Promising Candidates For Cancer Immunotherapy Research Articles

Characterization of the Anti-PD-1 Antibody REGN2810 and Its Antitumor Activity in Human PD-1 Knock-In Mice.

The Programmed Death-1 (PD-1) receptor delivers inhibitory checkpoint signals to activated T cells upon binding to its ligands PD-L1 and PD-L2 expressed on antigen-presenting cells and cancer cells, resulting in suppression of T-cell effector function and tumor immune evasion. Clinical antibodies blocking the interaction between PD-1 and PD-L1 restore the cytotoxic function of tumor antigen-specific T cells, yielding durable objective responses in multiple cancers. This report describes the preclinical characterization of REGN2810, a fully human hinge-stabilized IgG4(S228P) high-affinity anti-PD-1 antibody that potently blocks PD-1 interactions with PD-L1 and PD-L2. REGN2810 was characterized in a series of binding, blocking, and functional cell-based assays, and preclinical in vivo studies in mice and monkeys. In cell-based assays, REGN2810 reverses PD-1-dependent attenuation of T-cell receptor signaling in engineered T cells and enhances responses of human primary T cells. To test the in vivo activity of REGN2810, which does not cross-react with murine PD-1, knock-in mice were generated to express a hybrid protein containing the extracellular domain of human PD-1, and transmembrane and intracellular domains of mouse PD-1. In these mice, REGN2810 binds the humanized PD-1 receptor and inhibits growth of MC38 murine tumors. As REGN2810 binds to cynomolgus monkey PD-1 with high affinity, pharmacokinetic and toxicologic assessment of REGN2810 was performed in cynomolgus monkeys. High doses of REGN2810 were well tolerated, without adverse immune-related effects. These preclinical studies validate REGN2810 as a potent and promising candidate for cancer immunotherapy. Mol Cancer Ther; 16(5); 861-70. ©2017 AACR.

The antigenic identity of human class I MHC phosphopeptides is critically dependent upon phosphorylation status.

Dysregulated post-translational modification provides a source of altered self-antigens that can stimulate immune responses in autoimmunity, inflammation, and cancer. In recent years, phosphorylated peptides have emerged as a group of tumour-associated antigens presented by MHC molecules and recognised by T cells, and represent promising candidates for cancer immunotherapy. However, the impact of phosphorylation on the antigenic identity of phosphopeptide epitopes is unclear. Here we examined this by determining structures of MHC-bound phosphopeptides bearing canonical position 4-phosphorylations in the presence and absence of their phosphate moiety, and examining phosphopeptide recognition by the T cell receptor (TCR). Strikingly, two peptides exhibited major conformational changes upon phosphorylation, involving a similar molecular mechanism, which focussed changes on the central peptide region most critical for T cell recognition. In contrast, a third epitope displayed little conformational alteration upon phosphorylation. In addition, binding studies demonstrated TCR interaction with an MHC-bound phosphopeptide was both epitope-specific and absolutely dependent upon phosphorylation status. These results highlight the critical influence of phosphorylation on the antigenic identity of naturally processed class I MHC epitopes. In doing so they provide a molecular framework for understanding phosphopeptide-specific immune responses, and have implications for the development of phosphopeptide antigen-specific cancer immunotherapy approaches.

Delta One T Cells for Immunotherapy of Chronic Lymphocytic Leukemia: Clinical-Grade Expansion/Differentiation and Preclinical Proof of Concept.

