Potentiation of natural killer cells to overcome cancer resistance to NK cell-based therapy and to enhance antibody-based immunotherapy.

Natural killer (NK) cells are cellular components of the innate immune system that can recognize and suppress the proliferation of cancer cells. NK cells can eliminate cancer cells through direct lysis, by secreting perforin and granzymes, or through antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC involves the binding of the Fc gamma receptor IIIa (CD16), present on NK cells, to the constant region of an antibody already bound to cancer cells. Cancer cells use several mechanisms to evade antitumor activity of NK cells, including the accumulation of inhibitory cytokines, recruitment and expansion of immune suppressor cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), modulation of ligands for NK cells receptors. Several strategies have been developed to enhance the antitumor activity of NK cells with the goal of overcoming cancer cells resistance to NK cells. The three main strategies to engineer and boost NK cells cytotoxicity include boosting NK cells with modulatory cytokines, adoptive NK cell therapy, and the employment of engineered NK cells to enhance antibody-based immunotherapy. Although the first two strategies improved the efficacy of NK cell-based therapy, there are still some limitations, including immune-related adverse events, induction of immune-suppressive cells and further cancer resistance to NK cell killing. One strategy to overcome these issues is the combination of monoclonal antibodies (mAbs) that mediate ADCC and engineered NK cells with potentiated anti-cancer activity. The advantage of using mAbs with ADCC activity is that they can activate NK cells, but also favor the accumulation of immune effector cells to the tumor microenvironment (TME). Several clinical trials reported that combining engineered NK cells with mAbs with ADCC activity can result in a superior clinical response compared to mAbs alone. Next generation of clinical trials, employing engineered NK cells with mAbs with higher affinity for CD16 expressed on NK cells, will provide more effective and higher-quality treatments to cancer patients.

Development and Characterization of an Anti-Cancer Monoclonal Antibody for Treatment of Human Carcinomas.

NEO-201 is an IgG1 humanized monoclonal antibody (mAb) that binds to tumor-associated variants of carcinoembryonic antigen-related cell adhesion molecule (CEACAM)-5 and CEACAM-6. NEO-201 reacts to colon, ovarian, pancreatic, non-small cell lung, head and neck, cervical, uterine and breast cancers, but is not reactive against most normal tissues. NEO-201 can kill tumor cells via antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) to directly kill tumor cells expressing its target. We explored indirect mechanisms of its action that may enhance immune tumor killing. NEO-201 can block the interaction between CEACAM-5 expressed on tumor cells and CEACAM-1 expressed on natural killer (NK) cells to reverse CEACAM-1-dependent inhibition of NK cytotoxicity. Previous studies have demonstrated safety/tolerability in non-human primates, and in a first in human phase 1 clinical trial at the National Cancer Institute (NCI). In addition, preclinical studies have demonstrated that NEO-201 can bind to human regulatory T (Treg) cells. The specificity of NEO-201 in recognizing suppressive Treg cells provides the basis for combination cancer immunotherapy with checkpoint inhibitors targeting the PD-1/PD-L1 pathway.

An NK cell line (haNK) expressing high levels of granzyme and engineered to express the high affinity CD16 allele.

Natural killer (NK) cells are known to play a role in mediating innate immunity, in enhancing adaptive immune responses, and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) by reactivity of CD16 with the Fc region of human IgG1 antibodies. The NK-92 cell line, derived from a lymphoma patient, has previously been well characterized and adoptive transfer of irradiated NK-92 cells has demonstrated safety and shown preliminary evidence of clinical benefit in cancer patients. The NK-92 cell line, devoid of CD16, has now been engineered to express the high affinity (ha) CD16 V158 FcγRIIIa receptor, as well as engineered to express IL-2; IL-2 has been shown to replenish the granular stock of NK cells, leading to enhanced perforin- and granzyme-mediated lysis of tumor cells. The studies reported here show high levels of granzyme in haNK cells, and demonstrate the effects of irradiation of haNK cells on multiple phenotypic markers, viability, IL-2 production, and lysis of a spectrum of human tumor cells. Studies also compare endogenous irradiated haNK lysis of tumor cells with that of irradiated haNK-mediated ADCC using cetuximab, trastuzumab and pertuzumab monoclonal antibodies. These studies thus provide the rationale for the potential use of irradiated haNK cells in adoptive transfer studies for a range of human tumor types. Moreover, since only approximately 10% of humans are homozygous for the high affinity V CD16 allele, these studies also provide the rationale for the use of irradiated haNK cells in combination with IgG1 anti-tumor monoclonal antibodies.

