The PennVet Tumor Tissue Bank (PennVet TTB)

• Within the last 10 years there has been increasing interest in comparative oncology and an increasing demand for small animal tumor tissue samples and related biofluids to support basic and clinical research. This research aims to further our understanding of spontaneous tumor biology and to discover novel therapies for tumor irradication in both the human and veterinary clinics.

  In response to this demand, the PennVet Tumor Tissue Bank (PennVet TTB) has been established within the School of Veterinary Medicine which aims to provide researchers with ready access to spontaneously occurring primary and metastatic tumor specimens for different research applications ranging from genomics and proteomics to allogeneic cancer vaccine preparation.

  The mission of the PennVet TTB is to generate a repository of canine and feline primary tumor specimens and related serum/plasma/biofluids that are stored in multiple formats and linked to a searchable comprehensive clinical database.

  This repository provides investigators the resources required to perform basic and translational comparative cancer research and serves to promote natural and synergistic collaborations between individuals working at the bench and those working in the clinics.

  Furthermore, the availability of primary spontaneously occurring tumor specimens facilitates the translation of promising novel cancer therapeutics from the bench into randomized clinical trials that can benefit dogs and cats with cancer when conventional therapies prove ineffective.

  The development of a large and comprehensive TTB at the School of Veterinary Medicine aims to:

  1. Promote collaborations between researchers/clinician scientists within and between the Schools of Medicine and Veterinary Medicine
  2. Increase awareness of spontaneous, large animal models of cancer and encourage researchers to undertake comparative cancer research
  3. Function in concert with the VCIC to translate therapeutics that show efficacy in vitro into large animal phase I and II clinical trials 
  4. Accelerate discoveries in basic and translational veterinary oncology

  The directors of the PennVet TTB manage the bank and its database and oversee quality assurance and control of banked specimens. Standard Operating Procedures are in place for tumor specimen collection and processing.

  All samples are stored in the following formats: formalin fixed, paraffin-embedded (e.g. for histopathology), OCT embedded (e.g. for immunohistochemistry), flash frozen (e.g. for DNA and RNA extraction) and cryopreserved as viable single cell suspensions (e.g. for the generation of primary cell lines).

  Through the use of an advanced database management system, all specimens can be rapidly identified, retrieved, cross-referenced and tracked. Basic and translational researchers at PennVet, the University of Pennsylvania’s School of Medicine and other international institutions have utilized the tissues and cell lines stored within the bank to further our understanding of canine cancer and to develop novel therapies for use in the treatment of canine and feline cancers.

The use of RNA-loaded CD40 activated B cells as a cancer vaccine

• In human patients there is convincing evidence that the immune system plays an important role in the control of malignant disease. Therefore, vaccination strategies to boost anti-tumor immunity and generate immunological memory against multiple tumor targets are being investigated to improve the outcome of human cancer patients.

  One promising approach has been the use of dendritic cells loaded with tumor antigens to stimulate anti-tumor immunity. Dendritic cells (DCs) are professional antigen presenting cells (APCs) that are responsible for activating immune cells known as T cells in vivo.

  Preclinical and clinical evidence suggest that ex-vivo pulsing of dendritic cells with tumor lysates and re-injection of “loaded” dendritic cells can trigger polyvalent T cell responses against multiple tumor antigens and can overcome aspects of immune incompetence in cancer patients and result in clinical responses. However, DCs do not proliferate and obtaining large numbers of DC precursors required for a therapeutic vaccine from smaller canine and pediatric human patients is challenging.

  In collaboration with Dr. Robert Vonderheide at the Abramson Family Cancer Research Institute at the University of Pennsylvania, Drs. Mason and Sorenmo at PennVet are evaluating the use of tumor RNA-transfected CD40-activated autologous B lymphocytes (CD40-B) as an alternative APC vaccine to generate anti-tumor immunity in canine patients with spontaneously occurring lymphoma.

  Canine lymphoma is a common malignancy affecting the white blood cells known as lymphocytes. The disease has an estimated incidence of 30/100,000 dogs per year. Current therapies to treat canine lymphoma utilize various combinations of chemotherapeutic agents that induce remission in 75-85% of dogs.