The Vδ1+ subset of γδ T lymphocytes is a promising candidate for cancer immunotherapy, but the lack of suitable expansion/differentiation methods has precluded therapeutic application. We set out to develop and test (preclinically) a Vδ1+ T-cell-based protocol that is good manufacturing practice compatible and devoid of feeder cells for prompt clinical translation. We tested multiple combinations of clinical-grade agonist antibodies and cytokines for their capacity to expand and differentiate (more than 2-3 weeks) Vδ1+ T cells from the peripheral blood of healthy donors and patients with chronic lymphocytic leukemia (CLL). We characterized the phenotype and functional potential of the final cellular product, termed Delta One T (DOT) cells, in vitro and in vivo (xenograft models of CLL). We describe a very robust two-step protocol for the selective expansion (up to 2,000-fold in large clinical-grade cell culture bags) and differentiation of cytotoxic Vδ1+ (DOT) cells. These expressed the natural cytotoxicity receptors, NKp30 and NKp44, which synergized with the T-cell receptor to mediate leukemia cell targeting in vitro When transferred in vivo, DOT cells infiltrated tumors and peripheral organs, and persisted until the end of the analysis without showing signs of loss of function; indeed, DOT cells proliferated and produced abundant IFNγ and TNFα, but importantly no IL17, in vivo Critically, DOT cells were capable of inhibiting tumor growth and preventing dissemination in xenograft models of CLL. We provide a clinical-grade method and the preclinical proof of principle for application of a new cellular product, DOT cells, in adoptive immunotherapy of CLL. Clin Cancer Res; 22(23); 5795-804. ©2016 AACR.

Promising cell-based immunotherapy using gamma delta T cells: together is better.

Gamma delta T-cell response to cellular stress signals expressed by tumor cells makes them promising candidates for cancer immunotherapy. The proof of concept for clinical scale propagation of polyclonal γδ T-cell lines with efficient in vitro and in vivo response against cancer is an important step in this direction.

Induction of antigen-specific immune responses by dendritic cells transduced with a recombinant lentiviral vector encoding MAGE-A3 gene

Melanoma antigen gene A3 (MAGE-A3) is aberrantly expressed in a number of cancer types. Because of its high specificity, MAGE-A3 has shown to be a promising candidate for cancer immunotherapy. Dendritic cells (DCs) have emerged as the natural agents for antigen delivery. DCs transduced with antigen may increase immune response and maintain immune durability. The aim of this study was to investigate the roles of DCs transduced with lentiviral vectors (LVs) encoding full-length MAGE-A3 gene in cancer immunotherapy . A LV containing full-length MAGE-A3 gene (rLV/MAGE-A3) was constructed. Reverse transcriptase-polymerase chain reaction and direct DNA sequencing were performed to verify the construct. Human DCs derived from umbilical cord blood were then transduced with rLV/MAGE-A3. The potency of rLV/MAGE-A3-transduced DCs was examined by measurement of surface markers and mixed lymphocyte reaction. The MAGE-A3-specific T-cell response induced by DCs was detected using the lactate dehydrogenase release assay. rLV/MAGE-A3 was constructed successfully and used to transduce DCs efficiently. DCs transduced with rLV/MAGE-A3 stably expressed MAGE-A3 and yielded high percentage of cells expressing CD80, CD86, and HLA-DR. rLV/MAGE-A3 transduction did not impair DCs viability and maturation at a multiplicity of infection of 30. The rLV/MAGE-A3-transduced DCs induced MAGE-A3-specific T lymphocytes that exhibited a significant lysis activity against MAGE-A3-bearing tumor cell lines (HuH-7 and SGC-7901). DC-directed rLV/MAGE-A3 efficiently induced antigen-specific immune responses, indicating the possibility of DC-based MAGE-A3 antigen vaccine as a promising strategy for treatment of MAGE-A3-associated cancer.

Enhancing The Efficacy and Specificity Of Antibody-Based T Cell Retargeting Strategies Against Hematological Malignancies

