First-in-human phase 1 clinical trial of anti-core 1 O-glycans targeting monoclonal antibody NEO-201 in treatment-refractory solid tumors.

BACKGROUND: NEO201 is a humanized IgG1 monoclonal antibody (mAb) generated against tumor-associated antigens from patients with colorectal cancer. NEO-201 binds to core 1 or extended core 1 O-glycans expressed by its target cells. Here, we present outcomes from a phase I trial of NEO-201 in patients with advanced solid tumors that have not responded to standard treatments. METHODS: This was a single site, open label 3 + 3 dose escalation clinical trial. NEO-201 was administered intravenously every two weeks in a 28-day cycle at dose level (DL) 1 (1 mg/kg), DL 1.5 (1.5 mg/kg) and DL 2 (2 mg/kg) until dose limiting toxicity (DLT), disease progression, or patient withdrawal. Disease evaluations were conducted after every 2 cycles. The primary objective was to assess the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of NEO-201. The secondary objective was to assess the antitumor activity by RECIST v1.1. The exploratory objectives assessed pharmacokinetics and the effect of NEO-201 administration on immunologic parameters and their impact on clinical response. RESULTS: Seventeen patients (11 colorectal, 4 pancreatic and 2 breast cancers) were enrolled; 2 patients withdrew after the first dose and were not evaluable for DLT. Twelve of the 15 patients evaluable for safety discontinued due to disease progression and 3 patients discontinued due to DLT (grade 4 febrile neutropenia [1 patient] and prolonged neutropenia [1 patient] at DL 2, and grade 3 prolonged (> 72 h) febrile neutropenia [1 patient] at DL 1.5). A total of 69 doses of NEO-201 were administered (range 1-15, median 4). Common (> 10%) grade 3/4 toxicities occurred as follows: neutropenia (26/69 doses, 17/17 patients), white blood cell decrease (16/69 doses, 12/17 patients), lymphocyte decrease (8/69 doses, 6/17 patients). Thirteen patients were evaluable for disease response; the best response was stable disease (SD) in 4 patients with colorectal cancer. Analysis of soluble factors in serum revealed that a high level of soluble MICA at baseline was correlated with a downregulation of NK cell activation markers and progressive disease. Unexpectedly, flow cytometry showed that NEO-201 also binds to circulating regulatory T cells and reduction of the quantities of these cells was observed especially in patients with SD. CONCLUSIONS: NEO-201 was safe and well tolerated at the MTD of 1.5 mg/kg, with neutropenia being the most common adverse event. Furthermore, a reduction in the percentage of regulatory T cells following NEO-201 treatment supports our ongoing phase II clinical trial evaluating the efficiency of the combination of NEO-201 with the immune checkpoint inhibitor pembrolizumab in adults with treatment-resistant solid tumors. TRIAL REGISTRATION: NCT03476681 . Registered 03/26/2018.

Identification of the O-Glycan Epitope Targeted by the Anti-Human Carcinoma Monoclonal Antibody (mAb) NEO-201.

Truncated O-glycans expressed in cancer cells support tumor progression, and they may serve as potential targets to improve the monitoring and treatment of cancers. Previously, we reported that NEO-201 binds to several tumors expressing tumor-associated CEACAM5 and CEACAM6 variants but does not bind to those expressed in healthy tissues. This specific binding may be associated with the presence of truncated O-glycans attached on the protein sequence of these variants. To evaluate the glycosylation pattern targeted by NEO-201 we performed an O-glycan array consisting of 94 O-glycans. O-glycan profiles were elucidated from the human pancreatic cancer cell line CFPAC-1, human hematological neoplastic cells (HL-60, U937, K562) and human neutrophils. The O-glycan array analysis showed that NEO-201 interacts with core 1-4 O-glycans and that the binding to a specific core 1 O-glycan was the strongest. The O-glycan profiling of the NEO-201-reactive cells CFPAC-1, HL-60, U937 and human neutrophils showed that cells recognized by NEO-201 express mostly core 1 and/or extended core 1 O-glycans. In addition, NEO-201 mediates antibody-dependent cell-mediated cytotoxicity (ADCC) against tumor cells expressing core 1 or extended core 1 O-glycan profiles. These results demonstrated that NEO-201 binds to core 1 and extended core 1 O-glycans expressed in its target cells. Since GalNAc residue can be added onto threonine and serine to form O-glycans, it is very likely that NEO-201 recognizes these O-glycans attached to any protein with amino acid regions containing serine and threonine.

