Several anti-PD1/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 PD1 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-L1/PD1 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 (a) the ability of avelumab to lyse a range of human tumor cells in the presence of PBMC or NK effectors; (b) IFNγ can enhance tumor cell PD-L1 expression and in some cases enhance ADCC tumor cell lysis; (c) purified NK cells are potent effectors for avelumab; (d) similar levels of avelumab-mediated ADCC lysis of tumor cells are seen using purified NK as effectors from either healthy donors or cancer patients; (e) 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 (f) 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.

Radiation therapy (RT) is used for local tumor control through direct killing of tumor cells. Radiation-induced cell death can trigger tumor antigen-specific immune responses, but these are often noncurative. Radiation has been demonstrated to induce immunogenic modulation (IM) in various tumor types by altering the biology of surviving cells to render them more susceptible to T cell-mediated killing. Little is known about the mechanism(s) underlying IM elicited by sub-lethal radiation dosing. We have examined the molecular and immunogenic consequences of radiation exposure in breast, lung, and prostate human carcinoma cells. Radiation induced secretion of ATP and HMGB1 in both dying and surviving tumor cells. In vitro and in vivo tumor irradiation induced significant upregulation of multiple components of the antigen-processing machinery and calreticulin cell-surface expression. Augmented CTL lysis specific for several tumor-associated antigens was largely dictated by the presence of calreticulin on the surface of tumor cells and constituted an adaptive response to endoplasmic reticulum stress, mediated by activation of the unfolded protein response.This study provides evidence that radiation induces a continuum of immunogenic alterations in tumor biology, from immunogenic modulation to immunogenic cell death. We also expand the concept of immunogenic modulation, where surviving tumor cells recovering from radiation-induced endoplasmic reticulum stress become more sensitive to CTL killing. These observations offer a rationale for the combined use of radiation with immunotherapy, including for patients failing RT alone.

Purpose: Many patients with clinically localized prostate cancer develop biochemical failure despite excellent local therapy perhaps due to occult metastatic disease. One potential solution is the utilization of a well-tolerated systemic therapy (e.g., vaccine) in concert with local therapy. Experimental Design:We present a randomized phase II clinical trial designed to determine if a poxviral vaccine encoding prostate-specific antigen (PSA) can induce a PSA-specific T-cell response when combined with radiotherapy in patients with clinically localized prostate cancer. Thirty patients were randomized in a 2:1 ratio into vaccine plus radiotherapy or radiotherapyonly arms. Those patients in the combination arm received a ''priming'' vaccine with recombinant vaccinia (r V) PSA plus r V containing theT-cell costimulatory molecule B7.1 (r V-B7.1) followed by monthly booster vaccines with recombinant fowlpox PSA. The vaccines were given with local granulocyte-macrophage colony-stimulating factor andlow-dose systemicinterleukin-2. Standard external beam radiation therapy was given between the fourth and the sixth vaccinations. Results: Seventeen of 19 patients in the combination arm completed all eight vaccinations and 13 of these 17 patients had increases in PSA-specificTcells of at least 3-fold versus no detectable increases in the radiotherapy-only arm (P <0.0005).There was also evidence ofde novo generation of T cells to well-described prostate-associated antigens not found in the vaccine, providing indirect evidence of immune-mediated tumor killing. The vaccine was well tolerated. Conclusion:This vaccine regimen can be safely given in patients undergoing radiation therapy for localized prostate cancer, with the majority of patients generating a PSA-specific cellular immune response to vaccine.

US National Institutes of Health.

Purpose: Identification of tumor antigens is essential in advancing immune-based therapeutic interventions in cancer. Particularly attractive targets are those molecules that are selectively expressed by malignant cells and that are also essential for tumor progression. Experimental Design and Results: We have used a computer-based differential display analysis tool for mining of expressed sequence tag clusters in the human Unigene database and identified Brachyury as a novel tumor antigen. Brachyury, a member of the T-box transcription factor family, is a key player in mesoderm specification during embryonic development. Moreover, transcription factors that control mesoderm have been implicated in the epithelial-mesenchymal transition (EMT), which has been postulated to be a key step during tumor progression to metastasis. Reverse transcription-PCR analysis validated the in silico predictions and showed Brachyury expression in tumors of the small intestine, stomach, kidney, bladder, uterus, ovary, and testis, as well as in cell lines derived from lung, colon, and prostate carcinomas, but not in the vast majority of the normal tissues tested. An HLA-A0201epitope of human Brachyury was identified that was able to expand T lymphocytes from blood of cancer patients and normal donors with the ability to lyse Brachyury-expressing tumor cells. Conclusions: To our knowledge, this is the first demonstration that (a) a T-box transcription factor and (b) a molecule implicated in mesodermal development, i.e., EMT, can be a potential target for human T-cell^mediated cancer immunotherapy.