New promising candidates for cancer immunotherapy are bispecific antibodies (bsAbs) which have already shown a convincing anti-tumor effect in first clinical trials. BsAbs are composed of two single-chain fragments variable (scFvs), derived from the variable heavy and light chain of a monoclonal Ab (mAb), which bind to the activating CD3-complex on T cells and a tumor-associated antigen (TAA) on cancer cells. Consequently, the cross-linkage of effector and target cells by bsAbs results in an efficient T cell-mediated cancer cell killing. However, several serious adverse effects were observed in patients after application of Ab constructs. Most critical is an overall activation of T cells which bears the risk of a systemic release of pro-inflammatory cytokines.With respect to the idea that the CD3 binding of an Ab construct is critical for T cell activation, the CD3 domain has to be optimized in order to prevent or even reduce unspecific T cell responses. For this reason, we cloned several anti-CD3 scFvs derived from different anti-CD3 mAb clones and compared their functionality and safety. The anti-CD3 scFv with lowest risk of side effects was introduced in different anti-TAA bsAbs. Functional and safety studies in vitro revealed that development of each bsAb requires extensive and time-consuming optimization steps to gain high efficacy but low risk of side effects. To improve the process of Ab engineering, we recently introduced a novel modular Ab platform that can be rapidly and cost-effectively adapted for redirection of T cells to any TAA (Arndt and Feldmann et al. accepted for publication in Leukemia 2013). In this novel modular system the dual specificity of a conventional bsAb is distributed to two separate Ab modules, (i) the effector module and (ii) the target module. Only the complex of both Ab modules mediates the cross-linkage of effector and target cells resulting in T cell activation and redirected cancer cell lysis similar to conventional T cell engaging bsAbs. The universal effector module is a well optimized bsAb balancing efficacy and safety. It binds CD3 on T cells and the E5B9 tag of the target module. The individual target module comprises an anti-TAA scFv and the peptide epitope E5B9. For treatment of lymphoid or myeloid malignancies a series of different target modules were designed. In vitro and in vivo data clearly underline that the combination of the established effector module with different target modules efficiently activated T cells against hematological malignancies. Both CD4+ and CD8+ T cells from healthy donors were efficiently activated to kill TAA-positive tumor cell lines at low effector to target cell ratios and Ab concentrations in a tumor-specific manner. Most importantly, we could demonstrate that patient-derived T cells were able to kill autologous malignant cells upon Ab-mediated cross-linkage. Another unique feature of the modular system is that multispecific or multifunctional target modules could be easily included to further improve the therapeutic effect. In order to increase anti-tumor specificity and reduce the risk of tumor escape variants, bispecific target modules that recognize different TAAs at the same time, were constructed. Cytotoxicity assays investigating dual targeting of double-positive tumor cells via the modular system demonstrated that target cell lysis can be considerably improved in comparison to single targeting modules. Moreover, proliferation and cytokine release of redirected T cells could be enhanced by supplying costimulatory immunoligands (e.g. 4-1BBL and Ox40L) via target modules.In summary, we developed a multifunctional and highly flexible modular system based on an anti-CD3 domain with lowest risk of side effects. In contrast to conventional anti-TAA-anti-CD3 bsAbs, the development of a novel modular system for different clinical indications is much easier and less time-consuming. Once the effector module containing the critical CD3 domain is optimized, the modular system can be flexibly applied to target any TAA simply by replacing the target module. Application of bispecific target modules or providing costimulatory signals via the modular system might prolong immune responses and further increase anti-tumor specificity and activity. Finally, the novel modular platform represents a valuable and promising tool for cancer immunotherapy. Disclosures:No relevant conflicts of interest to declare.

Preferential expression of cancer/testis genes in cancer stem‐like cells: proposal of a novel sub‐category, cancer/testis/stem gene

