Barbara J. Biller

Low-dose cyclophosphamide selectively decreases regulatory T cells and inhibits angiogenesis in dogs with soft tissue sarcoma.

BACKGROUND Low-dose, continuous (metronomic) chemotherapy improves tumor control by inhibiting tumor angiogenesis and suppressing regulatory T cells (Treg) in mice and humans. The effects of metronomic chemotherapy on Treg and tumor angiogenesis in dogs has not been investigated previously. OBJECTIVE To determine whether metronomic cyclophosphamide (CYC) therapy decreases Treg or exhibits antiangiogenic activity or both in dogs with soft tissue sarcoma (STS). We hypothesized that Treg numbers would be increased in dogs with STS and that continuous dosing of CYC would decrease Treg in a dose-dependent manner, as well as exhibit antiangiogenic activity. ANIMALS Eleven client-owned dogs with grade I or II STS. Twenty-one healthy dogs were used as controls. METHODS Prospective, open, clinical trial. Dogs with STS were enrolled in 2 dose cohorts and administered CYC at 12.5 or 15 mg/m(2) p.o. once daily for 28 days. Whole blood and tumor biopsy specimens were obtained on days 0, 14, and 28 to assess changes in T lymphocyte subsets by flow cytometry and tumor microvessel density (MVD), respectively. RESULTS Administration of CYC at 12.5 mg/m(2)/d significantly decreased the number of Treg from days 0 to 28, but there was no change in the percentage of Treg or tumor MVD. In dogs that received CYC at 15.0 mg/m(2)/d, both the number and percent of Treg as well as tumor MVD were significantly decreased over 28 days. CONCLUSIONS CYC administered at 15 mg/m(2)/d should be used in further studies examining the antitumor properties of low-dose CYC in dogs.

Changes in Regulatory T Cells in Dogs with Cancer and Associations with Tumor Type

BACKGROUND Regulatory T cells (Treg) have been shown to suppress antitumor immunity and often are increased in humans and rodents with cancer. However, Tregs have not been well studied in dogs with cancer and it is not known if certain tumor types are associated with increased Tregs. HYPOTHESIS We hypothesized that Treg percentages would be increased in dogs with cancer and that Treg percentages would be higher in dogs with certain types of cancer. ANIMALS The percentages and numbers of Tregs and nonregulatory T cells and B cells were assessed in 34 dogs with cancer and 9 age-matched control dogs. Dogs evaluated included 14 dogs with sarcoma, 7 dogs with carcinoma, 7 dogs with lymphoma, and 6 dogs with mast cell tumor. METHODS Numbers and percentages of Tregs, CD4+, and CD8+ T cells and B cells were determined using flow cytometry and compared between control dogs and dogs with cancer. RESULTS The percentage of Tregs was significantly increased overall in dogs with cancer compared with control dogs. When tumor types were compared, Treg percentages were significantly increased in dogs with carcinoma. The Treg/CD8 T cell ratio was significantly higher in dogs with cancer compared with control dogs and was also significantly increased in 2 dogs with T-cell lymphoma. CONCLUSIONS Treg percentages in blood were increased in dogs with cancer, particularly in dogs with carcinoma. The Treg/CD8 ratio also identified tumor-specific abnormalities in dogs with cancer. These findings indicate that tumor-specific factors may affect Tregs in dogs.

Clinical and Immunomodulatory Effects of Toceranib Combined with Low‐Dose Cyclophosphamide in Dogs with Cancer

