for other sequential therapy such as chemotherapy and bone marrow transplantation.

Natural killer (NK) cells play a pivotal role in monoclonal antibody-mediated immunotherapy through the antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. NK-92MI is an interleukin-2 (IL-2)-independent cell line, which was derived from NK-92 cells with superior cytotoxicity toward a wide range of tumor cells in vitro and in vivo. Nonetheless, the Fc-receptor (CD16) that usually mediates ADCC is absent in NK-92 and NK-92MI cells. To apply NK-92MI cell-based immunotherapy to cancer treatment, we designed and generated two chimeric receptors in NK-92MI cells that can bind the Fc portion of human immunoglobulins. The construct includes the low-affinity Fc receptor CD16 (158F) or the high-affinity Fc receptor CD64, with the addition of the CD8a extracellular domain, CD28 transmembrane domains, two costimulatory domains (CD28 and 4-1BB), and the signaling domain from CD3ζ. The resulting chimeric receptors, termed CD16-BB-ζ and CD64-BB-ζ, were used to generate modified NK-92MI cells expressing the chimeric receptor, which were named NK-92MIhCD16 and NK-92MIhCD64 cells, respectively. We found that NK-92MIhCD16 and NK-92MIhCD64 cells significantly improved cytotoxicity against CD20-positive non-Hodgkin's lymphoma cells in the presence of rituximab. These results suggest that the chimeric receptor-expressing NK-92MI cells may enhance the clinical responses to currently available anticancer monoclonal antibodies.

Yuan Yu,1–3 Jialu Li,1–3 Xuejun Zhu,4 Xiaowen Tang,2,5 Yangyi Bao,6 Xiang Sun,6 Yuhui Huang,1,2 Fang Tian,4 Xiaomei Liu,1,2 Lin Yang1–3 1The Cyrus Tang Hematology Center, 2Collaborative Innovation Center of Hematology, Soochow University, 3Suzhou Cancer Immunotherapy and Diagnosis Engineering Center, Suzhou, 4Central Laboratory, Department of Hematology, The Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital ofTraditional Chinese Medicine, Nanjing, 5Department of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, 6Department of Hematology-Oncology, The First People’s Hospital of Hefei, Hefei, People’s Republic of China Background: Nanobodies, named as VHHs (variable domain of heavy chain of HCAb [heavy-chain antibodies]), are derived from heavy-chain-only antibodies that circulate in sera of camelids. Their exceptional physicochemical properties, possibility of humanization, and unique antigen recognition properties make them excellent candidates for targeted delivery of biologically active components, including immunotoxins. In our previous efforts, we have successfully generated the monovalent and bivalent CD7 nanobody-based immunotoxins, which can effectively trigger the apoptosis of CD7-positive malignant cells. To pursue the possibility of translating those immunotoxins into clinics, we humanized the nanobody sequences (designated as dhuVHH6) as well as further truncated the Pseudomonas exotoxin A (PE)-derived PE38 toxin to produce a more protease-resistant form, which is named as PE-LR, by deleting majority of PE domain II. Methods and results: Three new types of immunotoxins, dhuVHH6-PE38, dVHH6-PE-LR, and dhuVHH6-PE-LR, were successfully constructed. These recombinant immunotoxins were expressed in Escherichia coli and showed that nanobody immunotoxins have the benefits of easy soluble expression in a prokaryotic expression system. Flow cytometry results revealed that all immunotoxins still maintained the ability to bind specifically to CD7-positive T lymphocyte strains without binding to CD7-negative control cells. Laser scanning confocal microscopy revealed that these proteins can be endocytosed into the cytoplasm after binding with CD7-positive cells and that this phenomenon was not observed in CD7-negative cells. WST-8 experiments showed that all immunotoxins retained the highly effective and specific growth inhibition activity in CD7-positive cell lines and primary T-cell acute lymphoblastic leukemia (T-ALL) cells. Further in vivo animal model experiments showed that humanized dhuVHH6-PE38 immunotoxin can tolerate higher doses and extend the survival of NOD-Prkdcem26Il2rgem26Nju (NCG) mice transplanted with CEM cells without any obvious decrease in body weight. Further studies on NCG mice model with patient-derived T-ALL cells, dhuVHH6-PE38 treatment, significantly prolonged mice survival with ~40% survival improvement. However, it was also noticed that although dhuVHH6-PE-LR showed strong antitumor effect in vitro, its in vivo antitumor efficacy was disappointing. Conclusion: We have successfully constructed a targeted CD7 molecule-modified nanobody (CD7 molecule-improved nanobody) immunotoxin dhuVHH6-PE38 and demonstrated its potential for treating CD7-positive malignant tumors, especially T-cell acute lymphoblastic leukemia. Keywords: CD7, humanized nanobody, T-cell acute lymphoblastic leukemia, patient-derived xenograft model, recombinant immunotoxins, Pseudomonas exotoxin A

We previously tested HER2-targeted antibody drug conjugates (ADC) in immunocompromised (SCID) mice, precluding evaluation of host immunity, impact on cancer stem cells (CSCs), and potential benefit when combined with PD-L1 blockade. In this study, we tested HER2-targeted ADC in two immunocompetent mouse tumor models. HER2-targeted ADC specifically inhibited the growth of HER2-expressing tumors, prolonged animal survival, and reduced HER2(+) and PD-L1(+) cells. ADC plus anti-PD-L1 antibody augmented therapeutic efficacy, modulated immune gene signatures, increased the number and function of CD3(+) and CD19(+) tumor-infiltrating lymphocytes (TILs), induced tumor antigen specific immunological memory, stimulated B cell activation, differentiation, and IgG1 production both systemically and in the tumor microenvironment. In addition, ADC therapy modulated T cell subsets and their activation in TILs. Furthermore, HER2-targeted ADC reduced the number and tumorigenicity of ALDH(hi) CSCs. This study demonstrates that HER2-targeted ADC effectively targets ALDH(hi) CSCs and this effect is augmented by co-administration of anti-PD-L1 antibody.

