In Vitro Expansion of Dendritic Cells Pulsed with Placental Peptides for the Generation of Antigen-Specific T Cells with Antitumor Activity.

*Neostem, Inc., New York, NY, Neostem Irvine, 18301 Von Karman Avenue, Irvine, CA 92612, USA; Tel.: +1 949 725 1750 ext. 107; Fax: +1 949 725 1756; rdillman@neostem.com

Melanoma cells that are proliferating and self-renewing in short-term cell culture, have the phenotypic and functional characteristics of tumor stem cells, and they express unique patient-specific neoantigens, as well as numerous common melanoma associated antigens. Patient-specific therapeutic vaccines, produced by Incubating autologous dendritic cells with autologous tumor cells from such cell lines (DC-TC), have yielded promising results in phase II trials in metastatic melanoma. In this study we examined the expression of genes on the tumor cell lines which had served as the sources of tumor associated antigens loaded onto autologous dendritic cells as part of a phase II trial in which there was an observed 5-year survival rate of 50%.[Dillman 2009] There was sufficient melanoma tumor cells to analyze for 50 of the 54 patients enrolled in the phase II trial. Microarray analysis was performed by Response Genetics, Inc. (Los Angeles) for 54,000 genes. The Excel data file was entered into an Affymetrix data processing algorithm using the R programming language. Gene ranking was performed using prediction analysis of microarrays.[Tibshirani 2002] Correlation with overall survival was done for 26 patients who survived 5 years or longer, and 24 patients who survived less than 5 years. Hierarchical clustering for over and under expression yielded 9 genes, VEPH1, ZNF280B, FGF13, ST6GALNAC3, QPCT, ZNF280B, BST, PBRM1 and C21orf91 which identified 11/11 patients who had a survival greater than 5 years, and 17/18 patients who died in less than 2 years. Using the top six genes, overall survival greater than 5 years was correctly predicted for 80.3% of patients, and survival less than 5 years was correctly predicted for 74.2% of patients. Gene array data obtained from self-renewing, proliferating, autologous tumor cells was prognostic for survival. It is unclear whether these genes are predictive of an effective immune response after repeated injections of a therapeutic dendritic cell-tumor cell vaccine. Citation Format: Andrew N. Cornforth, Gary Fogel, Denysha Carbonell, Robert O. Dillman. Microarray analysis of melanoma autologous tumor cell lines used as the source of tumor associated antigens in patient-specific dendritic cell immunotherapy phase II trial in patients with metastatic melanoma. [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 4845. doi:10.1158/1538-7445.AM2015-4845

Tumor Protection Following Vaccination With Low Doses of Lentivirally Transduced DCs Expressing the Self-antigen erbB2

Dendritic cells: at the clinical crossroads.

Dendritic Cells Loaded With mRNA Encoding Full-length Tumor Antigens Prime CD4+ and CD8+ T Cells in Melanoma Patients

Cancer immunotherapy targeting specific tumor-associated antigens (TAAs) is now being attempted. Dendritic cells (DCs) are potent antigen-presenting cells that play an important role in initiating and inducing antitumor immunity. DCs are used in cell therapy to present and prime TAAs to naive T cells for inducing antigen-specific antitumor immunity. Specific TAAs of gastrointestinal carcinomas (GICs) and hepatocellular carcinomas (HCCs) have not been identified. Immunization with fusion of DCs and cancer cells induce potent antitumor immunity against cancers whose TAA has not been identified. Immunotherapy with fusion of patients’ DCs and cancer cells might be used to treat GICs and HCCs. We present results of basic studies of immunotherapy using DC/cancer cell fusions against GIC and HCC.