BACKGROUND Tyrosine kinase inhibitors (TKIs) and metronomic dosing of cyclophosphamide (CYC) can improve tumor control by suppression of regulatory T cells (Treg) and restoration of T cell-mediated immune responses in mice and humans. The immunomodulatory effects of the TKI toceranib, as a single agent or in combination with metronomic CYC, have not been previously investigated in dogs. HYPOTHESIS The primary objectives of this study were to determine the effects of toceranib and metronomic CYC treatment on lymphocyte subsets including Treg and on interferon-gamma (IFN-γ) secretion in dogs with cancer. We hypothesized that toceranib would selectively decrease Treg numbers and increase IFN-γ production and that addition of CYC would further enhance these effects. ANIMALS Fifteen client-owned dogs with advanced tumors were entered into a prospective clinical trial. METHODS Dogs received toceranib at 2.75 mg/kg once every other day. After 2 weeks, oral CYC was added at 15 mg/m(2) daily. Numbers of Treg and lymphocyte subsets were measured in blood by flow cytometry during the 8-week study period. Serum concentrations of IFN-γ were measured by ELISA. RESULTS Administration of toceranib significantly decreased the number and percentage of Treg in the peripheral blood of dogs with cancer. Dogs receiving toceranib and CYC demonstrated a significant increase in serum concentrations of IFN-γ, which was inversely correlated with Treg numbers after 6 weeks of combination treatment. CONCLUSIONS In addition to antitumor effects, these data support further investigations into the immunomodulatory effects of toceranib, administered alone or in combination with CYC in dogs with cancer.

Evaluation of a Novel Tumor Vaccine in Dogs with Hemangiosarcoma

BACKGROUND Hemangiosarcoma (HSA) is a highly metastatic and often rapidly fatal tumor of dogs. At present, adjuvant chemotherapy is the only proven effective treatment for dogs with HSA, though the benefits from chemotherapy are modest. Administration of immunotherapy together with chemotherapy has also been reported to improve survival in dogs with HSA. Therefore, we evaluated safety and immunologic responses to a novel tumor vaccine administered together with doxorubicin chemotherapy in dogs with different stages of HSA. HYPOTHESIS That tumor vaccination could be safely and effectively combined with doxorubicin chemotherapy for treatment of dogs with HSA. ANIMALS Twenty-eight dogs with various stages of HSA were enrolled in the study. METHODS The HSA vaccine was prepared with lysates of allogeneic canine HSA cell lines mixed with an adjuvant composed of liposome-DNA complexes. Dogs received a series of 8 immunizations administered over a 22-week period, and most also received chemotherapy. Clinical adverse effects were noted, immune responses were measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry, and survival times were calculated. RESULTS The most common adverse effects observed in vaccinated dogs also treated with doxorubicin chemotherapy were diarrhea and anorexia. Vaccinated dogs were found to mount strong humoral immune responses against a control antigen and, most dogs also mounted antibody responses against canine HSA cells. Thirteen dogs with stage II splenic HSA that received the tumor vaccine plus doxorubicin chemotherapy had an overall median survival time of 182 days. CONCLUSIONS We conclude that an allogeneic tumor lysate vaccine is safe in dogs with HSA and can elicit humoral immune responses in dogs that are receiving concurrent doxorubicin chemotherapy.

Decreased ratio of CD8+ T cells to regulatory T cells associated with decreased survival in dogs with osteosarcoma.

BACKGROUND Increased numbers of regulatory T cells (Treg) and decreased ratios of CD8+ T cells to Treg have been shown to correlate with decreased survival times (ST) in humans with certain malignancies. A possible connection between Treg and ST in dogs with cancer has not been investigated previously. HYPOTHESIS The purpose of this study was to compare numbers of Treg and T lymphocyte subsets in dogs with osteosarcoma (OSA) to those of healthy dogs and to determine whether pretreatment values were associated with disease-free interval or with ST. We hypothesized that Treg numbers would be increased in dogs with cancer and that dogs with a high percentage of Treg would have a poorer prognosis. ANIMALS Twelve client-owned dogs with appendicular OSA were entered into a prospective clinical trial. Twenty-two healthy dogs were used as controls. METHODS The percentages and numbers of Treg and CD4+ and CD8+ T cells in blood, lymph nodes, and tumors were determined with flow cytometry and compared between dogs with OSA and control dogs. RESULTS Dogs with OSA had significantly fewer circulating CD8+ T cells and significantly more Treg compared with healthy dogs. The CD8/Treg ratio also was significantly lower in dogs with OSA compared with control dogs. In dogs with OSA, a decreased CD8/Treg ratio was associated with significantly shorter STs. CONCLUSIONS These data support a role for Treg in the immune control of canine OSA and suggest that determination of the CD8/Treg ratio may be useful for assessing outcomes.

