Fragment Antigen Research Articles

The reference intervals for CA125, CA15-3, CA19-9, CA72-4, AFP, CEA, NSE and CYFRA21-1

Tumor markers are noninvasive diagnostic tools for cancer. Their abnormal expression often occurs earlier than clinical symptoms or other detection signals. Appropriate reference intervals (RIs) of tumor markers are important for health evaluation, cancer diagnosis, therapy monitoring and prognosis assessment. In this study, we aimed to establish the RIs of cancer antigen 125 (CA125), CA15-3, CA19-9, CA72-4, alpha fetoprotein (AFP), carcino-embryonic antigen (CEA), neuron-specific enolase (NSE) and cytokeratin 19 fragment antigen 21–1 (CYFRA21-1) in apparently healthy Henan population. A total of 1705 apparently healthy participants (21–89 years) were recruited from five representative geographical regions in Henan province. Nonparametric 95th percentile intervals were used to define the RIs of CA125, CA15-3, CA19-9, CA72-4, AFP, CEA, NSE and CYFRA21-1. The test results of CA125, CA15-3, CA19-9, CA72-4, AFP, CEA, NSE and CYFRA21-1 can traceable to reference measurement procedures. The age- and gender-specific RIs of the tumor markers were established. We established age- and gender-specific RIs for CA125, CA15-3, CA19-9, CA72-4, AFP, CEA, NSE and CYFRA21-1. The newly established RIs should be more suitable for Henan population. It will be valuable for clinicians to make a medical diagnosis, therapeutic management decision and other physiological assessment.

Clinical efficacy and safety evaluation of pemetrexed combined with radiotherapy in treatment of patients with lung adenocarcinoma brain metastasis.

Clinical efficacy and adverse reactions of pemetrexed combined with stereotactic gamma-ray radiotherapy in the treatment of patients with lung adenocarcinoma brain metastasis in The First People's Hospital of Yunnan Province were evaluated. A total of 67 patients with lung adenocarcinoma brain metastasis in experimental group were treated with simple pemetrexed chemotherapy, and then with radiotherapy, followed by pemetrexed chemotherapy. Their treatment results were compared with those of 53 patients treated with simple gamma knife in control group. The results were analyzed by comparing the clinical efficacy, side reactions, serum level changes, and survival between the two groups. Among 67 patients in the experimental group, there were 16 cases of complete response (CR), 39 cases of partial response (PR), 7 cases of stable disease (SD) and 5 cases of progressive disease (PD), with an effective rate of 82.09% (55/67) and a tumor local control rate of 92.54% (62/67). Among 53 patients in the control group, there were 13 cases of CR, 20 cases of PR, 9 cases of SD and 11 cases of PD, with an effective rate of 62.26% (33/53) and a tumor local control rate of 79.25% (42/53). There were statistically significant differences in the effective rate and local control rate between the two groups (P<0.05). The 6-, 12- and 24-month survival rates in experimental group were higher than those in control group (P<0.05). The main adverse reactions after pemetrexed combined with radiotherapy were lower than those after simple radiotherapy (P<0.05). The expression levels of the tumor markers carcinoembryonic antigen (CEA) and cytokeratin fragment antigen 21-1 (CYFRA21-1) in the two groups of patients after treatment were lower than those before treatment (P<0.05). After treatment, the expression levels of serum CEA and CYFRA21-1 in the experimental group were significantly lower than those in the control group (P<0.05). Pemetrexed combined with radiotherapy in the treatment of lung adenocarcinoma brain metastasis is more effective than simple radiotherapy, with lighter adverse reactions, worthy of clinical application and promotion.

T Cell: Primary Culture from Peripheral Blood.

Peripheral blood mononuclear cells (PBMCs) can be used to assess cell-mediated immunity in general or via antigen-specific stimulation to detect previous exposure to a variety of antigens/allergens and to monitor the response to immunotherapies. Peripheral blood is the most common source of mononuclear cells for in vitro cultures, although mononuclear cells can be obtained from other sources involved in the allergic reaction. PBMCs from individuals previously exposed to an antigen proliferate in vitro when stimulated with the specific antigen. Proliferation is measured by the incorporation of (3H)-thymidine into newly synthesized DNA. This parameter is often used as an endpoint of lymphocyte stimulation induced by antigen or antigen fragments (e.g., synthetic peptides), mitogens, or anti-CD3/anti-CD28 combinations. The aim of this chapter is to describe the culture of T cells obtained from peripheral blood and the collection of cell supernatants for cytokine measurement.

