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Purpose: To perform a two-cohort, phase I safety and immunogenicity study of IMA950 in addition to standard chemoradiotherapy and adjuvant temozolomide in patients with newly diagnosed glioblastoma. IMA950 is a novel glioblastoma-specific therapeutic vaccine containing 11 tumor-associated peptides (TUMAP), identified on human leukocyte antigen (HLA) surface receptors in primary human glioblastoma tissue., Experimental Design: Patients were HLA-A*02-positive and had undergone tumor resection. Vaccination comprised 11 intradermal injections with IMA950 plus granulocyte macrophage colony-stimulating factor (GM-CSF) over a 24-week period, beginning 7 to 14 days prior to initiation of chemoradiotherapy (Cohort 1) or 7 days after chemoradiotherapy (Cohort 2). Safety was assessed according to NCI CTCAE Version 4.0 and TUMAP-specific T-cell immune responses determined. Secondary observations included progression-free survival (PFS), pretreatment regulatory T cell (T reg ) levels, and the effect of steroids on T-cell responses., Results: Forty-five patients were recruited. Related adverse events included minor injection site reactions, rash, pruritus, fatigue, neutropenia and single cases of allergic reaction, anemia and anaphylaxis. Two patients experienced grade 3 dose-limiting toxicity of fatigue and anaphylaxis. Of 40 evaluable patients, 36 were TUMAP responders and 20 were multi-TUMAP responders, with no important differences between cohorts. No effect of pretreatment Treg levels on IMA950 immunogenicity was observed, and steroids did not affect TUMAP responses. PFS rates were 74% at 6 months and 31% at 9 months., Conclusions: IMA950 plus GM-CSF was well-tolerated with the primary immunogenicity endpoint of observing multi-TUMAP responses in at least 30% of patients exceeded. Further development of IMA950 is encouraged. Clin Cancer Res; 22(19); 4776-85. ©2016 AACRSee related commentary by Lowenstein and Castro, p. 4760., Competing Interests: Statement: Norbert Hilf, Sarah Kutscher, Juha Lindner, Oliver Schoor and Harpreet Singh are current or past employees of Immatics Biotechnologies. Sarah Kutscher, Norbert Hilf, Oliver Schoor and Harpreet Singh have stock ownership interests in Immatics Biotechnologies. Sarah Kutscher, Norbert Hilf, Oliver Schoor, Harpreet Singh have intellectual property interests in Immatics Biotechnologies. Sarah Kutscher, Norbert Hilf, Oliver Schoor and Harpreet Singh have received either travel, accommodation or other expenses from Immatics Biotechnologies during the previous two years. Harpreet Singh has a leadership role at Immatics Biotechnologies. Other authors disclosed no conflicts of interest., (©2016 American Association for Cancer Research.)

DNA vaccination represents a smart and promising approach to cancer immunotherapy. DNA vaccines for cancer immunotherapy are designed to deliver one or several genes encoding tumor antigens, thereby eliciting or augmenting antigen-specific immune responses against antigens that play a central role in tumor initiation, progression and metastasis. Vaccine efficacy can be significantly improved by implementing strategies for enhancing antigen presentation and immunogenicity, such as new delivery systems, addition of molecular adjuvants and immunostimulatory signals, optimized prime-boost strategies or blockade of immune checkpoints. Taken into consideration that innate immune responses are important in the induction and enhancement of antigen-specific adaptive responses, manipulations that integrate these approaches in the vaccine design can achieve activation of protective adaptive immune responses, thereby overcoming the self-tolerance towards many tumor antigens. Such approaches are employed in a number of clinical trials for DNA cancer immunotherapy and hold promise for prophylactic and therapeutic vaccine development. In this context, strategies that improve immunogenicity and enhance the efficacy of DNA vaccines for cancer immunotherapy are discussed., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Low-grade gliomas (LGGs) are the most common brain tumors of childhood. Although surgical resection is curative for well-circumscribed superficial lesions, tumors that are infiltrative or arise from deep structures are therapeutically challenging, and new treatment approaches are needed. Having identified a panel of glioma-associated antigens (GAAs) overexpressed in these tumors, we initiated a pilot trial of vaccinations with peptides for GAA epitopes in human leukocyte antigen-A2+ children with recurrent LGG that had progressed after at least 2 prior regimens., Methods: Peptide epitopes for 3 GAAs (EphA2, IL-13Rα2, and survivin) were emulsified in Montanide-ISA-51 and administered subcutaneously adjacent to intramuscular injections of polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose every 3 weeks for 8 courses, followed by booster vaccines every 6 weeks. Primary endpoints were safety and T-lymphocyte responses against GAA epitopes. Treatment response was evaluated clinically and by MRI., Results: Fourteen children were enrolled. Other than grade 3 urticaria in one child, no regimen-limiting toxicity was encountered. Vaccination induced immunoreactivity to at least one vaccine-targeted GAA in all 12 evaluable patients: to IL-13Rα2 in 3, EphA2 in 11, and survivin in 3. One child with a metastatic LGG had asymptomatic pseudoprogression noted 6 weeks after starting vaccination, followed by dramatic disease regression with >75% shrinkage of primary tumor and regression of metastatic disease, persisting >57 months. Three other children had sustained partial responses, lasting >10, >31, and >45 months, and one had a transient response., Conclusions: GAA peptide vaccination in children with recurrent LGGs is generally well tolerated, with preliminary evidence of immunological and clinical activity., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Application of dendritic cells (DCs) pulsed with tumor-associated antigens is considered attractive in immunotherapy for hepatocellular carcinoma (HCC). In order to efficiently prime tumor-associated antigens specific for cytotoxic T lymphocytes (CTLs), it is important that DCs present tumor-associated antigens on MHC class I. MHC class I generally present endogenous antigens expressed in the cytosol. In this study, we developed a new antigen delivery tool based on cross presentation of exogenous antigens in DCs by using cytoplasmic transduction peptide (CTP). CTP protein could transduce FoxM1 tumor antigen into the cytosol of DCs, and CTP-FoxM1 fusion protein could stimulate activation and maturation of DCs. DCs pulsed with CTP-FoxM1 could induce specific CTLs. More importantly, the immunity induced by DCs loaded with CTP-FoxM1 could significantly inhibit tumor growth and metastasis in HCC-bearing mice, which was more potent than that induced by DCs loaded with FoxM1 or CTP, alone. Our results indicate that DCs pulsed with CTP-FoxM1 might be a promising vaccine candidate for HCC therapy and provide new insight into the design of DC-based immunotherapy., Competing Interests: The authors declare no conflicts of interest.