Dendritic cell (DC)-based vaccine strategies aimed at targeting cancer stem–like cells (CSC) may be most efficacious if deployed in the adjuvant setting. In this study, we offer preclinical evidence that this is the case for a CSC-DC vaccine as tested in murine models of SCC7 squamous cell cancer and D5 melanoma. Vaccination of mice with an ALDHhigh SCC7 CSC-DC vaccine after surgical excision of established SCC7 tumors reduced local tumor relapse and prolonged host survival. This effect was augmented significantly by simultaneous administration of anti-PD-L1, an immune checkpoint inhibitor. In the minimal disease setting of D5 melanoma, treatment of mice with ALDHhigh CSC-DC vaccination inhibited primary tumor growth, reduced spontaneous lung metastases, and increased host survival. In this setting, CCR10 and its ligands were downregulated on ALDHhigh D5 CSCs and in lung tissues, respectively, after vaccination with ALDHhigh D5 CSC-DC. RNAi-mediated attenuation of CCR10 blocked tumor cell migration in vitro and metastasis in vivo. T cells harvested from mice vaccinated with ALDHhigh D5 CSC-DC selectively killed ALDHhigh D5 CSCs, with additional evidence of humoral immunologic engagement and a reduction in ALDHhigh cells in residual tumors. Overall, our results offered a preclinical proof of concept for the use of ALDHhigh CSC-DC vaccines in the adjuvant setting to more effectively limit local tumor recurrence and spontaneous pulmonary metastasis, as compared with traditional DC vaccines, with increased host survival further accentuated by simultaneous PD-L1 blockade. Cancer Res; 76(16); 4661–72. ©2016 AACR.

BACKGROUND Radiotherapy is one of the primary treatments for esophageal squamous cell carcinoma (ESCC). Identification of novel radio-sensitizing agents will improve the therapeutic outcome of radiotherapy. This study aimed to determine the radio-sensitizing effect of the antipsychotic agent thioridazine in ESCC and explored the underlying mechanisms. MATERIAL AND METHODS ECA-109 and TE-1 ESCC cells were treated with thioridazine and radiotherapy alone and in combination. Cell survival was measured by MTT assay. Cell cycle and apoptosis were monitored by flow cytometry. Western blot analysis was used to analyze the expression of phospho-PI3K, phosphor-AKT, phospho-mTOR, Caspase-3, Caspase-9, Bax, Bcl-2, Bal-xl, Bak, and p53. The xenograft mouse model was used to study the in vivo anticancer effect of thioridazine and irradiation. RESULTS Combined treatment with thioridazine and irradiation significantly reduced viability of ESCC cells compared with thioridazine or irradiation treatment alone. Thioridazine and irradiation treatment induced G0/G1 phases cell cycle arrest through down-regulation of CDK4 and cyclinD1. In addition, thioridazine and irradiation treatment induced apoptosis through up-regulation of cleaved capase-3 and 9, as well as an increase in the expression of Bax and Bak and a decrease in the expression of Bcl-2 and Bcl-xl. Furthermore, thioridazine and irradiation treatment inhibited the PI3K-AKT-mTOR pathway and up-regulated the expression of p53. In xenograft mice, thioridazine and irradiation reduced ESCC tumor growth. CONCLUSIONS Thioridazine sensitizes ESCC cells to radiotherapy. Thioridazine may play a role in ESCC radiation therapy as a promising radiosensitizer.

In the past decades, chemotherapy has shown encouraging but limited efficacy treating triple negative breast cancer (TNBC). Targeting cancer stem cells (CSCs) has been recognized as a promising method to inhibit tumor growth and dissemination. This study aimed to determine whether an anti-psychotic drug thioridazine (THZ) could sensitize metastatic TNBC cells to anti-cancer drug carboplatin (CBP). To this end, metastatic TNBC (4T1)-bearing animals were treated with THZ, CBP and their combination respectively. MTT, flow cytometry, electron microscope and western blot were performed to evaluate the treated 4T1 cells. Tumor size and lung metastasis were monitored. The percentage of residual 4T1 cancer stem cells and the tumorigenecity of residual tumor cells from each treated group were compared. We found that both mono- and combination treatments suppressed 4T1 cells and induced apoptosis in a dose-dependent manner. These effects involved mitochondrial dysfunction and mitochondria-mediated apoptosis. The structure of mitochondrial membrane was impaired as observed by electron microscope, and the mitochondrial membrane stability was interrupted as evident by down-regulated Bcl-2 & Bcl-xL expression, and up-regulated BAX expression. Both extrinsic and intrinsic apoptosis were demonstrated by enrichment of activated caspase 3, 8, 9. Tumor growth and lung metastasis in 4T1-bearing animals were markedly inhibited by CBP combined with THZ. We also found suppression of PI3k/AKT/mTOR pathway and activation of ER stress by CSP + THZ both in vitro and in vivo. Moreover, angiogenesis was inhibited, demonstrating an important mechanism for tumor metastasis inhibition. More importantly, THZ demonstrated the capability of destroying CSCs and this effect was amplified when combined with CBP. The tumorigenecity of CBP + THZ combination-treated residual tumor cells was significantly impaired. These results show that THZ could inhibit 4T1 tumor cell proliferation and induce apoptosis and significantly destroy the stability of mitochondrial membrane, causing extrinsic and intrinsic apoptosis when combined with CBP. A signature of dysangiogenesis indicated that angiogenesis might be involved in this process. These findings suggest that the anti-psychotic drug THZ is a promising chemosensitizer in TNBC tumor and TNBC patients may benefit from THZ + CBP administration treatment by targeting cancer stem cells. Keywords: Triple-negative breast cancer (TNBC); cancer stem cells (CSCs); thioridazine (THZ); carboplatin (CBP). Citation Format: Yi Wang, Leiming Xia, Li Gong, Yang Xia, Liu Liu, Yuanyuan Liu, Wicha Max, Alfred E. Chang, Qiao Li, Yangyi Bao. Synergistic inhibition of thioridazine combined with carboplatin on triple negative breast cancer by targeting cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3803.

