Colin C. Malone

Characterization of human tumor-infiltrating lymphocytes expanded in hollow-fiber bioreactors for immunotherapy of cancer

We attempted to grow tumor-infiltrating lymphocytes (TIL) from 34 fresh tumors of eight different histologies using flasks for the initiation phase and hollow fiber bioreactors to expand TIL to therapeutic numbers. Overall success rate was 76% (26/34) including melanoma (9/14, 64%) and renal cell carcinoma (11/11, 100%). The mean number of days required to reach successful initiation (1 x 10(9) TIL) for all tumor types was 29 +/- 16 days (mean +/- S.D.). Therapeutic doses of TIL required an average of 88 +/- 23 days (initiation plus expansion) with an average TIL number of 3.2 x 10(10) +/- 2.8 x 10(10). TIL phenotype was predominantly CD4+ in 53% (16/30) and CD8+ in 47% (14/30), renal cell carcinoma samples accounted for 12/14 of the predominantly CD8+ TIL. Cells bearing the natural killer (NK) phenotype represented only 0-7% of TIL while LAK phenotype represented 0-68% (mean 11 +/- 15%); LAK was the predominant phenotype in one patient with kidney cancer. Cytotoxicity tests showed consistent NK and LAK activity in addition to cytolysis of autologous tumor. Autologous tumor cell restricted cytolysis was noted for three TIL cultures. The overall success rate and characteristics of TIL were similar to our results with TIL expanded in semi-permeable plastic bags. This work confirms that hollow-fiber bioreactors are a suitable alternative to semi-permeable bags and roller bottle systems for the expansion of human TIL for therapeutic use in cancer patients.

Cellular and vaccine therapeutic approaches for gliomas

Despite new additions to the standard of care therapy for high grade primary malignant brain tumors, the prognosis for patients with this disease is still poor. A small contingent of clinical researchers are focusing their efforts on testing the safety, feasibility and efficacy of experimental active and passive immunotherapy approaches for gliomas and are primarily conducting Phase I and II clinical trials. Few trials have advanced to the Phase III arena. Here we provide an overview of the cellular therapies and vaccine trials currently open for patient accrual obtained from a search of http://www.clinicaltrials.gov. The search was refined with terms that would identify the Phase I, II and III immunotherapy trials open for adult glioma patient accrual in the United States. From the list, those that are currently open for patient accrual are discussed in this review. A variety of adoptive immunotherapy trials using ex vivo activated effector cell preparations, cell-based and non-cell-based vaccines, and several combination passive and active immunotherapy approaches are discussed.

Characterization of cancer cell lines established from two human metastatic breast cancers

SummaryCell lines are valuable resources for the study of the malignancy and potential therapy of human breast cancer. A major problem with adapting fresh breast tumor specimens to grow in vitro is contamination by fibroblasts. Previously, we have reported a technique to overcome this problem (Nayak, S. K; Dillman, R. O. Clin. Biotechnol. 3:237–242; 1991). We have recently established two new breast cancer cell lines, HH315 and HH375, that were derived from abdominal and supraclavicular lymph node metastases from two patients. They were characterized by (1) growth kinetics; (2) staining with monoclonal antibodies (MoAbs) to cytokeratin-19, epithelial membrane antigen (EMA), anticarcinoembryonic antigen (CEA), breast cancer antigen 1 (BRST-1), breast cancer antigen 2 (BRST-2), Her2/neu, and p53; (3) expression of domains of urinary plasminogen activator (uPA), neural cell adhesion molecule (NCAM), and haptoglobin (Hp) (Harvey et al., 1997); and (4) karyotypic analysis. Growth kinetic studies showed that doubling times for both lines ranged from 48 to 96 h. These two cell lines were found to have characteristics of the metastatic breast cancer cells. Both lines stained positive with MoAbs to cytokeratin-19 and EMA, thus confirming their epithelial origin. They also strongly reacted with the pan-breast carcinoma MoAbs BRST-1 and BRST-2, and carcinoembryonic CEA MoAb. Both cell lines overexpressed the oncogene proteins Her2/neu and p53. The tumor cells were negative for estrogen and progesterone receptors. HH315 cells were poorly differentiated, whereas the HH375 cells exhibited adenocarcinoma morphology. Both cell lines showed intense cell surface and some cytoplasmic staining for uPA, NCAM, and Hp domains, which is a characteristic of malignant neoplasms (Harvey et al., 1997). The HH375 cell line showed two cell types, of which 60% were hyperdiploids with 60–70 chromosomes and 5–10 marker chromosomes. The remaining cells were polyploid with more than 200 chromosomes. Cell line HH315 consisted of only a polyploid population. These cell lines may be useful in breast cancer research.