Leber congenital amaurosis (LCA) is a severe disorder resulting in visual impairment usually starting in the first year of life. The most frequent genetic cause of LCA is an intronic mutation in CEP290 (c.2991 + 1655A > G) that creates a cryptic splice donor site resulting in the insertion of a pseudoexon (exon X) into CEP290 mRNA. Previously, we showed that naked antisense oligonucleotides (AONs) effectively restored normal CEP290 splicing in patient-derived lymphoblastoid cells. We here explore the therapeutic potential of naked and adeno-associated virus (AAV)-packaged AONs in vitro and in vivo In both cases, AON delivery fully restored CEP290 pre-mRNA splicing, significantly increased CEP290 protein levels and rescued a ciliary phenotype present in patient-derived fibroblast cells. Moreover, administration of naked and AAV-packaged AONs to the retina of a humanized mutant Cep290 mouse model, carrying the intronic mutation, showed a statistically significant reduction of exon X-containing Cep290 transcripts, without compromising the retinal structure. Together, our data highlight the tremendous therapeutic prospective of AONs for the treatment of not only CEP290-associated LCA but potentially many other subtypes of retinal dystrophy caused by splicing mutations., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Nanoimmunotherapy, the application of nanotechnology for sustained and targeted delivery of antigens to dendritic cells (DCs), has attracted much attention in stimulating antigen-specific immune response for antitumor therapy. In order to in situ deliver antigens to DCs for efficient antigen presentation and subsequent induction of strong cytotoxic T lymphocytes (CTL) response, here we developed a multi-peptide (TRP2180-188 and HGP10025-33) and toll-like receptor 4 agonist (monophosphoryl lipid A) codelivery system based on lipid-coated zinc phosphate hybrid nanoparticles (LZnP NPs). This delivery system equips with the chelating property of zinc to realize the high encapsulation efficiency with antigenic peptides and the influence on immune system with adjuvant-like feature. The combination of H-2K(b) and H-2D(b)-restricted peptides could provide multiple epitopes as the target of specific MHC alleles, making tumor more difficult to escape from the surveillance of immune system. The formulated LZnP nano-vaccine with the size of 30nm and outer leaflet lipid exhibited antitumor immunity as the secretion of cytokines in vitro and increased CD8(+) T cell response from IFN-γ ELISPOT analysis ex vivo. The antitumor effects were further evidenced from the prophylactic, therapeutic and metastatic melanoma tumor models compared with free antigens and single peptide-loaded nano-vaccines. These results validate the benefit of LZnP-based vaccine for antitumor immunity and indicate that co-delivery of tumor antigens along with adjuvant may be an optimized strategy for tumor immunotherapy., (Copyright © 2016. Published by Elsevier B.V.)

Objective: No efficient medical treatment is available for severe acute hepatitis (SAH) except N-acetylcysteine for acetaminophen-induced acute liver failure. The human C-type lectin Reg3α, referred to as ALF-5755, improved survival in an animal model of acute liver failure and was well tolerated in a phase 1 trial in humans. We performed a phase 2a trial of ALF5755 in non-acetaminophen induced SAH., Design: double-blind, randomized, placebo-controlled study. The primary end-point was the improvement in the coagulation protein synthesis assessed by the change of Prothrombin (PR) during the 72 hours following treatment initiation calculated as PRH0 minus PRH72 divided by 72 (PR slope H0H72). Intention to treat (ITT) and per-protocol (PP) analysis of the entire group and the Hepatitis B virus (HBV)/AIH (auto-immune hepatitis) sub-group were done separately., Results: 57 patients were included. Twenty-eight received ALF-5755, 29 the placebo. Etiologies were: Hepatitis A (n = 10), HBV (n = 13), AIH (n = 9), drug-induced (n = 8), other (n = 17). On the whole group, nor the PR slope H0H72 (0.18±0.31 vs 0.25±0.32), nor the transplant-free survival rate at day 21 (75 vs 86%) differed between groups. Conversely, in the HBV-AIH subgroup, in which ALF was more severe, PR slope H0-H72 was higher in the ALF-5755 arm, the difference being significant in PP analysis (0.048±0.066 vs -0.040±0.099, p = 0.04); the median length of hospitalization was lower in the ALF-5755 group (8 vs 14 days, p = 0.02)., Conclusion: ALF-5755 was not efficient in a ITT analysis performed on the whole sample; however it led to a significant, although moderate, clinical benefit in a PP analysis of the sub-group of patients with HBV or AIH related SAH. As HBV is the major cause of SAH in Asia and Africa and AIH a growing cause, this study emphasizes the need to pursuit the evaluation of this novel medical treatment of SAH., Trial Registration: ClinicalTrials.gov NCT01318525.

Combination of tumor antigens with immunostimulants is a promising approach in cancer immunotherapy. We assessed animal model toxicity of AS15 combined with various tumor antigens: WT1 (rabbits), or p501, dHER2 and recPRAME (cynomolgus monkeys), administered in seven or 20 dose regimens versus a saline control. Clinical and ophthalmological examinations, followed by extensive post-mortem pathological examinations, were performed on all animals. Blood hematology and biochemistry parameters were also assessed. Antigen-specific antibody titers were determined by enzyme-linked immunosorbent assay. Additional assessments in monkeys included electrocardiography and immunohistochemical evaluations of the p501 expression pattern. Transient increases in body temperature were observed 4 h or 24 h after injections of recPRAME + AS15 and dHER2 + AS15. Edema and erythema were observed up to 1 week after most injections of recPRAME + AS15 and all injections of dHER2 + AS15. No treatment-related effects were observed for electrocardiography parameters. Mean fibrinogen levels were significantly higher in all treated groups compared to controls, but no differences could be observed at the end of the treatment-free period. Transient but significant differences in biochemistry parameters were observed post-injection: lower albumin/globulin ratios (p501 + AS15), and higher bilirubin, urea and creatinine (dHER2 + AS15). Pathology examinations revealed significant increases in axillary lymph node mean weights (recPRAME + AS15) compared to controls. A 100% seroconversion rate was observed in all treated groups, but not in controls. p501 protein expression was observed in prostates of all monkeys from studies assessing p501 + AS15. These results suggest a favorable safety profile of the AS15-containing candidate vaccines, supporting the use of AS15 for clinical development of potential anticancer vaccines., (Copyright © 2015 The Authors. Journal of Applied Toxicology Published by John Wiley & Sons Ltd.)

Gliomas are the most common primary brain tumors of the central nervous system, and carry a grim prognosis. Novel approaches utilizing the immune system as adjuvant therapy are quickly emerging as viable and effective options. Immunotherapeutic strategies being investigated to treat glioblastoma include: vaccination therapy targeted against either specific tumor antigens or whole tumor lysate, adoptive cellular therapy with cytotoxic T lymphocytes, chimeric antigen receptors and bi-specific T-cell engaging antibodies allowing circumvention of major histocompatibility complex restriction, aptamer therapy with aims for more efficient target delivery, and checkpoint blockade in order to release the tumor-mediated inhibition of the immune system. Given the heterogeneity of glioblastoma and its ability to gain mutations throughout the disease course, multifaceted treatment strategies utilizing multiple forms of immunotherapy in combination with conventional therapy will be most likely to succeed moving forward., (© 2016 Elsevier B.V. All rights reserved.)