BACKGROUND Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have been widely used in the treatment of non-small cell lung cancer (NSCLC) patients with sensitive EGFR mutations. However, the survival of patients with EGFR-TKI administration is limited by the inevitable development of acquired drug resistance. Recently, multi-targeted drugs combination has been shown to be a promising strategy to improve the efficacy of EGFR-TKI treatment and enable the reduction of drug resistance in NSCLC. MATERIAL AND METHODS Humanized NSCLC cell lines PC9 and A549 were co-cultured with thalidomide and/or icotinib to test for anti-tumor efficiency. Cell proliferation was measured by MTT assay, cell apoptosis by flow cytometry and cell migration by wound healing assay. Western blot was performed to determine the expression of caspase-3, -8, -9, Bax, EGFR, VEGF-R, AKT, ERK, MMP2, MMP9, and NF-κB. The xenograft mouse model was used to explore the effects of thalidomide and icotinib in vivo. Immunohistochemical testing was used to determine the expression of Ki-67 and TUNEL staining in tumor tissues. RESULTS Treatments of thalidomide and/or icotinib reduced cell viability, induced apoptosis, and suppressed migration. Attenuation of pEGFR and pVEGF-R resulted in deactivation of ERK and AKT pathways, which eventually increased the anti-proliferative response. In PC9 xenograft model, combined administration of thalidomide and icotinib restrained tumor growth with remarkable reduced Ki-67 index and increased TUNEL positive cells. CONCLUSIONS Thalidomide sensitizes icotinib to increase apoptosis and prevent migration, and it may be a potentially promising anti-tumor drug in lung cancer multi-modality therapy.

We have previously reported that adoptive transfer of tumor-draining lymph node (TDLN) B cells confers tumor regression in a spontaneous pulmonary metastasis mouse model of breast cancer. In this study, we identified IL-10-producing cells within these B cells, and found that IL-10 removal, either by using IL-10−/− TDLN B cells or by systemic neutralization of IL-10, significantly augmented the therapeutic efficacy of adoptively transferred TDLN B cells. Depletion of IL-10 in B-cell adoptive transfers significantly increased cytotoxic lymphocytes (CTLs) and B-cell activity of peripheral blood mononuclear cells (PBMCs) and splenic cells in the recipient. Activated TDLN B cells express Fas ligand, which was further enhanced by co-culture of these TDLN B cells with 4T1 tumor cells. Effector B cells killed tumor cells directly in vitro in an antigen-specific and Fas ligand-dependent manner. Trafficking of TDLN B cells in vivo suggested that they were recruited to the tumor and lung as well as secondary lymphoid organs. These findings further define the biological function of antitumor effector B cells, which may offer alternative cellular therapies to cancer.

HER2-targeted antibody drug conjugate (ADC) was only tested in immunocompromised (SCID) host, which doesn’t allow the evaluation of the ADC-induced host immune responses. In addition, no potential impact of ADC on cancer stem cells (CSCs) has been reported. Furthermore, tumor cells with PD-L1 expression may escape immune attack by converting PD-1 positive immune effector cells into anergy. We hypothesized that HER2-targeted ADC could modulate host immune response, target HER2high CSCs, and the efficacy of ADC could be enhanced by anti-mPD-L1. To test this hypotheses, we used two immunocompetent murine models: mouse breast tumor D2/F2 and TNBC 4T1 with enforced expression of HER2, D2F2/E2 and HER2-4T1 respectively. Both D2F2/E2 and HER2-4T1 could generate tumor in immunocompetent Balb/c mice, which allowed us to test the efficacy and specificity of HER2-targeted ADC, and to evaluate host immune responses. In D2F2/E2 mouse model, we found that HER2-targeted ADC significantly inhibited D2F2/E2 tumor growth, but not the parental D2F2 tumors, in a dose dependent manner. The specificity was confirmed in HER2-4T1 model. In parallel, we observed that HER2-targeted ADC treatment reduced the number of HER2 positive cells and ALDHhighCSCs. We found that anti-mPD-L1 mAb significantly augmented the therapeutic efficacy of HER2-targeted ADC in both D2F2/E2 and HER2-4T1 tumor models. Mechanistically, HER2-targted ADC combined with anti-PD-L1 significantly modulated the gene signature of multiple immune factors in tumor microenvironment. At the cellular level, anti-mPD-L1 plus HER2-targeted ADC increased the number of CD3+ and CD19+ tumor infiltrating lymphocytes (TILs). Expanded CD3+ TILs from the tumor subjected to combined treatment killed tumor cells significantly more than mono-treatment. Expanded CD19+ TILs from tumor subjected to the combined therapy produced more IgG than all the controls. Importantly, the IgG could specifically bind to tumor cells, resulting in cytotoxicity of the tumor cells via ADCC. As a result, ADC plus anti-PD-L1 further reduced the number of HER2 positive cells. Importantly, the combined therapy not only reduced the number of ALDHhigh CSCs in the residual tumor, but also significantly paralyzed their tumoriginicity. Together, our data indicate that HER2-targeted ADC could induce significant host immune responses, which was evidenced by the regulation of gene signature and detection of anti-tumor CD3+ and CD19+ TILs. We first show that HER2-targeted ADC could target ALDHhigh cancer stem cells, and such effect was significantly enhanced by anti-mPD-L1 administration. Citation Format: Leiming Xia, Lu Wen, Gang Wu, Tao Zhang, Frank Comer, Mary Jane Masson Hinrichs, Michael Oberst, Steven R. Coats, Alfred E. Chang, Yuanyuan Liu, Yangyi Bao, Max Wicha, Qiao Li. HER2-targeted antibody drug conjugates (ADCs) induce host immunity against cancer stem cells and are enhanced by anti-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 4981.