Short-term EGFR blockade enhances immune-mediated cytotoxicity of EGFR mutant lung cancer cells: rationale for combination therapies.

The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) erlotinib has been approved for years as a first-line therapy for patients harboring EGFR-sensitizing mutations. With the promising implementation of immunotherapeutic strategies for the treatment of lung cancer, there is a growing interest in developing combinatorial therapies that could utilize immune approaches in the context of conventional or targeted therapies. Tumor cells are known to evade immune attack by multiple strategies, including undergoing phenotypic plasticity via a process designated as the epithelial-mesenchymal transition (EMT). As signaling through EGFR is a major inducer of EMT in epithelial cells, we have investigated the effect of EGFR inhibition with erlotinib on tumor phenotype and susceptibility to immune attack. Our data shows that short-term exposure of tumor cells to low-dose erlotinib modulates tumor plasticity and immune-mediated cytotoxicity in lung cancer cells harboring a sensitizing EGFR mutation, leading to a remarkable enhancement of tumor lysis mediated by innate NK cells and antigen-specific T cells. This effect positively correlated with the ability of short-term EGFR blockade to modulate tumor phenotype towards a more epithelial one, as well as to increase susceptibility to caspase-mediated apoptosis. The effect, however, was lost when erlotinib was utilized for long periods of time in vitro or in vivo, which resulted in gain of mesenchymal features and decreased (rather than increased) tumor lysis in response to immune effector mechanisms. Our data provides rationale for potential combinations of erlotinib and immunotherapies for the treatment of lung carcinomas in the early setting, before the establishment of tumor relapse with long-term EGFR inhibition.

The generation and analyses of a novel combination of recombinant adenovirus vaccines targeting three tumor antigens as an immunotherapeutic.

Phenotypic heterogeneity of human carcinoma lesions, including heterogeneity in expression of tumor-associated antigens (TAAs), is a well-established phenomenon. Carcinoembryonic antigen (CEA), MUC1, and brachyury are diverse TAAs, each of which is expressed on a wide range of human tumors. We have previously reported on a novel adenovirus serotype 5 (Ad5) vector gene delivery platform (Ad5 [E1-, E2b-]) in which regions of the early 1 (E1), early 2 (E2b), and early 3 (E3) genes have been deleted. The unique deletions in this platform result in a dramatic decrease in late gene expression, leading to a marked reduction in host immune response to the vector. Ad5 [E1-, E2b-]-CEA vaccine (ETBX-011) has been employed in clinical studies as an active vaccine to induce immune responses to CEA in metastatic colorectal cancer patients. We report here the development of novel recombinant Ad5 [E1-, E2b-]-brachyury and-MUC1 vaccine constructs, each capable of activating antigen-specific human T cells in vitro and inducing antigen-specific CD4+ and CD8+ T cells in vaccinated mice. We also describe the use of a combination of the three vaccines (designated Tri-Ad5) of Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-brachyury and Ad5 [E1-, E2b-]-MUC1, and demonstrate that there is minimal to no "antigenic competition" in in vitro studies of human dendritic cells, or in murine vaccination studies. The studies reported herein support the rationale for the application of Tri-Ad5 as a therapeutic modality to induce immune responses to a diverse range of human TAAs for potential clinical studies.

Antibody-Dependent Cellular Cytotoxicity Activity of a Novel Anti-PD-L1 Antibody Avelumab (MSB0010718C) on Human Tumor Cells.

