James J. Mulé

DESIGN AND FUNCTION OF A DENDRIMER-BASED THERAPEUTIC NANODEVICE TARGETED TO TUMOR CELLS THROUGH THE FOLATE RECEPTOR

AbstractPurpose. We sought to develop nanoscale drug delivery materials that would allow targeted intracellular delivery while having an imaging capability for tracking uptake of the material. A complex nanodevice was designed and synthesized that targets tumor cells through the folate receptor.nMethods. The device is based on an ethylenediamine core polyamidoamine dendrimer of generation 5. Folic acid, fluorescein, and methotrexate were covalently attached to the surface to provide targeting, imaging, and intracellular drug delivery capabilities. Molecular modeling determined the optimal dendrimer surface modification for the function of the device and suggested a surface modification that improved targeting.nResults. Three nanodevices were synthesized. Experimental targeting data in KB cells confirmed the modeling predictions of specific and highly selective binding. Targeted delivery improved the cytotoxic response of the cells to methotrexate 100-fold over free drug.nConclusions. These results demonstrate the ability to design and produce polymer-based nanodevices for the intracellular targeting of drugs, imaging agents, and other materials.

Dendritic cells genetically engineered to express IL-4 inhibit murine collagen-induced arthritis

Dendritic cells (DCs) are specialized antigen-presenting cells that migrate from the periphery to lymphoid tissues, where they activate and regulate T cells. Genetic modification of DCs to express immunoregulatory molecules would provide a new immunotherapeutic strategy for autoimmune and other diseases. We have engineered bone marrow-derived DCs that express IL-4 and tested the ability of these cells to control murine collagen-induced arthritis (CIA), a model for rheumatoid arthritis in which Th1 cells play a critical role. IL-4-transduced DCs inhibited Th1 responses to collagen type II in vitro. A single injection of IL-4-transduced DCs reduced the incidence and severity of CIA and suppressed established Th1 responses and associated humoral responses, despite only transient persistence of injected DCs in the spleen. In contrast, control DCs and IL-4-transduced T cells or fibroblastic cells failed to alter the course of the disease. The functional effects correlated well with the differential efficiency of DC migration from various sites of injection to lymphoid organs, especially the spleen. The ability of splenic T cells to produce IL-4 in response to anti-CD3 was enhanced after the administration of IL-4-transduced DCS: These results support the feasibility of using genetically modified DCs for the treatment of autoimmune disease.

Review: Gene-Modified Dendritic Cells for Use in Tumor Vaccines

Dendritic cells (DCs) are potent antigen-presenting cells capable of priming activation of naive T cells. Because of their immunostimulatory capacity, immunization with DCs presenting tumor antigens has been proposed as a treatment regimen for cancer. The results from translational research studies and early clinical trials point to the need for improvement of DC-based tumor vaccines before they become a more broadly applicable treatment modality. In this regard, studies suggest that genetic modification of DCs to express tumor antigens and/or immunomodulatory proteins may improve their capacity to promote an antitumor response. Because the DC phenotype is relatively unstable, nonperturbing methods of gene transfer must be employed that do not compromise viability or immunostimulatory capacity. DCs expressing transgenes encoding tumor antigens have been shown to be more potent primers of antitumor immunity both in vitro and in animal models of disease; in some measures of immune priming, gene-modified DCs exceeded their soluble antigen-pulsed counterparts. Cytokine gene modification of DCs has improved their capacity to prime tumor antigen-specific T cell responses and promote antitumor immunity in vivo. Here, we review the current status of gene-modified DCs in both human and murine studies. Although successful results have been obtained to date in experimental systems, we discuss potential problems that have already arisen and may yet be encountered before gene-modified DCs are more widely applicable for use in human clinical trials.

Treatment of solid tumours in children with tumour-lysate-pulsed dendritic cells

Dendritic cells are potent stimulators of antigen-specific immune responses, including antitumour responses. We explored the use of tumour-lysate-pulsed dendritic cells in children with relapsed solid tumours. Dendritic cell treatment in children was feasible and apparently not toxic. The treatment was able to produce significant tumour regression in a child with metastatic fibrosarcoma.