Mucin1 (MUC1) proteins represent a family of high molecular weight trans-membrane glycoproteins that protect epithelial cells and mediate signal transduction by communication with extracellular stimuli. On the other hand, MUC1 has been observed to be overexpressed and glycosylated in adenocarcinoma, making it an ideal molecular target for cancer treatment. MUC1 based antibody and specific chimeric antigen receptors (CARs) modified T/NK cells exhibit strong antibody-dependent or direct-cell cytotoxicity to MUC1 positive tumor cells. However, treatment failure with MUC1-targeted therapy was usually caused by tumor microenvironment (TME)-driven immune suppression. PD-L1 has been identified as a negative checkpoint molecule that promotes immune evasion of tumor cells. The interaction of PD-1 and PD-L1 inhibits the function of tumor-infiltrating lymphocytes (TILs) or infused T/NK cells while activating the negative immune-regulatory cells in TME, such as regulatory T-cells and MDSCs. To overcome these barriers, we engineered clinically applicable NK-92 cells by lentiviral gene transfer to express chimeric antigen receptors comprising an anti-MUC1 scFv antibody fusion protein with CD28-CD137 as a signaling moiety and truncated PD-1 peptide. NK92 cells expressing anti-MUC1 CAR specifically and efficiently lysed MUC1 positive tumor cells in vitro and in vivo. The safety of NK-92 cell administration has been demonstrated by numerous phase I clinical trials. In our study, 13 patients with different kinds of tumors (lung cancer, pancreatic cancer, colon cancer and ovarian cancer) with positive PD-L1 and MUC-1 expression were enrolled. CAR-NK cells were infused (1×109 cells/time) into these patients. Of 13 patients, 3 patients were withdrawn, 9 patients (69.2%) showed stable disease and 1 patient showed progressive disease. The cytokine level and hematologic changes were monitored to evaluate the safety. Severe cytokine storm and/or bone marrow suppression were not observed. From this phase I clinical trial we found that CAR-NK therapy has a broad prospect of application as a new cellular immunotherapy due to its stable clinical efficacy, mild side effects and ease of preparation. Citation Format: Qiao Li, Yi Wang, Ming Lin, Leiming Xia, Yangyi Bao, Xiang Sun, Lin Yang. Phase I clinical trial with PD-1/MUC1 CAR-pNK92 immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A014.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

// Yang Xia 1, 2, * , Huimin Tao 1, 3, 8, * , Yangyang Hu 1, 3, * , Quanning Chen 1, 4, * , Xin Chen 1, 5 , Leiming Xia 1, 2 , Li Zhou 1, 6 , Yi Wang 2 , Yangyi Bao 2 , Shiang Huang 3 , Xiubao Ren 6 , Steven K. Lundy 7 , Fu Dai 2, ** , Qiao Li 1, ** , Alfred E. Chang 1, ** 1 Department of Surgery, University of Michigan, Ann Arbor, Michigan, USA 2 The No.1 People’s Hospital of Hefei, Hefei, China 3 Hubei Province Stem Cell Research & Appling Center, Wuhan Union Hospital, Wuhan, China 4 Department of General Surgery, Tongji Hospital of Tongji University, Shanghai, China 5 Department of Oncology, Wuhan University, Renmin Hospital, Wuhan, China 6 Department of Biotherapy, Tianjin University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key laboratory of Cancer Immunology and Biotherapy, Tianjin, China 7 Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA 8 Current address: Fuda Cancer Hospital, Jinan University School of Medicine and Fuda Cancer Institute, Guangzhou, China * These authors have contributed equally to this work ** Joint senior authors Correspondence to: Qiao Li, email: qiaoli@umich.edu Alfred E. Chang, email: aechang@umich.edu Fu Dai, email: hfsyydf@sina.com Keywords: B cells, adoptive immunotherapy, IL-2, CXCR4/CXCL12, perforin Abbreviations: TDLN, tumor-draining lymph node; CM, complete medium; s.c., subcutaneous; i.v., intravenously; i.p., intraperitoneal Received: December 06, 2015 Accepted: July 26, 2016 Published: August 09, 2016 ABSTRACT We previously reported that antitumor B cells directly kill tumor cells via the Fas/FasL pathway and are regulated by IL-10. In this study, we defined additional mechanisms involved in B cell antitumor immunity. Administration of IL-2 significantly augmented the therapeutic efficacy of adoptively transferred tumor-draining lymph node (TDLN) B cells which express IL- 2R. Culture supernatant of purified B splenocytes harvested from the mice that received adoptive transfer of 4T1 TDLN B cells plus IL-2 administration produced larger amounts of IgG which bound to 4T1, resulting in 4T1 lysis. Furthermore, we detected CXCR4 expression on 4T1 TDLN B cells, and 4T1 tumor cells produced its ligand CXCL12. Transwell experiments demonstrated the chemoattraction of CXCR4-expressing 4T1 TDLN B cells towards CXCL12- producing 4T1 cells. Blockade of CXCR4 using a CXCR4-specific inhibitor, AMD3100, significantly reduced the killing of 4T1 tumor cells by 4T1 TDLN B cells. Blockade of FasL and CXCR4 concurrently inhibited B cell-mediated direct killing of tumor cells in an additive manner, indicating that both Fas/FasL and CXCL12/CXCR4 pathways are involved in the direct killing of 4T1 cells by 4T1 TDLN B cells. TDLN B cells produced perforin. Additional transwell experiments showed that effector B cells could directly kill tumor cells in cell-cell contact via the Fas/FasL and CXCR4/CXCL12 pathways as well as perforin, while without cell contact, perforin secreted by B cells led to tumor cell cytotoxicity. These findings underscore the diversity of function by which B cells can play an important role in the host immune response to tumor.

