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In normal cells, binding of the transmembrane protein CD47 to signal regulatory protein-α (SIRPα) on macrophages induces an antiphagocytic signal. Tumor cells hijack this pathway and overexpress CD47 to evade immune destruction. Macrophage antitumor activity can be restored by simultaneously blocking the CD47-SIRPα signaling axis and inducing a prophagocytic signal via tumor-opsonizing antibodies. We identified a novel, fully human mAb (BMS-986351) that binds SIRPα with high affinity. BMS-986351 demonstrated broad binding coverage across SIRPα polymorphisms and potently blocked CD47-SIRPα binding at the CD47 binding site in a dose-dependent manner. In vitro, BMS-986351 increased phagocytic activity against cell lines from solid tumors and hematologic malignancies, and this effect was markedly enhanced when BMS-986351 was combined with the opsonizing antibodies cetuximab and rituximab. A phase I dose-escalation/-expansion study of BMS-986351 for the treatment of advanced solid and hematologic malignancies is underway (NCT03783403)., Significance: Increasing the phagocytotic capabilities of tumor-associated macrophages by modulating macrophage-tumor cell surface signaling via the CD47-SIRPα axis is a novel strategy. Molecules targeting CD47 have potential but its ubiquitous expression necessitates higher therapeutic doses to overcome potential antigen sink effects. The restricted expression pattern of SIRPα may limit toxicities and lower doses of the SIRPα antibody BMS-986351 may overcome target mediated drug disposition while maintaining the desired pharmacology., (© 2024 The Authors; Published by the American Association for Cancer Research.)

Human B cells and their expressed antibodies are crucial in conferring immune protection. Identifying pathogen-specific antibodies following infection is possible due to enhanced humoral immunity against well-described molecules on the pathogen surface. However, screening for cancer-reactive antibodies remains challenging since target antigens are often not identified a priori and the frequency of circulating B cells recognizing cancer cells is likely very low. We investigated whether combined ex vivo culture of human B cells with three innate stimuli, interleukin-17 (IL-17), B-cell activation factor (BAFF), and the toll-like receptor 9 (TLR-9) agonist DNA motif CpG ODN 2006 (CpG), each known to activate B cells through different signalling pathways, promote cell activation, proliferation, and antibody production. Combined IL-17+BAFF+CpG prolonged B-cell survival and increased proliferation compared with single stimuli. IL-17+BAFF+CpG triggered higher IgG secretion, likely by activating differentiated, memory and class-switched CD19+CD20+CD27+IgD- B cells. Regardless of anti-FOLR antibody seropositive status, IL-17+BAFF+CpG combined with a monovalent tumour-associated antigen (folate receptor alpha [FOLR]) led to secreted antibodies recognizing the antigen and the antigen-expressing IGROV1 cancer cells. In a seropositive individual, FOLR stimulation favoured class-switched memory B-cell precursors (CD27-CD38-IgD-), class-switched memory B cells and anti-FOLR antibody production, while IL-17+BAFF+CpG combined with FOLR, promoted class-switched memory B-cell precursors and antibody-secreting (CD138+IgD-) plasma cells. Furthermore, IL-17+BAFF+CpG stimulation of peripheral blood B cells from patients with melanoma revealed tumour cell-reactive antibodies in culture supernatants. These findings suggest that innate signals stimulate B-cell survival and antibody production and may help identify low-frequency antigen-reactive humoral responses., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Immunology.)

Immunotherapy targeting the PD-1-PD-L1 axis yielded good results in treating different immunologically ''hot'' tumors. A phase II study revealed good therapeutic activity of pembrolizumab in selected prostatic carcinoma (PC)-patients. We performed a systematic literature review (PRISMA guidelines), which analyzes the immunohistochemical expression of PD-L1 in human PC samples and highlights the pre-analytical and interpretation variables. Interestingly, 29% acinar PCs, 7% ductal PCs, and 46% neuroendocrine carcinomas/tumors were PD-L1+ on immunohistochemistry. Different scoring methods or cut-off criteria were applied on variable specimen-types, evaluating tumors showing different clinic-pathologic features. The positivity rate of different PD-L1 antibody clones in tumor cells ranged from 3% (SP142) to 50% (ABM4E54), excluding the single case tested for RM-320. The most tested clone was E1L3N, followed by 22C3 (most used for pembrolizumab eligibility), SP263, SP142, and 28-8, which gave the positivity rates of 35%, 11-41% (depending on different scoring systems), 6%, 3%, and 15%, respectively. Other clones were tested in <200 cases. The PD-L1 positivity rate was usually higher in tumors than benign tissues. It was higher in non-tissue microarray specimens (41-50% vs. 15%), as PC cells frequently showed heterogenous or focal PD-L1-staining. PD-L1 was expressed by immune or stromal cells in 12% and 69% cases, respectively. Tumor heterogeneity, inter-institutional preanalytics, and inter-observer interpretation variability may account for result biases.

A syngeneic mouse model bearing a transplanted tumor is indispensable for the evaluation of the efficacy of immune checkpoint inhibitors (ICIs). However, few syngeneic mouse models of liver cancer are available. We established liver tumor cell lines (MHCF1 and MHCF5) from hepatitis C virus transgenic mice fed an atherogenic high-fat diet. MHCF1 and MHCF5 were successfully transplanted into the subcutaneous space of syngeneic C57BL/6 mice, in addition, they efficiently developed orthotopic tumors in the liver of syngeneic C57BL/6 mice. MHCF5 grew rapidly and showed a more malignant phenotype compared with MHCF1. Histologically, MHCF1-derived tumors were a combined type of hepatocellular carcinoma and MHCF5-derived tumors showed a sarcomatous morphology. Interestingly, MHCF1 and MHCF5 showed different sensitivity against an anti-PD1 antibody and MHCF5-derived tumors were resistant to this antibody. CD8 T cells infiltrated the MHCF1-derived tumors, but no CD8 T cells were found within the MHCF5-derived tumors. Gene expression profiling and whole-exon sequencing revealed that MHCF5 displayed the features of an activated cancer stem cell-like signature of sonic hedgehog and Wnt signaling. Therefore, these cell lines could be useful for the identification of new biomarkers and molecular mechanisms of ICI resistance and the development of new drugs against liver cancer.

The status of humoral immunity of cancer patients is not clear compared to cellular immunity because the ability of specific antibody production is difficult to analyze in vitro. We previously developed a humanized mouse model to evaluate antigen-specific antibody production by transplanting human peripheral blood mononuclear cells (PBMCs) into NOG-hIL-4-Tg mice (hu-PBL hIL-4 NOG). In this study, these mice were transplanted with PBMCs derived from breast cancer patients (BC) and immunized with a human epidermal growth factor receptor 2 (HER2) peptide, CH401MAP, to analyze humoral immunity of BCs. The hu-PBL hIL-4 NOG mice recapitulated immune environment of BCs as the ratio of CD8+/CD4+T cells was lower and that of PD-1 + T cells was higher compared to healthy donors (HDs). Diverse clusters were detected in BC-mouse (BC-M) plasma components involving immunoglobulins and complements unlike HD-M, and there was a significant diversity in CH401MAP-specific IgG titers in BC-M. The number of B cell clones producing high CH401MAP-specific IgG was not increased by immunization in BC-M unlike HD-M. These results demonstrated that the humoral immunity of BCs appeared as diverse phenotypes different from HDs in hu-PBL hIL-4 NOG mice, which may provide important information for the study of personalized medicine.

