OESCA Supported Health Research Projects
Grant Title: 01415: Development of Anti-IgE Peptide for Treatment of Canine Allergy
Principal Investigator: Dr. Bruce Hammerberg, DVM PhD
Research Institution: North Carolina State University
Start - End Dates: 1/1/2011 - 12/31/2012
Original Project Description:
Treatment of chronic allergic diseases in dogs, often seen as recurring dermatitis, frequently results in less than optimal outcomes. When the disease can be linked to exposure to specific allergens, such house dust mites, desensitization injections can be effective in some individuals when carried out over an extended time; however, most cases are not resolved by desensitization and require a combination of allergen avoidance and anti-inflammatory drugs. The prolonged use of these drugs, such as corticosteroids, can result in severe side effects. These same challenges exist for human allergy suffers, but recently there has been a major breakthrough in the development of a new, safe and effective therapy using a monoclonal antibody that specifically binds and neutralizes human IgE that is responsible for activating inflammation-producing cells. This new product is called Xolair® and it has been used safely by millions of allergy patients for more than 5 years. Our laboratory has developed a monoclonal antibody that specifically binds canine IgE in the same manner as the monoclonal antibody used to develop Xolair®. There are two obstacles remaining in providing the canine equivalent to Xolair® for treatment of allergies in dogs and they are the Objectives of this proposal: 1. Modifying the monoclonal antibody to reduce the dog's natural response to clear this protein; and, 2. Developing cost effective production of the modified antibody. Our Approach is to: 1. Generate a single chain recombinant peptide from the IgE-binding region of our canine IgE-specific monoclonal antibody that is small in size and of limited antigenic potential; and 2. Develop a transgenic plant (eg. tobacco) containing the gene for this recombinant peptide using well established techniques that will allow production of the therapeutic peptide in kilogram quantities. The expected outcome will be to provide a new, safe and highly effective treatment option for canine allergic diseases that is affordable to use for maintenance therapy.
Objective 1: To create a recombinant, nonanaphylactic, single-chain antibody fragment (scFv) with high affinity for canine IgE from the variable region gene sequences of mAb 5.91 clones.
Objective 2: To generate a plant-derived recombinant, nonanaphylactic, single-chain antibody fragment with high affinity for IgE that can be scaled up for production at kilogram amounts.
Report to Grant Sponsor from Investigator:
1) To create a recombinant, non-anaphylactic, single-chain antibody fragment (scFv) with high affinity for canine IgE from the variable region gene sequences of mAb 5.91.
The sequence for the light chain variable region of mAb 5.91 was completed in April, 2011. The sequence for the heavy chain variable region was completed in October, 2011. Linkage of the two sequences and expression of a recombinant scFv of mAb 5.91 with confirmation of high affinity binding to canine IgE was completed in November, 2011.
A Fab fragment was produced from the whole molecule mAb 5.91 and used in flow cytometry assays as a model for the recombinant scFv version of the antibody by May, 2011. Whole blood from allergic dogs was processed and assayed. Results showed that the whole mAb 5.91 molecule reduced the amount of binding of canine IgE to the monocyte cell population from 15% to 7.7%. Moreover, the intact mAb 5.91 was able to bind the free IgE to prevent it from binding cell surface receptors. However, whole molecule mAb 5.91 complexed with canine IgE bound to 13.7% of the lymphocyte cell population possibly reacting with IgG Fc receptors. The Fab fragment of mAb 5.91, pre-incubated with canine IgE, reduced the binding of canine IgE to the monocyte cell population from 15% to 5.6%. This demonstrated that the Fab fragment of mAb 5.91 was even more effective in reducing the binding of IgE to the monocyte cell population than the intact mAb 5.91. There was no evidence of Fab fragment complexed with canine IgE binding to lymphocytes as previously seen with intact mAb 5.91.
These preliminary results indicate that the recombinant scFv form of the mAb 5.91 will be more effective at blocking IgE binding to cell surface receptors as well as decreasing the potential of cross reactivity of the lymphocyte cell population with the IgG Fc receptors than the original mAb 5.91.
2) To generate a plant-derived recombinant, nonanaphylactic, scFv with high affinity for IgE that can be scaled up for production at kilogram amounts. To be completed in the second year. Gene constructs of the newly made 5.91-scFv were designed to target the chloroplast and ER regions of the tobacco leaf cells. Both gene constructs were inserted into a PVX pGR106 amplicon vector and amplified in E.coli. The purified 5.91scFv-pGR106 constructs are being used to transform Agrobacterium tumefaciens strain GV3101. However, problems have been encountered during transformation attempts of Agrobacterium tumefaciens with the purified 5.91scFv-pGR106 constructs. A second round of transformation is being performed at this time.