Epitope Mapping via Phage Display from Single-Gene Libraries.

Antibodies are widely used in a large variety of research applications, for diagnostics and therapy of numerous diseases, primarily cancer and autoimmune diseases. Antibodies are binding specifically to target structures (antigens). The antigen-binding properties are not only dependent on the antibody sequence, but also on the discrete antigen region recognized by the antibody (epitope). Knowing the epitope is valuable information for the improvement of diagnostic assays or therapeutic antibodies, as well as to understand the immune response of a vaccine. While huge progress has been made in the pipelines for the generation and functional characterization of antibodies, the available technologies for epitope mapping are still lacking effectiveness in terms of time and effort. Also, no technique available offers the absolute guarantee of succeeding. Thus, research to develop and improve epitope mapping techniques is still an active field. Phage display from random peptide libraries or single-gene libraries are currently among the most exploited methods for epitope mapping. The first is based on the generation of mimotopes and it is fastened to the need of high-throughput sequencing and complex bioinformatic analysis. The second provides original epitope sequences without requiring complex analysis or expensive techniques, but depends on further investigation to define the functional amino acids within the epitope. In this book chapter, we describe how to perform epitope mapping by antigen fragment phage display from single-gene antigen libraries and how to construct these types of libraries. Thus, we also provide figures and analysis to demonstrate the actual potential of this technique and to prove the necessity of certain procedural steps.

Protective antigen domain 4 of Bacillus anthracis as a candidate for use as vaccine for anthrax.

Existing research for using the protective antigen (PA) of Bacillus anthracis as a vaccine component shows that protection against anthrax may be obtained using fragments of this protein. The aim of the research is to check whether the selected protein fragment of the protective antigen (domain 4) encoded by an appropriate nucleotide sequence of gene pag of B.anthracis, was expressed in the bacterial system of E. coli. In order to examine the selected sequence of the pag gene, a PCR reaction and a highly effective TOPO cloning strategy were used, followed by purification of the recombinant proteins and their detection by a western-blot method. In the planning of the PA4 antigen expression a higher level of effectiveness in production of small protein - domain 4 - was anticipated. As a result, the 139 amino acids protein fragment of B. anthracis PA (domain 4) was isolated. The research may have found the basis for in vivo research aimed at finding potential anthrax vaccine components.

Dissociation of the Dimer of the Intrinsically Disordered Domain of RNase Y upon Antibody Binding

Although RNase Y acts as the key enzyme initiating messenger RNA decay in Bacillus subtilis and likely in many other Gram-positive bacteria, its three-dimensional structure remains unknown. An antibody belonging to the rare immunoglobulin G (IgG) 2b λx isotype was raised against a 12-residue conserved peptide from the N-terminal noncatalytic domain of B. subtilis RNase Y (BsRNaseY) that is predicted to be intrinsically disordered. Here, we show that this domain can be produced as a stand-alone protein called Nter-BsRNaseY that undergoes conformational changes between monomeric and dimeric forms. Circular dichroism and size exclusion chromatography coupled with multiangle light scattering or with small angle x-ray scattering indicate that the Nter-BsRNaseY dimer displays an elongated form and a high content of α-helices, in agreement with the existence of a central coiled-coil structure appended with flexible ends, and that the monomeric state of Nter-BsRNaseY is favored upon binding the fragment antigen binding (Fab) of the antibody. The dissociation constants of the IgG/BsRNaseY, IgG/Nter-BsRNaseY, and IgG/peptide complexes indicate that the affinity of the IgG for Nter-BsRNaseY is in the nM range and suggest that the peptide is less accessible in BsRNaseY than in Nter-BsRNaseY. The crystal structure of the Fab in complex with the peptide antigen shows that the peptide adopts an elongated U-shaped conformation in which the unique hydrophobic residue of the peptide, Leu6, is completely buried. The peptide/Fab complex may mimic the interaction of a microdomain of the N-terminal domain of BsRNaseY with one of its cellular partners within the degradosome complex. Altogether, our results suggest that BsRNaseY may become accessible for protein interaction upon dissociation of its N-terminal domain into the monomeric form.