Antibodies against inhibitory immune checkpoint molecules (ICPMs), referred to as immune checkpoint inhibitors (ICIs), have gained a prominent place in cancer therapy. Several ICIs in clinical use have been engineered to be devoid of effector functions because of the fear that ICIs with preserved effector functions could deplete immune cells, thereby curtailing antitumor immune responses. ICPM ligands (ICPMLs), however, are often overexpressed on a sizeable fraction of tumor cells of many tumor types and these tumor cells display an aggressive phenotype with changes typical of tumor cells undergoing an epithelial-mesenchymal transition. Moreover, immune cells expressing ICPMLs are often endowed with immunosuppressive or immune-deviated functionalities. Taken together, these observations suggest that compounds with the potential of depleting cells expressing ICPMLs may become useful tools for tumor therapy. In this article, we summarize the current state of the art of these compounds, including avelumab, which is the only ICI targeting an ICPML with preserved effector functions that has gained approval so far. We also discuss approaches allowing to obtain compounds with enhanced tumor cell-depleting potential compared to native antibodies. Eventually, we propose treatment protocols that may be applied in order to optimize the therapeutic efficacy of compounds that deplete cells expressing ICPMLs.

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.

Radionuclide molecular imaging of cancer-specific targets is a promising method to identify patients for targeted antibody therapy. Radiolabeled full-length antibodies however suffer from slow clearance, resulting in high background radiation. To overcome this problem, a pretargeting system based on complementary peptide nucleic acid (PNA) probes has been investigated. The pretargeting relies on sequential injections of primary, PNA-tagged antibody and secondary, radiolabeled PNA probe, which are separated in time, to allow for clearance of non-bound primary agent. We now suggest to include a clearing agent (CA), designed for removal of primary tumor-targeting agent from the blood. The CA is based on the antibody cetuximab, which was conjugated to PNA and lactosaminated by reductive amination to improve hepatic clearance. The CA was evaluated in combination with PNA-labelled trastuzumab, T-ZHP1, for radionuclide HER2 pretargeting. Biodistribution studies in normal mice demonstrated that the CA cleared ca. 7 times more rapidly from blood than unmodified cetuximab. Injection of the CA 6 h post injection of the radiolabeled primary agent [ 131 I]I-T-ZHP1 gave a moderate reduction of the radioactivity concentration in the blood after 1 h from 8.5 ± 1.8 to 6.0 ± 0.4%ID/g. These proof-of-principle results could guide future development of a more efficient CA.

Targeted clearance of colorectal cancer stem cells (CCSCs) has become a novel strategy for tumor immunotherapy. Molecule mucin1 (MUC1) is one of targetable cell surface antigens in CCSCs. However, the critical role of MUC1 in anti-tumor effects of CCSC vaccine remains unclear. In the present study, we showed that MUC1 may be required for CCSC vaccine to exert tumor immunity. CD133 + CCSCs were isolated from CT26 cell line using a magnetic-activated cell sorting system, and MUC1 shRNA or recombinant plasmid was further used to decrease or increase the expression of MUC1 in CD133 + CCSCs. Mice were subcutaneously immunized with the CCSC lysates, MUC1 knockin CCSCs, and MUC1 knockdown CCSCs respectively, followed by a challenge with CT26 cells. We found that CCSC vaccine significantly reduced the tumor growth via a target killing of CCSCs as evidenced by a decrease of CD133 + cells and ALDH + cells in tumors. Moreover, CCSC vaccine markedly increased the cytotoxicity of NK cells and the splenocytes, and promoted the release of IFN-γ, Perforin, and Granzyme B, and also reduced the TGF-β1 expression. Additionally, CCSC vaccination enhanced the antibody production and decreased the myeloid derived suppressor cells and Treg subsets. More importantly, MUC1 knockdown partly impaired the anti-tumor efficacy of CCSC vaccine, whereas MUC1 overexpression dramatically enhanced the CCSC vaccine immunity. Overall, these results reveal a novel role and molecular mechanisms of MUC1 in CCSC vaccine against colorectal cancer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: Ovarian cancer has only a 17% 5-year survival rate in patients diagnosed with late stage disease. Tumor-associated glycoprotein-72 (TAG72), expressed in 88% of all stages of ovarian cancer, is an excellent candidate for antibody-targeted therapy, as it is not expressed in normal human adult tissues, except in the secretory endometrium., Methods: Using the clinically relevant anti-TAG72 murine monoclonal antibody CC49, we evaluated antibody drug conjugates (ADCs) incorporating the highly potent, synthetic antimitotic agent monomethylauristatin E (MMAE). MMAE was conjugated to CC49 via reduced disulfides in the hinge region, using three different types of linker chemistry, vinylsulfone (VS-MMAE), bromoacetamido (Br-MMAE), and maleimido (mal-MMAE)., Results: The drug antibody ratios (DARs) of the three ADCs were 2.3 for VS-MMAE, 10 for Br-MMAE, and 9.5 for mal-MMAE. All three ADCs exhibited excellent tumor to blood ratios on PET imaging, but the absolute uptake of CC49-mal-MMAE (3.3%ID/g) was low compared to CC49-Br-MMAE (6.43%ID/g), at 142 hours. Blood clearance at 43 hours was 38% for intact CC49, about 24% for both CC49-VS-MMAE and CC49-Br-MMAE, and 7% for CC49-mal-MMAE. CC49-VS-MMAE was not further studied due to its low DAR, while CC49-mal-MMAE was ineffective in the OVCAR3 xenograft likely due to its rapid blood clearance. In contrast, CC49-Br-MMAE treated mice exhibited an average of a 15.6 day tumor growth delay and a 40% increase in survival vs controls with four doses of 7.5 or 15 mg/kg of CC49-Br-MMAE., Conclusion: We conclude that CC49-Br-MMAE with a high DAR and stable linker performs well in a difficult to treat solid tumor model., (© 2020 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

The identification of residual leukemia following therapy, termed minimal or measurable residual disease (MRD), has emerged as one of the most important prognostic factors for patients with acute leukemia, including acute myeloid leukemia (AML). Flow cytometry is a preferred method for MRD detection due to its general applicability and the rapid results that it makes available. In this article, the basic protocol outlines a simple and efficient method for the labeling of hematopoietic cells from bone marrow or peripheral blood with a panel of monoclonal antibodies designed both to highlight patterns of normal maturation and allow identification of neoplastic hematopoietic progenitor populations with a high degree of sensitivity and specificity. The method was developed in a clinical laboratory setting for the diagnosis of myeloid stem cell disorders and neoplasms, and has been extensively validated both technically and clinically for the detection of MRD in AML. © 2020 The Authors. Basic Protocol: Staining and flow cytometry for AML minimal residual disease detection Support Protocol: Analysis and interpretation of data for AML minimal residual disease detection., (© 2020 The Authors.)

High expression levels of the tumor-associated antigen MUC1 have been correlated with tumor aggressiveness, poor response to therapy, and poor survival in several tumor types, including breast, pancreatic, and epithelial ovarian cancer. Herein, we report the synthesis, characterization, and in vivo evaluation of a novel radioimmunoconjugate for the immuno-positron emission tomography (immunoPET) imaging of MUC1 expression based on the AR20.5 antibody. To this end, we modified AR20.5 with the chelator desferrioxamine (DFO) and labeled it with the positron-emitting radiometal zirconium-89 (t 1/2 ~3.3 d) to produce [ 89 Zr]Zr-DFO-AR20.5. In subsequent in vivo experiments in athymic nude mice bearing subcutaneous MUC1-expressing ovarian cancer xenografts, [ 89 Zr]Zr-DFO-AR20.5 clearly delineated tumor tissue, producing a tumoral activity concentration of 19.1 ± 6.4 percent injected dose per gram (%ID/g) at 120 h post-injection and a tumor-to-muscle activity concentration ratio of 42.4 ± 10.6 at the same time point. Additional PET imaging experiments in mice bearing orthotopic MUC1-expressing ovarian cancer xenografts likewise demonstrated that [ 89 Zr]Zr-DFO-AR20.5 enables the visualization of tumor tissue-including metastatic lesions-with promising tumor-to-background contrast.

