Prognostic significance of spontaneous antibody responses against tumor‐associated antigens in malignant melanoma patients

Distribution, patterns and prognostic impact of spontaneous antibody responses against different tumor‐associated antigens (TAAs) in malignant melanoma patients are unknown so far and were investigated in this study for the first time in a large cohort at different stages of the disease, identifying new prognostic biomarkers for malignant melanoma. Serum samples from 365 melanoma patients (97 Stage I melanoma patients, 87 Stage II, 92 Stage III and 89 Stage IV) and 100 age and gender matched healthy control donors were analyzed. Samples were drawn at the time of diagnosis (Stages I–III) or at time of diagnosis of distant metastasis (Stage IV). Applying a novel multiplex assay, humoral immune responses against 29 TAAs were determined and the association between response and patient survival was investigated. Antibody responses were mainly found in melanoma patients and all tested antigens elicited immune responses in all disease stages. Antibody responses against single antigens were either associated with poor prognosis and/or shorter progression‐free survival (PFS) or had no influence. While in Stages I–III significant associations were observed between an antibody response and overall survival or PFS, among Stage IV patients, no significant association was found. Multivariate analyses identified specific humoral immune responses as prognostic factors independently of age, chemotherapy and immunotherapy. Antibody responses against specific TAA in Stage I‐III melanoma patients correlate with poor prognosis and/or shorter PFS. These results may help to design clinical studies in order to evaluate the potential of these responses as prognostic serological biomarkers.     

Inducible Hsp70 as target of anticancer immunotherapy: Identification of HLA-A*0201-restricted epitopes

The design of a broad application tumor vaccine requires the identification of tumor antigens expressed in a majority of tumors of various origins. We questioned whether the major stress-inducible heat shock protein Hsp70 (also known as Hsp72), a protein frequently overexpressed in human tumors of various histological origins, but not in most physiological normal tissues, constitutes a tumor antigen. We selected the p391 and p393 peptides from the sequence of the human inducible Hsp70 that had a high affinity for HLA-A*0201. These peptides were able to trigger a CTL response in vivo in HLA-A*0201-transgenic HHD mice and in vitro in HLA-A*0201+ healthy donors. p391- and p393-specific human and murine CTL recognized human tumor cells overexpressing Hsp70 in a HLA-A*0201-restricted manner. Tetramer analysis of TILs showed that these Hsp70 epitopes are targets of an immune response in many HLA-A*0201+ breast cancer patients. Hsp70 is a tumor antigen and the Hsp70-derived peptides p391 and p393 could be used to raise a cytotoxic response against tumors of various origins.     

The forkhead box M1 transcription factor as a candidate of target for anti‐cancer immunotherapy

The present study attempted to identify a target antigen for immunotherapy for cholangiocarcinoma. Forkhead box M1 (FOXM1) was selected as a candidate antigen based on the data of previous cDNA microarray analysis of clinical samples of cholangiocarcinoma. The level of FOXM1 mRNA was more than 4 times higher in cancer cells in comparison to adjacent normal epithelial cells, in all of 24 samples of cholangiocarcinoma tissues. An immunohistochemical analysis also detected FOXM1 protein in the cancer cells but not in the normal cells. Twenty‐three human FOXM1‐derived peptides predicted to bind to HLA‐A2 were analyzed to determine their ability to induce HLA‐A2‐restricted T cells in HLA‐A2 transgenic mice. FOXM1_362‐370 (YLVPIQFPV), FOXM1_373‐382 (SLVLQPSVKV), and FOXM1_640‐649 (GLMDLSTTPL) peptides primed HLA‐A2‐restricted cytotoxic T lymphocytes (CTLs) in the HLA‐A2 transgenic mice. Human CTL lines reactive to these 3 peptides could also be established from HLA‐A2‐positive healthy donors and cancer patients. Natural processing of the 3 epitopes from FOXM1 protein was confirmed by specific killing of HLA‐A2‐positive FOXM1‐transfectants by peptide‐induced CTLs. FOXM1 is expressed in various types of cancers and it is also functionally involved in oncogenic transformation and the survival of cancer cells. Therefore, FOXM1 may be a suitable target for immunotherapy against various cancers including cholangiocarcinoma.     

