Intratumoral expression of IL-12 from lentiviral or RNA vectors acts synergistically with TLR4 agonist (GLA) to generate anti-tumor immunological memory.

Systemic interleukin-12 (IL12) anti-tumor therapy is highly potent but has had limited utility in the clinic due to severe toxicity. Here, we present two IL12-expressing vector platforms, both of which can overcome the deficiencies of previous systemic IL12 therapies: 1) an integrating lentiviral vector, and 2) a self-replicating messenger RNA formulated with polyethyleneimine. Intratumoral administration of either IL12 vector platform resulted in recruitment of immune cells, including effector T cells and dendritic cells, and the complete remission of established tumors in multiple murine models. Furthermore, concurrent intratumoral administration of the synthetic TLR4 agonist glucopyranosyl lipid A formulated in a stable emulsion (GLA-SE) induced systemic memory T cell responses that mediated complete protection against tumor rechallenge in all survivor mice (8/8 rechallenged mice), whereas only 2/6 total rechallenged mice treated with intratrumoral IL12 monotherapy rejected the rechallenge. Taken together, expression of vectorized IL12 in combination with a TLR4 agonist represents a varied approach to broaden the applicability of intratumoral immune therapies of solid tumors.

ZVex™, a dendritic-cell-tropic lentivector, primes protective antitumor T cell responses that are significantly boosted using heterologous vaccine modalities.

Therapeutic cancer vaccines must induce high levels of tumor-specific cytotoxic CD8 T cells to be effective. We show here that tumor-antigen specific effector and memory T cell responses primed with a non-integrating, dendritic-cell targeted lentiviral vector (ZVex™) could be boosted significantly by either adjuvanted recombinant protein, adenoviral vectors, or self-replicating RNA. These heterologous prime-boost regimens also provided significantly better protection in murine tumor models. In contrast, homologous prime-boost regimens, or using the lentiviral vector as a boost, resulted in lower T cell responses with limited therapeutic efficacy. Heterologous prime-boost regimens that utilize ZVex as the prime may be attractive modalities for therapeutic cancer vaccines.

Intratumoral immune activation with TLR4 agonist synergizes with effector T cells to eradicate established murine tumors.

Effective T cell-based immunotherapy of solid malignancies requires intratumoral activity of cytotoxic T cells and induction of protective immune memory. A major obstacle to intratumoral trafficking and activation of vaccine-primed or adoptively transferred tumor-specific T cells is the immunosuppressive tumor microenvironment (TME), which currently limits the efficacy of both anti-tumor vaccines and adoptive cell therapy (ACT). Combination treatments to overcome TME-mediated immunosuppression are therefore urgently needed. We combined intratumoral administration of the synthetic toll-like receptor 4 agonist glucopyranosyl lipid A (oil-in-water formulation, G100) with either active vaccination or adoptive transfer of tumor-specific CD8 T cells to mice bearing established melanomas or orthotopically inoculated glioblastomas. In combination with cancer vaccines or ACT, G100 significantly increased expression of innate immune genes, infiltration and expansion of activated effector T cells, antigen spreading, and durable immune responses. Complete tumor regression of both injected and non-injected tumors was observed only in mice receiving combination immunotherapy. TLR4-based intratumoral immune activation may be a viable approach to enhance the efficacy of therapeutic cancer vaccines and ACT in patients.

Distinct mechanisms of B and T lymphocyte accumulation generate tumor-draining lymph node hypertrophy.

Tumor-draining lymph nodes (TDLNs) often enlarge in human cancer patients and in murine tumor models, due to lymphocyte accumulation and lymphatic sinus growth. B lymphocytes within TDLNs can drive lymph node hypertrophy in response to tumor growth, however little is known about the mechanisms directing the preferential accumulation of B lymphocytes relative to T cells in enlarging TDLNs. To define why B and T lymphocytes accumulate in TDLNs, we quantified lymphocyte proliferation, apoptosis, entry, and exit in TDLNs versus contralateral non-TDLNs (NTDLNs) in a footpad B16-F10 melanoma mouse model. B and T lymphocyte proliferation and apoptosis were increased as the TDLNs enlarged, although relative rates were similar to those of NTDLNs. TDLN entry of B and T lymphocytes via high endothelial venules was also modestly increased in enlarged TDLNs. Strikingly, the egress of B cells was strongly reduced in TDLNs versus NTDLNs, while T cell egress was modestly decreased, indicating that regulation of lymphocyte exit from TDLNs is a major mechanism of preferential B lymphocyte accumulation. Surface sphingosine-1-phosphate receptor 1 (S1PR1) which binds S1P and signals lymphocyte egress, exhibited greater downregulation in B relative to T lymphocytes, consistent with preferential retention of B lymphocytes in TDLNs. TDLN lymphocytes did not activate surface CD69 expression, indicating a CD69-independent mechanism of downregulation of S1PR1. B and T cell trafficking via afferent lymphatics to enter TDLNs also increased, suggesting a pathway for accumulation of tumor-educated lymphocytes in TDLNs. These mechanisms regulating TDLN hypertrophy could provide new targets to manipulate lymphocyte responses to cancer.

Regulatory B cells preferentially accumulate in tumor-draining lymph nodes and promote tumor growth.