Purpose of Review: The aim of this study is to provide a comprehensive overview of the current insight about the clinical utility of cancer-associated autoantibodies (CAAs) as biomarkers in paraneoplastic myositis syndrome (PMS). In addition, the possible mechanisms of the relationship between malignancy and myositis onset are discussed., Recent Findings: It has become increasingly clear that a subgroup of the myositis-specific autoantibodies could be considered as CAAs because they are closely related to the PMS. Increased risk of cancer was found in patients with antitranscriptional intermediary factor 1-γ (TIF1-γ), antinuclear matrix protein-2 (NXP-2), anti3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) or antismall ubiquitin-like modifier 1-activating enzyme (SAE) antibodies. However, the diagnosing sensitivity and specificity of these CAAs for PMS are different among different cohort studies. Abnormally expressed or mutated autoantigen genes in tumor could possibly induce cross immunity against self-proteins and subsequently lead to the development of PMS., Summary: Anti-TIF1-γ, anti-NXP-2, anti-HMGCR and anti-SAE antibodies may act as CAAs in PMS. It is necessary to closely screen and monitor for cancer in patients with CAAs. The recent studies of the relationship between CAAs and PMS provided important new insights into the disease mechanisms.

Objective: To date, The Cancer Genome Atlas (TCGA) has provided the most extensive molecular characterization of invasive cervical cancer (ICC). Analysis of reverse phase protein array (RPPA) data from TCGA samples showed that cervical cancers could be stratified into 3 clusters exhibiting significant differences in survival outcome: hormone, EMT, and PI3K/AKT. The goals of the current study were to: 1) validate the TCGA RPPA results in an independent cohort of ICC patients and 2) to develop and validate an algorithm encompassing a small antibody set for clinical utility., Methods: Subjects consisted of 2 ICC patient cohorts with accompanying RPPA and clinical-pathologic data: 155 samples from TCGA (TCGA-155) and 61 additional, unique samples (MCW-61). Using data from 173 common RPPA antibodies, we replicated Silhouette clustering analysis in both ICC cohorts. Further, an index score for each patient was calculated from the survival-associated antibodies (SAAs) identified using Random survival forests (RSF) and the Cox proportional hazard regression model. Kaplan-Meier survival analysis and the log-rank test were performed to assess and compare cluster or risk group survival outcome., Results: In addition to validating the prognostic ability of the proteomic clusters reported by TCGA, we developed an algorithm based on 22 unique antibodies (SAAs) that stratified women with ICC into low-, medium-, or high-risk survival groups., Conclusions: We provide a signature of 22 antibodies which accurately predicted survival outcome in 2 separate groups of ICC patients. Future studies examining these candidate biomarkers in additional ICC cohorts is warranted to fully determine their clinical potential., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Multidrug resistance is one of the leading causes of chemotherapy failure in cancer patients. Early detection and capture of drug-resistant tumor cells can facilitate the monitoring of the therapy process and improve the prognosis of patients. In this study, novel P-glycoprotein (P-gp) antibody modified porous hydrogel particles are proposed for drug-resistant tumor cells capture. The hydrogel particles employ a highly biocompatible hydrogel, methacrylate gelatin (GelMA), as the carrier and replicate from the silica colloidal crystal beads. By the modification of P-gp antibody probes on their surfaces, the hydrogel particles are endowed with the ability to capture drug-resistant tumor cells, which overexpress specific components of P-gp on their membranes. Additionally, the acquired ordered porous nanostructure of the particles can provide not only more surface area for antibody immobilization but also a nanopatterned platform for highly efficient target cell capture. The above advantages make the porous hydrogel particles ideal for efficient capture and detection of the drug-resistant tumor cells, which can be expected to facilitate the point-of-care pharmacotherapy and promisingly improve the patient outcomes., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Little is known about the in vivo impacts of targeted therapy on melanoma cell abundance and protein expression. Here, 21 antibodies were added to an established melanoma mass cytometry panel to measure 32 cellular features, distinguish malignant cells, and characterize dabrafenib and trametinib responses in BRAF V600mut melanoma. Tumor cells were biopsied before neoadjuvant therapy and compared to cells surgically resected from the same site after 4 weeks of therapy. Approximately 50,000 cells per tumor were characterized by mass cytometry and computational tools t-SNE/viSNE, FlowSOM, and MEM. The resulting single-cell view of melanoma treatment response revealed initially heterogeneous melanoma tumors were consistently cleared of Nestin-expressing melanoma cells. Melanoma cell subsets that persisted to week 4 were heterogeneous but expressed SOX2 or SOX10 proteins and specifically lacked surface expression of MHC I proteins by MEM analysis. Traditional histology imaging of tissue microarrays from the same tumors confirmed mass cytometry results, including persistence of NES- SOX10+ S100β+ melanoma cells. This quantitative single-cell view of melanoma treatment response revealed protein features of malignant cells that are not eliminated by targeted therapy., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Lymph nodes (LNs) are central in the generation of adaptive immune responses. Follicular helper CD4 T (Tfh) cells, a highly differentiated CD4 population, provide critical help for the development of antigen-specific B cell responses within the germinal center. Throughout the past decade, numerous studies have revealed the important role of Tfh cells in Human Immunodeficiency Virus (HIV) pathogenesis as well as in the development of neutralizing antibodies post-infection and post-vaccination. It has also been established that tumors influence various immune cell subsets not only in their proximity, but also in draining lymph nodes. The role of local or tumor associated lymph node Tfh cells in disease progression is emerging. Comparative studies of Tfh cells in chronic infections and cancer could therefore provide novel information with regards to their differentiation plasticity and to the mechanisms regulating their development.

A promising approach capitalizing on the specific and highly sensitive characteristics of the body's own immune system is demonstrated in the context of revealing pancreatic ductal adenocarcinoma cancer (PDAC). IgA from a local biofluid called gastrointestinal lavage fluid (GLF) is used to investigate glycan reactivity to show the potential of this approach. IgA antibody responses, just as with IgG, result in amplification of a small signal which aids in detecting changes from a healthy state. IgA from GLF was screened against glycan arrays containing 609 glycan structures to investigate differential binding patterns associated with the disease. Samples included PDAC (n = 14) and non-PDAC (n = 6). Non-PDAC conditions included samples from healthy patients and the potentially confounding conditions of colon cancer and its precancerous lesion, colon adenoma. Results demonstrated characteristic reactivity in the PDAC sample group to a glycan structure. Also, IgA non-reactive motifs arose showing remarkable consistency within and between sample groups. While sample sizes are too small to identify putative biomarkers, these data show the use of IgA from GLF to be a promising avenue of research for local disease biomarker discovery.

Methods for isolation and quantification of circulating tumor cells (CTCs) are attracting more attention every day, as the data for their unprecedented clinical utility continue to grow. However, the challenge is that CTCs are extremely rare (as low as 1 in a billion of blood cells) and a highly sensitive and specific technology is required to isolate CTCs from blood cells. Methods utilizing microfluidic systems for immunoaffinity-based CTC capture are preferred, especially when purity is the prime requirement. However, antibody immobilization strategy significantly affects the efficiency of such systems. In this study, two covalent and two bioaffinity antibody immobilization methods were assessed with respect to their CTC capture efficiency and selectivity, using an anti-epithelial cell adhesion molecule (EpCAM) as the capture antibody. Surface functionalization was realized on plain SiO 2 surfaces, as well as in microfluidic channels. Surfaces functionalized with different antibody immobilization methods are physically and chemically characterized at each step of functionalization. MCF-7 breast cancer and CCRF-CEM acute lymphoblastic leukemia cell lines were used as EpCAM positive and negative cell models, respectively, to assess CTC capture efficiency and selectivity. Comparisons reveal that bioaffinity based antibody immobilization involving streptavidin attachment with glutaraldehyde linker gave the highest cell capture efficiency. On the other hand, a covalent antibody immobilization method involving direct antibody binding by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)-N-hydroxysuccinimide (NHS) reaction was found to be more time and cost efficient with a similar cell capture efficiency. All methods provided very high selectivity for CTCs with EpCAM expression. It was also demonstrated that antibody immobilization via EDC-NHS reaction in a microfluidic channel leads to high capture efficiency and selectivity.