Several anti-PD-1/PD-L1 monoclonal antibodies (mAb) are currently providing evidence of clinical benefit in subsets of cancer patients. The mode of action of these mAbs is to inhibit PD-1 on immune cells interacting with PD-L1 on tumor cells. These mAbs are either designed or engineered to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), which, however, has been implicated as an important mechanism in several highly effective mAb-mediated cancer therapies. A fully human anti-PD-L1 mAb would potentially be able to block PD-1/PD-L1 interactions and also mediate the ADCC lysis of tumor cells. MSB0010718C (designated avelumab) is a fully human IgG1 anti-PD-L1 mAb. The studies reported here demonstrate (i) the ability of avelumab to lyse a range of human tumor cells in the presence of PBMC or NK effectors; (ii) IFNγ can enhance tumor cell PD-L1 expression and, in some cases, enhance ADCC tumor cell lysis; (iii) purified NK cells are potent effectors for avelumab; (iv) similar levels of avelumab-mediated ADCC lysis of tumor cells are seen using purified NK as effectors from either healthy donors or cancer patients; (v) very low levels of avelumab-mediated lysis are seen using whole PBMCs as targets; this finding complements results seen in analyses of PBMC subsets of patients receiving avelumab; and (vi) the addition of IL12 to NK cells greatly enhances avelumab-mediated ADCC. These studies thus provide an additional mode of action for an anti-PD-L1 mAb and support the rationale for further studies to enhance avelumab-mediated ADCC activity.

Identification and characterization of a cytotoxic T-lymphocyte agonist epitope of brachyury, a transcription factor involved in epithelial to mesenchymal transition and metastasis.

The transcription factor brachyury is a major driver of epithelial to mesenchymal transition in human carcinoma cells. It is overexpressed in several human tumor types versus normal adult tissues, except for testes and thyroid. Overexpression is associated with drug resistance and poor prognosis. Previous studies identified a brachyury HLA-A2 cytotoxic T-lymphocyte epitope. The studies reported here describe an enhancer epitope of brachyury. Compared to the native epitope, the agonist epitope: (a) has enhanced binding to MHC class I, (b) increased the IFN-γ production from brachyury-specific T cells, (c) generated brachyury-specific T cells with greater levels of perforin and increased proliferation, (d) generated T cells more proficient at lysing human carcinoma cells endogenously expressing the native epitope, and (e) achieved greater brachyury-specific T-cell responses in vivo in HLA-A2 transgenic mice. These studies also report the generation of a heat-killed recombinant Saccharomyces cerevisiae (yeast) vector expressing the full-length brachyury gene encoding the agonist epitope. Compared to yeast-brachyury (native) devoid of the agonist epitope, the yeast-brachyury (agonist) enhanced the activation of brachyury-specific T cells, which efficiently lysed human carcinoma cells. In addition to providing the rationale for the recombinant yeast-brachyury (agonist) as a potential vaccine in cancer therapy, these studies also provide the rationale for the use of the agonist in (a) dendritic cell (DC) vaccines, (b) adjuvant or liposomal vaccines, (c) recombinant viral and/or bacterial vaccines, (d) protein/polypeptide vaccines, (e) activation of T cells ex vivo in adoptive therapy protocols, and (f) generation of genetically engineered targeted T cells.

Therapeutic cancer vaccines.

Therapeutic cancer vaccines have the potential of being integrated in the therapy of numerous cancer types and stages. The wide spectrum of vaccine platforms and vaccine targets is reviewed along with the potential for development of vaccines to target cancer cell "stemness," the epithelial-to-mesenchymal transition (EMT) phenotype, and drug-resistant populations. Preclinical and recent clinical studies are now revealing how vaccines can optimally be used with other immune-based therapies such as checkpoint inhibitors, and so-called nonimmune-based therapeutics, radiation, hormonal therapy, and certain small molecule targeted therapies; it is now being revealed that many of these traditional therapies can lyse tumor cells in a manner as to further potentiate the host immune response, alter the phenotype of nonlysed tumor cells to render them more susceptible to T-cell lysis, and/or shift the balance of effector:regulatory cells in a manner to enhance vaccine efficacy. The importance of the tumor microenvironment, the appropriate patient population, and clinical trial endpoints is also discussed in the context of optimizing patient benefit from vaccine-mediated therapy.

Overexpression of the EMT driver brachyury in breast carcinomas: association with poor prognosis.