Advanced-stage non-small cell lung cancer (NSCLC) and small cell lung cancer are cancers in which chemotherapy produces a survival benefit, although it is small. We now know that anti-PD-1/PD-L1 has substantial clinical activity in both of these diseases, with an overall response rate (ORR) of 15%-20%. These responses are frequently rapid and durable, increase median overall survival (OS) compared with chemotherapy, and produce long-term survivors. Despite these very significant results, many patients do not benefit from anti-PD-1/PD-L1. This is because of the potential for malignancies to co-opt myriad immunosuppressive mechanisms other than aberrant expression of PD-L1. Conceptually, these can be divided into three categories. First, for some patients there is likely a failure to generate sufficient functional tumor antigen-specific T cells. Second, for others, tumor antigen-specific T cells may be generated but fail to enter into the tumor parenchyma. Finally, there are a large number of immunosuppressive mechanisms that have the potential to be operational within the tumor microenvironment: surface membrane immune checkpoint proteins PD-1, CTLA-4, LAG3, TIM3, BTLA, and adenosine A2AR; soluble factors and metabolic alterations interleukin (IL)-10, transforming growth factor (TGF)-β, adenosine, IDO, and arginase; and inhibitory cells, cancer-associated fibroblasts (CAFs), regulatory T cells, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages. In this article, we discuss three strategies to generate more tumor-reactive T cells for patients: anti-CTLA-4, therapeutic tumor vaccination, and adoptive cellular therapy, with T cells redirected to tumor antigens using T-cell receptor (TCR) or chimeric antigen receptor (CAR) gene modification. We also review some of the various strategies in development to thwart tumor microenvironment immunosuppressive mechanisms. Strategies to drive more T cells into tumors remain a significant challenge.

Introduction: Methods to induce T cell responses to protein vaccines have not been optimized. The immunostimulant AS15 has been administered with the recombinant MAGE-A3 protein (recMAGE-A3) i.m. but not i.d. or s.c. This study tests hypotheses that the i.d./s.c. route is safe and will increase CD4(+) and CD8(+) T cell responses to MAGE-A3., Patients and Methods: Twenty-five patients with resected stage IIB-IV MAGE-A3(+) melanoma were randomized to immunization with recMAGE-A3 combined with AS15 immunostimulant (MAGE-A3 immunotherapeutic) either i.m. (group A, n = 13) or i.d./s.c. (group B, n = 12). Adverse events were recorded. Ab responses to MAGE-A3 were measured by ELISA. T cell responses to overlapping MAGE-A3 peptides were assessed in PBMC and a sentinel immunized node (SIN) after 1 in vitro stimulation with recMAGE-A3, by IFN-γ ELISPOT assay and by flow cytometry for multifunctional (TNF-α/IFN-γ) responses., Results: Both routes of immunization were well tolerated without treatment-related grade 3 adverse events. All patients had durable Ab responses. For all 25 patients, the T cell response rate by ELISPOT assay was 30 % in SIN (7/23) but only 4 % (1/25) in PBMC. By flow cytometry, multifunctional CD8(+) T cell responses were identified in one patient in each group; multifunctional CD4(+) T cell response rates for groups A and B, respectively, were 31 and 64 % in SIN and 31 and 50 % in PBMC., Conclusion: The MAGE-A3 immunotherapeutic was well tolerated after i.d./s.c. administration, with trends to higher CD4(+) T cell response rates than with i.m. administration. This study supports further study of AS15 by i.d./s.c. administration., Competing Interests: Dr. Slingluff has the following relationships directly related to this work: The recombinant MAGE-A3 protein, overlapping peptides, and AS15 were provided to the University of Virginia for this trial by GlaxoSmithKline, and the trial was funded largely by a grant to the University of Virginia from GlaxoSmithKline. The remaining authors have no conflicts.

The PRAME tumor antigen is a potential target for immunotherapy. We assessed the immunogenicity, the antitumor activity, and the safety and the tolerability of a recombinant PRAME protein (recPRAME) combined with the AS15 immunostimulant (recPRAME+ AS15) in preclinical studies in mice and Cynomolgus monkeys. Four groups of 12 CB6F1 mice received 4 injections of phosphate-buffered saline (PBS), recPRAME, AS15, or recPRAME+AS15. Immunized mice were injected with tumor cells expressing PRAME (CT26-PRAME) 2 weeks or 2 months after the last injection. The mean tumor surface was measured twice a week. Two groups of 10 monkeys received 7 injections of saline or recPRAME+ AS15. T-cell responses were measured by flow cytometry using intracellular cytokine staining (ICS). In CB6F1 mice, repeated injections of recPRAME+ AS15 induced high PRAME-specific antibody titers and mostly CD4+ T cells producing cytokines. This immune response was long-lasting in these animals and was associated with protection against a challenge with PRAME-expressing tumor cells (CT26-PRAME) applied either 2 weeks or 2 months after the last injection; these data indicate the induction of an immune memory. In HLA-A02.01/HLA-DR1 transgenic mice, recPRAME+ AS15 induced both CD4+ and CD8+ T-cell responses, indicating that this antigen can be processed by the human leukocyte antigen and is potentially immunogenic in humans. In addition, a repeated-dose toxicity study in monkeys showed that 7 biweekly injections of recPRAME+ AS15 were well tolerated, and induced PRAME-specific antibodies and T cells. In conclusion, these preclinical data indicate that repeated injections of the PRAME cancer immunotherapeutic are immunogenic and have an acceptable safety profile.

Introduction: To assess the safety and immunogenicity of MAGE-A3 immunotherapeutic in patients with stage IB-III MAGE-A3-positive non-small-cell lung cancer (NSCLC) who were or were not undergoing standard cisplatin/vinorelbine chemotherapy., Methods: This open, prospective, multicenter, parallel-group phase I study (NCT00455572) enrolled patients with resected (cohorts 1-3) or unresectable (cohort 4) MAGE-A3-positive NSCLC. MAGE-A3 immunotherapeutic (300 μg recombinant MAGE-A3 formulated with AS15) was administered (eight doses, 3 weeks apart) concurrent with (cohort 1), after (cohort 2), or without (cohort 3) standard-adjuvant chemotherapy, or after standard radiotherapy and/or chemotherapy (cohort 4)., Results: Sixty-seven patients received greater than or equal to 1 dose of MAGE-A3 immunotherapeutic. Grade 3/4 adverse events (AEs) were reported for 16 out of 19 (84%), 2 out of 18 (11%), 5 out of 18 (28%), and 1 out of 12 (8%) patients in cohorts 1, 2, 3, and 4, respectively. Many grade 3/4 AEs in cohort 1 (e.g., neutropenia) were typical of chemotherapy. Six patients, including three in cohort 1, reported study treatment-related grade 3/4 AEs (injection-site reactions or musculoskeletal/back pain, which resolved within 5 days). One patient (in cohort 4) died, but this and the other serious adverse events were not study treatment related. MAGE-A3-specific antibody responses to immunotherapy were induced in all patients evaluated in all cohorts. MAGE-A3-specific CD4 T-cell responses to immunotherapy were detected in 4 out of 11 (36%), 4 out of 15 (27%), 2 out of 8 (25%), and 5 out of 6 (83%) evaluated patients in cohorts 1, 2, 3, and 4, respectively; and CD8 T-cell responses were only detected in four patients., Conclusion: In resected and unresectable NSCLC patients and irrespective of whether standard chemotherapy was concurrent or not, MAGE-A3 immunotherapeutic is well tolerated and induces MAGE-A3-specific immune responses.