Immobilization and unbinding investigation of the antigen-antibody complex using theoretical and experimental techniques

Experimental results for the antibody known as immunoglobulin G – IgG interacting with phenobarbital were obtained via atomic force microscopy (AFM) and thereafter investigated using computer simulation modeling tools. Using molecular dynamics simulation and docking calculations, the energetically stable configurations of an immobilized antibody over a silicon surface were searched. Six stable configurations of the immobilized antibody over the silicon nitride surface covered by linker molecules were found. Although, only three of them (P1, P2, P5) maintained the Fragment antigen binding available for antigen interaction. Therefore, these configurations were equilibrated after reaching 100 ns molecular dynamics trajectory. The average interaction energy between the surface and the immunoglobulin G - IgG antibody in the P1, P2 and P5 configurations were −62.4 ± 2.4 kcal/mol; −54.3 ± 5.7 kcal/mol, and −360.9 ± 4.2 kcal/mol respectively. Phenobarbital was docked within the Fab domain of P1, P2, and P5 immobilized configurations and equilibrated with molecular dynamics for binding energy estimation. Then, steered molecular dynamics was performed to evaluate unbinding energy pathway between phenobarbital and IgG in each of the three-oriented IgG configurations. No significant differences were observed in the rupture force values (EP1 = 591 ± 13 pN, EP2 = 605 ± 18 pN, and EP5 = 610 ± 45 pN). In comparison, the average AFM experimental results were (641.6 ± 363.3 pN). Therefore, it is worth noting that P5 is the configuration with highest protein-surface interaction. Therefore, the force value calculated for the P5 orientation is statistically more favorable and it is the one to be compared to the experimental data. The agreement between experimental and theoretical results indicates a favorable presented for this study opening new perspectives for antigen-antibody evaluation.

Identifying the Best Marker Combination in CEA, CA125, CY211, NSE, and SCC for Lung Cancer Screening by Combining ROC Curve and Logistic Regression Analyses: Is It Feasible?

The detection of serum biomarkers can aid in the diagnosis of lung cancer. In recent years, an increasing number of lung cancer markers have been identified, and these markers have been reported to have varying diagnostic values. A method to compare the diagnostic value of different combinations of biomarkers needs to be established to identify the best combination. In this study, automatic chemiluminescence analyzers were employed to detect the serum concentrations of carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), cytokeratin 19 fragment (CY211), neuron-specific enolase (NSE), and squamous cell carcinoma antigen (SCC) in 780 healthy subjects, 650 patients with pneumonia, and 633 patients with lung cancer. Receiver operating characteristic (ROC) curve and logistic regression analyses were also used to evaluate the diagnostic value of single and multiple markers of lung cancer. The sensitivities of the five markers alone were lower than 65% for lung cancer screening in healthy subjects and pneumonia patients. SCC was of little value in screening lung cancer. After combining two or more markers, the areas under the curves (AUCs) did not increase with the increase in the number of markers. For healthy subjects, the best marker for lung cancer screening was the combination CEA + CA125, and the positive cutoff range was 0.577 CEA + 0.035 CA125 > 2.084. Additionally, for patients with pneumonia, the best screening markers displayed differences in terms of sex but not age. The best screening marker for male patients with pneumonia was the combination CEA + CY211 with a positive cutoff range of 0.008 CEA + 0.068 CY211 > 0.237, while that for female patients with pneumonia was CEA > 2.73 ng/mL, which could be regarded as positive. These results showed that a two-marker combination is more suitable than a multimarker combination for the serological screening of tumors. Combined ROC curve and logistic regression analyses are effective for identifying the best markers for lung cancer screening.

Effect of MRI combined with serum levels of CA153, CEA and CYFRA21-1 on the diagnostic efficacy of breast cancer patients