B7-H3 as a Novel CAR-T Therapeutic Target for Glioblastoma

Apoptotic Blebs From Leukemic Cells as a Source of Tumor Associated Antigen for Monocyte-Derived Dendritic Cell Loading

See article in J. Gastroenterol. Hepatol. 2004; 19: 388–396 Hepatocellular carcinoma (HCC) is one of the most difficult cancers to treat. Although HCC patients undergo medical and surgical treatment for primary tumor lesions, intrahepatic and extrahepatic recurrence frequently occurs, thus limiting patient survival.1,2 Therefore, to improve the prognosis of patients, new strategies such as cytokine-based gene therapy and immunotherapy have been launched for treating HCC in animal models, and the results are promising.3–8 Because of the poor outcome of several types of immunotherapy applied to HCC,9 HCC tumor cells are generally considered poorly immunogenic. Accumulating evidence suggests that the reason why the host immune system cannot eradicate tumors is not the absence of recognizable tumor-associated antigens (TAA) on HCC tumor cells, but rather the inability of TAA of HCC to elicit an effective immune response.10,11 To overcome this problem, two immunotherapy approaches have been adopted in treating HCC in animal models. The whole scenario is illustrated in Figure 1: one approach involved engineering tumor cells to generate systemic tumor immunity by gene transfer of immunostimulatory cytokines such as interleukin (IL)-12, with or without cotranfection of B7 family costimulatory molecules (Fig. 1e);5,12,13 the other involved engineering non-tumor cells such as autologous fibroblasts with IL-12 that were implanted in the peritumor area (Fig. 1).14 The B7 family costimulatory molecules are a family of proteins that interact with counter receptors: CD28 and cytotoxic T-lymphocyte-associated antigen-4 (CTLA4; CD152) on T cells providing signals that modulate the immune response. Transfection of B7-1 (CD80) and B7-2 (CD86), which make tumor cells, including HCC, more immunogenic and cotransfected with IL-12, has been proven beneficial in certain models,12 but not in others.13 Mechanisms involved in the antitumor effects of interleukin (IL)-12 gene therapy for cancers: a hepatocellular carcinoma (HCC) model. (1) IL-12 engineered tumor cells to generate systemic tumor immunity with or without (e) co-transfection of B7 family costimulatory molecules. (2) IL-12 engineered non-tumor cells such as autologous fibroblasts that were implanted into the peritumor area.14 (a) The intratumoral injection of dendritic cells (DC) engineered to secrete IL-12, which may activate DC1 cells and (a1) in turn activate T-helper (Th)1 cells.21 (b) IL-12 gene therapy may combine with (b,d) other T-cell therapies or (c) other cytokine therapies. (f) IL-12 may trigger γ-interferon (IFN)-γ production from different cellular sources including cytotoxic T lymphocytes (CTL) and (h) natural killer (NK) cells and (a1) Th1 cells. (i,g) As a result, immune and non-immune therapeutic mechanisms were turned on. The combination of IL-12 with other immunostimulating genes or adoptive T-cell therapy has been observed to enhance efficacy in a synergistic fashion.31 IP-10, IFN-γ-inducible protein 10; Mig, monokine induced by IFN-γ; MØ, macrophages. The mechanisms of antitumor activity using IL-12 gene therapy are not yet completely understood. All the available data have been generated with transplantable cell lines that represent various carcinomas, sarcomas, melanomas and lymphomas, but are not with spontaneously arising tumors. Generally, IL-12 gene transfer leads to significant antitumor activity in these experimental studies, and complete tumor eradication has often been reported,3–6 but there is room for improvement. IL-12, a heterodimeric cytokine, plays a central role in promoting type 1 T-helper cell (Th1) responses and, hence, cell-mediated immunity.15 IL-12 can be used to upregulate artificially the functions of dendritic cells (DC) to promote cellular immune responses.15,16 The intratumoral injection of DC engineered to secrete IL-12 (Fig. 1a) by means of adenoviral17 or retroviral18 transfection could eradicate the established tumors derived from at least five different tumor cell lines. Transfection of the IL-12 gene into DC probably acted at several levels:19 stimulating T cells, lymphokine-activating killer cells, natural killer (NK) cells, and macrophages, as well as DC themselves in an autocrine fashion. DC captures tumor antigens inside the malignant tissue and migrates avidly to draining lymph nodes where antigens are present while high local levels of IL-12 are being produced.16,19 There is a large body of literature that involves animal models of tumor immunity in which DC have been loaded with TAA that induced protective antitumor responses.16,19,20 Accordingly, the immune mechanisms of IL-12-mediated tumor killing might be mainly attributed to the activation of DC, from which IL-12 might function by: (i) stimulating γ-interferon (IFN-γ) production from NK and T cells(Fig. 1h); and (ii) promoting the cellular immune responses by facilitating the proliferation and activation of Th1 cells (Fig. 1, a1).21 The non-immune mechanisms of IL-12-mediated tumor killing have been identified and the most prominent is its ability to inhibit tumor angiogenesis in vivo (Fig. 1g).22 In addition to enhancing tumor killing, the activation of Th1 responses has been shown to play an important role in the successful treatment of viral hepatitis B and C,23–26 as well as in viral clearance of acute hepatitis C.27 Because of the enhancement of Th1 responses, IL-12 cytokine and/or IL-12 gene therapy are beginning clinical testing as a single agent.28–30 Its combinations with immunotherapy approaches such as gene transfer of other cytokines, chemokines, costimulatory molecules and/or adoptive T-cell therapy are: (i) at hand (Fig. 1a–e), and have achieved successful eradication of HCC in animal models;3–8 and (ii) found to produce synergistic rather than additive effects, and clinical trials are being evaluated.31–34 IL-12 has interacted synergistically with the costimulatory molecule B7.1/CD80 to enhance protective antitumor immunity.3,12 B7.1-mediated antitumor activity in these studies was largely attributed to the stimulation of NK cells and CD8+ cytotoxic T cells, whereas the requirement for CD4+ T cells in tumor rejection was highly dependent on the tumor model.35 Moreover, the role of CD4+CD25+ T cells, also referred to as regulatory or suppressor T cells, in the enhancement or the suppression of antitumor immunity is being investigated.36,37 In a recent study, Peron et al. developed an ex vivo gene therapy model to treat murine HCC using genetically modified cells with retroviral vector expressing IL-12 only, without B7.1.38 The results of this study showed a marked antitumor effect of direct liver expression of IL-12, which could be expressed by tumor cells themselves (Fig. 1,[1] without e) or the modified syngenic fibroblasts in a 7-day therapy model (Fig. 1,[2]). Meanwhile, the antitumor effect was associated with a prominent liver infiltration of CD4+ T cells, CD8+ T cells, NK cells and macrophages, but without elevation in serum liver enzymes. This effect was maintained in nude mice and NK cell-depleted nude mice. These data may further endorse IL-12 gene therapy in clinical trials for treating human HCC. This report was supported in part by a grant (CMFHT 9102) from Chi-Mei Foundation Medical Center.