HER2 represents an ideal target for cancer immunotherapy. HER2-targeted antibody drug conjugate (ADC) was tested in immunocompromised (SCID) host, which doesn't allow the evaluation of the ADC-induced immune responses. In addition, no potential impact of ADC on cancer stem cells (CSCs) has been reported. Furthermore, tumor cells with PD-L1 expression may escape immune attack by converting PD-1 positive immune effector cells into anergy. We hypothesized that HER2-targeted ADC could modulate host immune response, target HER2high CSCs, and the efficacy of ADC could be enhanced by anti-PD-L1. To test this hypothesis, we used two immunocompetent murine models: mouse breast tumor D2/F2 and TNBC 4T1 with enforced expression of HER2, D2F2/E2 and HER2-4T1 respectively. Both D2F2/E2 and HER2-4T1 could generate tumor in immunocompetent Balb/c mice, which allowed us to test the efficacy and specificity of HER2-targeted ADC, and to evaluate host immune responses. In D2F2/E2 mouse model, we found that HER2-targeted ADC significantly inhibited D2F2/E2 tumor growth, but not the parental D2F2 tumors, in a dose dependent manner. The specificity was confirmed in HER2-4T1 model because we observed significant suppression of HER2-targted ADC on HER2-4T1 tumor growth, but not on the parental 4T1 tumor. In parallel, we observed that HER2-targeted ADC treatment reduced the number of HER2 positive cells, and ALDHhigh cells. ALDHhigh cells have been identified as cancer stem cells in multiple malignancies. We have characterized the stemness of ALDHhigh 4T1 cells. In the present study, we confirmed that the ALDHhigh sub-population of D2F2/E2 cells are highly tumorigenic compared to ALDHlow cells. Next, we tested and observed that PD-L1 expression increased in D2F2/E2 and HER2-4T1 fresh tumor cells than in cell lines. Anti-PD-L1 mAb significantly augmented the therapeutic efficacy of HER2-targeted ADC in both D2F2/E2 and HER2-4T1 tumor models. In addition, anti-PD-L1 plus HER2-targeted ADC further reduced the number of HER2 positive cells, and ALDHhigh cells, but increased the number of CD3+ and CD19+ tumor infiltrating lymphocytes (TILs). Expanded CD3+ TILs from the tumor subjected to HER2-targeted ADC plus anti-PD-L1 treatment killed tumor cells significantly more than mono-treatment. Expanded CD19+ TILs from tumor subjected to HER2-targeted ADC plus anti-PD-L1 produced more IgG than all the controls. Importantly, the IgG could specifically bind to tumor cells, resulting in the highest cytotoxicity of the tumor cells via ADCC. Together, our data indicate that HER2-targeted ADC could induce significant host immune responses, which was evidenced by the detection of anti-tumor CD3+ and CD19+ TILs. We first show that HER2-targeted ADC could target ALDHhigh cancer stem cells, and such effect was significantly enhanced by anti-PD-L1 administration. Citation Format: Leiming Xia, Lu Wen, Ming Lin, Comer Frank, Hinrichs Mary Jane, Oberst Michael, Coats Steven, Yangyi Bao, Alfred E. Chang, Max Wicha, Qiao Li. HER2-targeted antibody drug conjugates induce host immunity and target cancer stem cells and the efficacy can be enhanced by anti-PD-L1 [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-297.

In patients with acute myeloid leukemia, an about 20-40% of the patients show no response to current therapy, and about 50-70% of the patients relapse after complete response (CR). These patients represent the majority of the refractory or relapsed acute myeloid leukemia (R/R AML). The 1-year survival rate of those patients is limited to Key words: Refractory or relapsed acute myeloid leukemia (R/R AML), CD33, Chimeric antigen receptor (CAR), NK cells, Immunotherapy. Citation Format: Leiming Xia, Xiang Sun, Liu Liu, Yi Wang, Tan Li, Bin Li, Gregory Wolf, Qiao Li, Lin Yang, Yangyi Bao. A clinical study on CD33-directed chimeric antigen receptormodified NKcells in patient with refractory or relapsed acute myeloid leukemia [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 CT060.

Richter syndrome (RS) indicates the transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma (mostly DLBCL). RS is a rare complication with unfavorable clinical outcome. There is no effective treatment for it currently. As a new type of cell-based immune therapy, chimeric antigen receptor modified T (CAR-T) cell treatment is now widely used in treating hematological malignancies, especially in CD19+ B cell malignancy. Therefore, CD19-directed chimeric antigen receptor modified T cells (CART-19) treatment is worthwhile to be developed as a new method for Richter Syndrome’s patients. In this study, the patient with Richter Syndrome expressing high level of CD19 was enrolled in a clinical trial. The patient had received a series of standard treatments with no therapeutic effect before 4.55 x 108 autologous CART-19 cells were infused, of which approximately 50% were transduced with the CAR. After CART-19 infusion, the main side effects were repeated fever, pancytopenia and significant rises of serum cytokines, including IFN-γ, IL-6 and IL-10. However, before discharge, the level of cytokines reduced to normal level. In addition, we detected K+, Ca2+, creatinine and glutamic-pyruvic transaminase, and found all of these were normal, which suggested that there was no significant tumor necrosis after treatment. The treatment included two courses. During the first course, the patient was infused with 4 x 107 CART-19 cells twice, followed by 1 x 108 cells in the 3rd infusion and then 1x106 cells in the 4th infusion. After these infusions, the proportion of B cells in the patient’s peripheral blood decreased from 72% to 40%, accompanied by reduced size of cervical, mediastinal and axillary lymph nodes. Eighteen days later, the patient was infused in the second course with 3.7x107 CART-19 cells twice, followed by 1x108 cells twice. After the second course, the proportion of B cells maintained at approximate 85%, and the lymph nodes enlargement was observed as disease progression. Based on the recent therapeutic landscape for hematological malignancies including B cell lymphomas, CD19-CAR T cell therapy may offer a new therapeutic option for RS, which aims to reduce the patients’ tumor burden, prolong their survival time, and provide opportunities for combined therapy with chemotherapy and bone marrow transplantation. Note: This abstract was not presented at the meeting. Citation Format: Leiming Xia, Qian Chen, Qiao Li, Tan Li, Yi Wang, Yangyi Bao. The clinical study on CD19-directed chimeric antigen receptor-modified T cells in patient with Richter Syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT041. doi:10.1158/1538-7445.AM2017-CT041