  However, regardless of the chemotherapeutic induction protocol, 85% of these dogs will relapse with clinical signs of systemic disease within 10-12 months of diagnosis. Thus there is a clear need for alternative therapies to maintain remission in canine patients with lymphoma.

  Basic research performed at Penn has shown that many millions of B cells (known as canine CD40-B cells) can be generated from 4-5mls of peripheral blood taken from canine patients with spontaneously occurring lymphoma. Furthermore, these cells can be loaded with antigen-specific RNA and used to induce antigen-specific T cell responses in vitro.

  The use of genetic material from the tumor (tumor RNA) as the antigenic payload permits an MHC-independent, multiple-antigen targeting approach particularly important in canine tumors where few tumor-associated antigens have been described. These findings were recently published in the journal Gene Therapy [Mason et al. Gene Therapy. 2008 Mar 13 (Epub ahead of print)] and have been translated into the veterinary clinics where tumor-RNA loaded CD40-B cells are now being evaluated in a phase I clinical trial in canine patients with lymphoma.

  Preliminary results from this phase I trial indicate that the use of RNA loaded CD40-B cells is safe and efficacious in prolonging clinical remission times in ~ 40% of patients. Studies are now underway to investigate whether the immunological response induced by CD40-B cells differs between responders and non-responders.

  Importantly, demonstrating the efficacy of this novel approach to induce therapeutic anti-tumor immunity against lymphoma aims to provide proof of principle that cCD40-B cells can be used as an effective cell based vaccine. This will pave the way to using this technology to treat other types of cancer since RNA can be made from any tumor type and be introduced into cCD40-B cells to stimulate anti-tumor immunity against as yet unidentified tumor-associated antigens.

  This work has been generously funded by the Alliance for Cancer Gene Therapy, the Barry and Savannah Poodle Memorial Fund and the Portuguese Water Dog Foundation.

Generation of canine single chain Fragment variable antibody libraries for the identification and targeting of tumor-associated antigens in the dog

• The use of monoclonal antibodies and antibody fragments to directly target tumors has shown promising results in clinical trials in humans. However, these exciting targeted approaches have not been possible in canine cancer patients since tumor antigens have not been identified, monoclonal antibodies of canine origin are not available and the efficacy of xenogeneic antibodies in the dog is limited by neutralizing antibody responses.

  Recently, it has been shown by screening combinatorial antibody libraries generated from human cancer patients that these patients break self-tolerance and generate antibody responses against their own tumors and that screening these antibody responses can lead to the identification of antibodies and antibody fragments that can be used therapeutically to target cancer in vivo.

  Researchers in Dr. Mason’s laboratory at PennVet have developed a system to clone the rearranged variable heavy (VH) and variable light (VL) immunoglobulin chains within circulating B lymphocytes of healthy dogs and dogs with spontaneously occurring cancers. They have used this system to generate combinatorial single chain (scFv) antibody libraries that provide a “fossil record” of the patient’s immunological repertoire.

  Using phage display technology, these scFv libraries are being analyzed for their ability to bind specifically to tumor antigens. Tumor specific canine scFvs can then be isolated and used to target tumor antigens in vivo. Furthermore, tumor antigens that are recognized by scFvs can be isolated, analyzed and identified.

  Dr. Mason is currently exploring this novel targeting approach to treat canine hemangiosarcoma, a highly aggressive, fatal malignancy of vascular endothelial cells that affects large breed dogs. The overall hypothesis is that as in human cancer patients, canine cancer patients generate antibody responses against their own TAAs. These tumor-specific antibodies can be used either alone or linked to a chemotherapeutic agent to specifically target primary and metastatic neoplastic lesions in vivo.

  Dr. Mason’s laboratory is using standard molecular techniques to recapitulate the immune repertoire of canine patients with hemangiosarcoma and screen the resulting combinatorial antibody libraries for single chain fragments that specifically target malignant endothelial cells. These fragments are being isolated, amplified and screened in vitro for their ability to recognize and kill autologous and allogeneic canine HSA cell lines.

  This work aims to develop the first canine-derived, tumor-specific targeting approach for the treatment of HSA in dogs and aims to provide proof of principal for this approach that can then be employed to generate targeting reagents for many other tumor types.