Evaluation of the Anti-Tumor Activity of the Humanized Monoclonal Antibody NEO-201 in Preclinical Models of Ovarian Cancer.

Purpose: Despite high initial response rates with cytoreductive surgery, conventional chemotherapy and the incorporation of biologic agents, ovarian cancer patients often relapse and die from their disease. New approaches are needed to improve patient outcomes. This study was designed to evaluate the antitumor activity of NEO-201 monoclonal antibody (mAb) in preclinical models of ovarian cancer where the NEO-201 target is highly expressed. Experimental Design: Functional analysis of NEO-201 against tumor cell lines was performed by antibody-dependent cellular cytotoxicity (ADCC) assays. Binding of NEO-201 to tumor tissues and cell lines were determined by immunohistochemistry (IHC) and flow cytometry, respectively. Further characterization of the antigen recognized by NEO-201 was performed by mass spectrometry. Ovarian cancer models were used to evaluate the anti-tumor activity of NEO-201 in vivo. NEO-201 at a concentration of 250 g/mouse was injected intraperitoneally (IP) on days 1, 4, and 8. Human PBMCs were injected IP simultaneously as effector cells. Results: Both IHC and flow cytometry revealed that NEO-201 binds prominently to the colon, pancreatic, and mucinous ovarian cancer tissues and cell lines. Immunoprecipitation of the antigen recognized by NEO-201 was performed in human ovarian, colon, and pancreatic cancer cell lines. From these screening, carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and CEACAM6 were identified as the most likely targets of NEO-201. Our results confirmed that NEO-201 binds different types of cancers; the binding is highly selective for the tumor cells without cross reactivity with the surrounding healthy tissue. Functional analysis revealed that NEO-201 mediates ADCC killing against human ovarian and colorectal carcinoma cell lines in vitro. In addition, NEO-201 inhibited tumor growth in the presence of activated human PBMCs in orthotopic mouse models of both primary and metastatic ovarian cancer. Importantly, NEO-201 prolonged survival of tumor-bearing mice. Conclusions: These data suggested that NEO-201 has an antitumor activity against tumor cells expressing its antigen. Targeting an antigen expressed in tumors, but not in normal tissues, allows patient selection for optimal treatment. These findings strongly indicate that NEO-201 warrants clinical testing as both a novel therapeutic and diagnostic agent for treatment of ovarian carcinomas. A first in human clinical trial evaluating NEO-201 in adults with chemo-resistant solid tumors is ongoing at the NIH clinical Center.

Phase II Study of Ensituximab, a Novel Chimeric Monoclonal Antibody, in Adults with Unresectable, Metastatic Colorectal Cancer.

PURPOSE: Patients with metastatic colorectal cancer refractory to chemotherapy have limited treatment options. Ensituximab (NEO-102) is a novel chimeric mAb targeting a variant of MUC5AC with specificity to colorectal cancer. PATIENTS AND METHODS: Single-arm, phase II trial assessed the efficacy and safety of ensituximab in patients with advanced, refractory cancer who expressed MUC5AC antigen in tumor tissue. Ensituximab was administered intravenously every 2 weeks with 3 mg/kg as recommended phase II dose (RP2D). A minimum sample size of 43 patients was required on the basis of the assumption that ensituximab would improve median overall survival (OS) by 7 months using a one-sided significance level of 10% and 80% power. Written informed consent was obtained from all patients. RESULTS: Sixty-three patients with advanced, refractory colorectal cancer were enrolled and 53 subjects were treated in phase II arm. Median age was 58 years and 46% of the patients were female. Among 57 evaluable patients, median OS was 6.8 months. No responses were observed, and stable disease was achieved in 21% of the patients. The most common treatment-related adverse events (AE) at RP2D included fatigue (38%), anemia (30%), nausea (15%), vomiting (11%), increased bilirubin (9%), constipation (8%), decreased appetite (6%), and diarrhea (6%). Serious AEs at least possibly related to ensituximab occurred in 4 patients and included anemia, nausea, increased bilirubin, and hypoxia. No patients discontinued treatment due to drug-related AEs. CONCLUSIONS: Ensituximab was well tolerated and demonstrated modest antitumor activity in patients with heavily pretreated refractory colorectal cancer.