We have performed an in-depth single-cell phenotypic characterization of high-grade serous ovarian cancer (HGSOC) by multiparametric mass cytometry (CyTOF). Using a CyTOF antibody panel to interrogate features of HGSOC biology, combined with unsupervised computational analysis, we identified noteworthy cell types co-occurring across the tumors. In addition to a dominant cell subset, each tumor harbored rarer cell phenotypes. One such group co-expressed E-cadherin and vimentin (EV), suggesting their potential role in epithelial mesenchymal transition, which was substantiated by pairwise correlation analyses. Furthermore, tumors from patients with poorer outcome had an increased frequency of another rare cell type that co-expressed vimentin, HE4, and cMyc. These poorer-outcome tumors also populated more cell phenotypes, as quantified by Simpson's diversity index. Thus, despite the recognized genomic complexity of the disease, the specific cell phenotypes uncovered here offer a focus for therapeutic intervention and disease monitoring., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cancers display distinctive carbohydrate molecules (glycans) on their surface proteins and lipids. mAb A4, an in-house generated monoclonal IgM antibody, is capable of distinguishing malignant ovarian carcinoma cells from benign ovarian epithelia by binding specifically to cancer cell-associated glycans. However, the structural details of the glycan targets of mAb A4 have been elusive. Here we developed a novel approach of isolating and fractionating glycan molecules released from glycoproteins in cancer cell lysates using HILIC-UPLC, and used them as probes on a microarray for affinity-based identification of the binding targets, allowing full-size, difficult to synthesize, cancer-associated glycans to be directly studied. As a result of this "shotgun" glycomics approach, we corroborate the previously assigned specificity of mAb A4 by showing that mAb A4 binds primarily to large (>15 glucose units), sialylated N-glycans containing the H-type 1 antigen (Fuc-α1,2-Gal-β1,3-GlcNAc). Although mAb A4 was also capable of directly binding to type 1 N-acetyl-lactosamine, this epitope was mostly shielded by sialylation and thus relatively inaccessible to binding. Knowledge of the structure of mAb A4 antigen will facilitate its clinical development as well as its use as a diagnostic biomarker.

Background: The structural perturbations in DNA molecule may be caused by a break in a strand, a missing base from the backbone, or a chemically changed base. These alterations in DNA that occurs naturally can result from metabolic or hydrolytic processes. DNA damage plays a major role in the mutagenesis, carcinogenesis, aging and various other patho-physiological conditions. DNA damage can be induced through hydrolysis, exposure to reactive oxygen species (ROS) and other reactive carbonyl metabolites including 4-hydroxynonenal (HNE). 4-HNE is an important lipid peroxidation product which has been implicated in the mutagenesis and carcinogenesis processes., Methods: The present study examines to probe the presence of auto-antibodies against 4-hydroxynonenal damaged DNA (HNE-DNA) in various cancer subjects. In this study, the purified calf thymus DNA was damaged by the action of 4-HNE. The DNA was incubated with 4-HNE for 24 h at 37°C temperature. The binding characteristics of cancer auto-antibodies were assessed by direct binding and competitive inhibition ELISA., Results: DNA modifications produced hyperchromicity in UV spectrum and decreased fluorescence intensity. Cancer sera exhibited enhanced binding with the 4-HNE modified calf thymus DNA as compared to its native conformer. The 4-HNE modified DNA presents unique epitopes which may be one of the factors for the auto-antibody induction in cancer patients., Conclusion: The HNE modified DNA presents unique epitopes which may be one of the factors for the autoantibody induction in cancer patients., (© 2017 Wiley Periodicals, Inc.)

Background: Epithelioid peritoneal malignant mesothelioma (EPMM) is the most common subtype of this aggressive tumor. We compared two antibodies against PD-L1, a recent theranostic biomarker, and evaluated the prognostic value of PD-L1 expression by mesothelial and immune cells in EPMM., Methods: Immunohistochemistry was performed on 45 EPMM. Clinical and pathological data were extracted from the RENAPE database. Using E1L3N and SP142 clones, inter-observer agreement, PD-L1 expression by mesothelial and immune cells and inter-antibody agreement were evaluated. The prognostic relevance of PD-L1 expression was evaluated in 39 EPMM by univariate and multivariate analysis of overall survival (OS) and progression-free survival (PFS)., Results: Inter-observer agreement on E1L3N immunostaining was moderate for mesothelial and immune cells, and fair for mesothelial and poor for immune cells using SP142. Using E1L3N, 31.1% of mesothelial and 15.6% of immune cells expressed PD-L1, and 22.2% of mesothelial and 26.7% of immune cells using SP142. Inter-antibody agreement was moderate. In most positive cases, 1-5% of tumor cells were positive. Using E1L3N, PD-L1 expression by lymphocytes was associated with better OS and PFS by both univariate and multivariate analysis. Cytoreductive surgery with hyperthermic intraperitoneal chemotherapy predicted better prognosis than other treatments. Solid subtype was an independent prognostic factor for worse OS., Conclusion: E1L3N appeared easier to use than SP142 to evaluate PD-L1 expression. A minority of EPMM expressed PD-L1, and only a few cells were positive. PD-L1 expression by immune cells evaluated with E1L3N was an independent prognostic factor in EPMM., (Copyright © 2017 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Microfluidic technologies have miniaturized a variety of biomedical applications, and these chip-based systems have several significant advantages over their large-scale counterparts. Recently, this technology has been used for automating labor-intensive and time-consuming screening processes, whereby affinity reagents, including aptamers, peptides, antibodies, polysaccharides, glycoproteins, and a variety of small molecules, are used to probe for molecular biomarkers. When compared to conventional methods, the microfluidic approaches are faster, more compact, require considerably smaller quantities of samples and reagents, and can be automated. Furthermore, they allow for more precise control of reaction conditions (e.g., pH, temperature, and shearing forces) such that more efficient screening can be performed. A variety of affinity reagents for targeting cancer cells or cancer biomarkers are now available and will likely replace conventional antibodies. In this review article, the selection of affinity reagents for cancer cells or cancer biomarkers on microfluidic platforms is reviewed with the aim of highlighting the utility of such approaches in cancer diagnostics.

Background: To date, there are no effective therapeutic targeting agents for triple-negative breast cancer (TNBC), and PD-L1 has presented potential as an effective marker of immunotherapeutic agents. The aim of this study was to evaluate the expression of PD-L1 by three different immunohistochemical antibodies in TNBC., Methods: Interpretation of all three PD-L1 antibodies showed good concordance among three readers (kappa value >0.610) in both cancer cells and immune cells. Using a tissue microarray (TMA) constructed from 218 cases of TNBC, we performed immunohistochemical staining using three of the most popular commercially used PD-L1 monoclonal antibodies (clones 28-8, E1L3N and SP142) in cancer cells and immune cells., Results: Using various cut-off values of previous studies (1, 5, 10 and 50 %), the expression rates in cancer cells were: PD-L1 (E1L3N) (14.7, 14.7, 11.0, 2.3 %), PD-L1 (28-8) (13.3, 12.4, 10.1, 1.8 %), and PD-L1 (SP142) (11.5, 11.0, 6.9, 0.5 %), respectively. At the 5 % cut-off value, the discordance rate among the three antibodies was 6.0-10.6 % and was highest between PD-L1 (SP142) and the other two antibodies. The expression rates in immune cells were PD-L1 (E1L3N) (37.6 %), PD-L1 (28-8) (36.7 %), and PD-L1 (SP142) (19.3 %), and the discordance rate among the three antibodies ranged from 13.8 to 24.8 % and was also highest between PD-L1 (SP142) and the other two antibodies. Among stromal histologic types, higher PD-L1 expression in cancer cells and immune cells was measured in inflammatory-type (p < 0.05). The absence of PD-L1 (28-8) staining in immune cells was associated with shorter disease free survival (DFS) and overall survival (OS) (p = 0.043, and p = 0.021) in univariate analyses, and with shorter OS in multivariate Cox analysis (hazard ratio: 5.429, 95 % CI 1.214-24.28, p = 0.027)., Conclusions: PD-L1 detection in cancer cells and immune cells varied by antibody clone. The greatest amount of staining occurred with PD-L1 (E1L3N), followed by PD-L1 (28-8) and PD-L1 (SP142). The concordance rate among monoclonal PD-L1 antibodies was higher between PD-L1 (28-8) and PD-L1 (E1L3N). To determine the gold standard antibody and the most appropriate cut-off value, further study of the clinical trial group treated with PD-L1 inhibitor is necessary.