Comparative analysis of murine dendritic cells derived from spleen and bone marrow

In order to improve upon preclinical tumor vaccine strategies that employ dendritic cells (DC), we now have compared short-term cultures of spleen- and GMCSF/IL-4-stimulated bone marrow (BM) to determine if differences exist in phenotype and function of murine DC derived from primary and secondary hematolymphoid organs. Although cultures of BM contained a lower percentage of DC compared to spleen, their capacity to stimulate a primary allogeneic mixed leukocyte reaction (MLR) and to uptake fluorescent dextran was substantially greater. In addition, the overall yields of DC per animal was at least twofold greater from BM compared to spleen. Cultures of BM harvested at day 3, 6, or 9 stimulated comparable levels of primary allo-MLR on a per-cell basis. However, there was a consistent loss (at least twofold) of all cells occurring beyond day 6 as compared with cell yields from earlier time points. Importantly, we also improved on methods to rapidly obtain highly enriched DC (>90%) from BM, which has obviated the reported prior need for complex antibody and complement treatments to remove contaminating mature T and B lymphocytes, la-bearing cells, and granulocytes before DC generation. In contrast, although similar purity of DC with similar phenotype and function could be obtained from the spleen, substantial loss in yield occurred, suggesting a further difference in DC between the two tissue sources. The overall yield of DC derived from spleen and BM cultures could be substantially increased by in vivo pretreatment of the donor animals with recombinant Flt3-L. Collectively, these studies demonstrate that notable differences exist in DC preparations derived from spleen vs. BM and that BM provides the preferred source of DC that can be rapidly enriched to high purity for use in further vaccine development.

Inducible expression of macrophage receptor Marco by dendritic cells following phagocytic uptake of dead cells uncovered by oligonucleotide arrays.

The efficient Ag presenting and immunostimulatory capacity of dendritic cells (DCs) has led to the use of tumor Ag-pulsed DCs in treatment regimens for cancer. Although vaccine studies involving tumor lysate-pulsed DCs have been performed, little, if any, information is available on the effects of phagocytic uptake of tumor lysate on DC biology and function. We have investigated gene expression pattern differences between unpulsed DCs and tumor lysate-pulsed-DCs, using Affymetrix MG-U74Av2 oligonucleotide arrays, which contain ∼12,000 genes and expressed sequence tags. Upon 24 h tumor lysate pulsing, the levels of 87 transcripts increased at least 3-fold while the levels of 121 transcripts were reduced by one-third or more, with accompanying p values <0.01. Most of these genes encoded proteins important for DC effector functions including cytokines, chemokines, and receptors, such as IL-12p40, macrophage inflammatory protein-2, and IL-6; Ag presentation, such as carboxypeptidase D and H2-DM; cell adhesion (e.g., EGF-like module containing, mucin-like, hormone receptor-like sequence 1, rhoB); and T cell activation. Interestingly, we observed a high level of expression of a novel member of the class A scavenger receptor family, macrophage receptor with collagenous structure (Marco). Marco is thought to play an important role in the immune response by mediating binding and phagocytosis, but also in the formation of lamellipodia-like structures and of dendritic processes. Therefore, we have identified a repertoire of genes that likely play a role in DC function.

Chemokine gene modification of human dendritic cell-based tumor vaccines using a recombinant adenoviral vector

Previous animal studies conducted in our laboratory have shown that tumor antigen-pulsed dendritic cells (TP-DC) can mediate antitumor effects in vivo. However, durable and complete regression of established tumors has been difficult to achieve through the administration of TP-DC alone. To better augment immune priming to tumors in vivo, we have hypothesized that it is necessary to achieve an increased number of host-derived, naïve T cells at the site of TP-DC vaccine injections. To accomplish this goal, we have embarked on a series of studies that utilize defined chemokines. One of these molecules, secondary lymphoid tissue chemokine (SLC), has been shown to be uniquely chemoattractant for naïve T cells and dendritic cells. We propose that gene modification of DC-based tumor vaccines to produce human SLC will enhance T-cell recruitment and immune priming to tumor-associated antigens, and thereby translate into improved antitumor vaccine efficacy in vivo. Utilizing an E1-, E3-deleted adenoviral vector containing the gene for human SLC, we have been able to transduce human DC to produce biologically active human SLC that chemoattracts human T cells in vitro. SLC production by transduced DC was markedly enhanced upon DC maturation. Additionally, these SLC-secreting DC were found to be viable to a large extent despite the cytopathic effect inherent in adenoviral gene transfer and, most importantly, functional as determined by their ability to prime autologous T cells to a known melanoma-associated antigen, MART-1. Based on these encouraging results, we plan to initiate Phase I clinical studies utilizing DC-SLC to treat patients with advanced solid tumors.