The Monoclonal Antibody NEO-201 Enhances Natural Killer Cell Cytotoxicity Against Tumor Cells Through Blockade of the Inhibitory CEACAM5/CEACAM1 Immune Checkpoint Pathway.

Background: Natural killer (NK) cells are essential to innate immunity and participate in cancer immune surveillance. Heterophilic interactions between carcinoembryonic antigen (CEA) on tumor cells and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) on NK cells inhibit NK cell cytotoxicity against tumor cells. NEO-201 is a humanized IgG1 monoclonal antibody that recognizes members of CEACAM family, expressed specifically on a variety of human carcinoma cell lines and tumor tissues. This investigation was designed to determine whether the binding of NEO-201 with CEACAM5 on tumor cells can block the CEACAM5/CEACAM1 interaction to restore antitumor cytotoxicity of NK cells. Materials and Methods: In vitro functional assays, using various human tumor cell lines as target cells and NK-92 cells as effectors, were conducted to assess the ability of NEO-201 to block the interaction between CEACAM5 on tumor cells and CEACAM1 on NK cells to enhance the in vitro killing of tumor cells by NK-92. NK-92 cells were used as a model of direct NK killing of tumor cells because they lack antibody-dependent cellular cytotoxicity activity. Results: Expression profiling revealed that various human carcinoma cell lines expressed different levels of CEACAM5+ and NEO-201+ cells. Addition of NEO-201 significantly enhanced NK-92 cell cytotoxicity against highly CEACAM5+/NEO-201+ expressing tumor cells, suggesting that its activity is correlated with the level of CEACAM5+/NEO-201+ expression. Conclusions: These findings demonstrate that NEO-201 can block the interaction between CEACAM5 on tumor cells and CEACAM1 on NK cells to reverse CEACAM1-dependent inhibition of NK cytotoxicity.

An IL-15 Superagonist, ALT-803, Enhances Antibody-Dependent Cell-Mediated Cytotoxicity Elicited by the Monoclonal Antibody NEO-201 Against Human Carcinoma Cells.

BACKGROUND: A major mechanism of action for therapeutic antibodies is antibody-dependent cell-mediated cytotoxicity (ADCC). ALT-803 is an interleukin-15 superagonist complex that enhances ADCC against human carcinoma cells in vitro and exerts an antitumor activity in murine, rat, and human carcinomas in vivo. The authors investigated the ability of ALT-803 to modulate ADCC mediated by the humanized IgG1 monoclonal antibody (mAb) NEO-201 against human carcinoma cells. MATERIALS AND METHODS: ALT-803 modulating activity on ADCC mediated by NEO-201 was evaluated on several NEO-201 ligand-expressing human carcinoma cells. Purified human natural killer (NK) cells from multiple healthy donors were treated with ALT-803 before their use as effectors in ADCC assay. Modulation of NK cell phenotype and cytotoxic function by exposure to ALT-803 was evaluated by flow cytometry and gene expression analysis. RESULTS: ALT-803 significantly enhanced ADCC mediated by NEO-201. ALT-803 also upregulated NK activating receptors, antiapoptotic factors, and factors involved in the NK cytotoxicity, as well as downregulated gene expression of NK inhibiting receptors. CONCLUSIONS: These findings indicate that ALT-803 can enhance ADCC activity mediated by NEO-201, by modulating NK activation and cytotoxicity, suggesting a possible clinical use of ALT-803 in combination with NEO-201 for the treatment of human carcinomas.