It is now well established that the immune system can recognize developing cancers and that therapeutic manipulation of immunity can induce tumor regression. The capacity to manifest remarkably durable responses in some patients has been ascribed in part to T cells that can (a) kill tumor cells directly, (b) orchestrate diverse antitumor immune responses, (c) manifest long-lasting memory, and (d) display remarkable specificity for tumor-derived proteins. This specificity stems from fundamental differences between cancer cells and their normal counterparts in that the former develop protein-altering mutations and undergo epigenetic and genetic alterations, resulting in aberrant protein expression. These events can result in formation of tumor antigens. The identification of mutated and aberrantly expressed self-tumor antigens has historically been time consuming and laborious. While mutant antigens are usually expressed in a tumor-specific manner, aberrantly expressed antigens are often shared between cancers and, therefore, in the past, have been the major focus of therapeutic cancer vaccines. However, advances in next-generation sequencing and epitope prediction now permit the rapid identification of mutant tumor neoantigens. This review focuses on a discussion of mutant tumor neoantigens and their use in personalizing cancer immunotherapies.

Converging data indicate that the immune system is able to recognize cancer epitopes as non-self and mount an immune reaction that may erase, or temporarily block, tumor growth. The immune pressure supports the amplification of immune resistant tumor clones, creating an immune suppressive environment that leads to the formation of a clinically relevant tumor. These general observations also apply to brain tumors and specifically to gliomas. Cancer immunotherapy strategies are aimed at reverting such immune suppression. Two approaches are already used in the clinics. The first one, peptide immunotherapy, has been oriented to the most aggressive glioma, glioblastoma (GBM) where, in the context of EGFR (epidermal growth factor receptor) amplification, a large deletion arises and creates a novel, cancer-specific antigen, EGFRvIII. The second one is dendritic cell immunotherapy. Dendritic cells are potent antigen presenting cells that can be pulsed with autologous tumor lysate or peptide pp65 from cytomegalovirus (CMV) that is present in GBM but not in normal brain. Antigen presentation by dendritic cells is bolstered by preconditioning their injection site with the tetanus/diphtheria toxoid. The third approach is adoptive cell therapy (ACT) in which tumor-specific T cells can be amplified ex vivo and subsequently re-injected to the patient to lyse cells expressing tumor antigens, increasing survival durably in a fraction of melanoma patients. ACT may also be based on T cell transduction of tumor specific receptors or chimeric antigen receptors (CARs). CARs are powerful tools for immunotherapy but off-target toxicity may be an issue as they do not request MHC presentation for activation. Upcoming clinical trial results will clarify the most effective direction for cancer immunotherapy in gliomas and other cancers with poor prognosis.

The MAGE-A3 recombinant protein combined with AS15 immunostimulant (MAGE-A3 Cancer Immunotherapeutic) is under development by GlaxoSmithKline for the treatment of lung cancer and melanoma. We performed non-clinical safety studies evaluating potential local and systemic toxic effects induced by MAGE-A3 Cancer Immunotherapeutic in rabbits (study 1) and cynomolgus monkeys (study 2). Animals were allocated to two groups to receive a single (rabbits) or 25 repeated (every 2 weeks) injections (monkeys) of MAGE-A3 Cancer Immunotherapeutic (treatment groups) or saline (control groups). All rabbits were sacrificed 3 days post-injection and monkeys 3 days following last injection (3/5 per gender per group) or after a 3-month treatment-free period (2/5 per gender per group). Local and systemic reactions and MAGE-A3-specific immune responses (monkeys) were assessed. Macroscopic and microscopic (for rabbits, injection site only) post-mortem examinations were performed on all animals. No systemic toxicity or unscheduled mortalities were recorded. Single (rabbits) and repeated (monkeys; up to four times at the same site) injections were well tolerated. Following five to seven repeated injections, limb circumferences increased up to 26% (5 h post-injection), but returned to normal after 1-8 days. Three days after the last injection, enlargements of iliac, popliteal, axillary and inguinal lymph nodes, and increased incidence or severity of mononuclear inflammatory cell infiltrates was observed in injected muscles of treated monkeys. No treatment-related macroscopic findings were recorded after the treatment-free period. MAGE-A3-specific antibody and T-cell responses were raised in all treated monkeys, confirming test item exposure. Single or repeated intramuscular injections of MAGE-A3 Cancer Immunotherapeutic were well tolerated in rabbits and monkeys., (Copyright © 2014 GlaxoSmithKline Vaccines. Journal of Applied Toxicology published by John Wiley & Sons, Ltd.)

Carbonic anhydrase IX (CA IX) is an attractive target for cancer therapy. Many anti-CA IX antibodies have been reported but few have been shown to possess inhibition activity. Furthermore, effective use of CA IX-inhibition antibodies for cancer immunotherapy has not been well-validated since data are mainly limited to in vitro assays. In this study, we established that chKM4927, an anti-CA IX chimeric antibody, recognizes CA IX and has CA IX-specific inhibition activity. ChKM4927 also retains antibody-dependent cellular cytotoxicity (ADCC) activity against CA IX-expressing cancer cells. Compared to controls, chKM4927 treatment (10 mg/kg) showed anti-tumor activity in the VMRC-RCW xenograft model in vivo. ChKM4927-attenuated ADCC activity showed equally effective anti-tumor activity. These results suggest that the CA IX-inhibition antibody chKM4927 has an anti-tumor effect in the VMRC-RCW xenograft model via an ADCC-independent mechanism., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Performance enhancement of the immune system can now be generated through ex vivo gene modification of T cells in order to redirect native specificity to target tumor antigens. This approach combines the specificity of antibody therapy, the expanded response of cellular therapy and the memory activity of vaccine therapy. Recent clinical trials of chimeric antigen receptor (CAR) T cells directed toward CD19 as a stand-alone therapy have shown sustained complete responses in patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia. As these drug products are individually derived from a patient's own cells, a different manufacturing approach is required for this kind of personalized therapy compared with conventional drugs. Key steps in the CAR T-cell manufacturing process include the selection and activation of isolated T cells, transduction of T cells to express CARs, ex vivo expansion of modified T cells and cryopreservation in infusible media. In this review, the steps involved in isolating, genetically modifying and scaling-out the CAR T cells for use in a clinical setting are described in the context of in-process and release testing and regulatory standards.

Dendritic cells (DCs) are potent antigen-presenting cells that can be used in cancer vaccines. Thus, various strategies have been developed to deliver tumor-associated antigens via DCs. One strategy includes administering DC-tumor fusion cells (DC-tumor FCs) to induce antitumor immune responses in cancer patients. However, clinical trials using this strategy have fallen short of expectations. Several factors might limit the efficacy of these anticancer vaccines. To induce efficient antitumor immune responses and enhance potential clinical benefits, DC-tumor FC-based cancer vaccines require manipulations that improve immunogenicity for both DCs and whole tumor cells. This review addresses recent progress in improving clinical outcomes using DC-tumor FC-based cancer vaccines.