On combining antineoplastic drugs with tumor vaccines

Over the past 2 decades, the classical paradigm of trimodal cancer therapy has been expanded to include immunotherapy, encompassing both passive, adoptive T-cell transfer techniques as well as active vaccination strategies. As the mainstay of anticancer therapy, antineoplastic drugs have long been used for their direct tumoricidal properties, while the immunosuppressive adverse effects have been merely tolerated and supported. With the advent of the increasing use of immunotherapy in the clinical setting, investigators have sought to determine ways in which to combine accepted chemotherapeutic regimens with innovative immunotherapeutic techniques, and have discovered that the lymphodepletion that results from antineoplastic drug administration may be, in some cases, advantageous in eliciting clinically relevant responses to cancer immunotherapy. As well, several of these drugs have been found, paradoxically, to actually augment antitumor immunity. There is a paucity of preclinical and clinical data to date on combining chemotherapy and antitumor vaccines, as this is a strategy in its infancy. However, it may ultimately be found that chemotherapy combined with vaccine therapy offers therapeutic advantages over single-modality treatment. Here we will explore the available data regarding the mechanisms behind enhancement of antitumor efficacy through the combination of antineoplastic drugs with tumor vaccines.

Identification of a T-cell receptor from a therapeutic murine T-cell clone.

Tumor-infiltrating lymphocytes (TIL) have been successfully used for the treatment of metastatic malignancies in clinical trials and in experimental animal models. Tumor-specific reactivity by TIL is mediated via receptors expressed on the surface of T cells (TcRs), which recognize tumor-associated antigens (TAA) presented in the context of MHC molecules on the surface of tumor cells. The current study was performed to identify the TcR alpha and beta chains from a tumor-specific therapeutic TIL clone that can be used to develop a preclinical animal model for genetically modifying lymphocytes and hematopoietic progenitors with TcR genes. TIL 205 was generated from a subcutaneous implant of MCA-205 fibrosarcoma and at 21 days was cloned by limiting dilution. TIL clone 8, obtained from a culture seeded at one cell/well, mediated specific lysis and specific secretion of gamma-interferon to MCA-205 and WP6, a subclone of MCA 205. No reactivity was observed against other syngeneic sarcoma lines. Anchor polymerase chain reaction analysis determined that antigen recognition by clone 8 was mediated by a TcR consisting of V alpha 3/J alpha 27 and V beta 8.2/D beta 2.1/D beta 2.4. Immunofluorescent staining with V beta subfamily specific monoclonal antibodies revealed that > 95% of the T cells in TIL clone 8 expressed V beta 8.2, confirming that TIL clone 8 was indeed a clone. In contrast, approximately 30% of the T cells in the parental TIL 205 expressed V beta 8.2. The transfer of as few as 500,000 TIL clone 8 cells in conjunction with the systemic administration of recombinant human interleukin-2 mediated regression of established 3-day WP6 lung metastases. Thus, clone 8 recognizes a biologically relevant tumor rejection antigen, making the V alpha 3/J alpha 27-V beta 8.2/D beta 2.1/J beta 2.4 TcR isolated from this clone useful as a probe for cloning the tumor-rejection antigen in the WP6 tumor as well as modeling, in mice, the TcR-based gene therapies being developed for humans.

Nitric oxide inhibition selectively increases MMP-9 activation in vitro by aortic smooth muscle cells

IL-10, when cultured with LPS stimulated macrophages, significantly inhibited TNF-a release and PCA by 75% and 71%, respectively (p , 0.05). However, serum from IL-10 treated shock and clamp rats, when cultured with LPS stimulated macrophages, resulted in a 120% rise in PCA (p , 0.05), despite a 99% (p , 0.05) inhibition of TNF-a release. Serum from untreated shock and clamp animals did not affect PCA.