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.

Purpose: Docetaxel has activity against androgen-independent prostate cancer and preclinical studies have shown that taxane-based chemotherapy can enhance antitumor response of vaccines. The primary objective of this study was to determine if concurrent docetaxel (with dexamethasone) had any effect on generating animmune responseto the vaccine. Secondary endpoints were whether vaccine could be given safely with docetaxel and the clinical outcome of the treatment regimen. Experimental Design: The vaccination regimen was composed of (a) recombinant vaccinia virus (rV) that expresses the prostate-specific antigen gene (rV-PSA) admixed with (b) rV that expresses the B7.1 costimulatory gene (rV-B7.1), and (c) sequential booster vaccinations with recombinant fowlpox virus (rF-) containing the PSA gene (rF-PSA). Patients received granulocyte macrophage colony-stimulating factor with each vaccination. Twenty-eight patients with metastatic androgen-independent prostate cancer were randomized to receive either vaccine and weekly docetaxel or vaccine alone. Patients on the vaccine alone arm were allowed to cross over to receive docetaxel alone at time of disease progression. The ELISPOT assay was used to monitor immune responses for PSA-specificTcells. Results: The median increase in these T-cell precursors to PSA was 3.33-fold in both arms following 3 months of therapy. In addition, immune responses to other prostate cancerâ ssociated tumor antigens were also detected postvaccination. Eleven patients who progressed on vaccine alone crossed over to receive docetaxel at time of progression. Median progressionfree survival on docetaxel was 6.1months after receiving vaccine compared with 3.7 months with the same regimen in a historical control. Conclusion: This is the first clinical trial to show that docetaxel can be administered safely with immunotherapy without inhibiting vaccine specific T-cell responses. Furthermore, patients previously vaccinated with an anticancer vaccine may respond longer to docetaxel compared with a historical control of patients receiving docetaxel alone. Larger prospective clinical studies will be required to validate these findings.Adenocarcinoma of the prostate is the most common noncutaneous malignancy diagnosed in American males and the second leading cause of cancer death. One of six men will develop clinically significant prostate cancer in his lifetime.During 2005, an estimated 232,900 men will be diagnosed with prostate cancer and 30,350 will die from the disease in the United States (1). Overall survival for patients with metastatic androgen-independent prostate cancer has been improved with a docetaxel-based regimen. The clinical benefit shown in these recent studies is an f3-month increase in survival using an every-3-week regimen of docetaxel (2, 3). However, compared with the every-3-week schedule, weekly docetaxel is associated with significantly less grade 3 or 4 hematologic toxicity. Other studies have looked at combining weekly docetaxel with other agents (4, 5). A random...

Antitumor immune response and chemotherapy-induced immunomodulation in colon cancer patients represented the rationale to design new strategies, like GOLFIG chemoimmunotherapy (gemcitabine, oxaliplatin, 5-fluorouracil/folinic acid, granulocyte macrophage colony-stimulating factor, and aldesleukine), that resulted a safe and very active regimen. Antitumor activity and immunity feedback to GOLFIG were strictly correlated with the best outcome observed in patients with autoimmunity signs, increase of central memory T cells, and decrease of regulatory T cells (Treg) in the peripheral blood. We thus investigated a potential correlation between the Treg tumor infiltration at diagnosis and the clinical outcome in a current randomized phase 3 trial aimed to compare the GOLFIG regimen with the standard FOLFOX chemotherapy (GOLFIG-2). An immunohistochemistry study was carried out to quantify the infiltration of Treg/FoxP3+ T lymphocytes in tumor samples of 57 patients enrolled in the GOLFIG-2 trial. Treg tumor infiltration scores were correlated with overall survival, treatment-relative survival, and progression-free survival (PFS). Higher Treg tumor infiltration scores were associated with a better prognosis in the whole series (Treg high score vs. low score: overall survival=mean 43.2 mo vs. 28.6 mo, P=0.0005) and a better outcome after treatment (Treg high score vs. low score: PFS=mean 15.8 mo vs. 8.8 mo, P=0.0009; treatment-relative survival=mean 23.1 mo vs. 18.2 mo, P=0.004). PFS was significantly longer in GOLFIG high versus all other subgroups (mean 18.1 mo vs. 9.9 mo, P=0.01). Our results suggest that a higher FoxP3+ T-lymphocyte tumor infiltration score is a favorable prognostic factor in colon cancer patients undergoing chemo or chemoimmunotherapy.