Induction of remission results in spontaneous enhancement of anti-tumor cytotoxic T-lymphocyte activity in dogs with B cell lymphoma

Characterization of the tumor microenvironment, particularly the immune cells that infiltrate tumors, provides important predictive and prognostic information in humans with lymphoma and other types of cancer. Tumor associated T lymphocytes have not been previously described in dogs with lymphoma. Therefore, we investigated the phenotype and function of T cells in the lymph nodes of dogs with B cell Non-Hodgkins lymphoma (NHL), as well as the function of T cells in circulation of these dogs. We found that CD4+ and CD8+ T lymphocytes were few in number and minimally responsive to mitogenic stimuli compared to T cells in lymph nodes of normal dogs. Additionally, regulatory T cells (Treg) were significantly increased in tumor tissues compared to lymph nodes of healthy dogs. To better understand cell mediated antitumor immune responses we developed a non-radioactive assay to measure cytotoxic T lymphocyte (CTL) mediated killing of autologous tumor cells. Using this assay, we found that spontaneous CTL activity in the blood of dogs with lymphoma improved significantly following induction of tumor remission using doxorubicin. Coincident with the improvement in CTL activity, circulating Treg numbers were significantly decreased compared to pretreatment levels. We conclude from these studies that CTL activity in dogs with lymphoma can be significantly improved following induction of tumor remission using chemotherapy, as assessed using a new non-radioactive CTL assay.

Defining the Pharmacodynamic Profile and Therapeutic Index of NHS-IL12 Immunocytokine in Dogs with Malignant Melanoma

Background Interleukin (IL)-12 is a pro-inflammatory cytokine that mediates T-helper type 1 responses and cytotoxic T-cell activation, contributing to its utility as anti-cancer agent. Systemic administration of IL-12 often results in unacceptable toxicity; therefore, strategies to direct delivery of IL-12 to tumors are under investigation. The objective of this study was to assist the preclinical development of NHS-IL12, an immunocytokine consisting of an antibody, which targets necrotic tumor regions, linked to IL-12. Specifically this study sought to evaluate the safety, serum pharmacokinetics, anti-tumor activity, and immune modulation of NHS-IL12 in dogs with naturally occurring cancers. Methodology/Principal Findings A rapid dose-escalation study of NHS-IL12 administered subcutaneously to dogs with melanoma was conducted through the Comparative Oncology Trials Consortium (COTC). Eleven dogs were enrolled in four dose-escalation cohorts; thereafter, an additional seven dogs were treated at the defined tolerable dose of 0.8 mg/m2. The expanded cohort at this fixed dose (ten dogs in total) was accrued for further pharmacokinetics and pharmacodynamics assessment. NHS-IL12 levels, serum cytokine concentrations, and peripheral blood mononuclear cell characterization (post-treatment) and draining lymph node immune profiling, and tumor biopsies (pre- and post-treatment) were collected. Adverse events included thrombocytopenia, liver enzymopathies, fever, and vasculitis. Correlation between interferon (IFN)-γ induction, adverse events, and NHS-IL12 exposure (maximum concentration and area under the concentration-time curve) were dose-dependent. Serum IL-10 levels and intratumoral CD8+ populations increased after treatment. Partial responses, according to Response Evaluation Criteria in Solid Tumors (RECIST) criteria, were observed in two dogs treated with NHS-IL12 0.8 mg/m2 and 1.6 mg/m2. Conclusions/Significance NHS-IL12 was administered safely to dogs with melanoma and both immunologic and clinical activity was observed. This study successfully defined a narrow therapeutic window for systemic delivery of NHS-IL12 via the subcutaneous route. Results will inform the design and implementation of first-in-human clinical trials of NHS-IL12 in cancer patients.

Metronomic chemotherapy in veterinary patients with cancer: rethinking the targets and strategies of chemotherapy.