Our previous studies found that B16-F10 melanoma growth in the rear footpad of immunocompetent mice induces marked B cell accumulation within tumor-draining popliteal lymph nodes (TDLN). This B cell accumulation drives TDLN remodeling that precedes and promotes metastasis, indicating a tumor-promoting role for TDLN B cells. Here we show that phenotypic characterization of lymphocytes in mice bearing B16-F10 melanomas identifies preferential accumulation of T2-MZP B cells in the TDLN. Comparison of non-draining LNs and spleens of tumor-bearing mice with LNs and spleens from naïve mice determined that this pattern of B cell accumulation was restricted to the TDLN. B cell-deficient and immunocompetent mice reconstituted with T2-MZP B cells but not with other B cell subsets displayed accelerated tumor growth, demonstrating that T2-MZP B cells possess regulatory activity in tumor-bearing mice. Unlike splenic regulatory B cells, however, these TDLN B cells did not exhibit increased IL-10 production, nor did they promote Treg generation in the TDLN. These findings demonstrate that tumors initially signal via the lymphatic drainage to stimulate the preferential accumulation of T2-MZP regulatory B cells. This local response may be an early and critical step in generating an immunosuppressive environment to permit tumor growth and metastasis.

Evaluation of reference genes for quantitative PCR analysis of mouse lymphocytes.

Normalization to a reference gene is the method of choice for quantitative PCR analysis. The stability of reference genes is critical for accurate gene expression analysis, as significant variations in reference gene expression can alter experimental results and conclusions. In this study, we evaluated the expression stability of five commonly used reference genes found in mouse lymphocytes. Using NormFinder and BestKeeper algorithms, we consistently show that ubiquitin C (Ubc) is the optimal reference gene for normalizing qPCR data obtained from mouse lymphocytes, whereas beta-actin (Actb) is not a suitable reference gene due to its extensive variability in expression. Our findings emphasize the importance of validating reference genes for qPCR analyses. We provide a shortlist of reference genes to use for normalization and recommend freely available software programs as a rapid approach to validate potential reference genes.

Multiple BH3 mimetics antagonize antiapoptotic MCL1 protein by inducing the endoplasmic reticulum stress response and up-regulating BH3-only protein NOXA.

BH3 mimetics are small molecules designed or discovered to mimic the binding of BH3-only proteins to the hydrophobic groove of antiapoptotic BCL2 proteins. The selectivity of these molecules for BCL2, BCL-X(L), or MCL1 has been established in vitro; whether they inhibit these proteins in cells has not been rigorously investigated. In this study, we used a panel of leukemia cell lines to assess the ability of seven putative BH3 mimetics to inhibit antiapoptotic proteins in a cell-based system. We show that ABT-737 is the only BH3 mimetic that inhibits BCL2 as assessed by displacement of BAD and BIM from BCL2. The other six BH3 mimetics activate the endoplasmic reticulum stress response inducing ATF4, ATF3, and NOXA, which can then bind to and inhibit MCL1. In most cancer cells, inhibition of one antiapoptotic protein does not acutely induce apoptosis. However, by combining two BH3 mimetics, one that inhibits BCL2 and one that induces NOXA, apoptosis is induced within 6 h in a BAX/BAK-dependent manner. Because MCL1 is a major mechanism of resistance to ABT-737, these results suggest a novel strategy to overcome this resistance. Our findings highlight a novel signaling pathway through which many BH3 mimetics inhibit MCL1 and suggest the potential use of these agents as adjuvants in combination with various chemotherapy strategies.

Vinblastine induces acute, cell cycle phase-independent apoptosis in some leukemias and lymphomas and can induce acute apoptosis in others when Mcl-1 is suppressed.

Chemotherapeutic agents modify intracellular signaling that culminates in the inhibition of Bcl-2 family members and initiates apoptosis. Inhibition of the extracellular signal-regulated kinase by PD98059 dramatically accelerates vinblastine-mediated apoptosis in ML-1 leukemia with cells dying in 4 hours from all phases of the cell cycle. Inhibition of protein synthesis by cycloheximide also markedly accelerated vinblastine-induced apoptosis, showing that the proteins required for this acute apoptosis are constitutively expressed. Vinblastine induced the rapid induction of Mcl-1 that was inhibited by PD98059 and cycloheximide. No change in Bcl-2 or Bcl-X was observed. We hypothesize that ML-1 cells use Mcl-1 for protection from the rapid vinblastine-induced apoptosis. This was confirmed by targeting Mcl-1 with short hairpin RNA. We also investigated the response of 13 other leukemia and lymphoma cell lines and cells from seven chronic lymphocytic leukemia patients. Four cell lines and all chronic lymphocytic leukemia cells were killed in 6 hours by vinblastine alone. Two additional cell lines were sensitized to vinblastine by PD98059, which suppressed Mcl-1. This acute apoptosis either alone or in combination with PD98059 required vinblastine-mediated activation of c-Jun-NH(2)-terminal kinase. PD98059 did not suppress Mcl-1 in other cell lines whereas sorafenib did, but this did not sensitize the cells to vinblastine, suggesting that the acute apoptosis varies depending on which Bcl-2 protein mediates protection. Most of the cell lines were sensitized to vinblastine by cycloheximide, suggesting that inhibition of a short-lived protein in addition to Mcl-1 can acutely sensitize cells. These results suggest several clinical strategies that might provide an effective therapy for selected patients. Mol Cancer Ther; 9(4); 791-802. (c)2010 AACR.