Cancer/testis (CT) antigens encoded by CT genes are immunogenic antigens, and the expression of CT gene is strictly restricted to only the testis among mature organs. Therefore, CT antigens are promising candidates for cancer immunotherapy. In a previous study, we identified a novel CT antigen, DNAJB8. DNAJB8 was found to be preferentially expressed in cancer stem-like cells (CSCs)/cancer-initiating cells (CICs), and it is thus a novel CSC antigen. In this study, we hypothesized that CT genes are preferentially expressed in CSCs/CICs rather than in non-CSCs/-CICs and we examined the expression of CT genes in CSCs/CICs. The expression of 74 CT genes was evaluated in side population (SP) cells (=CSC) and main population (MP) cells (=non-CSC) derived from LHK2 lung adenocarcinoma cells, SW480 colon adenocarcinoma cells and MCF7 breast adenocarcinoma cells by RT-PCR and real-time PCR. Eighteen genes (MAGEA2, MAGEA3, MAGEA4, MAGEA6, MAGEA12, MAGEB2, GAGE1, GAGE8, SPANXA1, SPANXB1, SPANXC, XAGE2, SPA17, BORIS, PLU-1, SGY-1, TEX15 and CT45A1) showed higher expression levels in SP cells than in MP cells, whereas 10 genes (BAGE1, BAGE2, BAGE4, BAGE5, XAGE1, LIP1, D40, HCA661, TDRD1 and TPTE) showed similar expression levels in SP cells and MP cells. Thus, considerable numbers of CT genes showed preferential expression in CSCs/CICs. We therefore propose a novel sub-category of CT genes in this report: cancer/testis/stem (CTS) genes.

TAP expression level in tumor cells defines the nature and processing of MHC class I peptides for recognition by tumor‐specific cytotoxic T lymphocytes

We identified that the antigen preprocalcitonin (ppCT) is recognized on a human lung carcinoma by a cytotoxic T lymphocyte clone derived from autologous tumor-infiltrating lymphocytes. The antigenic peptide ppCT(16-25) is encoded by the gene calcitonin-related polypeptide alpha (CALCA), which codes for CT and is overexpressed in several lung carcinomas compared with normal tissues. The ppCT peptide is derived from the C-terminal region of the signal peptide and is processed independently of proteasomes and the transporter associated with antigen processing (TAP)1/TAP2 heterodimeric complexes. Instead, processing occurs within the endoplasmic reticulum by a novel mechanism involving signal pepsidase (SP) and signal peptide peptidase (SPP). Although lung cancer cells bearing the ppCT(16-25) epitope displayed low levels of TAP, restoration of TAP expression by interferon (IFN)-γ treatment or by TAP1/TAP2 gene transfer inhibited ppCT antigen presentation. Thus, the ppCT(16-25) human tumor epitope requires low TAP expression for efficient presentation. These results indicate that emerging SP-generated peptides represent alternative T cell targets that permit cytotoxic T lymphocytes to destroy TAP-impaired tumors, a process that helps to overcome tumor escape from CD8(+) T cell immunity. Additionally, our data suggest that ppCT is a promising candidate for cancer immunotherapy.

Interleukin-12: Clinical usage and molecular markers of cancer susceptibility

The last decade has seen the emergence of immunomodulators as therapeutic agents in cancer treatment. Interleukins (ILs) are a category of small cell-signaling molecules that organize communication and interaction between immune cells and therefore they could be used as perfect immunomodulators. IL-12 is a promising candidate for cancer immunotherapy since it plays a major role in development of antitumor immune response. Numerous studies report that IL-12 promotes an effective destruction of cancer cells both in vivo and in vitro. In addition, IL-12 has anti-angiogenic activity and it is able to dramatically decrease tumor-supportive activities of tumor-associated macrophages. The first part of the review is devoted to immunobiology of IL-12. Signaling pathways of IL-12 as well as clinical trials of this cytokine are discussed. The second part of the review is concerned on the inherited variations in IL-12A and IL-12B genes that could modulate cancer susceptibility, and as a consequence, possess predictive, therapeutic, or prognostic significance. It is known that functional single nucleotide polymorphisms (SNPs) in IL-12A and IL-12B genes may dramatically affect on protein expression level, or alter its functions, which may lead to immune disorders, autoimmune diseases, and eventually contribute to cancer occurrence. The list of genetic polymorphisms for further investigations might include the following: IL-12B_+1188A/C (rs3212227), IL-12A_+277G/A (rs568408), IL-12A_-798T/A (rs582054), IL-12A_-504T/G (rs190533), IL-12A_-1148T/C (rs2243123), and IL-12B_+16974 A/C. Perhaps, some of these SNPs may become an attractive target for oncogenomics and possibly could be used in programs of early cancer diagnosis as well as cancer prevention in the nearest future.

