Selective Expansion of Chimeric Antigen Receptor-targeted T-cells with Potent Effector Function using Interleukin-4

Polyclonal T-cells can be directed against cancer using transmembrane fusion molecules known as chimeric antigen receptors (CARs). Although preclinical studies have provided encouragement, pioneering clinical trials using CAR-based immunotherapy have been disappointing. Key obstacles are the need for robust expansion ex vivo followed by sustained survival of infused T-cells in patients. To address this, we have developed a system to achieve selective proliferation of CAR(+) T-cells using IL-4, a cytokine with several pathophysiologic and therapeutic links to cancer. A chimeric cytokine receptor (4 alpha beta) was engineered by fusion of the IL-4 receptor alpha (IL-4R alpha) ectodomain to the beta(c) subunit, used by IL-2 and IL-15. Addition of IL-4 to T-cells that express 4 alpha beta resulted in STAT3/STAT5/ERK phosphorylation and exponential proliferation, mimicking the actions of IL-2. Using receptor-selective IL-4 muteins, partnering of 4 alpha beta with gamma(c) was implicated in signal delivery. Next, human T-cells were engineered to co-express 4 alpha beta with a CAR specific for tumor-associated MUC1. These T-cells exhibited an unprecedented capacity to elicit repeated destruction of MUC1-expressing tumor cultures and expanded through several logs in vitro. Despite prolonged culture in IL-4, T-cells retained specificity for target antigen, type 1 polarity, and cytokine dependence. Similar findings were observed using CARs directed against two additional tumor-associated targets, demonstrating generality of application. Furthermore, this system allows rapid ex vivo expansion and enrichment of engineered T-cells from small blood volumes, under GMP-compliant conditions. Together, these findings provide proof of principle for the development of IL-4-enhanced T-cell immunotherapy of cancer

Metastatic cells from pleural effusions isolated from breast cancer patients can form mammospheres

Cells were isolated from pleural effusions (PEs) and placed in non-differentiating medium in non-adherent culture flasks (see Materials and methods). PE14, PE6, PE21, and PE8 show four representative cultures. PE mammospheres were disrupted and the cells plated onto glass coverslips in medium supplemented with 1% foetal calf serum. After 5 days of adherent culture, the cells were stained with antibodies to MUC1 (HMFG2), CK5 (D5/6), CK14 (LL002), and CK19 (BA17). Breast cancer cell lines were also placed in non-differentiating medium in non-adherent culture flasks. Mammospheres could be seen developing in MCF7 and SKBR3 cultures, while MDAMB231 produced loosely adhered clumps of cells.<p><b>Copyright information:</b></p><p>Taken from "Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells"</p><p>http://breast-cancer-research.com/content/10/3/R52</p><p>Breast Cancer Research : BCR 2008;10(3):R52-R52.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2481500.</p><p></p

CD24 expression by pleural effusion cells as detected by antibodies SWA11 and ML5

Cells isolated from pleural effusions were stained with unconjugated SWA11 and ML5, and antibody binding was detected by fluorescein isothiocyanate-conjugated rabbit anti-mouse secondary antibody (see Materials and methods). Filled histograms, 2° antibody only; thin grey line, SWA11; thick black line, ML5. PE, pleural effusion.<p><b>Copyright information:</b></p><p>Taken from "Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells"</p><p>http://breast-cancer-research.com/content/10/3/R52</p><p>Breast Cancer Research : BCR 2008;10(3):R52-R52.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2481500.</p><p></p

Flexible Targeting of ErbB Dimers That Drive Tumorigenesis by Using Genetically Engineered T Cells

Pharmacological targeting of individual ErbB receptors elicits antitumor activity, but is frequently compromised by resistance leading to therapeutic failure. Here, we describe an immunotherapeutic approach that exploits prevalent and fundamental mechanisms by which aberrant upregulation of the ErbB network drives tumorigenesis. A chimeric antigen receptor named T1E28z was engineered, in which the promiscuous ErbB ligand, T1E, is fused to a CD28 + CD3ζ endodomain. Using a panel of ErbB-engineered 32D hematopoietic cells, we found that human T1E28z+ T cells are selectively activated by all ErbB1-based homodimers and heterodimers and by the potently mitogenic ErbB2/3 heterodimer. Owing to this flexible targeting capability, recognition and destruction of several tumor cell lines was achieved by T1E28z+ T cells in vitro, comprising a wide diversity of ErbB receptor profiles and tumor origins. Furthermore, compelling antitumor activity was observed in mice bearing established xenografts, characterized either by ErbB1/2 or ErbB2/3 overexpression and representative of insidious or rapidly progressive tumor types. Together, these findings support the clinical development of a broadly applicable immunotherapeutic approach in which the propensity of solid tumors to dysregulate the extended ErbB network is targeted for therapeutic gain