Cancer chemotherapy in dogs and cats has traditionally involved administration of chemotherapy agents at the maximum tolerated dose. Cytotoxic chemotherapy has an acceptably low risk of serious toxicity, but an obligatory rest period must be included to allow for recovery of drug-sensitive normal cell populations. This rest period can also allow significant recovery of tumor cells. Metronomic chemotherapy is characterized by more frequent administration of lower doses of oral drugs and appears to halt or slow tumor progression through multiple mechanisms. This approach may be at least as effective as conventional chemotherapy with a lower risk of toxicity.

Abstract A07: Phase I evaluation of anti-human leukocyte antigen-DR monoclonal antibody therapy in spontaneous canine lymphoma

IMMU-114 (also known as hL243) is a humanized anti-HLA-DR monoclonal antibody (mAb) with potent anti-tumor activity against a broad range of human hematopoietic malignancies. As MHC Class II antigens are also expressed on canine lymphocytes, IMMU-114 has previously been found to recognize and kill malignant lymphocytes collected from dogs with spontaneous B cell lymphoma. Canine lymphoma is a highly relevant animal model of B-cell non-Hodgkin9s lymphoma (NHL) in humans with many advantages over rodent models. In addition, clinical trials in pet dogs can be designed to address therapeutic endpoints relevant to phase II/III studies in both dogs and humans. This study is being undertaken to determine the safety, pharmacodynamic and pharmacokinetic profiles of IMMU-114 in dogs with heavily pre-treated B cell lymphoma (NHL). Our hypothesis is that the mAb can safely be administered to tumor-bearing dogs and that it will be preferentially cytotoxic to B lymphocytes. Our overall objectives are to characterize the toxicity and biologic profile of IMMU-114 to advance the therapy of canine lymphoma and to better inform human clinical trials using anti-HLA-DR mAbs. Thirteen pet dogs with B cell lymphoma demonstrating binding to IMMU-114 have been enrolled in an on-going phase I clinical trial at the Flint Animal Cancer Center at Colorado State University. Enrollment follows a standard 3 x 3 dose escalation to identify the maximally tolerated dose (MTD) of the mAb, starting at a dose of 3 mg/kg, and escalating thus far to 6, 12, and 24 mg/kg. Data collection includes pre and post-infusion peripheral blood mononuclear cells (PBMCs), serum, serial lymph node measurements, complete blood counts and serum chemistry profiles. Preliminary pharmacodynamic evaluation shows a significant and transient dose-dependent decrease in the percentage and absolute number of B lymphocytes in the blood of treated dogs 24 hours post infusion. This corresponds to a transient increase in the relative percentage of T lymphocytes. Neutropenia and other bone marrow toxicities have not been observed. Infusion related side effects appear common but have been mild to moderate in nature with 10/13 patients experiencing grade 1 or grade 2 allergic reactions (fever, urticaria, and hypersalivation) and 2/13 with grade 1 nausea. No dose limiting toxicities have been observed; therefore the MTD has not yet been reached. Thus far 2/13 dogs (15%) have had prolonged disease stabilization and received multiple weekly infusions of 12 mg/kg while 11/13 dogs have had progressive disease (lymph node enlargement) within one week post therapy. Pharmacokinetic data obtained at the end of infusion, 2, 24 hours and 1 and 2 weeks post infusion showed minimally detectable levels at doses of 3 and 6 mg/kg. At 12 mg/kg, 2 animals had evidence of persistent antibody levels in serum for 24 hours, followed by return to pre-treatment levels within 6 days. Two dogs currently treated at 24 mg/kg maintained hL243 levels in their blood over 1 day, with residual antibody still found in the blood 6 days later Four of the 13 dogs developed anti-hL243 responses 2 to 4 weeks after treatment initiation. These findings provide a rationale for larger clinical studies to investigate the efficacy of anti-HLA-DR mAbs for both veterinary and human applications. Citation Format: Barbara J. Biller, Rodney Page, Robert Sharkey, David M. Goldenberg. Phase I evaluation of anti-human leukocyte antigen-DR monoclonal antibody therapy in spontaneous canine lymphoma. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A07.