  It is intended that canine scFvs that target malignant endothelial cells will be employed in a phase I clinical trial to evaluate the safety and efficacy of this novel approach to treat dogs with spontaneously occurring HSA.

  This work is generously funded by the Canine Health Foundation (American Kennel Club)  and a Chase Foundation grant from the American College of Veterinary Internal Medicine.

  A manuscript describing this work has recently been published:

  Generation and validation of canine single chain variable fragment phage display libraries. Braganza A, Wallace K, Pell L, Parrish CR, Siegel DL, Mason NJ.  Vet Immunol Immunopathol.2010 Aug 7 [Epub] 

Ex vivo expansion and genetic manipulation of T cells for adoptive immunotherapy of canine cancer

• Adoptive immunotherapy (AI) using autologous, genetically modified T lymphocytes to recognize and kill malignant cells holds great promise for cancer therapy in humans and in dogs with spontaneously occurring cancer. Highly efficient retroviral gene transfer into peripheral T cells requires robust expansion of functional T cells ex vivo.

  Previously, non-physiologic methods that require exogenous T cell growth factors such as IL-2 to expand canine T cells ex-vivo have been reported as potential systems for AI. Using these methods, retroviral transduction frequencies of canine T cells have not exceeded 40%.

  Researchers in Dr. Mason’s laboratory at PennVet have developed an artificial antigen presenting cell (aAPC) that supports ex-vivo expansion and high frequency retroviral transduction of canine T cells, providing the first building blocks required to make AI for the treatment of canine cancer a reality. In this system, the human erythroleukemic K562 cell line has been stably transduced to express canine co-stimulatory molecules that are required for physiologic activation of T cells.

  White blood cells taken from small volumes of peripheral blood from healthy client-owned volunteer dogs can be expanded in vitro using this system, without the requirement for exogenous IL-2. Furthermore, these expanded cells can be stably transduced with retroviral vectors allowing the introduction of genetic constructs that optimize the T cells ability to recognize and kill transformed cells.

  One strategy used to circumvent immune evasion by neoplastic cells is to genetically modify T cells to express chimeric immunoreceptors (CIRs) that consist of a cell surface expressed single chain Fv (scFv), composed of the VH and VL domains of an antigen-specific immunoglobulin, that is linked in turn to a flexible hinge, transmembrane domain and intracellular signaling domains that lie within the cytosolic tail of the CIR (Fig.2).

  These surrogate CIRs can bestow primary T cells with MHC independent antigen recognition capabilities and antigen-dependent T cell activation, cytokine production and cytotoxic killing in the absence of co-stimulatory ligands.

  Adoptive transfer of ex-vivo expanded, tumor-specific re-directed T cells have shown promising anti-tumor effects in phase I clinical trials in human oncology patients.

  Thus, with the design of optimal CIR for the canine T cells, the ability to achieve high frequency gene transfer into peripheral dog T cells and refined ex-vivo canine T cell expansion techniques large numbers of autologous re-directed tumor-specific T cells can be produced, offering exciting possibilities for the treatment of primary and metastatic neoplasia in the dog.

  This project is currently funded through a University of Pennsylvania’s Research Foundation Grant.

Characterization of NF-kappa(k)B activity and the effects of its inhibition by NEMO binding domain peptide in spontaneously occurring canine cancer

• NF-kappa(k)B is a highly evolutionary conserved family of transcription factors that play important roles in the regulation of genes involved in immune responses, inflammation, stress responses, cellular proliferation, differentiation and apoptosis.

  However, the role of NF-kappa(k)B in oncogenesis has only been recognized recently. NF-kappa(k)B activation is highly regulated and in the inactive state, NF-kappa(k)B proteins are sequestered in the cytoplasm through their association with the inhibitory IkB and p100 proteins.

  Impaired regulation of NF-kappa(k)B activation can lead to loss of its highly controlled inducibility and constitutive activity. This results in aberrant expression of anti-apoptotic and pro-survival genes and genes that are involved in cell cycle control and migration, processes that occur in the initiation and progression of cancer. Indeed, NF-kappa(k)B is constitutively active in many solid tumors, where it contributes to malignant cell proliferation, growth and survival.