Identification of SPARC as a candidate target antigen for immunotherapy of various cancers

To establish efficient anticancer immunotherary, it is important to identify tumor‐associated antigens (TAAs) directing the immune system to attack cancer. A genome‐wide cDNA microarray analysis identified that secreted protein acidic and rich in cysteine (SPARC) gene is overexpressed in the gastric, pancreatic and colorectal cancer tissues but not in their noncancerous counterparts. This study attempted to identify HLA‐A24 (A*2402)‐restricted and SPARC‐derived CTL epitopes. We previously identified H‐2K^d‐restricted and SPARC‐derived CTL epitope peptides in BALB/c mice, of which H‐2K^d‐binding peptide motif is comparable with that of HLA‐A24 binding peptides. By using these peptides, we tried to induce HLA‐A24 (A*2402)‐restricted and SPARC‐reactive human CTLs and demonstrated an antitumor immune response. The SPARC‐A24‐1_143–151 (DYIGPCKYI) and SPARC‐A24‐4_225–234 (MYIFPVHWQF) peptides‐reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLA‐A24 (A*2402) positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both SPARC and HLA‐A24 (A*2402). Furthermore, the adoptive transfer of the SPARC‐specific CTLs could inhibit the tumor growth in nonobese diabetic/severe combined immunodeficient mice bearing human cancer cells expressing both HLA‐A24 (A*2402) and SPARC. These findings suggest that SPARC is a potentially useful target candidate for cancer immunotherapy.     

Heat shock protein DNAJB8 is a novel target for immunotherapy of colon cancer‐initiating cells

The aim of the present study was to establish cancer stem‐like cell/cancer‐initiating cell (CSC/CIC)‐targeting immunotherapy. The CSC/CIC are thought to be essential for tumor maintenance, recurrence and distant metastasis. Therefore they are reasonable targets for cancer therapy. In the present study, we found that a heat shock protein (HSP) 40 family member, DnaJ (Hsp40) homolog, subfamily B, member 8 (DNAJB8), is preferentially expressed in CSC/CIC derived from colorectal cancer (CRC) cells rather than in non‐CSC/CIC. Overexpression of DNAJB8 enhanced the expression of stem cell markers and tumorigenicity, indicating that DNAJB8 has a role in CRC CSC/CIC. A DNAJB8‐specific cytotoxic T lymphocyte (CTL) response could be induced by a DNAJB8‐derived antigenic peptide. A CTL clone specific for DNAJB8 peptide showed higher killing activity to CRC CSC/CIC compared with non‐CSC/CIC, and CTL adoptive transfer into CRC CSC/CIC showed an antitumor effect in vivo. Taken together, the results indicate that DNAJB8 is expressed and has role in CRC CSC/CIC and that DNAJB8 is a novel target of CRC CSC/CIC‐targeting immunotherapy.     

Identification of HLA‐A2‐ and A24‐restricted T‐cell epitopes derived from SOX6 expressed in glioma stem cells for immunotherapy

Malignant gliomas are the most aggressive human primary brain tumors and are currently incurable. Immunotherapies have the potential to target glioma and glioma stem cells (GSCs) that are resistant to conventional therapies. We previously identified SOX6 as a human glioma antigen and demonstrated that vaccination with SOX6 DNA induced cytotoxic T lymphocytes (CTLs) specific for glioma, thereby exerting therapeutic antitumor responses in glioma‐bearing mice. In this study, we attempted to identify SOX6‐derived peptides as specific targets for effective and safe T‐cell‐mediated immunotherapy targeting SOX6‐positive glioma and GSCs. In vitro stimulation with human leukocyte antigen (HLA)‐A*2402 (A24)‐restricted peptides, RFENLGPQL (SOX6₅₀₄) and PYYEEQARL (SOX6₆₂₈) or the HLA‐A*0201 (A2)‐restricted peptide, ALFGDQDTV (SOX6₄₄₇) was capable of inducing SOX6 peptide‐specific CTLs in peripheral blood mononuclear cells derived from healthy donors and glioma patients. These CTLs were able to lyse a majority of glioma cell lines and a GSC line derived from human glioblastoma in an HLA Class I‐restricted and an antigen‐dependent manner. Furthermore, peptide vaccines of SOX6₆₂₈, which was conserved in the murine SOX6 protein and expected to bind to major histocompatibility complex (MHC) H‐2^d, induced CTLs specific for SOX6₆₂₈ in H‐2^d mice. Normal autologous cells from mice, in which SOX6‐specific immune responses were generated, were not destroyed. These results suggest that these SOX6 peptides are potnetially immunogenic in HLA‐A24 or ‐A2 positive glioma patients and should be considered as a promising strategy for safe and effective T‐cell‐based immunotherapy of patients with gliomas.     