BACKGROUND: The epithelial-mesenchymal transition (EMT) has been implicated as an important process in tumor cell invasion, metastasis, and drug resistance. The transcription factor brachyury has recently been described as a driver of EMT of human carcinoma cells. METHODS: Brachyury mRNA and protein expression was analyzed in human breast carcinomas and benign tissues. The role of brachyury in breast tumor prognosis and drug resistance and the ability of brachyury-specific T cells to lyse human breast carcinoma cells were also evaluated. Kaplan-Meier analyses were used to evaluate the association between brachyury expression and survival. All statistical tests were two-sided. RESULTS: The level of brachyury expression in breast cancer cells was positively associated with their ability to invade the extracellular matrix, efficiently form mammospheres in vitro, and resist the cytotoxic effect of docetaxel. A comparison of survival among breast cancer patients treated with tamoxifen in the adjuvant setting who had tumors with high vs low brachyury mRNA expression demonstrated that high expression of brachyury is associated as an independent variable with higher risk of recurrence (hazard ratio [HR] = 7.5; 95% confidence interval [CI] = 2.4 to 23.5; P = 5.14×10(-4)) and distant metastasis (HR = 15.2; 95% CI = 3.5 to 66.3; P = 3.01×10(-4)). We also demonstrated that brachyury-specific T cells can lyse human breast carcinoma cells. CONCLUSIONS: The studies reported here provide the rationale for the use of a vaccine targeting brachyury for the therapy of human breast cancer, either as a monotherapy or in combination therapies.

A pan inhibitor of DASH family enzymes induces immunogenic modulation and sensitizes murine and human carcinoma cells to antigen-specific cytotoxic T lymphocyte killing: implications for combination therapy with cancer vaccines.

Recent studies have suggested that pan inhibitors of dipeptidyl peptidase-4 activity and/or structure homologs (DASH), including ARI-4175, can mediate tumor regression by immune-mediated mechanisms. This study assessed the potential of combining ARI-4175 with cancer vaccines. We evaluated ARI-4175's effect on immunogenic modulation, ability to sensitize tumor cells to antigen-specific CTL killing, effect on immune-cell subsets and function, and antitumor activity in 2 tumor models, both as a monotherapy and in combination with a recombinant viral or dendritic cell (DC)-based tumor-cell vaccine. ARI-4175's effects on the growth, surface phenotype, and antigen-specific CTL-mediated lysis of murine and human carcinoma cell lines were assessed in vitro. In vivo, C57BL-6 mice were treated orally with ARI-4175, after which splenocytes were assessed by flow cytometry and functional assays. Antitumor studies were performed in murine models of colon carcinoma (MC38-CEA(+) in CEA-transgenic C57BL-6 mice) and rhabdomyosarcoma (M3-9-M in C57BL-6 mice). Mice received oral ARI-4175 alone or in combination with a vaccine consisting of recombinant vaccinia/fowlpox CEA-TRICOM (colon model) or a DC-based tumor-cell vaccine (rhabdomyosarcoma model). Exposure to ARI-4175 had no effect on the proliferation or viability of carcinoma cells in vitro; however, it did alter tumor phenotype, making murine and human tumor cells more sensitive to antigen-specific CTL killing. Assessment of immune-cell subsets and function indicated that ARI-4175 increased levels of natural killer cells and DCs. Detrimental immune effects, including reduced T effector cells and increased immunosuppressive cells (Tregs, MDSCs), were normalized when treatment stopped, suggesting that scheduling is critical when combining this agent with vaccine. As a monotherapy, ARI-4175 had potent antitumor activity in both tumor models, and had even greater effects when combined with a vaccine (either DC-based or poxviral vector based). These findings provide the rationale for the combined use of cancer immunotherapy with DASH enzyme inhibitors such as ARI-4175.

WEE1 inhibition alleviates resistance to immune attack of tumor cells undergoing epithelial-mesenchymal transition.

Aberrant expression of the T-box transcription factor brachyury in human carcinomas drives the phenomenon of epithelial-mesenchymal transition (EMT), a phenotypic modulation that facilitates tumor dissemination and resistance to conventional therapies, including chemotherapy and radiotherapy. By generating isogenic cancer cell lines with various levels of brachyury expression, we demonstrate that high levels of brachyury also significantly reduce the susceptibility of cancer cells to lysis by both antigen-specific T cells and natural killer cells. Our results indicated that resistance of brachyury-high tumor cells to immune-mediated attack was due to inefficient caspase-dependent apoptosis, manifested as inefficient nuclear lamin degradation in the presence of activated effector caspases. We correlated this phenomenon with loss of cell-cycle-dependent kinase 1 (CDK1), which mediates lamin phosphorylation. In support of a causal connection, pretreatment of tumor cells with a specific inhibitor of WEE1, a negative regulator kinase of CDK1, could counter the defective apoptosis of tumor cells expressing high levels of brachyury. Thus, our findings suggested that reconstituting CDK1 activity to threshold levels may be sufficient to restore immunosurveillance of mesenchymal-like cancer cells that have escaped previous immune detection or eradication.