  NF-kappa(k)B also regulates the expression of a number of genes that are important in the process of tumor metastases and constitutive activation of NF-kappa(k)B has been reported in highly metastatic tumor cell lines. Finally, recent studies have indicated that constitutive NF-kappa(k)B activation in malignant cells is responsible for their observed chemoresistance.

  As such, attention has focused on the use of NF-kappa(k)B inhibitors as potential therapeutic agents to prevent cancer progression and metastasis and increase sensitivity to currently used chemotherapeutics. Recent studies have shown that inhibitors of NF-kappa(k)B activation suppress the development of carcinogen-induced tumors, inhibit cancer cell growth, induce apoptosis in malignant cell populations and may sensitize malignant cells to the apoptotic effects of more conventional chemotherapeutic agents.

  The Nemo Binding Domain (NBD) peptide is an established selective inhibitor of IKK and inhibits NF-kappa(k)B activation. This leads to inhibition of cell proliferation and induction of apoptosis. Importantly, it has been shown in vivo that the appropriate dosage of NBD peptide does not affect basal NF-kappa(k)B activity while NF-kappa(k)B activity that is inducible by proinflammatory stimuli is effectively blocked.

  This indicates that the normal cellular functions of NF-kappa(k)B are unaffected by NBD peptide and suggests that activation via TNF and EGF which contribute to oncogenesis may be selectively blocked by NBD peptide.

  Basic and translational researchers Dr. Michael May and Dr. Nicola Mason at PennVet are working together to evaluate the role of NF-kappa(k)B in canine oncogenesis and the potential therapeutic use of NBD peptide to inhibit NF-kappa(k)B in the treatment of canine cancer.

  Using low passage primary tumor cell lines, generated from spontaneously occurring canine tumors obtained from the PennVet Tumor Tissue Bank, Drs. Mason and May are characterizing the role of NF-kappa(k)B family members and the effects of the NBD peptide on NF-kappa(k)B activation, anti-apoptotic, tumor suppressor and cell cycle gene expression and in vitro viability, growth and proliferation in a variety of canine cancers.

  The overall hypothesis is that NBD peptide will inhibit constitutive NF-kappa(k)B activation resulting in decreased cell growth, proliferation and survival. It is anticipated that this work will lead to a pilot clinical trial to evaluate the safety and efficacy of the NBD peptide as an adjunct therapy in the treatment of a wide range of canine cancers that include transitional cell carcinoma, mesothelioma and lymphoma.

  This work is supported by the NIH/Merck Summer Research Program 2008.

Investigation of canine regulatory T cell biology and its implications for cancer immunotherapy and transplantation tolerance

• The presence of regulatory or suppressor T cells are a significant barrier to the success of immunotherapeutic approaches to treat cancer in humans. However, the significance of these cells and the role that they play in canine cancers has yet to be formally demonstrated.

  Researchers in Dr. Mason’s laboratory at PennVet are seeking to characterize the biology of canine regulatory T cells and to understand how they may be manipulated for therapeutic purposes. While the potent immunosuppressive function of these cells is detrimental to cancer immunotherapy, it may be beneficially employed for the treatment of autoimmune disease and strategies to prevent organ rejection after transplantation.

  Dr. Mason’s group have identified a subset of canine peripheral CD4+ T cells that have the phenotypic characteristics of natural regulatory T cells including surface expression of the IL-2 receptor and intracellular expression of the transcription factor, FoxP3.

  The low frequency of circulating naturally occurring regulatory T cells indicates that ex vivo activation and expansion of these cells will be required to generate sufficient numbers for immunosuppressive therapies required for autoimmunity and prevention of organ transplant rejection.

  In the laboratory, we have generated a system that allows for polyclonal expansion of canine regulatory T cells that are capable of suppressing autologous T cell proliferation.

  It is the overall hypothesis of this project that regulatory T cells isolated and expanded from patients undergoing organ transplant can be used to suppress allo-reactive donor T cells and induce tolerance to the donor graft.

  Further studies are required to elucidate the mechanisms responsible for canine regulatory T cell mediated suppression prior to the therapeutic use of these cells in phase I clinical trials to prevent organ rejection in transplantation medicine and suppress aberrant immune responses in autoimmune disease.