Combined Alloreactive CTL Cellular Therapy with Prodrug Activator Gene Therapy in a Model of Breast Cancer Metastatic to the Brain

Purpose: Individual or combined strategies of cellular therapy with alloreactive CTLs (alloCTL) and gene therapy using retroviral replicating vectors (RRV) encoding a suicide prodrug activating gene were explored for the treatment of breast tumors metastatic to the brain. Experimental Design: AlloCTL, sensitized to the HLA of MDA-MB-231 breast cancer cells, were examined in vitro for antitumor functionality toward breast cancer targets. RRV encoding the yeast cytosine deaminase (CD) gene was tested in vivo for virus spread, ability to infect, and kill breast cancer targets when exposed to 5-fluorocytosine (5-FC). Individual and combination treatments were tested in subcutaneous and intracranial xenograft models with 231BR, a brain tropic variant. Results: AlloCTL preparations were cytotoxic, proliferated, and produced IFN-γ when coincubated with target cells displaying relevant HLA. In vivo, intratumorally placed alloCTL trafficked through one established intracranial 231BR focus to another in contralateral brain and induced tumor cell apoptosis. RRV-CD efficiently spread in vivo, infected 231BR and induced their apoptosis upon 5-FC exposure. Subcutaneous tumor volumes were significantly reduced in alloCTL and/or gene therapy–treated groups compared to control groups. Mice with established intracranial 231BR tumors treated with combined alloCTL and RRV-CD had a median survival of 97.5 days compared with single modalities (50–83 days); all experimental treatment groups survived significantly longer than sham-treated groups (median survivals 31.5 or 40 days) and exhibited good safety/toxicity profiles. Conclusion: The results indicate combining cellular and suicide gene therapies is a viable strategy for the treatment of established breast tumors in the brain. Clin Cancer Res; 19(15); 4137–48. ©2013 AACR.

Radial mobility and cytotoxic function of retroviral replicating vector transduced, non-adherent alloresponsive T lymphocytes.

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

Abstract 1660: Retroviral replicating vector transduced alloresponsive T lymphocytes retain mobility and cytotoxic functionalities

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The radial monolayer cell migration assay was originally developed to measure the infiltrative properties of adherent tumor cells on individual extracellular matrix (ECM) proteins, or on ECM extracted from tumor cell monolayers. For this assay a single cell suspension of tumor cells is seeded using a cell sedimentation manifold (CSM), precisely into the center of Teflon-masked, ECM-coated slide wells. The adherent tumor cells establish a focal, circular monolayer from which horizontal motility can be microscopically-viewed and photographed over time. We adapted use of the CSM to evaluate the in vitro mobility of non-adherent effector T cells on confluent tumor cell monolayers or on ECM extracts from them. Cytotoxic T lymphocytes (CTL) sensitized to the human leukocyte antigens (HLA) on 13-06-MG glioma cells or on brain-tropic breast MDA-MB-231 carcinoma cells were generated by one-way mixed lymphocyte tumor cell reactions. These alloresponsive CTL were then transduced with retroviral replicating vectors (RRV) coding for green fluorescent protein (GFP). Using the CSM, the RRV-GFP transduced CTL were seeded into the center of wells with a confluent monolayer of glioma cells that were partially HLA matched to 13-06-MG. By light and fluorescence microscopy, we visualized not only the a) mobility of individual GFP+ T cells, but b) the cytotoxicity engendered to the underlying adherent glioma cell monolayer by the overlaid nonadherent CTL, in addition to c) transfer of RRV from the CTL to tumor cells. The in vitro cytotoxic functionality of the RRV-transduced CTL was confirmed by real-time xCELLigence impedance cytotoxicity assays. In other experiments, the RRV-GFP transduced CTL were also stained with a vital fluorescent red dye, CMPTX, to distinguish transduced from nontransduced CTL. The CTL were placed into the center of wells coated with ECM harvested from MDA-MB-231BR cells and radial movement of the transduced and nontransduced CTL was also observed over 72 hr time. We conclude that effector T cells transduced with RRV maintain mobility and cytotoxic functionality, and further, are capable of transmitting RRV to tumor cells. Thus, transduced alloresponsive CTL can facilitate immunogene therapy by their capability of gene delivery to infiltrating tumor cells in the brain. Citation Format: Kate L. Erickson, Colin C. Malone, Michelle J. Hickey, Geoffrey C. Owens, Yuki Kato, Robert M. Prins, Noriyuki Kasahara, Carol A. Kruse. Retroviral replicating vector transduced alloresponsive T lymphocytes retain mobility and cytotoxic functionalities. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1660. doi:10.1158/1538-7445.AM2014-1660