A combination trial of vaccine plus ipilimumab in metastatic castration-resistant prostate cancer patients: immune correlates.

We recently reported the clinical results of a Phase I trial combining ipilimumab with a vaccine containing transgenes for prostate-specific antigen (PSA) and for a triad of costimulatory molecules (PROSTVAC) in patients with metastatic castration-resistant prostate cancer. Thirty patients were treated with escalating ipilimumab and a fixed dose of vaccine. Of 24 chemotherapy-naïve patients, 58 % had a PSA decline. Combination therapy did not exacerbate the immune-related adverse events associated with ipilimumab. Here, we present updated survival data and an evaluation of 36 immune cell subsets pre- and post-therapy. Peripheral blood mononuclear cells were collected before therapy, at 13 days and at 70 days post-initiation of therapy, and phenotyped by flow cytometry for the subsets of T cells, regulatory T cells, natural killer cells, and myeloid-derived suppressor cells. Associations between overall survival (OS) and immune cell subsets prior to treatment, and the change in a given immune cell subset 70 days post-initiation of therapy, were evaluated. The median OS was 2.63 years (1.77-3.45). There were trends toward associations for longer OS and certain immune cell subsets before immunotherapy: lower PD-1(+)Tim-3(NEG)CD4EM (P = 0.005, adjusted P = 0.010), higher PD-1(NEG)Tim-3(+)CD8 (P = 0.002, adjusted P = 0.004), and a higher number of CTLA-4(NEG) Tregs (P = 0.005, adjusted P = 0.010). We also found that an increase in Tim-3(+) natural killer cells post- versus pre-vaccination associated with longer OS (P = 0.0074, adjusted P = 0.015). These results should be considered as hypothesis generating and should be further evaluated in larger immunotherapy trials.

Identification and characterization of agonist epitopes of the MUC1-C oncoprotein.

The MUC1 tumor-associated antigen is overexpressed in the majority of human carcinomas and several hematologic malignancies. Much attention has been paid to the hypoglycosylated variable number of tandem repeats (VNTR) region of the N-terminus of MUC1 as a vaccine target, and recombinant viral vector vaccines are also being evaluated that express the entire MUC1 transgene. While previous studies have described MUC1 as a tumor-associated tissue differentiation antigen, studies have now determined that the C-terminus of MUC1 (MUC1-C) is an oncoprotein, and its expression is an indication of poor prognosis in numerous tumor types. We report here the identification of nine potential CD8⁺ cytotoxic T lymphocyte epitopes of MUC1, seven in the C-terminus and two in the VNTR region, and have identified enhancer agonist peptides for each of these epitopes. These epitopes span HLA-A2, HLA-A3, and HLA-A24 major histocompatibility complex (MHC) class I alleles, which encompass the majority of the population. The agonist peptides, compared to the native peptides, more efficiently (a) generate T-cell lines from the peripheral blood mononuclear cells of cancer patients, (b) enhance the production of IFN-γ by peptide-activated human T cells, and (c) lyse human tumor cell targets in an MHC-restricted manner. The agonist epitopes described here can be incorporated into various vaccine platforms and for the ex vivo generation of human T cells. These studies provide the rationale for the T-cell-mediated targeting of the oncogenic MUC1-C, which has been shown to be an important factor in both drug resistance and poor prognosis for numerous tumor types.

Phase I study of intraprostatic vaccine administration in men with locally recurrent or progressive prostate cancer.