Over the years, the role of B cells in the host immune response to malignancy has been overshadowed by our focus on T cells. The role played by B cells in cancer immunology is complex and controversial. The observation made by our lab that activated B cells alone can mediate tumor regression in the adoptive immunotherapy of solid tumors is innovative (JI 2009; CCR 2011; JSO 2012; EJI 2015). One novel mechanism by which activated B cells mediate tumor regression is via direct tumor cell cytotoxicity in the absence of antibodies. We previously reported that antitumor B cells directly kill tumor cells via the Fas/FasL pathway and are regulated by IL-10. In this study, we defined additional mechanisms involved in B cell antitumor immunity. Administration of IL-2 significantly augmented the therapeutic efficacy of adoptively transferred tumor-draining lymph node (TDLN) B cells which express IL-2R. Culture supernatant of purified B splenocytes harvested from the mice that received adoptive transfer of 4T1 TDLN B cells plus IL-2 administration produced larger amounts of IgG which bound to 4T1, resulting in 4T1 lysis. Furthermore, we detected CXCR4 expression on 4T1 TDLN B cells, and 4T1 tumor cells produced its ligand CXCL12. Transwell experiments demonstrated the chemotraction of CXCR4-expressing 4T1 TDLN B cells towards CXCL12-producing 4T1 cells. Blockade of CXCR4 using a CXCR4-specific inhibitor, AMD3100, significantly reduced the killing of 4T1 tumor cells by 4T1 TDLN B cells. Blockade of FasL and CXCR4 concurrently inhibited B cell-mediated direct killing of tumor cells in an additive manner, indicating that both Fas/FasL and CXCL12/CXCR4 pathways are involved in the direct killing of 4T1 cells by 4T1 TDLN B cells. TDLN B cells produced perforin. Additional transwell experiments showed that effector B cells could directly kill tumor cells in cell-cell contact via the Fas/FasL and CXCR4/CXCL12 pathways as well as perforin, while without cell contact, perforin secreted by B cells led to tumor cell cytotoxicity. These findings underscore the diversity of function by which B cells can play an important role in the host immune response to tumor, and clearly indicated that transferred effector B cells can act independently of T cells in causing tumor destruction in adoptive immunotherapy. Citation Format: Leiming Xia, Yang Xia, Quanning Chen, Yi Wang, Yangyi Bao, Steven K. Lundy Lundy, Fu Dai, Alfred E Chang, Qiao Li. Adoptively transferred B cells directly kill tumor cells via the CXCR4/CXCL12 & perforin pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1621. doi:10.1158/1538-7445.AM2017-1621

In a phase I clinical trial, we used anti-CD3 x anti-HER2 bispecific antibody (HER2Bi) armed T cells. Utilizing HER2-directed T cells benefitted both HER2+ patients and patients with 1 or 2+ HER2 expression, ones that would be considered “HER2-negative” by classical criteria. We have also shown that the level of cancer stem cell (CSC) marker ALDH in HER2+ breast cancer cells (ALDHhighHER2+) is much higher than that in HER2- breast cancer cells (ALDHlowHER2-), and that in luminal breast cancers that are considered HER2-, HER2 is actually selectively expressed in the ALDHhigh CSC population. These observations might account for the surprising result that HER2Bi-armed T cells, while intended to target HER2, seemed to benefit HER2- patients after adoptive transfer. In this study, we found that the HER2Bi-armed T cells used in the clinical trial killed ALDHhigh human breast CSCs isolated from MCF7 (HER2-) tumor significantly more than ALDHlow MCF7 cells in vitro, while the same HER2Bi-armed T cells killed ALDHhigh human breast CSCs (ALDHhighHER2+) isolated from BT474 (HER2+) tumor equally to ALDHlow BT474 cells (ALDHlowHER2+). We then tested the “mouse HER2” (neu) expression on ALDHhigh vs. ALDHlow 4T1 cells (mouse TNBC), and found that mHER2 was selectively expressed in the ALDHhigh 4T1 CSC population. These results replicated our findings in human breast cancers that HER2 is selectively expressed on CSCs, even in HER2- murine tumors, such as 4T1. For mHER2 targeting in animal models, we generated anti-mouse HER2-CD3 bispecific (mHER2Bi) that binds to mouse HER2 and mouse CD3. In vitro, the mHER2Bi-armed T cells killed ALDHhigh 4T1 CSCs significantly more than ALDHlow 4T1 cells. In vivo, adoptive transfer of mHER2Bi-armed T cells for HER2 targeted therapy showed antitumor effect in mHER2- 4T1-bearing host. To boost the therapeutic efficacy of mHER2-targeted T cells, we administrated anti-mouse PD-L1 during mHER2Bi-armed T cell adoptive transfer. This combination decreased metastases significantly more than the use of either strategy alone. Collectively, these studies have generated evidence providing proofs of principle that due to the selective expression of HER2 on CSCs, HER2-targeted T cell therapy could benefit HER2- host as well as HER2+ hosts via immune destruction of HER2+ CSCs, and use of anti-PD-L1 as an adjuvant could significantly boost the efficacy of HER2-targeted T cell therapy. Citation Format: Yang Xia, Yangyang Hu, Yi Wang, Yangyi Bao, Fu Dai, Shiang Huang, Elaine Hurt, Robert E. Hollingsworth, Archana Thakur, Lawrence Lum, Alfred E. Chang, Max S. Wicha, Qiao Li. Adjuvant effect of anti-PD-L1 in boosting HER2-targeted T cell adoptive immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 540.