Heat shock protein 70 and tumor‐infiltrating NK cells as prognostic indicators for patients with squamous cell carcinoma of the head and neck after radiochemotherapy: A multicentre retrospective study of the German Cancer Consortium Radiation Oncology Group (DKTK‐ROG)

Tumor cells frequently overexpress heat shock protein 70 (Hsp70) and present it on their cell surface, where it can be recognized by pre‐activated NK cells. In our retrospective study the expression of Hsp70 was determined in relation to tumor‐infiltrating CD56⁺ NK cells in formalin‐fixed paraffin embedded (FFPE) tumor specimens of patients with SCCHN (N = 145) as potential indicators for survival and disease recurrence. All patients received radical surgery and postoperative cisplatin‐based radiochemotherapy (RCT). In general, Hsp70 expression was stronger, but with variable intensities, in tumor compared to normal tissues. Patients with high Hsp70 expressing tumors (scores 3–4) showed significantly decreased overall survival (OS; p = 0.008), local progression‐free survival (LPFS; p = 0.034) and distant metastases‐free survival (DMFS; p = 0.044), compared to those with low Hsp70 expression (scores 0–2), which remained significant after adjustment for relevant prognostic variables. The adverse prognostic value of a high Hsp70 expression for OS was also observed in patient cohorts with p16‐ (p = 0.001), p53‐ (p = 0.0003) and HPV16 DNA‐negative (p = 0.001) tumors. The absence or low numbers of tumor‐infiltrating CD56⁺ NK cells also correlated with significantly decreased OS (p = 0.0001), LPFS (p = 0.0009) and DMFS (p = 0.0001). A high Hsp70 expression and low numbers of tumor‐infiltrating NK cells have the highest negative predictive value (p = 0.00004). In summary, a strong Hsp70 expression and low numbers of tumor‐infiltrating NK cells correlate with unfavorable outcome following surgery and RCT in patients with SCCHN, and thus serve as negative prognostic markers.     

Development of a novel,quantitative protein microarray platform for the multiplexed serological analysis of autoantibodies to cancer‐testis antigens

The cancer‐testis antigens are a group of unrelated proteins aberrantly expressed in various cancers in adult somatic tissues. This aberrant expression can trigger spontaneous immune responses, a phenomenon exploited for the development of disease markers and therapeutic vaccines. However, expression levels often vary amongst patients presenting the same cancer type, and these antigens are therefore unlikely to be individually viable as diagnostic or prognostic markers. Nevertheless, patterns of antigen expression may provide correlates of specific cancer types and disease progression. Herein, we describe the development of a novel, readily customizable cancer‐testis antigen microarray platform together with robust bioinformatics tools, with which to quantify anti‐cancer testis antigen autoantibody profiles in patient sera. By exploiting the high affinity between autoantibodies and tumor antigens, we achieved linearity of response and an autoantibody quantitation limit in the pg/mL range—equating to a million‐fold serum dilution. By using oriented attachment of folded, recombinant antigens and a polyethylene glycol microarray surface coating, we attained minimal non‐specific antibody binding. Unlike other proteomics methods, which typically use lower affinity interactions between monoclonal antibodies and tumor antigens for detection, the high sensitivity and specificity realized using our autoantibody‐based approach may facilitate the development of better cancer biomarkers, as well as potentially enabling pre‐symptomatic diagnosis. We illustrated the usage of our platform by monitoring the response of a melanoma patient cohort to an experimental therapeutic NY‐ESO‐1‐based cancer vaccine; inter alia, we found evidence of determinant spreading in individual patients, as well as differential CT antigen expression and epitope usage.     

The cancer testis antigens CABYR-a/b and CABYR-c are expressed in a subset of colorectal cancers and hold promise as targets for specific immunotherapy

Introduction: Calcium-binding tyrosine phosphorylation-regulated protein (CABYR) is expressed in the human germ line but not in adult human tissues, thus, it is considered a cancer testis protein. The aim of this study is to evaluate the CABYR isoforms: a/b and c mRNA expression in colorectal cancer (CRC) and to determine if these proteins hold promise as vaccine targets.Materials and Methods: CABYR mRNA expression in a set of normal human tissues, including the testis, were determined and compared using semi-quantitative PCR. As regards the tumor and normal mucosal samples from study patients, RNA was extracted and cDNA generated after which quantitative PCR was carried out. Analysis of CABYR protein expressions by immunohistochemistry in tumor and normal colon tissues was also performed.Results: A total of 47 paired CRC and normal tissue specimens were studied. The percent of patients with a relative expression ratio of malignant to normal (M/N) tissues over 1 was 70% for CABYR a/b and 72% for CABYR c. The percent with both a M/N ratio over 1 and expression levels over 0.1% of testis was 23.4% for CABYR-a/b and 25.5% for CABYR c. CABYR expression in tumors was further confirmed by immunohistochemistry.Conclusions: CABYR a/b and c hold promise as specific immunotherapy targets, however, a larger and more diverse group of tumors (Stage 1-4) needs to be assessed and evaluation of blood for anti-CABYR antibodies is needed to pursue this concept.     