Objective To investigate the effect of color magnetic resonance imaging (MRI) combined with serum levels of carbohydrate antigen 153 (CA153), carcinoembryonic antigen (CEA) and cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) on the diagnostic efficacy of breast cancer patients. Methods From December 2015 to December 2017, 80 cases of breast cancer in our hospital were selected as the observation group. Another 98 patients with benign breast diseases as control group A, and 94 healthy women as control group B. The serum levels of CA153, CEA and CYFRA21-1in the three groups were measured by electrochemiluminescence and the level of the above serum indexes in patients with different disease stages (stage Ⅰ to Ⅱ, Ⅲ to Ⅳ) in the observation group were compared. The 3 groups were examined by color Doppler high frequency ultrasound. The specificity, sensitivity and accuracy of color Doppler ultrasound combined with serum CA153, CEA and CYFRA21-1 or detecting alone in the diagnosis of breast cancer analyzed. Results There were significant differences in the levels of serum CA153, CEA and CYFRA21-1 in the 3 groups (P<0.05), and the level of serum CA153, CEA and CYFRA21-1 in the observation group was higher than that in the control group A and the control group B (P<0.05). The levels of serum CA153, CEA and CYFRA21-1 in patients in stage Ⅲ to Ⅳ of the observation group were higher than those in patients in stage Ⅰ to Ⅱ (P<0.05). The sensitivity and accuracy of color Doppler ultrasound combined with serum CA153, CEA and CYFRA21-1 in the diagnosis of breast cancer were 96.25% (77/80) and 84.93% (231/272), respectively, which were all higher than those of serum CA153, CEA and CYFRA21-1 alone (P<0.05). And the sensitivity of combined diagnosis was higher than that of color Doppler high frequency ultrasound alone (P<0.05). Conclusions Color Doppler ultrasound combined with serum levels of CA153, CEA and CYFRA21-1 can significantly improve the sensitivity and accuracy of breast cancer diagnosis, reduce the risk of missed diagnosis, and provide a strong basis for diagnosis and treatment of breast cancer. Key words: Breast neoplasms/DI; Magnetic resonance imaging; CA-15-3 Antigen/BL; Carcinoembryonic antigen/BL; Keratin-19/BL; Antigens, neoplasm

Combined detection of estrogen and tumor markers is an important reference factor in the diagnosis and prognosis of lung cancer.

The correlation between lung cancer tumor markers and sex differences in lung cancer remains a clinical problem that is worthy of further study. This study investigated the significance of the combined detection of 17β-estrogen (E2) and tumor markers in the diagnosis and prognosis of lung cancer. A total of 174 patients, including 117 patients with non-small-cell lung cancer (NSCLC) and 57 patients with benign pulmonary lesions (BPL), were enrolled. An enzyme-linked immunosorbent assay was used to detect the expression of E2, carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) in patients with NSCLC and BPL to analyze the correlation between E2 and CEA, NSE or CYFRA21-1 expression, and its correlation with clinicopathological features and prognosis. The expression of tumor markers was then examined in different lung cancer cells (A549, H1795, H460, and SK-MES-1). The expression of tumor markers was detected by a real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. The expressions of p-p44/42 mitogen-activated protein kinase (MAPK) and phospho-AKT (p-AKT) were detected by Western blot analysis. The expression levels of E2, CEA, NSE, and CYFRA21-1 in patients with NSCLC were significantly higher than those in patients with BPL ( P < .05); E2 was positively correlated with tumor markers ( P < .01). Patients with a high expression of E2 and tumor markers showed a poor prognosis( P < .05). RT-quantitative PCR and Western blot analysis showed that the expression levels of CEA, NSE, CYFRA21-1, p-p44/42 MAPK, and p-AKT in the E2 group were higher than those in the other groups ( P < .05). These studies indicate that the interaction of E2 and tumor markers can significantly improve the role of tumor markers in the diagnosis and prognosis of lung cancer.

Abstract 2227: Multi-analyte profiling reveals relationships among circulating biomarkers in colorectal cancer