Melanoma antigen family A, 3 (MAGE-A3) is a cancer-testis antigen whose expression has been demonstrated in a wide array of malignancies including melanoma, brain, breast, lung and ovarian cancer. In addition, its ability to elicit spontaneous humoral and cellular immune responses has been shown in cancer patients. As antigen-specific immune responses can be stimulated by immunization with MAGE-A3, several clinical trials have used MAGE-A3 vaccines to observe clinical responses. The frequent expressions of this antigen in various tumors and its immunogenicity in cancer patients have led to application of this antigen in cancer immunotherapy. However, the results of recent clinical trials indicate that there is a need for research in the vaccine design, adjuvant selection as well as patient selection criteria.

Importance: In conjunction with chemotherapy, immunotherapy with dendritic cells (DCs) may eliminate minimal disease burden by generating cytotoxic T lymphocytes. Enhanced cytosolic bioavailability of tumor-specific antigens improves access to human leukocyte antigen (HLA) class I molecules for more efficient cytotoxic T lymphocyte generation. Various cell-penetrating domains (CPDs) are known to ferry covalently linked heterologous antigens to the intracellular compartment by traversing the plasma membrane., Objective: To determine whether generating melanoma antigen family A, 3 (MAGE-A3), a tumor-specific cancer-testis antigen, as a fusion protein with CPD will enhance the cytosolic bioavailability of MAGE-A3., Design: MAGE-A3 was amplified by polymerase chain reaction using complementary DNA from renal tissue and cloned in frame with a CPD (YARKARRQARR) at the amino-terminal end and hexahistidine at the carboxy-terminal end to generate CPD-MAGE-A3 in a pQE-70 expression vector. Cultures were grown in Escherichia coli BL21 Star (DE3-pLysS) cells followed by nickel-nitrilotriacetic acid affinity purification of recombinant proteins., Main Outcomes and Measures: Measurement of DC membrane penetration of CPD-MAGE-A3 vs MAGE-A3 and determination of the effect of CPD-MAGE-A3 pulsing on DC phenotypic expression of cell-surface antigens., Results: Media composition and isopropyl-d-thiogalactosidase induction were optimized to achieve high levels of protein expression followed by purification. Western blot analysis with MAGE-A3 antibodies recognized both MAGE-A3 and CPD-MAGE-A3 proteins, while CPD antibodies recognized only CPD-MAGE-A3. Purified CPD-MAGE-A3 exhibited more efficient DC membrane penetration than did MAGE-A3 alone as confirmed by immunofluorescence analysis. High-level expression of several unique DC markers (CD80, CD83, CD86, and HLA-DR) by flow cytometry was consistent with a mature DC phenotype, indicating that pulsing with CPD-MAGE-A3 did not alter specific cell-surface antigens required for T-cell activation., Conclusions and Relevance: We have demonstrated for the first time, to our knowledge, that cloning and purification of MAGE-A3 with CPD enhances its cytosolic bioavailability in DCs without altering cell-surface antigens, potentially making it a more potent therapeutic cancer vaccine compared with existing MAGE-A3 protein and peptide vaccines.

Glioblastoma is the most aggressive primary tumor of the central nervous system with a medium overall survival of 7-15 months after diagnosis. Since tumor cells penetrate the surrounding brain tissue, complete surgical resection is impossible and tumor recurrence is almost a certainty. New treatment modalities are therefore needed, and these should be able to trace, identify, and kill dispersed tumor cells with great accuracy. Immunological approaches in principle meet these needs. Unfortunately, due to profound tumor-associated mechanisms of immunosuppression and -evasion, immunotherapeutic strategies like peptide vaccination have so far not been translated into clinical success. If future, peptide-based vaccination approaches shall be successful in glioblastoma therapy, multiple questions need to be solved including identification of suitable antigens, route and mode of vaccination, preparation of the tumor-bearing "host" and antagonizing, as much as this is possible, glioblastoma-associated mechanisms of immune evasion and poor vaccination response. In this review we will address the immunological challenges of glioblastoma and discuss key aspects that have rendered successful immunotherapy difficult in the past., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: Active immunotherapy is a promising antitumoral strategy; however its use in combination with chemotherapy in dogs with large B-cell lymphoma (DLBCL) remains largely untested. Heat shock proteins (HSP) bind the small peptides they chaperone (HSPPC), allowing for immunization of the host against a large repertoire of tumor-associated antigens. Hydroxylapatite vehicles HSPPCs and acts as an immunologic adjuvant. The aim of this study was to show that an autologous vaccine with hydroxylapatite and tumor-derived HSPPCs is safe and therapeutically effective in dogs with DLBCL., Experimental Design: Nineteen dogs with naturally occurring DLBCL were entered into a prospective randomized placebo-controlled double-blinded trial of HSPPCs-hydroxylapatite plus chemotherapy versus chemotherapy alone. Endpoints included time to progression (TTP), lymphoma-specific survival (LSS), and incidence of toxicoses., Results: Median first TTP after randomization to the vaccine arm was 304 days versus 41 days for the control arm (P = 0.0004). There was also a statistically significant difference in duration of second remission between the two groups (P = 0.02). Median LSS was 505 days for the vaccinated dogs versus 159 days for the unvaccinated dogs (P = 0.0018). Six vaccinated dogs achieved molecular remission, as shown by clonal immunoglobulin H (IgH) rearrangement. Toxicoses were comparable between the two treatment arms., Conclusions: The results of this trial demonstrate that the autologous vaccine tested here is safe and efficacious in prolonging TTP and LSS in dogs with DLBCL when used in combination with dose-intense chemotherapy. On the basis of these results, additional evaluation of this novel therapeutic strategy is warranted in human DLBCL., (©2013 AACR.)

Four different carbonic anhydrases are expressed in the human nephron, the functional unit of the kidney. These are specifically expressed in different nephron segments, emphasizing the critical role carbonic anhydrases play in maintaining the homeostasis of this crucial organ.Whereas the localization of carbonic anhydrases in the kidney has been long established, interest in carbonic anhydrases has increased dramatically for renal cancer, in particular for the clear cell variant of renal cell carcinoma (ccRCC) because carbonic anhydrase IX is specifically expressed in ccRCC. Therefore carbonic anhydrase IX is being studied as potential diagnostic and therapeutic target, despite carbonic anhydrase IX expression in non-renal tissues.

Unlabelled: For the last thirty years immunotherapy has become an integral part of treatment of some cancers. The most effective approach in this context would be the use of complex immunostimulatory factors including tumor antigen in different forms, interleukins that stimulate differentiation., Background: The aim of the study was to compare cellular immune response on specific and combined immunotherapy in patients with cervical cancer., Patients and Methods: 76 cervical cancer patients undergoing combined radiotherapy were included into the study. Plus to basic radiation treatment two types of immunotherapy was performed: first--autolymphocytes reinfusion after in vitro incubation with tumor antigen (tumor tissue homogenate) and interleukin-2, second--combination of above mentioned immunotherapy, interleukin-2 and systemic enzyme., Results: This has resulted in increase of cellular immunity parameters in the main group with combined immunotherapy an excess of cells with killer activity to ensure an effective antitumor immune response and, accordingly, the clinical efficacy of specific methods and adoptive immunotherapy in patients with cervical cancer., Conclusion: Immunotherapy usage in the form of a preparation of interleukin-2 and a system enzymotherapy in a combination with a specific immunotherapy is possible. This way of treatment is recommended in cases of decreasing of cellular immunity indicators.