The primary end point of this study was to determine the safety and feasibility of intraprostatic administration of PSA-TRICOM vaccine [encoding transgenes for prostate-specific antigen (PSA) and 3 costimulatory molecules] in patients with locally recurrent or progressive prostate cancer. This trial was a standard 3 + 3 dose escalation with 6 patients each in cohorts 4 and 5 to gather more immunologic data. Nineteen of 21 patients enrolled had locally recurrent prostate cancer after definitive radiation therapy, and 2 had no local therapy. All cohorts received initial subcutaneous vaccination with recombinant vaccinia (rV)-PSA-TRICOM and intraprostatic booster vaccinations with recombinant fowlpox (rF)-PSA-TRICOM. Cohorts 3-5 also received intraprostatic rF-GM-CSF. Cohort 5 received additional subcutaneous boosters with rF-PSA-TRICOM and rF-GM-CSF. Patients had pre- and post-treatment prostate biopsies, and analyses of peripheral and intraprostatic immune cells were performed. There were no dose-limiting toxicities, and the maximum tolerated dose was not reached. The most common grade 2 adverse events were fever (38%) and subcutaneous injection site reactions (33%); the single grade 3 toxicity was transient fever. Overall, 19 of 21 patients on trial had stable (10) or improved (9) PSA values. There was a marked increase in CD4+ (p = 0.0002) and CD8+ (p = 0.0002) tumor infiltrates in post- versus pre-treatment tumor biopsies. Four of 9 patients evaluated had peripheral immune responses to PSA or NGEP. Intraprostatic administration of PSA-TRICOM is safe and feasible and can generate a significant immunologic response. Improved serum PSA kinetics and intense post-vaccination inflammatory infiltrates were seen in the majority of patients. Clinical trials examining clinical end points are warranted.

Effect of talactoferrin alfa on the immune system in adults with non-small cell lung cancer.

BACKGROUND: Talactoferrin alfa (talactoferrin), an agent with immune-stimulating properties, has demonstrated safety and preliminary efficacy in clinical trials. METHODS: Ten patients (five males and five females) with stage IV non-small cell lung cancer (NSCLC) in a single-arm pilot study received orally administered talactoferrin (1.5 g, b.i.d.) for up to 24 weeks. Radiographic and immunologic studies were performed at baseline and at weeks 6 and 12. Circulating immune cells (natural killer cells [NKCs], CD4+, CD8+, and regulatory T cells) and systemic cytokine levels were measured to assess immune response. RESULTS: Patients enrolled in the study had received a median of four prior chemotherapy regimens, and all patients were symptomatic. Talactoferrin was well tolerated, with no grade 3 or 4 toxicities. Median time to progression (TTP) and overall survival were 6 weeks and 14.5 weeks, respectively. The four patients with ≥9 weeks TTP had evidence of immunologic activity (three with increased NKC activity). CONCLUSIONS: The median of four previous chemotherapy regimens, with elevated levels of interleukin (IL) 6 and tumor necrosis factor-alfa in most patients, suggests these patients were poor candidates for immunotherapy.

Chemotherapy-induced immunogenic modulation of tumor cells enhances killing by cytotoxic T lymphocytes and is distinct from immunogenic cell death.

Certain chemotherapeutic regimens trigger cancer cell death while inducing dendritic cell maturation and subsequent immune responses. However, chemotherapy-induced immunogenic cell death (ICD) has thus far been restricted to select agents. In contrast, several chemotherapeutic drugs modulate antitumor immune responses, despite not inducing classic ICD. In addition, in many cases tumor cells do not die after treatment. Here, using docetaxel, one of the most widely used cancer chemotherapeutic agents, as a model, we examined phenotypic and functional consequences of tumor cells that do not die from ICD. Docetaxel treatment of tumor cells did not induce ATP or high-mobility group box 1 (HMGB1) secretion, or cell death. However, calreticulin (CRT) exposure was observed in all cell lines examined after chemotherapy treatment. Killing by carcinoembryonic antigen (CEA), MUC-1, or PSA-specific CD8(+) CTLs was significantly enhanced after docetaxel treatment. This killing was associated with increases in components of antigen-processing machinery, and mediated largely by CRT membrane translocation, as determined by functional knockdown of CRT, PERK, or CRT-blocking peptide. A docetaxel-resistant cell line was selected (MDR-1(+), CD133(+)) by continuous exposure to docetaxel. These cells, while resistant to direct cytostatic effects of docetaxel, were not resistant to the chemomodulatory effects that resulted in enhancement of CTL killing. Here, we provide an operational definition of "immunogenic modulation," where exposure of tumor cells to nonlethal/sublethal doses of chemotherapy alters tumor phenotype to render the tumor more sensitive to CTL killing. These observations are distinct and complementary to ICD and highlight a mechanism whereby chemotherapy can be used in combination with immunotherapy.

Brachyury, a driver of the epithelial-mesenchymal transition, is overexpressed in human lung tumors: an opportunity for novel interventions against lung cancer.