Tumoricidal Potential of Native Human Blood Dendritic Cells: Direct Tumor Cell Killing and Activation of NK Cell-Mediated Cytotoxicity.

The highly efficient nature of dendritic cells (DC) as antigen-presenting cells raises the possibility of uncovering in tumor-bearing hosts very low levels of T cell reactivity to poorly immunogenic tumors that are virtually undetectable by other means. Here, we demonstrate the in vitro and in vivo capacities of murine bone marrow-derived, cytokine-driven DC to elicit potent and specific anti-tumor responses when pulsed with whole tumor lysates. Stimulation of naive spleen-derived T cells by tumor lysate-pulsed DC generated tumor-specific proliferative cytokine release and cytolytic reactivities in vitro. In addition, in two separate strains of mice with histologically distinct tumors, s.c. injections of DC pulsed with whole tumor lysates effectively primed these animals to reject subsequent lethal challenges with viable parental tumor cells and, important to note, also mediated significant reductions in the number of metastases established in the lungs. Tumor rejection depended on host-derived CD8(+) T cells and, to a lesser extent, CD4(+) T cells. Spleens from mice that had rejected their tumors contained specific precursor cytotoxic T lymphocytes. The use of whole tumor lysates as a source of tumor-associated antigen(s) for pulsing of DC circumvents several limitations encountered with other methods as well as provides certain distinct advantages, which are discussed. These data serve as rationale for our recent initiation of a phase I clinical trial of immunization with autologous tumor lysate-pulsed DC in adult and pediatric cancer patients.