Epigenetic priming of both tumor and NK cells augments antibody-dependent cellular cytotoxicity elicited by the anti-PD-L1 antibody avelumab against multiple carcinoma cell types.

Checkpoint inhibitors targeting the PD-1/PD-L1 axis are promising immunotherapies shown to elicit objective responses against multiple tumor types, yet these agents fail to benefit most patients with carcinomas. This highlights the need to develop effective therapeutic strategies to increase responses to PD-1/PD-L1 blockade. Histone deacetylase (HDAC) inhibitors in combination with immunotherapies have provided preliminary evidence of anti-tumor effects. We investigated here whether exposure of either natural killer (NK) cells and/or tumor cells to two different classes of HDAC inhibitors would augment (a) NK cell‒mediated direct tumor cell killing and/or (b) antibody-dependent cellular cytotoxicity (ADCC) using avelumab, a fully human IgG1 monoclonal antibody targeting PD-L1. Treatment of a diverse array of human carcinoma cells with a clinically relevant dose of either the pan-HDAC inhibitor vorinostat or the class I HDAC inhibitor entinostat significantly enhanced the expression of multiple NK ligands and death receptors resulting in enhanced NK cell‒mediated lysis. Moreover, HDAC inhibition enhanced tumor cell PD-L1 expression both in vitro and in carcinoma xenografts. These data demonstrate that treatment of a diverse array of carcinoma cells with two different classes of HDAC inhibitors results in enhanced NK cell tumor cell lysis and avelumab-mediated ADCC. Furthermore, entinostat treatment of NK cells from healthy donors and PBMCs from cancer patients induced an activated NK cell phenotype, and heightened direct and ADCC-mediated healthy donor NK lysis of multiple carcinoma types. This study thus extends the mechanism and provides a rationale for combining HDAC inhibitors with PD-1/PD-L1 checkpoint blockade to increase patient responses to anti-PD-1/PD-L1 therapies.

ADCC employing an NK cell line (haNK) expressing the high affinity CD16 allele with avelumab, an anti-PD-L1 antibody.

NK-92 cells, and their derivative, designated aNK, were obtained from a patient with non-Hodgkin lymphoma. Prior clinical studies employing adoptively transferred irradiated aNK cells have provided evidence of clinical benefit and an acceptable safety profile. aNK cells have now been engineered to express IL-2 and the high affinity (ha) CD16 allele (designated haNK). Avelumab is a human IgG1 anti-PD-L1 monoclonal antibody, which has shown evidence of clinical activity in a range of human tumors. Prior in vitro studies have shown that avelumab has the ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of human tumor cells when combined with NK cells. In the studies reported here, the ability of avelumab to enhance the lysis of a range of human carcinoma cells by irradiated haNK cells via the ADCC mechanism is demonstrated; this ADCC is shown to be inhibited by anti-CD16 blocking antibody and by concanamycin A, indicating the use of the granzyme/perforin pathway in tumor cell lysis. Studies also show that while NK cells have the ability to lyse aNK or haNK cells, the addition of NK cells to irradiated haNK cells does not inhibit haNK-mediated lysis of human tumor cells, with or without the addition of avelumab. Avelumab-mediated lysis of tumor cells by irradiated haNK cells is also shown to be similar to that of NK cells bearing the V/V Fc receptor high affinity allele. These studies thus provide the rationale for the clinical evaluation of the combined use of avelumab with that of irradiated adoptively transferred haNK cells.

Identification and characterization of enhancer agonist human cytotoxic T-cell epitopes of the human papillomavirus type 16 (HPV16) E6/E7.