Abstract Introduction: Blood-based tests hold great promise as cancer diagnostics but until now have largely been restricted to the analysis of a single class of molecules (eg, circulating tumor DNA, platelet mRNA, circulating proteins). The ability to analyze multiple analytes simultaneously from the same biological sample may increase the sensitivity and specificity of such tests by exploiting independent information between signals. Here, we describe an experimental and analytical system that we developed and implemented for the integrated analysis of multiple analytes from a single blood sample, which revealed examples of both correlations and orthogonality among individual analytes. Methods: De-identified blood samples were obtained from healthy individuals, as well as individuals with pre-malignant conditions and stage I-IV colorectal cancer (CRC). After plasma separation, multiple types of analytes were assayed: cell-free DNA (cfDNA) content was assessed by low-coverage whole-genome sequencing (lcWGS) and whole-genome bisulfite sequencing (WGBS), cell-free microRNA (cf-miRNA) was assessed by small-RNA sequencing, and levels of circulating proteins were measured by quantitative immunoassay. Results: lcWGS of plasma cfDNA was able to identify CRC samples with high tumor fraction (&amp;gt;20%) on the basis of copy number variation (CNV) across the genome. High tumor fractions, while more frequent in late-stage cancer samples, were observed in some stage I and II patients. Aberrant signals in each of the three other analytes—cf-miRNA profiles discordant with those in healthy controls, genome-wide hypomethylation at LINE1 (long interspersed nuclear element 1) CpG loci, and elevated levels of circulating carcinoembryonic antigen (CEA) and cytokeratin fragment 21-1 (CYFRA 21-1) proteins—were also observed in cancer patients. Strikingly, aberrant profiles across analytes were indicative of high tumor fraction (as estimated from cfDNA CNV), rather than cancer stage. Conclusion: Our data suggest that tumor fraction is correlated with cancer stage but has a large potential range, even in early stage samples. Previous literature on blood-based screens for detection of cancer has displayed discordance in the claimed ability of different single analytes to detect early stage cancer. Tumor fraction may be able to explain the historical disagreement, as we found that aberrant profiles among cf-miRNA, cfDNA methylation, and circulating protein levels were more strongly associated with high tumor fraction than with late stage. These findings suggest that some positive “early stage” detection results may in fact be “high tumor fraction” detection results. Our results further demonstrate that assaying multiple analytes from a single sample may enable the development of classifiers that are reliable at low tumor fraction and for detecting pre-malignant or early-stage disease. Citation Format: Daniel Delubac, Eric Ariazi, Jonathan Berliner, Adam Drake, John Dulin, Riley Ennis, Erik Gafni, Kate Niehaus, Gabriel Otte, Jennifer Pecson, Girish Putcha, Corey Schaninger, Aarushi Sharma, Mike Singer, Abraham Tzou, Jill Waters, David Weinberg, Brandon White, Imran S. Haque. Multi-analyte profiling reveals relationships among circulating biomarkers in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2227.

Abstract 2712: Naptumomab Estafenatox induces T cell recognition, turning anti-PD-1 unresponsive "cold" tumors into "hot" responsive tumors

Abstract Tumor recognition is a key factor in checkpoint inhibitors (CPI) efficacy and acquired resistance. Lack or loss of tumor antigens expression or inefficient presentation prevents tumor cell recognition by T cells, inhibiting CPI anti-cancer effect. Naptumomab estafenatox (Nap) is a tumor-targeted superantigen (TTS) protein that increases tumor recognition by both coating tumor cells with bacterial-derived superantigens (SAg) as well as selectively expanding T cells lineages that can recognize it. Nap consists of a genetically engineered SAg, staphylococcal enterotoxin A (SEA/E-120), linked to a fragment antigen binding (Fab) moiety directed to the 5T4 oncofetal tumor-associated antigen expressed on many tumors. Here we present new pre-clinical results of synergistic anti-tumor effect of Nap (or its murine equivalent TTS) with anti-PD1, under conditions which mimic poor tumor recognition. In vitro studies with Nap investigated the effect of co-culturing SAg activated T cells with HCC827 NSCLC or MDA-MB 231 Triple-Negative Breast Cancer cells. Co-cultures of T cells with either cancer cell line resulted in an increase in tumor staining for PDL1, which was further elevated in the presence of Nap, probably due to increase in IFNγ secretion. The viability of HCC827 or MDA-MB-231 cells co-cultured with the SAg activated T cells was examined in the presence or absence of the PD1 inhibitor, Nap or the combination. Anti-PD1 alone had no effect on T cell mediated cytotoxicity, whereas Nap induced a significant cytotoxic effect, with the combination of anti-PD1 plus Nap producing the most significant reduction in tumor cell viability. Similar to the in vitro studies, the in vivo mouse studies using low immunogenic tumor models showed limited or no effect of anti-PD1 monotherapy. Although TTS alone increased T cells activation and infiltration into the tumor, the combination with anti-PD1 was significantly more effective in increasing serum cytokines, increasing the CD8:CD4 ratio in the tumor, reducing tumor burden and prolonging median survival. In summary, our studies show that combination of anti-PD1 with tumor-targeted superantigens overcomes the limited effect of anti-PD1 monotherapy in low immunogenic tumor models in vitro and in vivo. This novel combination may offer clinical benefit for CPI unresponsive patients, particularly in cases where tumor recognition is lost or restricted. Clinical phase 1-2 trials are planned with the combination of naptumomab estafenatox and anti-PD1/PDL1 blockade. Citation Format: Meir Azulay, Sveta Lifshits, Adam Fridman, Gunnar Hedlund, Marie Törngren, Michal Shahar. Naptumomab Estafenatox induces T cell recognition, turning anti-PD-1 unresponsive "cold" tumors into "hot" responsive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2712.