We have previously reported the development of a strategy to target the cancer stem cell (CSC) populations in melanoma and squamous cell carcinoma using CSC lysate-pulsed dendritic cells (DCs). Using mouse models we demonstrated that DCs pulsed with CSCs enriched by virtue of their expression of the CSC marker ALDH (termed CSC-DC) significantly inhibited tumor growth. However, CSC-DC vaccine therapy alone may not be sufficient to overcome the immunosuppressive components of the tumor microenvironment. CSC-mediated suppression of T cells has been reported in a process involving the PD-L1/PD-1 axis. Immunologically targeting CSCs while simultaneously blocking PD-L1/PD-1-mendiated immune suppression may significantly enhance the outcome of current immunotherapies of cancer. To test this hypothesis, we investigated the effect of using anti-PD-L1 to block the immunosuppression during the CSC-DC vaccination to augment the therapeutic efficacy of using each regimen alone. We used 4T1 cell line, a mammary carcinoma syngeneic to BALB/c mice, and found that 4T1 tumors contain approx. 6-10% ALDEFLUOR+/ALDHhigh cells, and that these cells are enriched for tumor initiating capacity compared to bulk or ALDEFLUOR-/ALDHlow cells. Inoculating 4T1 cells into the mammary fat pad induces the development of spontaneous pulmonary metastases. ALDHhigh 4T1 CSCs expressed PD-L1. 4T1 ALDHhigh CSC-DC vaccine + anti-PD-L1 administration significantly reduced pulmonary metastases and prolonged survival compared with 4T1 ALDHhigh CSC-DC vaccine or anti-PD-L1 administration alone. Anti-PD-L1 administration increased systematic anti-4T1 CSC immunity induced by CSC-DC vaccine. This is evident by the increased production of total IgG in the serum samples collected from the 4T1-bearing animals subjected to 4T1 CSC-DC vaccination with anti-PD-L1 administration. The CSC-reactivity/specificity of the immune sera was demonstrated by their specific binding to the ALDHhigh vs. ALDHlow 4T1 cells in flow cytometry. Importantly, the immunological consequence of such binding was Ab-mediated ALDHhigh 4T1 CSC lysis via complement dependent cytotoxicity. In addition, CTLs generated from the splenocytes harvested from the hosts subjected to 4T1 CSC-DC vaccination with anti-PD-L1 administration killed ALDHhigh 4T1 CSCs significantly more than ALDHlow 4T1 cells. These results are supportive of the conclusion that administration of anti-PD-L1 as an adjuvant could significantly boost the therapeutic efficacy of the CSC-DC vaccine, and that this effect was due to both antibody and T cell-mediated anti-CSC immunity. Citation Format: Yangyang Hu, Yang Xia, Xin Chen, Li Zhou, Yangyi Bao, Shiang Huang, Xiubao Ren, Elaine Hurt, Robert E. Hollingsworth, Alfred E. Chang, Max S. Wicha, Qiao Li. Increasing the efficacy of cancer stem cell-based vaccine in the PD-L1/PD-1 blockade. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3329.

BACKGROUND Significant progress has been made in the therapy of multiple myeloma (MM) with the autologous hematopoietic stem cell transplantation(ASCT) in recent years. But the relapse after transplantation is still the main problem because the grafts are often contaminated by tumor cells and have no effect of anti myeloma. The residual myeloma cells are reduced by increasing doses of cytotoxic drug in the pretreatment. But the related mortality of ASCT is increased because of side effects and complications. Therefore, the pretreatment of MM hasn't changed very much in recent 50 years. So it is urgent to find a new pretreatment to improve curative effect which has small side effects. Natural killer (NK) cells are the principal effector cells in the innate immune system. They have extensive biological functions including anti-tumor and antiviral effects, etc. There is no report about applying NK cells as a pretreatment for ASCT to treat MM so far. In this study, NK cells were added to the pretreatment of ASCT to treat MM We hope NK cells can increase the curative effect and reduce relapse rate, but not increase the side effects. Meanwhile, they can clear myeloma cells without killing by chemotherapy drugs and to decrease infection in transplantation. OBJECTIVE To observe the safety and effectiveness of the technology by applying NK cells in peritransplantation of MM ASCT. METHODS The ages of 8 MM patients including 7 males and 1 female were 42-62 years, the median age was 51 years, 2 patents of CR and 6 patents of PR before transplantation. The method of NK cell culture: membrane embedded active factor culture system. The pretreatment mainly based on standard Melphalan or busulfan. NK cells were infused to 2 cases back 6-7days after infusing stem cell, and to 6 cases back 24-48 hours before infusing stem cells and 48 hours after chemotherapy as a part of pretreatment in transplantation. Stem cell infusion volume: the number of CD34+ cell 2.2-4.0×106/kg, the number of mononuclear cells 4.3-5.8×106/kg. NK cell infusion volume: 16-160×106/kg. we observed the adverse reactions, recovery time of blood cells and the curative effects. RESULTS There were no instances of fever, rash, diarrhea,shock, and other adverse reactions in this study. Recovery time of hematopoietic cells was: 7-14d of granulocyte and 9d of the median time, 9-34d of platelet and 11d of the median time. Fever(38-39 degrees) occurred in 6 patients during the inhibitory period of bone marrow after ASCT. Infection is the major cause of fever in patients receiving ASCT. We evaluated therapeutic effect 3 months after transplantation was 3 cases of CR and 5 cases of PR in 8 patients. The effect 6 months after transplantation was 3 cases of CR and 3 cases of PR in 6 patients. 8 patients were given thalidomide for maintenance therapy. The follow-up time was 3-29 months, median follow-up time was 11 months. Up to the present, there was no death case. CONCLUSIONS It was safe and effective to infuse autologous NK cells back in peritransplantation of MM ASCT. Storm effect of inflammatory factors didn't occur during this study. We will expand the number of cases for further clinical research to observe the long-term therapeutic effects and adverse reactions. Disclosures No relevant conflicts of interest to declare.