Immunological responses in cancer patients after vaccination with the therapeutic telomerase-specific vaccine Vx-001.

Vx-001, an HLA-A*0201 restricted telomerase (TERT)-specific anti-tumor vaccine, is composed of the 9-mer cryptic TERT(572) peptide and its optimized variant TERT(572Y). We have previously shown that Vx-001 is non-toxic, highly immunogenic and in vaccinated NSCLC patients early specific immune response is associated with prolonged survival. The aim of the present study was to investigate the specific T-cell immune response against Vx-001. Fifty-five patients with chemo-resistant advanced solid tumors were vaccinated with TERT(572Y) (2 subcutaneous injections) followed by TERT(572) peptide (4 subcutaneous injections) every 3weeks. Specific immune response was evaluated by IFN-γ and perforin ELISpot and intracellular cytokine staining assays. TERT-reactive T cells were detected in 27 (51%) out of 53 evaluable patients after the 2nd vaccination and in 22 (69%) out of 32 evaluable patients after the completion of 6 vaccinations. Immune responses developed irrespective of the stage of disease and disease status before vaccination. Patients with disease progression at study entry who developed a post-vaccination-induced immunological response had a significant overall survival benefit compared to the post-vaccination non-responders. The Vx-001 vaccine is a promising candidate for cancer immunotherapy since it can induce a TERT-specific T-cell immune response that is associated with prolonged survival.

Novel centrosome protein, TCC52, is a cancer‐testis antigen

A novel centrosome protein, TCC52, was identified as a cancer-testis (CT) antigen. The TCC52 gene was tissue-restricted in normal tissues but highly expressed in lung cancer tissues and some cancer cell lines. Immunoglobulin G antibody specific to TCC52 was detected in serum samples from patients with prostate cancer (59.4%, 69/116), cholangiocarcinoma (17.6%, 6/34), laryngeal cancer (8%, 8/100) and lung cancer (5.6%, 4/71) in patients, rather than from healthy donors. Based on its restricted expression pattern and immunogenicity in some types of tumor, TCC52, as a novel CT antigen, would be a promising candidate for cancer immunotherapy.

Preprocalcitonin signal peptide generates a cytotoxic T lymphocyte-defined tumor epitope processed by a proteasome-independent pathway

We identified an antigen recognized on a human non-small-cell lung carcinoma by a cytotoxic T lymphocyte clone derived from autologous tumor-infiltrating lymphocytes. The antigenic peptide is presented by HLA-A2 and is encoded by the CALCA gene, which codes for calcitonin and for the alpha-calcitonin gene-related peptide. The peptide is derived from the carboxy-terminal region of the preprocalcitonin signal peptide and is processed independently of proteasomes and the transporter associated with antigen processing. Processing occurs within the endoplasmic reticulum of all tumoral and normal cells tested, including dendritic cells, and it involves signal peptidase and the aspartic protease, signal peptide peptidase. The CALCA gene is overexpressed in medullary thyroid carcinomas and in several lung carcinomas compared with normal tissues, leading to recognition by the T cell clone. This new epitope is, therefore, a promising candidate for cancer immunotherapy.

Expression of MAGE-A1 and MAGE-A3 in Bone Marrow from Monoclonal Gammapathy to Myeloma Patients.