Clinical significance of combined detection of interleukin-6 and tumour markers in lung cancer

Tumour markers play an important role in the early diagnosis and guidance of prognosis of lung cancer. This study focused on the significance of combined monitoring of interleukin (IL)-6 and tumour markers in improving the specificity and sensitivity of lung cancer diagnosis and disease. The expression of IL-6, carcinoembryonic antigen (CEA), neuron-specific enolase (NSE) and cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) in serum of patients with non-small cell lung cancer (NSCLC) (n = 138) was significantly higher compared to patients with benign pulmonary lesions (BPL) (n = 60) and was associated with the clinicopathological features of NSCLC patients. The simultaneous increase in the expression of IL-6 and tumour markers worsened the prognosis of patients. Lung cancer cells were grouped into the blank control group, IL-6 group, niclosamide group (IL-6 pathway inhibitor, NIC) and IL-6 + NIC group. The expression of CEA, NSE, CYFRA21-1, p-Erk1/2 and p-AKT in the IL-6 group was significantly higher compared to the other groups. Therefore, the combined detection of IL-6 and tumour markers is critical to improve the specificity and sensitivity of lung cancer diagnosis. This in-depth study not only helped to elucidate the mechanism of how IL-6 promotes lung cancer but also provided new ideas and entry points to resolve the low specificity and sensitivity of lung cancer-related tumour markers.

Tribody [(HER2)2xCD16] Is More Effective Than Trastuzumab in Enhancing γδ T Cell and Natural Killer Cell Cytotoxicity Against HER2-Expressing Cancer Cells.

An enhanced expression of human epidermal growth factor receptor 2 (HER2, ErbB2) often occurs in an advanced stage of breast, ovarian, gastric or esophageal cancer, and pancreatic ductal adenocarcinoma (PDAC). Commonly, HER2 expression is associated with poor clinical outcome or chemoresistance in ovarian and breast cancer patients. Treatment with humanized anti-HER2 monoclonal antibodies, such as trastuzumab or pertuzumab, has improved the outcome of patients with HER2-positive metastatic gastric or breast cancer, but not all patients benefit. In this study, the bispecific antibody [(HER2)2xCD16] in the tribody format was employed to re-direct CD16-expressing γδ T lymphocytes as well as natural killer (NK) cells to the tumor-associated cell surface antigen HER2 to enhance their cytotoxic anti-tumor activity. Tribody [(HER2)2xCD16] comprises two HER2-specific single chain fragment variable fused to a fragment antigen binding directed to the CD16 (FcγRIII) antigen expressed on γδ T cells and NK cells. Our results revealed the superiority of tribody [(HER2)2xCD16] compared to trastuzumab in triggering γδ T cell and NK cell-mediated lysis of HER2-expressing tumor cells, such as PDAC, breast cancer, and autologous primary ovarian tumors. The increased efficacy of [(HER2)2xCD16] can be explained by an enhanced degranulation of immune cells. Although CD16 expression was decreased on γδ T cells in several PDAC patients and the number of tumor-infiltrating NK cells and γδ T cells was impaired in ovarian cancer patients, [(HER2)2xCD16] selectively enhanced cytotoxicity of cells from these patients. Here, unique anti-tumor properties of tribody [(HER2)2xCD16] are identified which beyond addressing HER2 overexpressing solid tumors may allow to treat with similar immunoconstructs combined with the adoptive transfer of γδ T cells and NK cells refractory hematological malignancies. A major advantage of γδ T cells and NK cells in the transplant situation of refractory hematological malignancies is given by their HLA-independent killing and a reduced graft-versus-host disease.

The combination of the blood based tumor biomarkers cytokeratin 19 fragments (CYFRA 21-1) and carcinoembryonic antigen (CEA) as a potential predictor of benefit from adjuvant chemotherapy in early stage squamous cell carcinoma of the lung (SCC)