LAMP2A is the key protein of chaperone-mediated autophagy (CMA), downregulation of LAMP2A leads to CMA blockade. CMA activation has been implicated in cancer growth, but the exact mechanisms are unclear. Elevated expression of LAMP2A was found in 8 kinds of tumors (n=747), suggesting that LAMP2A may have an important role in cancer progression. Unsurprisingly, LAMP2A knockdown in gastric cancer (GC) cells hindered proliferation, accompanied with altered expression of cell cycle-related proteins and accumulation of RND3/RhoE. Interactomic and KEGG analysis revealed that RND3 was a putative CMA substrate. Further study demonstrated that RND3 silencing could partly rescue the proliferation arrest induced by LAMP2A knockdown; RND3 was increased upon lysosome inhibition via both chemicals and LAMP2A-shRNA; Furthermore, RND3 could interact with CMA components HSPA8 and LAMP2A, and be engulfed by isolated lysosomes. Thus, constant degradation of RND3 by CMA is required to sustain rapid proliferation of GC cells. At last, the clinical significance of LAMP2A was explored in 593 gastric noncancerous lesions and 173 GC tissues, the results revealed that LAMP2A is a promising biomarker for GC early warning and prognosis of female GC patients.

Autoimmune dementia and encephalopathies (ADE) are complex disorders that can cause immune-mediated cognitive deficits and have confusing nomenclature. Presentation varies from acute limbic encephalitis to subacute or chronic disorders of cognition mimicking neurodegenerative dementia. It may occur as a paraneoplastic phenomenon or an idiopathic autoimmune phenomenon. The presence of a personal/family history of autoimmunity, inflammatory spinal fluid, serologic evidence of autoimmunity (neural or nonorgan-specific), or mesial temporal magnetic resonance imaging abnormalities are clues to diagnosis. Bedside cognitive assessment and/or detailed neuropsychologic testing are useful. Neural-specific autoantibodies, mostly discovered in the past two decades, may bind antigens on the cell surface (e.g., N-methyl-d-aspartate receptor autoantibodies) and are likely to be pathogenic, with treatment aimed at antibody-depleting agents often with success, while antibodies binding intracellular antigens (e.g., antineuronal nuclear autoantibody type 1 (ANNA1 or anti-Hu)) are a marker of a T-cell-mediated process and treated with T-cell-depleting immunotherapies, with variable responses. Detection and treatment of cancer (when present) are essential. High-dose corticosteroids are the initial treatment in most patients and may serve as a diagnostic test when the diagnosis is uncertain. Repeat cognitive testing after immunotherapy helps document objective improvements. Maintenance immunotherapy is recommended in those at risk for relapse. Prognosis is variable, but paraneoplastic ADE with antibodies to intracellular antigens have a worse prognosis. The field is still developing and future studies should provide guidelines for diagnosis and treatments., (© 2016 Elsevier B.V. All rights reserved.)

Signaling mechanisms underlying self-renewal of leukemic stem cells (LSCs) are poorly understood, and identifying pathways specifically active in LSCs could provide opportunities for therapeutic intervention. T-cell immunoglobin mucin-3 (TIM-3) is expressed on the surface of LSCs in many types of human acute myeloid leukemia (AML), but not on hematopoietic stem cells (HSCs). Here, we show that TIM-3 and its ligand, galectin-9 (Gal-9), constitute an autocrine loop critical for LSC self-renewal and development of human AML. Serum Gal-9 levels were significantly elevated in AML patients and in mice xenografted with primary human AML samples, and neutralization of Gal-9 inhibited xenogeneic reconstitution of human AML. Gal-9-mediated stimulation of TIM-3 co-activated NF-κB and β-catenin signaling, pathways known to promote LSC self-renewal. These changes were further associated with leukemic transformation of a variety of pre-leukemic disorders and together highlight that targeting the TIM-3/Gal-9 autocrine loop could be a useful strategy for treating myeloid leukemias., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: A single-chain bispecific antibody (scBsAb; an engineered antibody), has promising clinical applications. Nonetheless, the effect of different interchain linkers on its activity is poorly understood., Methods: Gene synthesis was used to splice the anti-γ-seminoprotein single-chain antibody (anti-γ-Sm scFv) gene with the anti-CD3 single-chain antibody (anti-CD3 scFv) gene via different interchain peptide linkers. The Phyre2 software was used to predict spatial configuration of different scBsAbs. Eukaryotic expression vectors carrying scBsAbs were constructed by molecular cloning techniques and these plasmids were transfected into HeLa cells with liposomes. scBsAbs were purified by Ni(2+)-NTA agarose and analysed for antigen binding by an enzyme-linked immunosorbent assay (ELISA). Blood pharmacokinetics and inhibition of prostate tumour growth in nude mice were analysed in in vivo experiments., Results: Bioinformatics analysis and prediction showed that none of the three linkers, Fc, 205C', and HSA, had a significant effect on protein folding of anti-γ-Sm scFv or anti-CD3 scFv. Nevertheless, the spatial structures of the three linkers were noticeably different. Anti-γ-Sm × anti-CD3 scBsAb with an Fc, 205C', or HSA linker was successfully constructed, and these antibodies had similar protein expression levels. ELISA showed that all the three scBsAbs bound to Jurkat cells and the LNCaP membrane antigen, although binding of (205C')scBsAb was weaker than that of the two parental scFvs (P < 0.05). In contrast, binding strength of (HSA)scBsAb and (Fc)scBsAb was close to that of the parental scFvs (P > 0.05). Pharmacokinetic analysis showed that the half-clearance time of the elimination phase (T(1/2β)) for (HSA)scBsAb was the longest: up to 4.4 h. Compared with γ-Sm ScFv, the three scBsAbs all had a much stronger inhibitory effect on the growth of prostate cancer (P < 0.05), but there were no significant differences among the three scBsAbs (P > 0.05)., Conclusions: HSA is the optimal linker for the anti-γ-Sm × anti-CD3 scBsAb and may improve antigen-binding affinity of antibodies and prolong physiological retention time. Interchain linkers affect the function of scBsAbs; these effects may have important implications for construction of antibodies.