In cancer immunotherapy, effector B cells can inhibit spontaneous tumor metastases as well as local tumor growth in animal models. We have previously reported that antitumor effector B cells generated from tumor-draining lymph node (TDLN) could directly kill tumor cells via the Fas/FasL pathway and are regulated by IL-10. Trafficking of TDLN B cells in vivo suggested that they were recruited to the tumor and lung as well as secondary lymphoid organs. The number of TDLN B cells increased following transfer. As a result, adoptive transfer of TDLN B cells conferred tumor regression in a spontaneous pulmonary metastasis mouse model of breast cancer, 4T1. Removal of IL-10, either by using IL-10-/- TDLN B cells or by systemic neutralization of IL-10 using anti-IL-10 antibody, significantly augmented the therapeutic efficacy of adoptively transferred TDLN B cells. In recent studies, we found that tumor cell death mediated by effector B cells involves the CXCR4/CXCL12 as well as Fas/FasL interactions. Blockade of CXCR4 and FasL using a CXCR4-specific inhibitor, AMD3100, and anti-FasL antibody, respectively, reduced the killing of 4T1 tumor cells by 4T1 TDLN B cells. Of note, blockade of CXCR4 and FasL concurrently inhibited B cell-mediated direct killing of tumor cells in an additive fashion, suggesting that both CXCL12/CXCR4 and Fas/FasL pathways are involved in the direct killing of 4T1 tumor cells by 4T1 TDLN B cells. In other experiments, we found that TDLN B cells express IL-2 receptors (IL-2R) and the ability of the TDLN B cells to inhibit metastasis was significantly enhanced with exogenous IL-2 administration. Besides our previously reported Fas/FasL pathway and IL-10-mediated suppression, our recently identified CXCL12/CXCR4 pathway and IL-2-mediated augmentation of the biological function of effector B cells suggests these B cells may represent an important component of the host immune response against tumor. Citation Format: Yang Xia, Huimin Tao, Yangyang Hu, Li Zhou, Yangyi Bao, Steven K Lundy, Shiang Huang, Qiao Li, Alfred E Chang. Antitumor effector B cells directly kill tumor cells involving the CXCL12/CXCR4 pathway and their therapeutic efficacy is enhanced by IL-2. [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 1340. doi:10.1158/1538-7445.AM2015-1340

Using mouse models we reported that dendritic cells (DC) pulsed with cancer stem cells (CSC) enriched by virtue of their expression of the CSC marker aldehyde dehydrogenase (ALDH) significantly reduced development of pulmonary metastases and prolonged survival. In this recent study, we established the concept that the antigenicity/immunogenicity of ALDHhigh human head and neck squamous cell carcinoma (HNSCC) cancer stem cells is distinct from that of ALDHlow non-CSCs. To this end, we generated CSC-loaded DCs to sensitize autologous peripheral blood lymphocytes to react with CSCs using human HNSCC samples in vitro. From peripheral blood and paired tumor tissues collected from 9 patients with HNSCC, we obtained PBMCs and established 4 HNSCC tumor cell lines. DCs generated from the PBMC and pulsed with the lysate of ALDHhigh cells isolated from cultured HNSCC cells (CSC-DC) could sensitize autologous T, B lymphocytes in vitro, which was evident by cytokine production, CTL activity, and antibody secretion of these primed T, B cells in response to ALDHhigh CSCs. In contrast, DCs pulsed with lysate of ALDHlow cells from the same HNSCC patient (ALDHlow-DC) resulted in limited sensitization/priming of autologous T, B lymphocytes to produce IFNγ, lyse CSCs, and secrete IgM and IgG in response to ALDHhigh CSCs. These results demonstrated significant differences in the antigenicity/immunogenicity between ALDHhigh CSCs vs. ALDHlow cells isolated from the tumor specimen of patients with HNSCC, which indicates the existence of unique CSC antigens in the ALDHhigh population. In addition, this study demonstrates that it is feasible to generate DCs from the PBMCs and isolate ALDHhigh CSCs from cultured tumor cells of the patients with HNSCC to prepare CSC-DC vaccines for clinical application. Citation Format: Mark E.P. Prince, Li Zhou, Yang Xia, Huimin Tao, Jeffrey S. Moyer, Lin Lu, John Owen, Alfred E. Chang, Jianchuan Xia, Gregory Wolf, Max S. Wicha, Xiubao Ren, Yangyi Bao, Shiang Huang, Qiao Li. Evaluation of the immunogenicity of ALDHhigh human head and neck squamous cell carcinoma cancer stem cells in vitro. [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 1410. doi:10.1158/1538-7445.AM2015-1410

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in cancer cells but not in most normal cells, and is identified to be effective in various cancers, include myeloid leukemic cells[1]. Although some leukemia cell lines, K562 and KG-1, are sensitive to TRAIL, many showed certain degrees of resistance to TRAIL-mediated apoptosis[2,3], and the mechanism remains largely unknown, which forced us to find out ways to solve the problem. In this study, we investigated whether thioridazine, a phenothiazine derivative, could overcome the TRAIL resistance in K562 and KG-1 cells. Recently, we showed that Compared to treatment with thioridazine or TRAIL alone, co-treatment with thioridazine and TRAIL-induced apoptosis in K562 and KG-1 cells synergistically. This combination led to activation of caspase-8 and Bid, the cytosolic cumulation of cytochrome c from mitochondria as well as caspase-3 activated downstream. Treatment with thioridazine induced down-regulation of PI3K-AKT-NF-κB pathway. meanwhile, thioridazine dropped the level of NF-κB-dependent Bcl-xL, leading caspase activated and Bid cleaved. the expression of TRAIL-receptors in both K562 and KG-1 cells underwentthe treatment of thioridazine investigated that thioridazine significantly up-regulated DR5 by up to 51.22%, but not other TRAIL-receptors such as DR4, decoy receptor 1, and DcR2. Therefore, our results indicate that the combination of TRAIL with thioridazine overturn TRAIL resistance through Up-regulating the expression of DR5 and down-regulation of AKT protein, and combination treatment with thioridazine and TRAIL may be a novel therapeutic strategy in leukemia. Reference: Srivastava R K. TRAIL/Apo-2L: mechanisms and clinical applications in cancer.[J]. Neoplasia, 2001, 3(6):535-546. Nimmanapalli R, Porosnicu M, Nguyen D, et al. Cotreatment with STI-571 enhances tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL or apo-2L)-induced apoptosis of Bcr-Abl-positive human acute leukemia cells.[J]. Clinical Cancer Research An Official Journal of the American Association for Cancer Research, 2001, 7(2):350-357. Yang T, Lan J, Huang Q, et al. Embelin sensitizes acute myeloid leukemia cells to TRAIL through XIAP inhibition and NF-κB inactivation.[J]. Cell Biochemistry & Biophysics, 2015, 71(1):291-297. Disclosures No relevant conflicts of interest to declare.