Immune checkpoint blockade combined with IL‐6 and TGF‐β inhibition improves the therapeutic outcome of mRNA‐based immunotherapy

Improved understanding of cancer immunology has provided insight into the phenomenon of frequent tumor recurrence after initially successful immunotherapy. A delicate balance exists between the capacity of the immune system to control tumor growth and various resistance mechanisms that arise to avoid or even counteract the host's immune system. These resistance mechanisms include but are not limited to (i) adaptive expression of inhibitory checkpoint molecules in response to the proinflammatory environment and (ii) amplification of cancer stem cells, a small fraction of tumor cells possessing the capacity for self‐renewal and mediating treatment resistance and formation of metastases after long periods of clinical remission. Several individual therapeutic agents have so far been developed to revert T‐cell exhaustion or disrupt the cross‐talk between cancer stem cells and the tumor‐promoting microenvironment. Here, we demonstrate that a three‐arm combination therapy—consisting of an mRNA‐based vaccine to induce antigen‐specific T‐cell responses, monoclonal antibodies blocking inhibitory checkpoint molecules (PD‐1, TIM‐3, LAG‐3), and antibodies targeting IL‐6 and TGF‐β—improves the therapeutic outcome in subcutaneous TC‐1 tumors and significantly prolongs survival of treated mice. Our findings point to a need for a rational development of multidimensional anticancer therapies, aiming at the induction of tumor‐specific immunity and simultaneously targeting multiple resistance mechanisms.     

The efficacy and safety of anti‐PD‐1/PD‐L1 antibodies combined with chemotherapy or CTLA4 antibody as a first‐line treatment for advanced lung cancer

Checkpoint inhibitors show promising efficacy in advanced lung cancer, especially in non‐small‐cell lung cancer (NSCLC). This meta‐analysis was conducted to explore the therapeutic efficacy and safety of anti‐PD‐1/PD‐L1 antibodies combined with chemotherapy or CTLA4 antibody as first‐line treatments for patients with advanced lung cancer. A systematic search was performed in databases for this system review and quantitative meta‐analysis. Twelve trials were finally enrolled in the meta‐analysis. Our analyses revealed that the combined overall response rate (ORR) and disease control rate (DCR) for immune checkpoint inhibitors combined with chemotherapy for the treatment of NSCLC were 47.0% (95% CI: 34.2%‐60.2%) and 80.9% (95% CI: 69.4%‐88.7%), respectively. The combined ORR and DCR for CTLA4 antibody combined with chemotherapy for the treatment of small‐cell lung cancer (SCLC) were 65.4% (61.1%‐69.5%) and 87.6% (84.5%‐90.2%), respectively. The combined six‐month progression‐free survival rates (PFSRs_6m) for NSCLC and SCLC were 50.2% (95% CI: 21.9%‐78.4%) and 30.7% (21.2%‐40.3%), respectively, and the OSRs_1y were 56.4% (39.1%‐73.7%) and 36.9% (33.3%‐40.5%), respectively. In addition, the combined ORR and DCR for the checkpoint inhibitors plus CTLA4 antibody treatment group in NSCLC were 29.6% (95% CI: 11.4%‐57.8%) and 48.7% (16.8%‐81.7%), respectively. In subgroup analyses, a significant improvement in PFS was observed in NSCLC and SCLC, with a combined hazard ratio and 95% confidence interval of 0.841 (0.737‐0.961) and 0.856 (0.756‐0.968), respectively. In summary, synergistic activity and an acceptable safety profile were observed with checkpoint inhibitor plus chemotherapy combination treatment in lung cancer.     

Time‐domain in vivo near infrared fluorescence imaging for evaluation of matriptase as a potential target for the development of novel,inhibitor‐based tumor therapies