Human papillomavirus (HPV) is associated with the etiology of cervical carcinoma, head and neck squamous cell carcinoma, and several other cancer types. Vaccines directed against HPV virus-like particles and coat proteins have been extremely successful in the prevention of cervical cancer through the activation of host HPV-specific antibody responses; however, HPV-associated cancers remain a major public health problem. The development of a therapeutic vaccine will require the generation of T-cell responses directed against early HPV proteins (E6/E7) expressed in HPV-infected tumor cells. Clinical studies using various vaccine platforms have demonstrated that both HPV-specific human T cells can be generated and patient benefit can be achieved. However, no HPV therapeutic vaccine has been approved by the Food and Drug Administration to date. One method of enhancing the potential efficacy of a therapeutic vaccine is the generation of agonist epitopes. We report the first description of enhancer cytotoxic T lymphocyte agonist epitopes for HPV E6 and E7. While the in silico algorithm revealed six epitopes with potentially improved binding to human leukocyte antigen-A2 allele (HLA-A2)-Class I, 5/6 demonstrated enhanced binding to HLA-Class I in cell-based assays and only 3/6 had a greater ability to activate HPV-specific T cells which could lyse tumor cells expressing native HPV, compared to their native epitope counterparts. These agonist epitopes have potential for use in a range of HPV therapeutic vaccine platforms and for use in HPV-specific adoptive T- or natural killer-cell platforms.

Preclinical Characterization of a Novel Monoclonal Antibody NEO-201 for the Treatment of Human Carcinomas.

NEO-201 is a novel humanized IgG1 monoclonal antibody that was derived from an immunogenic preparation of tumor-associated antigens from pooled allogeneic colon tumor tissue extracts. It was found to react against a variety of cultured human carcinoma cell lines and was highly reactive against the majority of tumor tissues from many different carcinomas, including colon, pancreatic, stomach, lung, and breast cancers. NEO-201 also exhibited tumor specificity, as the majority of normal tissues were not recognized by this antibody. Functional assays revealed that treatment with NEO-201 is capable of mediating both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against tumor cells. Furthermore, the growth of human pancreatic xenograft tumors in vivo was largely attenuated by treatment with NEO-201 both alone and in combination with human peripheral blood mononuclear cells as an effector cell source for ADCC. In vivo biodistribution studies in human tumor xenograft-bearing mice revealed that NEO-201 preferentially accumulates in the tumor but not organ tissue. Finally, a single-dose toxicity study in non-human primates demonstrated safety and tolerability of NEO-201, as a transient decrease in circulating neutrophils was the only related adverse effect observed. These findings indicate that NEO-201 warrants clinical testing as both a novel diagnostic and therapeutic agent for the treatment of a broad variety of carcinomas.

The IDO1 selective inhibitor epacadostat enhances dendritic cell immunogenicity and lytic ability of tumor antigen-specific T cells.

Epacadostat is a novel inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1) that suppresses systemic tryptophan catabolism and is currently being evaluated in ongoing clinical trials. We investigated the effects of epacadostat on (a) human dendritic cells (DCs) with respect to maturation and ability to activate human tumor antigen-specific cytotoxic T-cell (CTL) lines, and subsequent T-cell lysis of tumor cells, (b) human regulatory T cells (Tregs), and (c) human peripheral blood mononuclear cells (PBMCs) in vitro. Simultaneous treatment with epacadostat and IFN-γ plus lipopolysaccharide (LPS) did not change the phenotype of matured human DCs, and as expected decreased the tryptophan breakdown and kynurenine production. Peptide-specific T-cell lines stimulated with DCs pulsed with peptide produced significantly more IFN-γ, TNFα, GM-CSF and IL-8 if the DCs were treated with epacadostat. These T cells also displayed higher levels of tumor cell lysis on a per cell basis. Epacadostat also significantly decreased Treg proliferation induced by IDO production from IFN-γ plus LPS matured human DCs, although the Treg phenotype did not change. Multicolor flow cytometry was performed on human PBMCs treated with epacadostat; analysis of 123 discrete immune cell subsets revealed no changes in major immune cell types, an increase in activated CD83+ conventional DCs, and a decrease in immature activated Tim3+ NK cells. These studies show for the first time several effects of epacadostat on human DCs, and subsequent effects on CTL and Tregs, and provide a rationale as to how epacadostat could potentially increase the efficacy of immunotherapeutics, including cancer vaccines.

Enhanced killing of chordoma cells by antibody-dependent cell-mediated cytotoxicity employing the novel anti-PD-L1 antibody avelumab.