PURPOSE: The epithelial-mesenchymal transition (EMT) is emerging as a critical factor for the progression and metastasis of carcinomas, as well as drug resistance. The T-box transcription factor Brachyury has been recently characterized as a driver of EMT in human carcinoma cells. The purpose of this study was to characterize Brachyury as a potential target for lung cancer therapy. EXPERIMENTAL DESIGN: The expression of Brachyury was evaluated by PCR and by immunohistochemistry in human lung tumors and adult normal tissues. Brachyury gene copy number and promoter methylation status were analyzed in tumor tissues with various levels of Brachyury expression. Lung carcinoma cells' susceptibility to T-cell lysis and EGF receptor (EGFR) kinase inhibition were also evaluated relative to the levels of Brachyury. RESULTS: Our results showed Brachyury protein expression in 41% of primary lung carcinomas, including 48% of adenocarcinomas and 25% of squamous cell carcinomas. With the exception of normal testis and some thyroid tissues, the majority of normal tissues evaluated in this study were negative for the expression of Brachyury protein. Brachyury-specific T cells could lyse Brachyury-positive tumors and the level of Brachyury corresponded to resistance of tumor cells to EGFR kinase inhibition. CONCLUSION: We hypothesize that the elimination of Brachyury-positive tumor cells may be able to prevent and/or diminish tumor dissemination and the establishment of metastases. The ability of Brachyury-specific T-cell lines to lyse Brachyury-positive tumor cells, in vitro, supports the development of Brachyury-based immunotherapeutic approaches for the treatment of lung cancer.

A pilot study of MUC-1/CEA/TRICOM poxviral-based vaccine in patients with metastatic breast and ovarian cancer.

PURPOSE: PANVAC is a recombinant poxviral vaccine that contains transgenes for MUC-1, CEA, and 3 T-cell costimulatory molecules. This study was conducted to obtain preliminary evidence of clinical response in metastatic breast and ovarian cancer patients. EXPERIMENTAL DESIGN: Twenty-six patients were enrolled and given monthly vaccinations. Clinical and immune outcomes were evaluated. RESULTS: These patients were heavily pretreated, with 21 of 26 patients having 3 or more prior chemotherapy regimens. Side effects were largely limited to mild injection-site reactions. For the 12 breast cancer patients enrolled, median time to progression was 2.5 months (1-37+) and median overall survival was 13.7 months. Four patients had stable disease. One patient had a complete response by RECIST and remained on study for 37 months or more, with a significant drop in serum interleukin (IL)-6 and IL-8 by day 71. Another patient with metastatic disease confined to the mediastinum had a 17% reduction in mediastinal mass and was on study for 10 months. Patients with stable or responding disease had fewer prior therapies and lower tumor marker levels than patients with no evidence of response. For the ovarian cancer patients (n = 14), the median time to progression was 2 months (1-6) and median overall survival was 15.0 months. Updated data are presented here for one patient treated with this vaccine in a previous trial, with a time to progression of 38 months. CONCLUSIONS: Some patients who had limited tumor burden with minimal prior chemotherapy seemed to benefit from the vaccine. Further studies to confirm these results are warranted.

A pilot safety trial investigating a vector-based vaccine targeting carcinoembryonic antigen in combination with radiotherapy in patients with gastrointestinal malignancies metastatic to the liver.

OBJECTIVE: Previous studies have demonstrated the ability of non-lethal doses of radiation to alter the phenotype of tumor cells to facilitate immune mediated killing. This pilot study evaluated the tolerability of a vector-based vaccine targeting carcinoembryonic antigen (CEA) in combination with radiation therapy in patients with gastrointestinal malignancies metastatic to the liver. METHODS: Patients enrolled had progressive CEA(+) tumors with metastatic liver lesions. Patients had received a median of three previous chemotherapy regimens, with a median of 2 months since their last chemotherapy regimen. Only 58% had metastatic disease limited to the liver. Vaccination commenced day 1 with biweekly boosters and split-course radiation (total 32 Gy) starting on day 21. Blood was collected at baseline and day 91 for immunological analysis. RESULTS/CONCLUSION: A total of 12 patients were enrolled. There were no grade 3 or greater toxicities or grade 2 or greater hepatic toxicities. Median time on-study was 3 months, with the longest time on treatment being 5 months (n = 2). Immunological analysis was limited to two patients; neither showed an increase above baseline in CEA-specific T cells post-therapy. CEA/TRICOM vaccination in combination with low-dose radiation therapy is safe. There was limited evidence of activity in this patient population.