Lung cancer is the main cause of cancer death worldwide. An advanced stage of the disease at the time of diagnosis, observed in the majority of cases, does not allow for introduction of radical treatment or makes the treatment ineffective. Lung cancer as a solid tumour with a very low antigenicity escapes immune surveillance, and cytotoxic cells attack. Cytotoxic lymphocytes play a key role in anticancer defence, but the population of these cells individually differs. Many suppressor and regulatory mechanisms inhibit the recognition of tumour antigens by dendritic and cytotoxic cell activation. The population of regulatory T cells (T regs) plays a crucial role in this inhibition of immune response. Their function depends on the expression of transcription factor Foxp3 and the presence of CTLA-4 molecules. The increased proportion of T regs and high expression of Foxp3 and CTLA-4 on lung cancer cells and tumour infiltrating lymphocytes were observed. Other components of immune response inhibition and tumour promotion are: Th17 cell population, M2 macrophage polarisation, the presence of myeloid derived suppressor cells (MDSCs) and a significantly elevated concentration of cytokines: TGF-b and IL-10 in the tumour microenvironment. The recognition of these mechanisms may have important therapeutic implications. Several types of agents which are capable of modulating the immune response have recently been used in many clinical trials conducted in lung cancer patients, some of them showing efficacy. Lung cancer immunotherapy has two main directions: the first goal is to improve the cytotoxic effect (for example by inhibition of CTLA-4, stimulation of dendritic cell function, inhibition of lymphocyte apoptosis), and the second way is the production of anti-cancer vaccines using known cancer antigens: MAGE A3, MUC1, EGF and TGF-b. Immunotherapy in lung cancer treatment has a character of personalised therapy - there is a need to specify the patient's immune status prior to treatment. The analysis of immune cells and mediators in the peripheral blood allows this, but the more valuable method seems to be bronchoalveolar lavage (BAL) examination with careful assessment of the tumour microenvironment.

Interest for cancer vaccination started more than 30 years ago after the demonstration that both in animal models and later on in patients it is possible to generate anti-tumor immune responses. The clinical application of this knowledge, however, was disappointing. In this review we summarize results on peptides epitopes recognized by T cells that have been studied thanks to their easy synthesis and the lack of significant side effects when administered in-vivo. To improve the clinical efficacy, peptides were modified in their aminoacid sequence to augment their immunogenicity. Peptides vaccines were recently shown to induce a high frequency of immune response in patients that were accompanied by clinical efficacy. These data are discussed at the light of recent progression of immunotherapy caused by the addition of check-point antibodies thus providing a general picture of the potential therapeutic efficacy of the peptide-based vaccines and their combination with other biological agents.

Recent studies have underlined the close link between immune response and prognosis of patients with colorectal cancer (CRC). Immune response understanding combined with biotechnology progress of the last years has allowed development of immunotherapy strategies in CRC. Immunotherapy strategies are divided in "active" or "passive" strategies (patients immune system stimulation or not) and considering the activation of antigen specific immune response or not. These immunotherapy strategies are well tolerated and induced cellular and humoral response correlated with clinical response. Many monoclonal antibodies targeting signalisation pathways or angiogenic growth factors have demonstrated their efficacy in CRC. Multiple vaccine strategies, using different tumour associated antigens, have demonstrated a biological efficacy but with poor clinical results. Results are more promising in adjuvant setting but need to be confirmed by randomized trials. Adoptive immunotherapy with transfer of tumour associated antigen specific T cell is probably the most promising strategy. Actually, except monoclonal antibodies, immunotherapy is not used in clinical practice in CRC due to the lack of results and absence of standardisation.

Lung cancer is the most common cause of cancer-related mortality worldwide and a therapeutic challenge. Recent success with antibodies blocking immune checkpoints in non-small-cell lung cancers (NSCLC) highlights the potential of immunotherapy for lung cancer treatment, and the need for trials of combination regimens of immunotherapy plus chemotherapy that lead to immunogenic cell death. Here, we review the development of immunogenic cytotoxic compounds, vaccines, and antibodies in NSCLC, in view of their integration into personalised oncology., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Introductions: The purpose of this study was to determine the anesthetic efficacy of lidocaine containing epinephrine compared with lidocaine containing epinephrine plus hyaluronidase (75 IU) when performing an inferior alveolar nerve block., Methods: Patients complaining of pain in the mandibular posterior teeth were selected. Based on their chief complaint, proper clinical and radiographic examinations were performed. Among them, 40 subjects diagnosed with irreversible pulpitis were selected. The inferior alveolar nerve block was induced using 3 mL 2% lidocaine with epinephrine. Hyaluronidase (75 IU) or a placebo was injected 30 minutes after the beginning of pulpal anesthesia (randomized and double-blind trial). The duration of the effect in the pulpal and gingival tissues was evaluated by the response to painful electrical stimuli applied to the adjacent premolar and by mechanical stimuli (pinprick) to the buccal gingiva, respectively., Results: In both pulpal and gingival tissues, the duration of the anesthetic effects with hyaluronidase was longer than with placebo., Conclusions: Hyaluronidase increased the duration of the effects of lidocaine in inferior alveolar nerve blocks., (Copyright © 2013. Published by Elsevier Inc.)

Purpose: Active immunization against the tumor-specific MAGE-A3 antigen is followed by a few but impressive and durable clinical responses. This randomized phase II trial evaluated two different immunostimulants combined with the MAGE-A3 protein to investigate whether a more robust and persistent immune response could be associated with increased clinical benefit., Patients and Methods: Patients with MAGE-A3-positive stage III or IV M1a melanoma were randomly assigned to receive the MAGE-A3 protein combined either with AS02B or with AS15 immunostimulant. Clinical end points were toxicity and rates of objective clinical responses, progression-free survival (PFS), and overall survival (OS)., Results: Seventy-five patients were treated, with 36 eligible patients per arm. Both treatments were well tolerated. In the AS15 arm, four objective responses were observed (three complete responses and one partial response) versus one partial response in the AS02B arm. In the AS15 and AS02B arms, the PFS rates after 6 months were 25% and 14%, respectively; and the median OS times were 33 months and 19.9 months, respectively, with a median observation period of 48 months. Antibodies against MAGE-A3, found in all patients, showed three-fold higher titers in the AS15 arm. The anti-MAGE-A3 cellular response was also more pronounced in the AS15 arm., Conclusion: In the MAGE-A3+AS15 arm, clinical activity was higher and the immune response more robust. Therefore, the AS15 immunostimulant was selected for combination with the MAGE-A3 protein in phase III trials.