Chordoma, a rare bone tumor derived from the notochord, has been shown to be resistant to conventional therapies. Checkpoint inhibition has shown great promise in immune-mediated therapy of diverse cancers. The anti-PD-L1 mAb avelumab is unique among checkpoint inhibitors in that it is a fully human IgG1 capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) of PD-L1-expressing tumor cells. Here, we investigated avelumab as a potential therapy for chordoma. We examined 4 chordoma cell lines, first for expression of PD-L1, and in vitro for ADCC killing using NK cells and avelumab. PD-L1 expression was markedly upregulated by IFN-γ in all 4 chordoma cell lines, which significantly increased sensitivity to ADCC. Brachyury is a transcription factor that is uniformly expressed in chordoma. Clinical trials are ongoing in which chordoma patients are treated with brachyury-specific vaccines. Co-incubating chordoma cells with brachyury-specific CD8+ T cells resulted in significant upregulation of PD-L1 on the tumor cells, mediated by the CD8+ T cells' IFN-γ production, and increased sensitivity of chordoma cells to avelumab-mediated ADCC. Residential cancer stem cell subpopulations of chordoma cells were also killed by avelumab-mediated ADCC to the same degree as non-cancer stem cell populations. These findings suggest that as a monotherapy for chordoma, avelumab may enable endogenous NK cells, while in combination with T-cell immunotherapy, such as a vaccine, avelumab may enhance NK-cell killing of chordoma cells via ADCC.

Tumor Cells Surviving Exposure to Proton or Photon Radiation Share a Common Immunogenic Modulation Signature, Rendering Them More Sensitive to T Cell-Mediated Killing.

PURPOSE: To provide the foundation for combining immunotherapy to induce tumor antigen-specific T cells with proton radiation therapy to exploit the activity of those T cells. METHODS AND MATERIALS: Using cell lines of tumors frequently treated with proton radiation, such as prostate, breast, lung, and chordoma, we examined the effect of proton radiation on the viability and induction of immunogenic modulation in tumor cells by flow cytometric and immunofluorescent analysis of surface phenotype and the functional immune consequences. RESULTS: These studies show for the first time that (1) proton and photon radiation induced comparable up-regulation of surface molecules involved in immune recognition (histocompatibility leukocyte antigen, intercellular adhesion molecule 1, and the tumor-associated antigens carcinoembryonic antigen and mucin 1); (2) proton radiation mediated calreticulin cell-surface expression, increasing sensitivity to cytotoxic T-lymphocyte killing of tumor cells; and (3) cancer stem cells, which are resistant to the direct cytolytic activity of proton radiation, nonetheless up-regulated calreticulin after radiation in a manner similar to non-cancer stem cells. CONCLUSIONS: These findings offer a rationale for the use of proton radiation in combination with immunotherapy, including for patients who have failed radiation therapy alone or have limited treatment options.

Inhibitors of histone deacetylase 1 reverse the immune evasion phenotype to enhance T-cell mediated lysis of prostate and breast carcinoma cells.

The clinical promise of cancer immunotherapy relies on the premise that the immune system can recognize and eliminate tumor cells identified as non-self. However, tumors can evade host immune surveillance through multiple mechanisms, including epigenetic silencing of genes involved in antigen processing and immune recognition. Hence, there is an unmet clinical need to develop effective therapeutic strategies that can restore tumor immune recognition when combined with immunotherapy, such as immune checkpoint blockade and therapeutic cancer vaccines. We sought to examine the potential of clinically relevant exposure of prostate and breast human carcinoma cells to histone deacetylase (HDAC) inhibitors to reverse tumor immune escape to T-cell mediated lysis. Here we demonstrate that prostate (LNCAP) and breast (MDA-MB-231) carcinoma cells are more sensitive to T-cell mediated lysis in vitro after clinically relevant exposure to epigenetic therapy with either the pan-HDAC inhibitor vorinostat or the class I HDAC inhibitor entinostat. This pattern of immunogenic modulation was observed against a broad range of tumor-associated antigens, such as CEA, MUC1, PSA, and brachyury, and associated with augmented expression of multiple proteins involved in antigen processing and tumor immune recognition. Genetic and pharmacological inhibition studies identified HDAC1 as a key determinant in the reversal of carcinoma immune escape. Further, our findings suggest that the observed reversal of tumor immune evasion is driven by a response to cellular stress through activation of the unfolded protein response. This offers the rationale for combining HDAC inhibitors with immunotherapy, including therapeutic cancer vaccines.