Proteolytic enzymes expressed on the surface of tumor cells, and thus easily accessible to external interventions, represent useful targets for anticancer and antimetastatic therapies. In our study, we thoroughly evaluated matriptase, a trypsin‐like transmembrane serine protease, as potential target for novel inhibitor‐based tumor therapies. We applied time‐domain near infrared fluorescence (NIRF) imaging to characterize expression and activity of matriptase in vivo in an orthotopic AsPC‐1 pancreatic tumor model in nude mice. We show strong and tumor‐specific binding of intravenously injected Cy5.5 labeled antimatriptase antibody (MT‐Ab*Cy5.5) only to primary AsPC‐1 tumors and their metastases over time within living mice, taking into account fluorescence intensities and fluorescence lifetimes of the applied probes. Specific binding of MT‐Ab*Cy5.5 to tumor sites was confirmed by ex vivo NIRF imaging of tumor tissue, NIRF microscopy and by coregistration of the in vivo acquired NIRF intensity maps to anatomical structures visualized by flat‐panel volume computed tomography (fpVCT) in living mice. Moreover, using an activatable synthetic substrate S*DY‐681 we could clearly demonstrate that matriptase is proteolytically active in vitro as well as in vivo in tumor‐bearing mice, and that application of synthetic active‐site inhibitors having high affinity and selectivity toward matriptase can efficiently inhibit its proteolytic activity for at least 24 hr. We thus successfully applied NIRF imaging in combination with fpVCT to characterize matriptase as a promising molecular target for inhibitor‐based cancer therapies.     

Significance of transforming growth factor β1 as a new tumor marker for colorectal cancer

Transforming growth factor β1 (TGF‐β1) is thought to be involved in cancer growth and progression. TGF‐β1 changes to its active form after being secreted in its latent form. Our aim was to clarify the significance of plasma concentrations of active and total TGF‐β1 of patients with colorectal cancer. Plasma concentrations of active and total TGF‐β1 in 45 patients with colorectal cancer and 23 healthy volunteers were measured using ELISA and the activation rate (ratio of active to total TGF‐β1) was determined. Plasma concentrations of active TGF‐β1 (21.9 ± 12.8 pg/ml) were significantly higher in patients with colorectal cancer than in healthy volunteers (9.9 ± 5.9 pg/ml; p < 0.001, Welch's t‐test). Concentration of total TGF‐β1 was also significantly higher for patients with colorectal cancer (18.0 ± 13.0 ng/ml vs. 11.1 ± 6.4 ng/ml; p < 0.01, Welch's t‐test). However, there was no significant difference in the TGF‐β1 activation rate between the 2 groups. There was a correlation between Dukes' stage and plasma concentration of active or total TGF‐β1 (p < 0.01, Spearman's rank correlation test) and on day 7 the active TGF‐β1 levels for patients recovering from curative resection were similar to those of the control group of healthy volunteers. These results suggest that active TGF‐β1 might be used as a tumor marker for colorectal cancer. © 2001 Wiley‐Liss, Inc.     

Selective targeting by preS1 domain of hepatitis B surface antigen conjugated with phosphorylcholine‐based amphiphilic block copolymer micelles as a biocompatible,drug delivery carrier for treatment of human hepatocellular carcinoma with paclitaxel

Using dithioester‐capped 2‐methacryloyloxyethyl phosphorylcholine (MPC) as a macro chain transfer agent, a diblock copolymer was synthesized with n‐butyl methacrylate (BMA) as hydrophobic core‐forming blocks. The MPC–BMA unit was copolymerized with an immobilizable unit, p‐nitrophenylcarbonyloxyethyl methacrylate (NPMA). The NPMA moiety then was modified by the addition of preS1 domain of hepatitis B surface antigen (HBsAg). This micelle‐forming nanoparticle, the poly (MPC‐co‐BMA‐co‐NPMA) (PMBN) conjugated with preS1 enables solubilization of paclitaxel (PTX) with increased hepatotropism. The 50% inhibitory concentration (IC₅₀) values of PTX and PTX/PMBN‐preS1 against the human hepatocellular carcinoma cell line, HepG2, were 1,008 and 131 nM, respectively (p < 0.05). Conjugation of preS1 to PMBN enhanced strongly the synergistic inhibitory effect of paclitaxel on HepG2 cells in vitro, whereas such a change in IC₅₀ was not detected against the human squamous cell carcinoma cell line, A431. Tumor growth rates of a HepG2 xenograft in Balb/c nude mice after intraperitoneal injection of PTX, PTX/PMBN and PTX/PMBN‐preS1 were +97.9%, ?74.3% and ?96.2%*, respectively (*p < 0.05 versus PTX). The local paclitaxel levels after administration of the PMBN‐preS1 conjugate were determined in the xenografts by high‐performance liquid chromatography and were 8 times higher than that after administration of paclitaxel alone. No side effects attributable to PMBN‐preS1 were observed histologically in vital organs, and body weight loss was significantly less in the PTX/PMBN‐preS1 group. These studies demonstrate that PMBN‐preS1 may be used as a human hepatocyte‐specific drug delivery carrier without serious adverse effects. © 2008 Wiley‐Liss, Inc.