Cancer immunotherapy aims to stimulate host antitumor immune responses or break tumor-related immune tolerance or both with the potential of curative treatment. Potential immunotherapy targets include Toll-like receptors (TLRs), which are key receptors of the innate immune system and play an important role in regulating adaptive immune responses. IMO-2125 is a potent and selective agonist of endosomal TLR9 which significantly induces IFN-α, and the maturation of dendritic cells (DC). In the setting of cancer immunotherapy, we hypothesize that intratumoral (i.t.) administration of IMO-2125 has the potential to stimulate DC maturation and T-cell activation in the tumor microenvironment, leading to increased local and systemic antitumor immune responses and tumor regression, and may potentiate the activity of checkpoint inhibitors. In the present study, we evaluated the antitumor immune activity of i.t. IMO-2125 in murine syngeneic colon carcinoma models. BALB/c mice were s.c. implanted with 3 x 106 CT26 and CT26.CL25 cells, a subclone of CT26 expressing a model antigen beta-galactosidase (beta-gal), on the right and left flanks, respectively. Treatment was initiated when tumor nodules reached 200 mm3. Treated mice received IMO-2125 at doses of 10, 50 and 100 µg per injection, placebo, or a control compound (n=8 each), by i.t. injection only in the CT26 tumor implanted in the right flank twice weekly for two weeks. To evaluate IMO-2125 in combination with an anti-CTLA-4 mAb, 50 µg IMO-2125 and 10 µg anti-mouse CTLA-4 mAb were co-injected into the right tumor. Over two weeks, IMO-2125 was administered five times and the anti-mouse CTLA-4 mAb was administered four times. Results showed that i.t. IMO-2125 treatment led to dose-dependent inhibition of both treated and distant tumor growth. In mice treated with 100 µg, there were reductions in tumor volume of 95.2% (p = 0.0058) and 91.2% (p = 0.0048) in the treated and distant tumors, respectively. At this dose, there was complete tumor regression in 5 out 8 mice (63%). Control compound showed no anti-tumor activity. Antitumor activity was correlated with increased CD8+ T cell infiltration into both treated and distant tumors. In vivo depletion of T cells showed that IMO-2125 i.t mediated antitumor effects depended on CD8+ T cells, while depletion of CD4+ T cells enhanced IMO-2125 mediated tumor regression through elimination of Tregs. Furthermore, treatment elicited tumor-specific cytotoxic T cells not only to CT26 associated antigen AH1 in the injected tumor and also to beta-gal presented only in distant CT26.CL25 tumors. The mice with complete regression were rechallenged with CT26/CT26.CL25 and rejected the implantation; however, they were not protected from rechallenge with A20 lymphoma, indicating IMO-2125 i.t. treatment resulted in persistent tumor memory. Treatment with a combination of i.t. IMO-2125 and i.t. anti CTLA-4 mAb resulted in more potent antitumor activity than either agent alone. In conclusion, i.t. administration of IMO-2125 changes the local tumor microenvironment by inducing Th1 type cytokines, thereby modulating levels of immune checkpoint expression, and exerting potent local and systemic antitumor activity. In addition, IMO-2125 treatment in combination with an anti-CTLA-4 mAb demonstrated more potent antitumor activity compared to either agent alone. IMO-2125 has been well tolerated and showed induction of systemic IFN-α in a human trial. Planning for a clinical trial of a combination of i.t. IMO-2125 and ipilumimab is ongoing. Citation Format: Daqing Wang, Fugang Zhu, Xianzhi Mao, Sudhir Agrawal. Intratumoral administration of IMO-2125, a novel TLR9 agonist, modulates the tumor microenvironment and exerts systemic antitumor activity alone and in combination with an anti-CTLA-4 mAb. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B094.