As tumor-associated antigens are not well characterized for the majority of human tumors, polyvalent vaccines prepared with whole-tumor antigens are an attractive approach for tumor vaccination. Vascular endothelial growth factor receptor-2 (VEGFR2), as a model antigen with which to explore the feasibility of immunotherapy, has shown great promise as a tumor vaccine. However, the efficacy of immunotherapy is often not ideal when used alone. In this study, we explored the therapeutic efficacy of an irradiated AdVEGFR2-infected cell vaccine-based immunotherapy in the weakly immunogenic and highly metastatic 4T1 murine mammary cancer model. An adenovirus encoding the VEGFR2 gene (AdVEGFR2) was constructed. Lethally irradiated, virus-infected 4T1 cells were used as vaccines. Vaccination with lethally irradiated AdVEGFR2-infected 4T1 cells inhibited subsequent tumor growth and pulmonary metastasis compared with challenge inoculations. Angiogenesis was inhibited, and the number of CD8+ T lymphocytes was increased within the tumors. Antitumor activity was also caused by the adoptive transfer of isolated spleen lymphocytes. In vitro, the expression of HMGB1 and HSP70 in the AdVEGFR2‑infected 4T1 cells was increased, and was involved in the activation of tumor antigen-specific T-cell immunity. Our results indicate that the immunotherapy based on irradiated AdVEGFR2-infected whole-cancer cell vaccines may be a potentially effective strategy for 4T1 cancer treatment.

Dendritic cell (DC) vaccination is emerging as a promising therapeutic option for malignant glioma patients. However, the optimal antigen formulation for loading these cells has yet to be established. The objective of this study was to compare the safety, feasibility, and immune responses of malignant glioma patients on 2 different DC vaccination protocols. Twenty-eight patients were treated with autologous tumor lysate (ATL)-pulsed DC vaccination, whereas 6 patients were treated with glioma-associated antigen (GAA) peptide-pulsed DCs. Safety, toxicity, feasibility, and correlative immune monitoring assay results were compared between patients on each trial. Because of HLA subtype restrictions on the GAA-DC trial, 6/15 screened patients were eligible for treatment, whereas 28/32 patients passed eligibility screening for the ATL-DC trial. Elevated frequencies of activated natural killer cells were observed in the peripheral blood from GAA-DC patients compared with the ATL-DC patients. In addition, a significant correlation was observed between decreased regulatory T lymphocyte (Treg) ratios (postvaccination/prevaccination) and overall survival (P = 0.004) in patients on both trials. In fact, Treg ratios were independently prognostic for overall survival in these patients, whereas tumor pathology was not in multivariate analyses. In conclusion, these results suggest that ATL-DC vaccination is associated with wider patient eligibility compared with GAA-DC vaccination. Decreased postvaccination/prevaccination Treg ratios and decreased frequencies of activated natural killer cells were associated with prolonged survival in patients from both trials, suggesting that these lymphocyte subsets may be relevant immune monitoring endpoints for immunotherapy protocols in malignant glioma patients.

Nine cancer patients were treated with adoptive cell therapy using autologous anti-MAGE-A3 T-cell receptors (TCR)-engineered T cells. Five patients experienced clinical regression of their cancers including 2 on-going responders. Beginning 1-2 days postinfusion, 3 patients (#'s 5, 7, and 8) experienced mental status changes, and 2 patients (5 and 8) lapsed into comas and subsequently died. Magnetic resonance imagining analysis of patients 5 and 8 demonstrated periventricular leukomalacia, and examination of their brains at autopsy revealed necrotizing leukoencephalopathy with extensive white matter defects associated with infiltration of CD3(+)/CD8(+) T cells. Patient 7, developed Parkinson-like symptoms, which resolved over 4 weeks and fully recovered. Immunohistochemical staining of patient and normal brain samples demonstrated rare positively staining neurons with an antibody that recognizes multiple MAGE-A family members. The TCR used in this study recognized epitopes in MAGE-A3/A9/A12. Molecular assays of human brain samples using real-time quantitative-polymerase chain reaction, Nanostring quantitation, and deep-sequencing indicated that MAGE-A12 was expressed in human brain (and possibly MAGE-A1, MAGE-A8, and MAGE-A9). This previously unrecognized expression of MAGE-A12 in human brain was possibly the initiating event of a TCR-mediated inflammatory response that resulted in neuronal cell destruction and raises caution for clinical applications targeting MAGE-A family members with highly active immunotherapies.

The development of immune monitoring assays is essential to determine the immune responses against tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs) and their possible correlation with clinical outcome in cancer patients receiving immunotherapies. Despite the wide range of techniques used, to date these assays have not shown consistent results among clinical trials and failed to define surrogate markers of clinical efficacy to antitumor vaccines. Multiparameter flow cytometry- (FCM-) based assays combining different phenotypic and functional markers have been developed in the past decade for informative and longitudinal analysis of polyfunctional T-cells. These technologies were designed to address the complexity and functional heterogeneity of cancer biology and cellular immunity and to define biomarkers predicting clinical response to anticancer treatment. So far, there is still a lack of standardization of some of these immunological tests. The aim of this review is to overview the latest technologies for immune monitoring and to highlight critical steps involved in some of the FCM-based cellular immune assays. In particular, our laboratory is focused on melanoma vaccine research and thus our main goal was the validation of a functional multiparameter test (FMT) combining different functional and lineage markers to be applied in clinical trials involving patients with melanoma.

Breast cancer is immunogenic, and infiltrating immune cells in primary breast tumors convey important clinical prognostic and predictive information. Furthermore, the immune system is critically involved in clinical responses to some standard cancer therapies. Early breast cancer vaccine trials have established the safety and bioactivity of breast cancer immunotherapy, with hints of clinical activity. Novel strategies for modulating regulators of immunity, including regulatory T cells, myeloid-derived suppressor cells and immune checkpoint pathways (monoclonal antibodies specific for the cytotoxic T-lymphocyte antigen-4 or programmed death), are now available. In particular, immune checkpoint blockade has enormous therapeutic potential. Integrative breast cancer immunotherapies that strategically combine established breast cancer therapies with breast cancer vaccines, immune checkpoint blockade or both should result in durable clinical responses and increased cures.

Studies have shown that cancer requires two conditions for tumor progression: cancer cell proliferation and an environment permissive to and conditioned by malignancy. Chemotherapy aims to control the number and proliferation of cancer cells, but it does not effectively control the two best-known conditions of the tumor-permissive environment: neoangiogenesis and tolerogenic immunity. Many malignant diseases exhibit poor outcomes after treatment with chemotherapy. Therefore, we investigated the potential benefits of adding an induction regimen of antiangiogenesis and antitumor immunity to chemotherapy in poor outcome disease. In a prospective, randomized trial, we included patients with advanced, unresectable pancreatic adenocarcinomas, non-small cell lung cancer, or prostate cancer. Two groups of each primary condition were compared: group 1 (G1), n = 30, was treated with the standard chemotherapy and used as a control, and group 2 (G2), n = 30, was treated with chemotherapy plus an induction regimen of antiangiogenesis and antitumor immunity. This induction regimen included a low dose of metronomic cyclophosphamide, a high dose of Cox-2 inhibitor, granulocyte colony-stimulating factor, a sulfhydryl (SH) donor, and a hemoderivative that contained autologous tumor antigens released from patient tumors into the blood. After treatment, the G2 group demonstrated significantly longer survival, lower blood level of neoangiogenesis and immune-tolerance mediators, and higher blood levels of antiangiogenesis and antitumor immunity mediators compared with the G1 group. Toxicity and quality of life were not significantly different between the groups. In conclusion, in several advanced malignancies of different primary localizations, an increase in survival was observed by adding an induction regimen of antiangiogenesis and antitumor immunity to standard chemotherapy.