Multiple myeloma (MM) is an incurable tumor characterized by multifocal clonal expansion of malignant plasma cells within bone marrow (BM), with sometimes extramedullary location, i.e. mainly in peripheral blood (PB), pleural effusion or ascites. Cancer/testis antigens (CTA) are a category of tumor antigens with normal expression restricted to a male germ cells in the testis but not in adult somatic tissues. CTA are promising candidates for cancer immunotherapy. Presently, 44 distinct CT-antigen families have been identified. Presence of RNA transcripts encoding members of the MAGE gene family, as well as NY-ESO-1, CT7/MAGE-C1 in myeloma tumor cells and cell lines has been documented. In MM, cancer germ-line specific genes are preferentially expressed in advanced disease (up to 50%) and in almost all cell lines. Most of the genes of the MAGE-A family were found to be expressed in myeloma cells, although with various incidences. The aim of this study was to evaluate the posibility of using these genes as molecular markers of the progression MGUS to MM and the early relapse of the MM. Due to rare extramedullary location of malignant plasma cells we tried to undercover if expression is detecable in PB simultaneously with expression in the BM. Total of 151 samples from BM were evaluated: 83 samples from advanced myeloma, 12 samples of patients with early stage of MM who did not required treatment (smoldering MM 4x, stage IA 8x), 41 samples of MGUS patiens and 15 samples of normal healthy donors served as control group. In 15 advanced myeloma patients we simultaneously assessed BM sample and PB sample. Total RNA was evaluated by RT-PCR and then by real-time PCR using FRET probes. For relative quantification we used G6PDH housekeeping gene as external standard. As positive control we used myeloma cell line U266. Only 4,8% (2 /41) samples from MGUS patient showed expression of MAGE-A1 or MAGE-A3. 50% (6/12) patients with early stage of MM (IA and smoldering) showed expression of MAGE-A1 or MAGE-A3. 39,7% (33/83) of the samples from the advanced myeloma patients expressed at least 1 of these genes, or both (20 cases). We tested 15 BM from normal donors. All were negative. In 15 advanced myeloma BM samples we have observed expression in 8/15 cases but no expression in PB sample. We have confirmed that expression of MAGE is not present in samples of healthy donors. There is an obvious correlation between expression of the MAGE genes and early-late stage of the disease. Our evaluation confirmed the detection of low expression levels of MAGE-type mRNA in BM from patients with MGUS and early stage of MM. It is possible that MAGE antigen monitoring may predict the evolution towards more advanced disease. But due to need of separated myeloma cells or BM samples this metod can not be used for monitoring minimal residual disease in patients with MM.

Characterization of a cancer/testis (CT) antigen gene family capable of eliciting humoral response in cancer patients.

Cancer/testis (CT) antigens are immunogenic proteins expressed in normal gametogenic tissues and in different types of tumors. CT antigens are promising candidates for cancer immunotherapy, and the identification of novel CT antigens is a prerequisite for the development of cancer vaccines. We have identified a CT antigen, named CTSP-1, with partial similarity to the breast differentiation antigen NY-BR-1. CTSP-1 presents several splicing and polyadenylation variants and has a very restricted expression pattern among normal tissues. CTSP-1 is exclusively expressed in normal testis and is aberrantly expressed in 47.6% (10 of 21) of tumor cell lines and in 44.4% (75 of 169) of tumors from different histological types. The highest percentages of positive expression were observed in melanomas (59.0%) followed by prostate (58.0%) and lung (57.0%) tumors. CTSP-1 is part of a highly conserved gene family, and members of this family also have a restricted expression pattern and similar protein structure. Antibodies against members of this gene family were detected in 10% (14 of 141) of plasma samples from patients with a wide spectrum of tumors. The highest percentages of antibody response were observed in patients with prostate (20.8%), thyroid (20.0%), and breast (16.6%) tumors. Because of its very restricted expression pattern in normal tissues and immunogenicity in different types of tumors, CTSP-1 should be considered a promising candidate for cancer immunotherapy.

