Functional characterization of an scFv-Fc antibody that immunotherapeutically targets the common cancer cell surface proteoglycan CSPG4.

Cell surface chondroitin sulfate proteoglycan 4 (CSPG4) is an attractive target for antibody-based cancer immunotherapy because of its role in tumor cell biology, its high expression on malignant cells including cancer-initiating cells, and its restricted distribution in normal tissues. The clinical use of CSPG4 has been hampered by the lack of a CSPG4-specific chimeric, humanized, or fully human monoclonal antibody. To overcome this limitation, we generated a CSPG4-specific fully human single-chain antibody termed scFv-FcC21 and characterized its specificity and antitumor activity. Viable CSPG4(+) melanoma cells were used in a screen of a human scFv phage display library that included CDR3 engineered to optimize antibody binding sites. The scFv antibody isolated was then recombinantly engineered with a human immunoglobulin G1 Fc region to construct the fully human antibody scFv-FcC21, which recognized tumors of neuroectodermal origin, various types of carcinomas, mesotheliomas, and sarcomas as well as myeloid leukemias. scFv-FcC21 inhibited in vitro growth and migration of tumor cells and in vivo growth of human tumor xenografts. These effects were mediated by inhibition of the activation of extracellular signal-regulated kinase and focal adhesion kinase signaling pathways that are critical for tumor cell growth and migration, respectively. Our findings define the CSPG4-specific fully human scFv-FcC21 antibody as a candidate therapeutic agent to target the many types of tumors that express CSPG4.

Targeting ALDH(bright) human carcinoma-initiating cells with ALDH1A1-specific CD8⁺ T cells.

PURPOSE: Cancer-initiating cells (CIC) are considered to represent the subpopulation of tumor cells that is resistant to conventional cancer treatments, highly tumorigenic in immunodeficient mice, and responsible for tumor recurrence and metastasis. Based on an elevated aldehyde dehydrogenase (ALDH) activity attributable to ALDH1/3 isoforms, ALDH(bright) cells have been identified and isolated from tumors and shown to have characteristics of CIC. The ALDH1A1 isoform was previously identified as a tumor antigen recognized by CD8(+) T cells. This study examines the ability of ALDH1A1-specific CD8(+) T cells to eliminate ALDH(bright) cells and control tumor growth and metastases. EXPERIMENTAL DESIGN: ALDH(bright) cells were isolated by flow cytometry using ALDEFLUOR from HLA-A2(+) human head and neck, breast, and pancreas carcinoma cell lines and tested for their tumorigenicity in immunodeficient mice. ALDH1A1-specific CD8(+) T cells were generated in vitro and tested for their ability to eliminate CICs in vitro and in vivo by adoptive transfer to immunodeficient mice bearing human tumor xenografts. RESULTS: ALDH(bright) cells isolated by flow cytometry from HLA-A2(+) breast, head and neck, and pancreas carcinoma cell lines at low numbers (500 cells) were tumorigenic in immunodeficient mice. ALDH(bright) cells present in these cell lines, xenografts, or surgically removed lesions were recognized by ALDH1A1-specific CD8(+) T cells in vitro. Adoptive therapy with ALDH1A1-specific CD8(+) T cells eliminated ALDH(bright) cells, inhibited tumor growth and metastases, or prolonged survival of xenograft-bearing immunodeficient mice. CONCLUSIONS: The results of this translational study strongly support the potential of ALDH1A1-based immunotherapy to selectively target CICs in human cancer.

A rapid, fully automated, molecular-based assay accurately analyzes sentinel lymph nodes for the presence of metastatic breast cancer.

OBJECTIVE: To develop a fully automated, rapid, molecular-based assay that accurately and objectively evaluates sentinel lymph nodes (SLN) from breast cancer patients. SUMMARY BACKGROUND DATA: Intraoperative analysis for the presence of metastatic cancer in SLNs from breast cancer patients lacks sensitivity. Even with immunohistochemical staining (IHC) and time-consuming review, alarming discordance in the interpretation of SLN has been observed. METHOD: A total of 43 potential markers were evaluated for the ability to accurately characterize lymph node specimens from breast cancer patients as compared with complete histologic analysis including IHC. Selected markers then underwent external validation on 90 independent SLN specimens using rapid, multiplex quantitative reverse transcription-polymerase chain reaction (QRT-PCR) assays. Finally, 18 SLNs were analyzed using a completely automated RNA isolation, reverse transcription, and quantitative PCR instrument (GeneXpert). RESULTS: : Following analysis of potential markers, promising markers were evaluated to establish relative level of expression cutoff values that maximized classification accuracy. A validation set of 90 SLNs from breast cancer patients was prospectively characterized using 4 markers individually or in combinations, and the results compared with histologic analysis. A 2-marker assay was found to be 97.8% accurate (94% sensitive, 100% specific) compared with histologic analysis. The fully automated GeneXpert instrument produced comparable and reproducible results in less than 35 minutes. CONCLUSIONS: A rapid, fully automated QRT-PCR assay definitively characterizes breast cancer SLN with accuracy equal to conventional pathology. This approach is superior to intraoperative SLN analysis and can provide standardized, objective results to assist in pathologic diagnosis.

Optimal healing environments in end-of-life care and beyond.

This paper is based on the premise that end-of-life care (EOLC) is the incarnation of an optimal healing environment (OHE). EOLC is characterized by factors that distinguish it from other forms of care or patient populations. These include: (1) formal EOLC did not evolve within the health care "industry," but was a reaction to that industry, created as an OHE; (2) patients nearing the end of life may be cared for in a formal "end-of-life" environment or may be located in other settings or systems; and (3) EOLC has a preordained outcome. Patients die in a variety of settings for medical, cultural, and accessibility reasons, and EOLC principles and practices are only beginning to be integrated into the full range of care settings. This paper proposes and defends the use of a single-question intervention to study the effect of EOLC care on its recipients, and considers the difficulty of establishing meaningful outcome variables. This paper also suggests that the principles of EOLC are well-suited to all phases of health services delivery and recommends the practical application of its elements throughout the medical services arena.