Lung cancer is the leading cause of cancer-related mortality worldwide, with non-small-cell lung cancer (NSCLC) accounting for approximately 85% of all cases. Most patients with NSCLC are diagnosed at an advanced stage and have a poor prognosis, with a 5-year survival rate of <5%. Despite the introduction of new chemotherapeutic agents and molecularly targeted drugs, outcomes remain poor, emphasising the need for new treatment approaches. Inducing or potentiating immune responses via immunotherapeutic manipulation is a viable treatment approach for lung cancer. Antigen-specific, tumour-cell, and dendritic cell-based vaccines have all been evaluated in lung cancer, and some have shown promising clinical activity in phase II trials. These include liposomal BLP25 vaccine (L-BLP25), which targets mucin 1, and melanoma-associated antigen 3 (MAGE-A3) antigen-specific cancer immunotherapeutic (ASCI), which targets MAGE-A3, a peptide expressed almost exclusively on tumour cells. MAGE-A3 ASCI is being evaluated in the adjuvant setting in a phase III trial of patients with early-stage NSCLC, while a phase III trial of L-BLP25 is enrolling patients with unresectable stage III NSCLC. T-cell modulating agents (e.g. antibodies against programmed death 1 and cytotoxic T-lymphocyte-associated antigen-4 [CTLA-4]) are also being investigated. For example, in patients with NSCLC treated with paclitaxel and carboplatin, the phased administration of ipilimumab (an antibody against CTLA-4) resulted in substantial improvements in immune-related progression-free survival compared with chemotherapy alone (5.7 versus 4.6 months; P = 0.05). Immunotherapy in lung cancer is starting to deliver promising results in clinical trials. However, further research will be required to establish the optimal timing of therapy (i.e. in the adjuvant or metastatic settings). In addition, it will be important to determine if immunotherapies are most effective when used alone or in combination with other agents.

The synthetic oligodeoxynucleotide CpG 7909, which contains unmethylated cytosine/guanine (CpG) motifs, has potent immunostimulatory effects when coadministered with NY-ESO-1 peptides or recombinant NY-ESO-1 protein, resulting in an enhanced cellular and humoral immune response against the vaccine antigen. In this study, we report the development of anti-CpG-ODN antibodies in 21 of 37 patients who received CpG 7909 either alone or as a vaccine adjuvant. Specific anti-CpG immunoglobulin G (IgG) antibody titers ranged from 1:400 to 1:100,000. The anti-CpG antibodies cross-reacted with other synthetic CpG-ODNs but not with the DNA of mixed bacterial vaccine and were shown to be phosphorothioate backbone specific. Vaccine-related severe side effects observed in some patients were most likely not related to the development of anti-CpG antibodies. In addition, anti-CpG antibodies did not have negative effects on the vaccine immune response. These results show that anti-CpG antibodies develop in humans against short unmethylated CpG dinucleotide sequences after administration of CpG 7909. Our data therefore substantiate the potency of CpG 7909 to directly stimulate human B-cells and suggest that anti-CpG antibody monitoring should be a part of ongoing and planned clinical trials with CpG-ODNs., (©2012 AACR.)

Peptides presented at the cell surface reflect the protein content of the cell; those on HLA class I molecules comprise the critical peptidome elements interacting with CD8 T lymphocytes. We hypothesize that peptidomes from ex vivo tumour samples encompass immunogenic tumour antigens. Here, we uncover >6000 HLA-bound peptides from HLA-A*02(+) glioblastoma, of which over 3000 were restricted by HLA-A*02. We prioritized in-depth investigation of 10 glioblastoma-associated antigens based on high expression in tumours, very low or absent expression in healthy tissues, implication in gliomagenesis and immunogenicity. Patients with glioblastoma showed no T cell tolerance to these peptides. Moreover, we demonstrated specific lysis of tumour cells by patients' CD8(+) T cells in vitro. In vivo, glioblastoma-specific CD8(+) T cells were present at the tumour site. Overall, our data show the physiological relevance of the peptidome approach and provide a critical advance for designing a rational glioblastoma immunotherapy. The peptides identified in our study are currently being tested as a multipeptide vaccine (IMA950) in patients with glioblastoma.

Soft tissue sarcomas are rare neoplasms, with approximately 9000 new cases in the USA every year. Unfortunately, during the past two decades, there has been little progress in the treatment of metastatic soft tissue sarcomas beyond the standard approaches of surgery, chemotherapy and radiation. Immunotherapy is a modality complementary to conventional therapy. It is appealing because functional antitumor activity could affect both local-regional and systemic disease, and act over a prolonged period of time. In this report, we review immunotherapeutic investigative strategies that are being developed, including several tumor vaccine, antigen vaccine and dendritic cell vaccine strategies.

Tumour and virus-infected cells are recognised by CD8+ cytotoxic T cells that, in response, are activated to eliminate these cells. In order to be activated, the clonotypic T-cell receptor (TCR) needs to encounter a specific peptide antigen presented by the membrane surface major histocompatibility complex (MHC) molecule. Cells that have undergone malignant transformation or viral infection present peptides derived from tumour-associated antigens or viral proteins on their MHC class I molecules. Therefore, disease-specific MHC-peptide complexes are desirable targets for immunotherapeutic approaches. One such approach transforms the unique fine specificity but low intrinsic affinity of TCRs to MHC-peptide complexes into high-affinity soluble antibody molecules endowed with a TCR-like specificity towards tumour or viral epitopes. These antibodies, termed TCR-like antibodies, are being developed as a new class of immunotherapeutics that can target tumour and virus-infected cells and mediate their specific killing. In addition to their therapeutic capabilities, TCR-like antibodies are being developed as diagnostic reagents for cancer and infectious diseases, and serve as valuable research tools for studying MHC class I antigen presentation.

Multiple myeloma remains an incurable disease although the prognosis has been improved by novel therapeutics and agents recently. Relapse occurs in the majority of patients and becomes fatal finally. Immunotherapy might be a powerful intervention to maintain a long-lasting control of minimal residual disease or to even eradicate disseminated tumor cells. Several tumor-associated antigens have been identified in patients with multiple myeloma. These antigens are expressed in a tumor-specific or tumor-restricted pattern, are able to elicit immune response, and thus could serve as targets for immunotherapy. This review discusses immunogenic antigens with therapeutic potential for multiple myeloma.

Cancer vaccines with optimal tumor-associated antigens show promise for anti-tumor immunotherapy. Recently, nano-sized vesicles, such as exosomes derived from tumors, were suggested as potential antigen candidates, although the total yield of exosomes is not sufficient for clinical applications. In the present study, we developed a new vaccine strategy based on nano-sized vesicles derived from primary autologous tumors. Through homogenization and sonication of tumor tissues, we achieved high yields of vesicle-bound antigens. These nanovesicles were enriched with antigenic membrane targets but lacked nuclear autoantigens. Furthermore, these nanovesicles together with adjuvant activated dendritic cells in vitro, and induced effective anti-tumor immune responses in both primary and metastatic melanoma mouse models. Therefore, autologous tumor-derived nanovesicles may represent a novel source of antigens with high-level immunogenicity for use in acellular vaccines without compromising safety. Our strategy is cost-effective and can be applied to patient-specific cancer therapeutic vaccination.