The impact of leukapheresis on immune-cell number and function in patients with advanced cancer.

BACKGROUND: Leukapheresis is often performed in cancer patients to harvest stem cells, manufacture therapeutic vaccines, or follow immunologic response to therapy. We have recently described the minimal impact of leukapheresis on normal donors. Here we provide additional immunologic data from patients with advanced cancer who underwent leukapheresis. METHODS: Using data from cancer patients on clinical trials who had leukapheresis (n = 64) or peripheral blood draws only (n = 90) as controls for immune analysis, we evaluated the impact of leukapheresis on number and function of lymphocytes. RESULTS: In the leukapheresis group, median age was 63.5 (range 38-82); 87.5 % were male. Comparing pre- and post-leukapheresis values within the groups, with each patient as its own control, there was no significant difference in enzyme-linked immunosorbent spot (ELISPOT), antivector humoral response, absolute lymphocyte count (ALC), or T cell number. Twelve patients completed three leukaphereses with subsequent ELISPOT analysis; seven had increased responses to flu (1.1- to 2.3-fold) with an even distribution around no change. Nineteen patients had matched ALC values after completing three leukaphereses with no significant change from baseline. CONCLUSIONS: These data provide evidence that leukapheresis has no detectable effects on a cancer patient's immune system in terms of number or function. These results contribute to a growing body of evidence refuting the hypothesis that a patient's immune competence is meaningfully affected by the procedure. Limitations include a restriction to 2-L leukapheresis procedure and small sample size.

Immune consequences of tyrosine kinase inhibitors that synergize with cancer immunotherapy.

Combination therapy for the treatment of cancer is becoming increasingly essential as we gain improved understanding of the complexity of cancer progression and the mechanisms by which cancer cells become resistant to single-agent therapy. Recent studies, both clinical and preclinical, have suggested that immunotherapy is a promising approach to the treatment of cancer; however, strategies to improve its clinical efficacy are still needed. A number of recent studies have indicated that antiangiogenic tyrosine kinase inhibitors (TKIs) target multiple components of the tumor microenvironment and are an ideal class of agents for synergizing with cancer immunotherapy. TKIs are well known to modulate tumor endothelial cells, leading to vascular normalization; however, these agents have also been recently shown to decrease tumor compactness and tight junctions, thereby reducing solid tumor pressure and allowing for improved perfusion of collapsed vessels and increased tumor oxygenation. In addition, some TKIs are capable of inducing immunogenic modulation, whereby tumor cells are sensitized to killing by T lymphocytes. Moreover, a number of TKIs have been shown to be involved in immune subset conditioning, increasing the frequency and function of effector immune elements, while decreasing the number and function of immune suppressor cells. The alteration of the immune landscape, direct modification of tumor cells, and improved vascular perfusion leads to improved antitumor efficacy when antiangiogenic TKIs are combined with immunotherapy. Collectively, the data presented in this review support the clinical combination of multi-targeted antiangiogenic TKIs, including but not limited to cabozantinib, sunitinib, and sorafenib, as well as to other antiangiogenic therapies, such as the anti-VEGF antibody bevacizumab, with cancer vaccines for improved treatment of solid tumors.

Detection and Characterization of Circulating Tumour Cells from Frozen Peripheral Blood Mononuclear Cells.