Proteins in serum or plasma hold great potential for use in disease diagnosis and monitoring. However, the correlation between tumor burden and protein biomarker concentration has not been established. Here, using an antibody colocalization microarray, the protein concentration in serum was measured and compared with the size of mammary xenograft tumors in 11 individual mice from the time of injection; seven blood samples were collected from each tumor-bearing mouse as well as control mice on a weekly basis. The profiles of 38 proteins detected in sera from these animals were analyzed by clustering, and we identified 10 proteins with the greatest relative increase in serum concentration that correlated with growth of the primary mammary tumor. To evaluate the diagnosis of cancer based on these proteins using either an absolute threshold (i.e. a concentration cutoff) or self-referenced differential threshold based on the increase in concentration before cell injection, receiver operating characteristic curves were produced for 10 proteins with increased concentration, and the area under curve was calculated for each time point based on a single protein or on a panel of proteins, in each case showing a rapid increase of the area under curve. Next, the sensitivity and specificity of individual and optimal protein panels were calculated, showing high accuracy as early as week 2. These results provide a foundation for studies of tumor growth through measuring serial changes of protein concentration in animal models., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Many forms of antibody-based targeted therapeutics, including antibody drug conjugates, utilize the internalizing function of the targeting antibody to gain intracellular entry into tumor cells. Ideal antibodies for developing such therapeutics should be capable of both tumor-selective binding and efficient endocytosis. The macropinocytosis pathway is capable of both rapid and bulk endocytosis, and recent studies have demonstrated that it is selectively up-regulated by cancer cells. We hypothesize that receptor-dependent macropinocytosis can be achieved using tumor-targeting antibodies that internalize via the macropinocytosis pathway, improving potency and selectivity of the antibody-based targeted therapeutic. Although phage antibody display libraries have been utilized to find antibodies that bind and internalize to target cells, no methods have been described to screen for antibodies that internalize specifically via macropinocytosis. We hereby describe a novel screening strategy to identify phage antibodies that bind and rapidly enter tumor cells via macropinocytosis. We utilized an automated microscopic imaging-based, High Content Analysis platform to identify novel internalizing phage antibodies that colocalize with macropinocytic markers from antibody libraries that we have generated previously by laser capture microdissection-based selection, which are enriched for internalizing antibodies binding to tumor cells in situ residing in their tissue microenvironment (Ruan, W., Sassoon, A., An, F., Simko, J. P., and Liu, B. (2006) Identification of clinically significant tumor antigens by selecting phage antibody library on tumor cells in situ using laser capture microdissection. Mol. Cell. Proteomics. 5, 2364-2373). Full-length human IgG molecules derived from macropinocytosing phage antibodies retained the ability to internalize via macropinocytosis, validating our screening strategy. The target antigen for a cross-species binding antibody with a highly active macropinocytosis activity was identified as ephrin type-A receptor 2. Antibody-toxin conjugates created using this macropinocytosing IgG were capable of potent and receptor-dependent killing of a panel of EphA2-positive tumor cell lines in vitro. These studies identify novel methods to screen for and validate antibodies capable of receptor-dependent macropinocytosis, allowing further exploration of this highly efficient and tumor-selective internalization pathway for targeted therapy development., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

It is postulated a conception of immunological disbalance between carcinogenesis inhibiting and stimulating antibodies (Ab). Inhibiting Ab prevent the carcinogens and estradiol but increase the progesterone penetration into the target cells. And vise versa do stimulating Ab. Inhibiting Ab could be blocked by corresponding antiidiotypic Ab. The processes of carcinogenesis initiation and promotion are intensified when stimulating Ab prevail over inhibiting ones., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: To study the expression of arginase-1 (Arg-1), glypican-3 (GPC3), hepatocyte paraffin antigen 1 (HepPar-1) and alpha-fetoprotein (AFP) in hepatocellular carcinoma (HCC), benign liver lesions (BLL) and metastatic carcinoma (MC), and their applications in diagnosis and differential diagnosis., Methods: Immunohistochemical study (EnVision method) for Arg-1, GPC3, HepPar-1 and AFP was carried out in three groups of liver lesions, including 85 cases of HCC, 35 cases of BLL and 19 cases of MC. The relationship between expression of Arg-1, GPC3, HepPar-1 and AFP and clinicopathologic features in HCC was also analyzed., Results: The positive expression rate of Arg-1 was 90.6% (79/85) in HCC and 100% (35/35) in BLL. Arg-1 expression was observed in 1 of the 19 cases of MC studied. The positive expression rate of GPC3 was 82.4% (70/85) in HCC, 5.3% (1/19) in MC and 0 (0/35) in BLL. The positive expression rate of AFP was 47.1% (40/85) in HCC and 0 in BLL or MC. The positive expression rate of HepPar-1 was 72.9% (62/85) in HCC, 100% (35/35) in BLL and 2/19 in MC. Arg-1 has a higher sensitivity in highlighting hepatocellular lesions than AFP and HepPar-1 (P=0.000 versus P=0.002). The specificity of GPC3 expression in HCC was 98.1%., Conclusions: Arg-1 is a sensitive hepatocellular marker in delineation of liver lesions.GPC3 is a relatively specific marker in diagnosis of HCC.

Objectives: Activating mutations in the epidermal growth factor receptor (EGFR) kinase domain are correlated with dramatic clinical responses in non-small cell lung cancer patients treated with EGFR-tyrosine kinase inhibitors (TKIs). The two most common EGFR mutations, representing 85-90% of EGFR mutations, are the E746&#95;A750 deletion in exon 19 and the L858R point mutation in exon 21. We conducted this study to evaluate the suitability of mutation-specific antibodies that can detect E746&#95;A750 deletion and L858R mutant EGFR proteins by immunohistochemistry (IHC)., Materials and Methods: In a cohort of consecutive patients with surgically resected lung adenocarcinomas (n=240), mutant EGFR protein expression was assessed by IHC using specific antibodies (clone SP111 and SP125) to the 2 major forms of EGFR mutations. Immunoreactivity was scored as 0-3, and the results were compared with the EGFR-mutational status., Results: With a cutoff value of IHC 2+ for SP 111 (anti-EGFR E746&#95;A750 del antibody) and SP 125 (anti-EGFR L858R antibody), both antibodies showed high specificity (99.0% and 89.7%, respectively) and sensitivity (70.6% and 80.4%, respectively). While cases with IHC scores of 3 using these 2 antibodies positively correlated with the EGFR-mutational status, cases with IHC scores lower than 3+ showed variable results regarding EGFR-mutational status., Conclusion: Although each antibody showed relatively high specificity, some EGFR-mutant cases were not detected by the mutation-specific antibodies. Various forms of exon 19 deletions, except E746&#95;A750, were rarely detected by the mutant-specific antibody. Therefore, IHC-negative cases require further molecular analysis to confirm the presence of EGFR mutations., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Object: For patients with glioblastoma multiforme, median survival time is approximately 14 months. Longer progression-free and overall survival times correlate with gross-total resection of tumor. The ability to identify tumor cells intraoperatively could result in an increased percentage of tumor resected and thus increased patient survival times. Available labeling methods rely on metabolic activity of tumor cells; thus, they are more robust in high-grade tumors, and their utility in low-grade tumors and metastatic tumors is not clear. The authors demonstrate intraoperative identification of tumor cells by using labeled tumor-specific antibodies., Methods: GL261 mouse glioma cells exhibit high expression of a membrane-bound protein called second tyrosinase-related protein (TRP-2). The authors used these cells to establish an intracranial, immunocompetent model of malignant glioma. Antibodies to TRP-2 were labeled by using Alexa Fluor 488 fluorescent dye and injected into the tail vein of albino C57BL/6 mice. After 24 hours, a craniotomy was performed and the tissue was examined in vivo by using an Optiscan 5.1 handheld portable confocal fiber-optic microscope. Tissue was examined ex vivo by using a Pascal 5 scanning confocal microscope., Results: Labeled tumor cells were visible in vivo and ex vivo under the respective microscopes., Conclusions: Fluorescently labeled tumor-specific antibodies are capable of binding and identifying tumor cells in vivo, accurately and specifically. The development of labeled markers for the identification of brain tumors will facilitate the use of intraoperative fluorescence microscopy as a tool for increasing the extent of resection of a broad variety of intracranial tumors.