ObjectivesTo determine whether the tumor biomarkers cytokeratin 19 fragment (CYFRA 21-1) and carcinoembryonic antigen (CEA), which are prognostic in early-stage non-small cell lung cancer (NSCLC), can predict which patients benefit from adjuvant chemotherapy (CTx). Materials and methodsSerum samples were collected preoperatively from patients with NSCLC who underwent resection. Samples were retrospectively analyzed for CYFRA 21-1 and CEA via electrochemiluminescence immunoassay. Recurrence-free survival (RFS) was compared for patients who received adjuvant CTx versus surgery alone, stratified based on the following prognostic classifications: (1) tumor stage (pT1‐2/N0 [stage I] or pT3/N0 or pT1‐2/N1 [stage II]), (2) biomarker-based risk score, (3) clinical characteristics. Absolute 2-year RFS rates were calculated via Kaplan-Meier estimations; statistical significance level: 0.05. Results227 patients were included (stage I: 69%; male: 67%; median age 65 years); 70 received adjuvant CTx. Median duration of sample collection was 58.8 months. All high-risk patients (by all three prognostic classifications) who received adjuvant CTx had a longer RFS versus those who received surgery alone. In patients with squamous cell carcinoma (SCC) classified as high risk by all three prognostic classifications, there was a benefit from adjuvant CTx versus surgery alone (tumor stage hazard ratio [HR] 4.9, p = 0.004; biomarker levels HR 9.4, p = 0.002; clinical characteristics HR 9.0, p = 0.003). None of the prognostic classifications were able to predict a benefit from adjuvant CTx in patients with adenocarcinoma. ConclusionBaseline CYFRA 21-1 and CEA levels may provide further information to help clinicians decide which patients with SCC should receive adjuvant CTx. Further evaluation of these biomarkers is warranted.

Acantholytic squamous cell carcinoma of the lung with marked lymphogenous metastases and high titers of myeloperoxidase-antineutrophil cytoplasmic antibodies: a case report

BackgroundAcantholytic squamous cell carcinoma (ASQCC), histologically characterized by intercellular bridge loosening, is recognized as a rare variant of squamous cell carcinoma (SQCC). ASQCC may demonstrate a worse prognosis than conventional SQCC. Pulmonary ASQCC is particularly rare; its biological behavior and prognostic data have not been reported.Case presentationWe report the clinical and autopsy findings of a 71-year-old Japanese man with pulmonary ASQCC. Pulmonary lesions, suggestive of idiopathic interstitial pneumonia, were radiologically observed 3 and 6 years prior to the patient’s most recent hospitalization; however, the patient did not undergo further medical examinations. Upon being discovered unconscious, the patient was admitted to our hospital. Dehydration and lower limb muscle weakness were noted, as were laboratory findings of coagulation abnormalities and renal dysfunction. Computed tomography helped confirm a 21-mm peripheral nodule in the upper left lobe of the lung, with associated swollen lymph nodes in the bilateral hilar, mediastinal, and para-aortic regions. Brain and spinal lesions, suggestive of neurological disturbances, were not found. Small cell lung carcinoma was suspected, upon admission, but high serum levels of squamous cell carcinoma antigen and cytokeratin-19 fragments were present. Therefore, advanced lung cancer, possibly SQCC, was diagnosed. The patient was treated with best supportive therapy, and died one month after admission. Hypercalcemia and high serum levels of parathyroid hormone-related protein (PTHrP) and myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA) titers were observed. Progressive renal insufficiency was absent due to improved renal function subsequent to hydration. An autopsy helped confirm the left lung tumor as an ASQCC associated with pulmonary lymphangitic carcinomatosis and multiple metastases in the lungs and lymph nodes. Skin lesions suggesting malignant tumors were absent. The metastatic lesions consisted largely of acantholytic tumor cells, and the lungs showed usual interstitial pneumonia pattern; vasculitis was absent.ConclusionsThis is the first reported case of pulmonary ASQCC resulting in an aggressive clinical course, with marked lymphogenous metastases and PTHrP-associated hypercalcemia. The high serum MPO-ANCA titers were clinicopathologically insignificant, but may have been related to the pulmonary interstitial lesion. Pulmonary ASQCC represents a highly malignant subset of lung cancer.

Evaluation of 177Lu[Lu]-CHX-A″-DTPA-6A10 Fab as a radioimmunotherapy agent targeting carbonic anhydrase XII