Extracorporeal photochemotherapy (ECP) is employed for the management of cutaneous T cell lymphoma (CTCL). ECP involves the extracorporeal exposure of white blood cells (WBCs) to a photosensitizer, 8-methoxypsoralen (8-MOP), in the context of ultraviolet A (UVA) radiation, followed by WBC reinfusion. Historically, the therapeutic activity of ECP has been attributed to selective cytotoxicity on circulating CTCL cells. However, only a fraction of WBCs is exposed to ECP, and 8-MOP is inactive in the absence of UVA light, implying that other mechanisms underlie the anticancer effects of ECP. Recently, ECP has been shown to enable the physiological differentiation of monocytes into dendritic cells (DCs) that efficiently cross-present tumor-associated antigens (TAAs) to CD8+ T lymphocytes to initiate cognate immunity. However, the source of TAAs and immunostimulatory signals for such DCs remains to be elucidated. Here, we demonstrate that 8-MOP plus UVA light reduces melanoma cell viability along with the emission of ICD-associated danger signals including calreticulin (CALR) exposure on the cell surface and secretion of ATP, high mobility group box 1 (HMGB1) and type I interferon (IFN). Consistently, melanoma cells succumbing to 8-MOP plus UVA irradiation are efficiently engulfed by monocytes, ultimately leading to cross-priming of CD8+ T cells against cancer. Moreover, malignant cells killed by 8-MOP plus UVA irradiation in vitro vaccinate syngeneic immunocompetent mice against living cancer cells of the same type, and such a protection is lost when cancer cells are depleted of calreticulin or HMGB1, as well as in the presence of an ATP-degrading enzyme or antibodies blocking type I IFN receptors. ECP induces bona fide ICD, hence simultaneously providing monocytes with abundant amounts of TAAs and immunostimulatory signals that are sufficient to initiate cognate anticancer immunity.

Objective To observe the roles of specific and non-specific cytotoxic activity in cyto-kine-induced killer cells (CIK) against gastric cancer (SGC-7901 ) cells in vitro. Methods Dendritic cells (DC) and CIK were induced from peripheral blood mononuclear cells (PBMC) from healthy donors. The DC were sensitized with tumor antigen extracted from gastric cancer SGC-7901 cells. The CIK were in-duced with DC to form DC-CIK,or induced with gastric cancer antigen-pulsed DC to form antigen-DC-CIK. On the 14th day of culture,CIK, DC-CIK and antigen-DC-CIK phenotypes (CD3 ,CD3/CD8 and CD3/CD56) ware analyzed by flow cytometry. CIK, DC-CIK and antigen-DC-CIK were used as the effector cells, and the SGC-7901 cells with or without blockade of major histocompatibility complex (MHC) molecule as the target cells. At 5: 1,10:1 and 20:1 effector-target ratio,the cytotoxic activity was detected by MTT. Results On the 14th day of culture,the proportion of CD3~+ CD8~+ and CD3~+ CD56~+ double positive cells in antigen-DC-CIK was significantly higher than that in DC-CIK and pure CIK (P <0.01 ). At 20:1 effector-target ratio in vitro,the cytotoxic activity of CIK, DC-CIK and antigen-DC-CIK against SGC-7901 cells was (46.6± 2.2) %, (52.6 ± 1.6) % and (72.5±2.1 ) % (P < 0.01 ), and after MHC molecule was blocked in SGC-7901 cells, the cytotoxic activity was (44.3 ± 1.1 ) %, (49.9 ±0. 9) % and (50.4 ± 1.9) % respectively. The proportion of specific cytotoxic activity in antigen-DC-CIK was higher than in DC-CIK and CIK. Conclusion The major eytotoxic activity of CIK against gastric cancer cells is non-specific, and specific cytotoxic activity only accounts for 4.9%. After induction of CIK by DC ,the total cytotoxic activity was enhanced and the ma-jor cytotoxic activity is non-specific, and specific cytotoxie activity was enhanced slightly to 5.1%. After in-duction of CIK by antigen-pulsed DC, the major cytotoxic activity is still non-specific, and the specific cyto-toxic activity was largely increased to 30.5%. Key words: Stomach neoplasm; Dendric cells; Killer cells; Antigen

The Signaling Adaptor Eps8 Is an Essential Actin Capping Protein for Dendritic Cell Migration