Melanoma has a propensity for lymph node metastasis. However, the incidence of lymphatic invasion detected by histology alone in primary melanoma is disproportionately low in comparison to the incidence of positive sentinel lymph nodes (SLN). With the discovery of lymphatic endothelial cell markers, such as podoplanin and LYVE-1, lymphatic vessels can be reliably detected in formalin-fixed paraffin-embedded (FFPE) tissues. There is a now consensus that lymphatic invasion detected by immunohistochemical stains in primary melanoma is much more common than previously reported by histological examination alone. Immunohistochemical stains show that lymphangiogenesis and lymphatic invasion in primary melanoma may occur intratumorally or peritumorally, and lymphatic invasion is common across the range of tumor thicknesses in primary vertical growth phase (VGP) melanomas. A number of studies have shown that lymphatic invasion in primary melanoma is associated with a positive sentinel lymph node biopsy and a worse clinical outcome. Although not currently a part of the standard of care for staging of melanoma, the detection of lymphatic invasion in primary melanoma using immunohistochemical markers may be helpful in planning of therapy in some cases and may find a routine role in primary melanoma microscopic attributes in future.

Cancer stem cells in a tumor mass form a very small subpopulation ranging from below 0.1% in a brain tumor but they have the crucial ability to become malignant. The goal of cancer therapy has been the total killing of tumor cells. However we should clarify that most of all tumor cells are differentiated cancer cells. Thus the elimination of 99.9% of tumor cells under histological criteria cannot ensure the cancer will be cured. Rather cancer cell biologists should turn their attention to reprogramming cancer stem cells to normal stem cells by which malignancy recuperates normal organogenesis from the aspect of the dichotomy of cancer stem cell. The cue points underlying the reverse cancer stem cell at blastogenesis in inflammation site is depending upon cell-to-cell recognition of the tumor-niche cells. Normalization of tumor-niche promises to lead cancer stem cell into normal stem cell owing to autonomous healing mechanisms that reside in the self-defense mechanisms in immunity and the cell competition mechanisms in the wound healing of the tissue cells. Among the cyto-skeletal proteins, vimentin becomes a target of self-restoration of cancer stem cell by means of immune surveillance. A human monoclonal antibody, CLN-IgG recognizes vimentin expressing on the cell surface of the malignant tumor. Since vimentin network resides in the cytoplasm connecting the plasma membrane with chromatin assembly in the nucleus, it is highly likely vimentin plays an important role in up-regulation and down-regulation through signal transduction between certain membrane receptors and gene expression with respect to the transformation of the cell. Aberrant arrangement of vimentin undergoes malignancy accompanied by epithelial-mesenchymal-transition relating to the aberrant apoptotic cellular behavior in the tumor-niche. Restraint of the aberrant expression of vimentin on the plasma membrane of the malignant cell evokes a pertinent signal transduction pathway for healing that is an indication there must be a reverse path that reprograms cancer stem cells to normal organogenesis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Antibody cancer therapies rely on systemically accessible targets and suitable antibodies that exert a functional activity or deliver a payload to the tumor site. Here, we present proof-of-principle of in vivo selection of human antibodies in tumor-bearing mice that identified a tumor-specific antibody able to deliver a payload and unveils the target antigen. By using an ex vivo enrichment process against freshly disaggregated tumors to purge the repertoire, in combination with in vivo biopanning at optimized phage circulation time, we have identified a human domain antibody capable of mediating selective localization of phage to human prostate cancer xenografts. Affinity chromatography followed by mass spectrometry analysis showed that the antibody recognizes the proteasome activator complex PA28. The specificity of soluble antibody was confirmed by demonstrating its binding to the active human PA28αβ complex. Whereas systemically administered control phage was confined in the lumen of blood vessels of both normal tissues and tumors, the selected phage spread from tumor vessels into the perivascular tumor parenchyma. In these areas, the selected phage partially colocalized with PA28 complex. Furthermore, we found that the expression of the α subunit of PA28 [proteasome activator complex subunit 1 (PSME1)] is elevated in primary and metastatic human prostate cancer and used anti-PSME1 antibodies to show that PSME1 is an accessible marker in mouse xenograft tumors. These results support the use of PA28 as a tumor marker and a potential target for therapeutic intervention in prostate cancer., Competing Interests: The authors declare no conflict of interest.

Background: Fludeoxyglucose positron emission topography ((18)F-FDG PET) is insufficiently sensitive at detecting small or low-grade breast tumors. The characterization of somatostatin receptors (SSTR) in tumors and the development of (68)Ga-DOTATOC PET for imaging could be of interest. The aim of this study was to validate an animal model expressing SSTR2 and to correlate the immunohistochemical (IHC) analysis with (18)F-FDG and (68)Ga-DOTATOC uptake in vivo., Materials and Methods: Ten nude mice were xenografted with the ZR-75-1 breast tumor cell line. Imaging was performed with (68)Ga-DOTATOC and (18)F-FDG and correlated to IHC analysis of SSTR2., Results: IHC analyses showed that the tumors expressed SSTR2. On PET imaging, the tumors were barely visible with (18)F-FDG, whereas with (68)Ga-DOTATOC, specific two-fold higher uptake was observed (p<0.005)., Conclusion: Our results suggest that (68)Ga-DOTATOC PET could be used for detection of breast tumors not detected with (18)F-FDG. SSTR2 status should be assessed to allow for individual treatment.

Immunocytokines (ICKs) targeting cytokines to the tumor environment using antibodies directed against a tumor-associated antigen often have a higher therapeutic index than the corresponding unconjugated cytokines. Various ICKs displaying significant antitumoral effects in several murine tumor models have already been developed, and some of them, in particular interleukin (IL)-2-based ICKs, are in Phase II clinical trials. Although sharing common biological activities with IL-2 in vitro, IL-15 is now considered as having a better potential in antitumor immunotherapeutical strategies and has been shown to be less toxic than IL-2 in preclinical studies. We previously developed the fusion protein RLI, linking a soluble form of human IL-15Rα-sushi+ domain to human IL-15. RLI showed better biological activities than IL-15 in vitro as well as higher antitumoral effects in vivo in murine and human cancer models. Here, we investigated, in the context of an ICK, the effect of associating RLI with an antibody targeting the GD2 ganglioside, a validated tumoral target expressed on many neurectodermal tumors. Anti-GD2-RLI fully retained the cytokine potential of RLI and the antibody effector functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity). It displayed strong antitumor activities in two syngeneic cancer models in immunocompetent mice (subcutaneous EL4 and metastatic NXS2). Its therapeutic potency was higher than those of RLI and anti-GD2 alone or in combination. We suggest that this is related to its bifunctional (cytokine and antibody) nature., (Copyright © 2013 UICC.)

Phage display is a powerful method for target discovery and selection of ligands for cancer treatment and diagnosis. Our goal was to select tumor-binding antibodies in cancer patients. Eligibility criteria included absence of preexisting anti-phage-antibodies and a Stage IV cancer status. All patients were intravenously administered 1 × 10(11) TUs/kg of an scFv library 1 to 4 h before surgical resection of their tumors. No significant adverse events related to the phage library infusion were observed. Phage were successfully recovered from all tumors. Individual clones from each patient were assessed for binding to the tumor from which clones were recovered. Multiple tumor-binding phage-antibodies were identified. Soluble scFv antibodies were produced from the phage clones showing higher tumor binding. The tumor-homing phage-antibodies and derived soluble scFvs were found to bind varying numbers (0-5) of 8 tested normal human tissues (breast, cervix, colon, kidney, liver, spleen, skin, and uterus). The clones that showed high tumor-specificity were found to bind corresponding tumors from other patients also. Clone enrichment was observed based on tumor binding and DNA sequence data. Clone sequences of multiple variable regions showed significant matches to certain cancer-related antibodies. One of the clones (07-2,355) that was found to share a 12-amino-acid-long motif with a reported IL-17A antibody was further studied for competitive binding for possible antigen target identification. We conclude that these outcomes support the safety and utility of phage display library panning in cancer patients for ligand selection and target discovery for cancer treatment and diagnosis.