IntroductionDue to their infiltrative growth behavior, gliomas have, even after surgical resection, a high recurrence tendency. The approach of intracavitary radioimmunotherapy (RIT) is aimed at inhibiting tumor re-growth by directly administering drugs into the resection cavity (RC). Direct application of the radioconjugate into the RC has the advantage of bypassing the blood-brain barrier, which allows the administration of higher radiation doses than systemic application. Carbonic anhydrase XII (CA XII) is highly expressed on glioma cells while being absent from normal brain and thus an attractive target molecule for RIT. We evaluated a CA XII-specific 6A10 Fab (fragment antigen binding) labelled with 177Lu as an agent for RIT. Methods6A10 Fab fragment was modified and radiolabelled with 177Lu and characterized by MALDI-TOF, flow cytometry and radio-TLC. In vitro stability was determined under physiological conditions. Biodistribution studies, autoradiography tumor examinations and planar scintigraphy imaging were performed on SCID-mice bearing human glioma xenografts. ResultsThe in vitro CA XII binding capacity of the modified Fab was confirmed. Radiochemical purity was determined to be >90% after 72 h of incubation under physiological conditions. Autoradiography experiments proved the specific binding of the Fab to CA XII on tumor cells. Biodistribution studies revealed a tumor uptake of 3.0%ID/g after 6 h and no detectable brain uptake. The tumor-to-contralateral ratio of 10/1 was confirmed by quantitative planar scintigraphy. ConclusionThe radiochemical stability in combination with a successful in vivo tumor uptake shows the potential suitability for future RIT applications with the 6A10 Fab.

Endocytosed soluble cowpox virus protein CPXV012 inhibits antigen cross-presentation in human monocyte-derived dendritic cells.

Viruses may interfere with the MHC class I antigen presentation pathway in order to avoid CD8+ T cell-mediated immunity. A key target within this pathway is the peptide transporter TAP. This transporter plays a central role in MHC class I-mediated peptide presentation of endogenous antigens. In addition, TAP plays a role in antigen cross-presentation of exogenously derived antigens by dendritic cells (DCs). In this study, a soluble form of the cowpox virus TAP inhibitor CPXV012 is synthesized for exogenous delivery into the antigen cross-presentation route of human monocyte-derived (mo)DCs. We show that soluble CPXV012 localizes to TAP+ compartments that carry internalized antigen and is a potent inhibitor of antigen cross-presentation. CPXV012 stimulates the prolonged deposition of antigen fragments in storage compartments of moDCs, as a result of reduced endosomal acidification and reduced antigen proteolysis when soluble CPXV012 is present. Thus, a dual function can be proposed for CPXV012: inhibition of TAP-mediated peptide transport and inhibition of endosomal antigen degradation. We propose this second function for soluble CPXV012 can serve to interfere with antigen cross-presentation in a peptide transport-independent manner.

Linear epitopes in African swine fever virus p72 recognized by monoclonal antibodies prepared against baculovirus-expressed antigen.

Protein p72 is the major capsid protein of African swine fever virus (ASFV) and is an important target for test and vaccine development. Monoclonal antibodies (mAbs) were prepared against a recombinant antigenic fragment, from amino acid (aa) 20-303, expressed in baculovirus. A total of 29 mAbs were recovered and tested by immunofluorescent antibody (IFA) staining on ASFV Lisbon-infected Vero cells. Six antibodies were IFA-positive and selected for further characterization. Epitope mapping was performed against overlapping polypeptides expressed in E. coli and oligopeptides. Based on oligopeptide recognition, the mAbs were divided into 4 groups: mAb 85 (aa 165-171); mAbs 65-3 and 6H9-1 (aa 265-280); mAbs 8F7-3 and 23 (aa 280-294); and mAb 4A4 (aa 290-303). All mAbs were located within a highly conserved region in p72. This panel of antibodies provides the opportunity to develop new assays for the detection of ASFV antibody and antigen.

Serum Carcinoembryonic Antigen Level Predicts Cancer-Specific Outcomes of Resected Non-Small Cell Lung Cancer With Interstitial Pneumonia.

BackgroundIt has been well accepted that the prognosis of non-small cell lung cancer (NSCLC) patients with interstitial pneumonia (IP) is significantly poor. However, there are only a few studies that indicated the prognostic factors, especially tumor markers, among NSCLC patients with IP.MethodsForty-one NSCLC patients with IP who underwent surgery at our institution were included. Patients died of other diseases including postoperative acute exacerbation (AE) of IP were excluded. Univariate and multivariate analyses were calculated by the Cox proportional hazards regression model.ResultsThe 5-year cancer-specific survival of overall and stage I patients were 37.4% and 39.2%, respectively. The 5-year cancer-specific survival of patients with high serum carcinoembryonic antigen (CEA) level was 9.4%, while that with normal serum CEA level was 55.6%. However, serum cytokeratin-19 fragment (CYFRA 21-1) and squamous cell carcinoma-related antigen (SCC) levels were not associated with patients’ survival. Furthermore, serum CEA level was significantly associated with poorer cancer-specific survival in univariate and multivariate analyses.ConclusionsThis study demonstrated that serum CEA level might serve as an efficient prognostic indicator after surgery in NSCLC with IP.