Retrospective analysis of patient tumour samples is a cornerstone of clinical research. CTC biomarker characterization offers a non-invasive method to analyse patient samples. However, current CTC technologies require prospective blood collection, thereby reducing the ability to utilize archived clinical cohorts with long-term outcome data. We sought to investigate CTC recovery from frozen, archived patient PBMC pellets. Matched samples from both mCRPC patients and mock samples, which were prepared by spiking healthy donor blood with cultured prostate cancer cell line cells, were processed "fresh" via Epic CTC Platform or from "frozen" PBMC pellets. Samples were analysed for CTC enumeration and biomarker characterization via immunofluorescent (IF) biomarkers, fluorescence in-situ hybridization (FISH) and CTC morphology. In the frozen patient PMBC samples, the median CTC recovery was 18%, compared to the freshly processed blood. However, abundance and localization of cytokeratin (CK) and androgen receptor (AR) protein, as measured by IF, were largely concordant between the fresh and frozen CTCs. Furthermore, a FISH analysis of PTEN loss showed high concordance in fresh vs. frozen. The observed data indicate that CTC biomarker characterization from frozen archival samples is feasible and representative of prospectively collected samples.

Immune impact induced by PROSTVAC (PSA-TRICOM), a therapeutic vaccine for prostate cancer.

PSA-TRICOM (PROSTVAC) is a novel vector-based vaccine designed to generate a robust immune response against prostate-specific antigen (PSA)-expressing tumor cells. The purpose of this report is to present an overview of both published studies and new data in the evaluation of immune responses to the PSA-TRICOM vaccine platform, currently in phase III testing. Of 104 patients tested for T-cell responses, 57% (59/104) demonstrated a ≥ 2-fold increase in PSA-specific T cells 4 weeks after vaccine (median 5-fold increase) compared with pre-vaccine, and 68% (19/28) of patients tested mounted post-vaccine immune responses to tumor-associated antigens not present in the vaccine (antigen spreading). The PSA-specific immune responses observed 28 days after vaccine (i.e., likely memory cells) are quantitatively similar to the levels of circulating T cells specific for influenza seen in the same patients. Measurements of systemic immune response to PSA may underestimate the true therapeutic immune response (as this does not account for cells that have trafficked to the tumor) and does not include antigen spreading. Furthermore, although the entire PSA gene is the vaccine, only one epitope of PSA is evaluated in the T-cell responses. Because this therapeutic vaccine is directed at generating a cellular/Th1 immune response (T-cell costimulatory molecules and use of a viral vector), it is not surprising that less than 0.6% of patients (2/349) tested have evidence of PSA antibody induction following vaccine. This suggests that post-vaccine PSA kinetics were not affected by PSA antibodies. An ongoing phase III study will evaluate the systemic immune responses and correlation with clinical outcomes.

Pan-Bcl-2 inhibitor, GX15-070 (obatoclax), decreases human T regulatory lymphocytes while preserving effector T lymphocytes: a rationale for its use in combination immunotherapy.

Bcl-2 inhibitors are currently being evaluated in clinical studies for treatment of patients with solid tumors and hematopoietic malignancies. In this study we explored the potential for combining the pan-Bcl-2 inhibitor GX15-070 (GX15; obatoclax) with immunotherapeutic modalities. We evaluated the in vitro effects of GX15 on human T cell subsets obtained from PBMCs in terms of activation, memory, and suppressive function. Our results indicated that in healthy-donor PBMCs, mature-activated T cells were more resistant to GX15 than early-activated T cells, and that GX15 preserved memory but not non-memory T cell populations. Furthermore, GX15 increased the apoptosis of regulatory T cells (Tregs), profoundly downregulated FOXP3 and CTLA-4 in a dose-dependent manner, and decreased their suppressive function. Treating PBMCs obtained from ovarian cancer patients with GX15 also resulted in increased CD8(+):Treg and CD4(+):Treg ratios. These results support preclinical studies in which mice vaccinated before treatment with GX15 showed the greatest reduction in metastatic lung tumors as a result of increased apoptotic resistance of mature CD8(+) T cells and decreased Treg function brought about by GX15. Taken together, these findings suggest that when a Bcl-2 inhibitor is combined with active immunotherapy in humans, such as the use of a vaccine or immune checkpoint inhibitor, immunotherapy should precede administration of the Bcl-2 inhibitor to allow T cells to become mature, and thus resistant to the cytotoxic effects of the Bcl-2 inhibitor.