Characterization of Monocyte‐Derived Dendritic Cells Maturated With IFN‐α

Dendritic cells (DC) are promising candidates for cancer immunotherapy. These cells can be generated from peripheral blood monocytes cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). In order to obtain full functional capacity, maturation is required, but the most potent reagents such as LPS or polyriboinosinic polyribocytidylic acid (Poly I:C) are not approved for clinical use. We tested the ability of type I interferon (IFN) to induce such maturation. We found that 24-h IFN-alpha co-culture of day 7 monocyte-derived DC generated with GM-CSF and IL-4 induces increased numbers of DC positive for CD54 and CD40 together with the co-stimulatory molecule CD80 but not the activation marker CD83. Also, IFN-alpha maturation leads to an increase in IP-10 and MCP-1 chemokine secretion, but only a minor increase in IL-12p40 secretion. In line with this, maturation with IFN-alpha has only a small effect on induction of autologous T-cell stimulatory capacity of the DC. However, an increase in DC allogeneic T-cell stimulatory capacity was observed. These data suggest that IFN-alpha has a potential as a maturation agent used in DC-based cancer vaccine trials, but not as a single reagent.

Safety and immunogenicity of the optimized cryptic peptide TERT572Y in patients with advanced malignancies: A Phase I clinical study

2579 Background: TERT572Y, an optimized cryptic peptide homologous to TERT induces efficient antitumoral T cell cytotoxic immunity but not autoreactivity in vivo in HLA-A*0201 transgenic mice and healthy blood donors and prostate cancer patients (J. Clin. Invest., 2004, 113,425). A phase I trial evaluating the safety and immunogenicity of the cryptic TERT572Y peptide was conducted in HLA-A*0201 cancer patients. Methods: Nineteen patients with chemotherapy refractory and progressing malignant tumors were enrolled in the study. The vaccination protocol consisted of two injections of optimized TERT572Y peptide followed by four injections of native TERT572 peptide. Peptides were injected emulsified in Montanide ISA51. Patients were vaccinated with escalated doses of peptide ranging from 2 to 6 mg. Toxicity and peptide-specific immune responses were evaluated. Results: Fourteen patients completed the entire vaccination program. Only grade I toxicity was observed, affecting 13 of the 19 patients. TERT572Y-specific cytotoxic T cells were detected in the peripheral blood of 13 out of 14 evaluate patients, as early as 3 weeks after the 2nd vaccine injection. CTLs were fully functional and killed TERT-overexpressing tumor cells. Four (29%) of 14 evaluable patients experienced stable disease for a median of 10 months. Conclusions: TERT572Y peptide vaccine is well tolerated and effective in eliciting a specific T cell immunity. This is the first clinical trial demonstrating that cryptic peptides are promising candidates for cancer immunotherapy. No significant financial relationships to disclose.

Phenotypic and Functional Characterization of Clinical Grade Dendritic Cells Generated from Patients with Advanced Breast Cancer for Therapeutic Vaccination

Dendritic cells (DC) are promising candidates for cancer immunotherapy. However, it is not known whether in vitro-generated monocyte-derived DC from cancer patients are altered compared with DC from healthy donors. In a clinical phase I/II study, monocyte-derived DC were generated in vitro utilizing granulocyte macrophage colony-stimulating factor and rh-interleukin-4 (IL-4) and used for cancer immunotherapy. In this study, we tested the effect of various maturation cocktails and performed a comparative evaluation of the DC phenotype and functional characteristics. Polyriboinosinic polyribocytidylic acid (Poly I:C) + tumour necrosis factor-alpha (TNF-alpha) induced significant IL-12 p70 secretion, which was increased after addition of a decoy IL-10 receptor. The lymph node homing chemokine receptor CCR-7 expression was induced by TNF-alpha + IL-1beta + IL-6 + prostaglandin E2 but was not induced by Poly I:C + TNF-alpha. In general, DC from patients had an intermediate maturity phenotype with a significantly higher expression of CD40 and CD54 compared with healthy donors. In vitro analyses showed an unimpaired capacity of the patient-derived DC for antigen-specific (cytomegalovirus, tetanus and keyhole limpet haemocyanin) T-cell stimulation, whereas the allostimulatory capacity of patient-derived DC was significantly decreased. These data suggest that patient-derived DC are more differentiated but are less sensitive to maturation-inducing agents than DC obtained from healthy individuals.