The immunoglobulins expressed by chronic lymphocytic leukemia (CLL) B cells are highly restricted, suggesting they are selected for binding either self or foreign antigen. Of the immunoglobulin heavy-chain variable (IGHV) genes expressed in CLL, IGHV1-69 is the most common, and often is expressed with little or no somatic mutation, and restricted IGHD and IGHJ gene usage. We found that antibodies encoded by one particular IGHV1-69 subset, designated CLL69C, with the HCDR3 encoded by the IGHD3-3 gene in reading frame 2 and IGHJ6, specifically bound to oxidation-specific epitopes (OSE), which are products of enhanced lipid peroxidation and a major target of innate natural antibodies. Specifically, CLL69C bound immunodominant OSE adducts termed MAA (malondialdehyde-acetaldehyde-adducts), which are found on apoptotic cells, inflammatory tissues, and atherosclerotic lesions. It also reacted specifically with MAA-specific peptide mimotopes. Light chain shuffling indicated that non-stochastically paired L chain of IGLV3-9 contributes to the antigen binding of CLL69C. A nearly identical CLL69C Ig heavy chain was identified from an MAA-enriched umbilical cord phage displayed Fab library, and a derived Fab with the same HCDR3 rearrangement displayed identical MAA-binding properties. These data support the concept that OSE (MAA-epitopes), which are ubiquitous products of inflammation, may play a role in clonal selection and expansion of CLL B cells.

Recruitment of immune cells to tumor cells targeted by a therapeutic antibody can heighten the antitumor efficacy of the antibody. For example, p185(her2/neu)-targeting antibodies not only downregulate the p185(her2/neu) kinase (ERBB2) but also trigger complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) through the antibody Fc region. Here, we describe a generalized strategy to improve immune cell recruitment to targeted cancer cells, using a modified scFv antibody we call a "Grababody" that binds the target protein and endogenous immunoglobulins. The model system we used to illustrate the use of this platform recognizes p185(her2/neu) and includes an IgG binding domain. The recombinant scFv Grababody that was created recruited circulating human IgGs and attracted immune cells carrying Fc receptors to tumor cells that expressed p185(her2/neu). The presence of the IgG binding domain significantly enhanced CDC and ADCC activity and improved antitumor activity in vivo. Our results illustrate a novel general approach to improve antibody-like proteins for therapeutic applications., (©2013 AACR.)

Cell surface antigens as biomarkers offer tremendous potential for early diagnosis, prognosis, and therapeutic response in a variety of diseases such as cancers. In this research, a simple, rapid, accurate, inexpensive, and easily available in vitro assay based on magnetic nanoparticles and magnetic cell separation principle was applied to identify and quantitatively analyze the cell surface antigen expression in the case of prostate cancer cells. Comparing the capability of the assay with flow cytometry as a gold standard method showed similar results. The results showed that the antigen-specific magnetic cell separation with antibody-coated magnetic nanoparticles has high potential for quantitative cell surface antigen detection and analysis.

Antibodies are under development to treat a variety of cancers, such as lymphomas, colon, and breast cancer. A major limitation to greater efficacy for this class of drugs is poor distribution in vivo. Localization of antibodies occurs slowly, often in insufficient therapeutic amounts, and distributes heterogeneously throughout the tumor. While the microdistribution around individual vessels is important for many therapies, the total amount of antibody localized in the tumor is paramount for many applications such as imaging, determining the therapeutic index with antibody drug conjugates, and dosing in radioimmunotherapy. With imaging and pretargeted therapeutic strategies, the time course of uptake is critical in determining when to take an image or deliver a secondary reagent. We present here a simple mechanistic model of antibody uptake and retention that captures the major rates that determine the time course of antibody concentration within a tumor including dose, affinity, plasma clearance, target expression, internalization, permeability, and vascularization. Since many of the parameters are known or can be estimated in vitro, this model can approximate the time course of antibody concentration in tumors to aid in experimental design, data interpretation, and strategies to improve localization., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

GRP78, a molecular chaperone with critical endoplasmic reticulum functions, is aberrantly expressed on the surface of cancer cells, including prostate and melanoma. Here it functions as a pro-proliferative and anti-apoptotic signaling receptor via NH(2)-terminal domain ligation. Auto-antibodies to this domain may appear in cancer patient serum where they are a poor prognostic indicator. Conversely, GRP78 COOH-terminal domain ligation is pro-apoptotic and anti-proliferative. There is no method to disrupt cell-surface GRP78 without compromising the total GRP78 pool, making it difficult to study cell-surface GRP78 function. We studied six cell lines representing three cancer types. One cell line per group expresses high levels of cell-surface GRP78, and the other expresses low levels (human hepatoma: Hep3B and HepG2; human prostate cancer: PC3 and 1-LN; murine melanoma: B16F0 and B16F1). We investigated the effect of Escherichia coli subtilase cytoxin catalytic subunit (SubA) on GRP78. We report that SubA specifically cleaves cell-surface GRP78 on HepG2, 1-LN, and B16F1 cells without affecting intracellular GRP78. B16F0 cells (GRP78(low)) have lower amounts of cleaved cell-surface GRP78. SubA has no effect on Hep3B and PC3 cells. The predicted 28-kDa GRP78 COOH-terminal fragment is released into the culture medium by SubA treatment, and COOH-terminal domain signal transduction is abrogated, whereas pro-proliferative signaling mediated through NH(2)-terminal domain ligation is unaffected. These experiments clarify cell-surface GRP78 topology and demonstrate that the COOH-terminal domain is necessary for pro-apoptotic signal transduction occurring upon COOH-terminal antibody ligation. SubA is a powerful tool to specifically probe the functions of cell-surface GRP78.

One of the deadly hallmarks of cancer is its ability to prosper within the constraints of the host immune system. Recent advances in immunoproteomics and high-throughput technologies have lead to profiling of the antibody repertoire in cancer patients. This in turn has lead to the identification of tumour associated antigens/autoantibodies. Autoantibodies are extremely attractive and promising biomarker entities, however there has been relatively little discussion on how to interpret the humoral immune response. It may be that autoantibody profiles hold the key to ultimately uncovering neoplastic associated pathways and through the process of immunosculpting the tumour may have yielded an immune response in the early stages of malignant tumour development. The aim of this review is to discuss the utility of the autoantibody response that is elicited as a result of malignancy and discuss the advantages and limitations of autoantibody profiling. This article is part of a Special Issue entitled: Translational Proteomics., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The occurrence of Ewing sarcoma-peripheral primitive neuroectodermal tumor as a primary intracranial tumor is very rare, with only 29 cases reported in the literature, 19 of which have included molecular studies. We present the clinical, radiologic and pathologic findings of an intracranial Ewing sarcoma in a 22-year-old woman arising from the dura over the right frontal convexity. The patient underwent craniotomy with gross total excision of the tumor. The tumor showed atypical histology and the diagnosis was confirmed by detection of a rearrangement of the EWSR1 gene by fluorescent in situ hybridization and identification of the diagnostic t(11;22)(q24;q12) translocation by reverse transcription-polymerase chain reaction. Additional features were detected in this tumor that are known to be associated with an unfavorable prognosis, including loss of p16 expression and gains of chromosomes 1q and 12. The patient experienced the most rapid downhill course reported to date for intracranial Ewing sarcoma, developing multiple extracranial metastases at 2 months and dying 6 months after the initial operation., (© 2011 Japanese Society of Neuropathology.)

This study describes a novel xenograft-based biomarker discovery platform and proves its usefulness in the discovery of serum markers for prostate cancer. By immunizing immuno-competent mice with serum from nude mice bearing prostate cancer xenografts, an antibody response against xenograft-derived antigens was elicited. By probing protein microarrays with serum from immunized mice, several prostate cancer-derived antigens were identified, of which a subset was successfully retrieved in serum from mice bearing prostate cancer xenografts and prevalidated in human serum samples of prostate cancer patients. Among the discovered and validated proteins were the members of the TAM receptor family (TYRO3, AXL, and MERTK), ACY1, and PSMA1. In conclusion, this novel method allows for the identification of low abundant cancer-derived serum proteins, circumventing dynamic range and host-response issues in standard patient cohort proteomics comparisons.