TEV-B is a transgenic tobacco plant that expresses a mutated P1/HC-Pro suppressor of Post Transcriptional Gene Silencing. It has been shown that this line of tobacco plants produces higher protein yields than wild type varieties of tobacco including Nicotiana benthamiana. TEVB seeds were planted on May 23rd and TEV-B plants should be ready for infection in July. TEV-B plants were Agroinfected with Agrobacterium tumefaciens GV3101 containing the gene 5.91scFv::chloro gene construct. Total protein was extracted from 2kg of transgenic leaf tissue. Crude extract was clarified from photosynthetic proteins and polyphenols that may interact with downstream applications.
Binding activity of the 5.91scFv in the extract was confirmed on ELISA and was later compared to the activity of 5.91Fab and intact mAb 5.91 in whole blood and canine mast cells flow cytometry assays.
Grant Progress Report: 1415 EY2 FINAL Summary.pdf
Identification and Characterization of a Canine Derived Single Chain Antibody that Binds and Neutralizes Canine VEGF
Grant Title: 01484: Identification and Characterization of a Canine Derived Single Chain Antibody that Binds and Neutralizes Canine VEGF
Principal Investigator: Dr. Nicola J Mason, BVetMed, PhD
Research Institution: University of Pennsylvania
Start - End Dates: 1/1/2011 - 6/30/2012
Original Project Description:
Canine hemangiosarcoma (HSA) is a common and highly aggressive tumor of blood vessels that is oftentimes fatal. At diagnosis most dogs have evidence of metastatic disease and despite chemotherapy, survival times rarely exceed 6 months. Novel approaches to the treatment of this disease are needed. The use of targeting antibodies against vascular endothelial growth factor (VEGF), a protein that promotes tumor growth and spread, plus chemotherapy has prolonged disease free survival in several different tumor types in man. It is the aim of this proposal to isolate a canine derived antibody fragment that can specifically bind to and neutralize the tumor promoting effects of canine VEGF. It is hypothesized that a VEGF specific antibody fragment that is canine in origin may be used in the veterinary clinics to retard or prevent the development of metastases in dogs with HSA. We have generated diverse libraries of canine antibody fragments that we will screen against canine VEGF to select fragments that specifically recognize canine VEGF. We will isolate VEGF specific antibody fragments using previously described techniques that are routinely performed in our laboratory and test their ability to inhibit the tumor promoting effects of VEGF in vitro. This work will build on our previous studies supported by the CHF that describe the technology to generate canine antibody fragment libraries. This canine-derived, tumor-specific targeting approach is the first of its kind in the veterinary field and if successful this agent may also be used to treat many other tumor types in the dog.
Objective 1: To isolate canine derived scFvs that bind specifically to canine VEGF
Objective 2: To isolate VEGF-specific scFvs that inhibit VEGF bioactivity.
Report to Grant Sponsor from Investigator:
In this proposal we set out to utilize our canine-derived scFv phage display platform technology to isolate canine antibody fragments that bind and neutralize Vascular Endothelial Growth Factor (VEGF). VEGF is a protein growth factor that promotes the growth and survival of new blood vessels. Tumors require the growth of new blood vessels to supply them with oxygen and nutrients that allow the tumor to grow. VEGF is a major contributor to this angiogenesis (growth of new blood vessels).
Bevacizumab (Avastin) is a humanized antibody that recognizes and neutralizes human VEGF. It is currently approved for the treatment of a number of different cancers including metastatic colon cancer, and non-small cell lung cancer. FDA approval and on-going clinical trials attest to the potential of VEGF inhibition as a treatment for many different cancer types in humans, however, the therapeutic effects of VEGF neutralization on cancer growth and metastasis have not been evaluated in the dog. Expression of VEGF has been reported in a wide range of different tumor types in the dog including hemangiosarcoma, malignant melanoma, soft tissue sarcomas, mast cell tumors, nasal carcinomas, intracranial neoplasias, and simple mammary gland adenocarcinomas and inflammatory mammary carcinoma. Serum levels of VEGF are increased in dogs with osteosarcoma, malignant melanoma and HSA and in dogs with osteosarcoma and malignant melanoma, serum levels correlate with disease free interval and survival times respectively. Together these reports suggest that VEGF plays an important role in the progression of these tumor types in the dog and as such represents a potential therapeutic target. However, to date there are no canine-derived antibodies that bind and neutralize the angiogenic activity of canine VEGF.
During this project, we identified 3 canine-derived scFv or antibody fragments that bind to canine VEGF. Furthermore results from cell-based assays in vitro suggest that these antibody fragments might inhibit the growth stimulating effects of VEGF on healthy endothelial cells and on a canine hemangiosarcoma cell line in vitro. These results suggest that not only do these antibody fragments bind VEGF but they also might inhibit its biological activity, which would be essential for any therapeutic effect. We have since developed several sophisticated techniques including surface plasmon resonance (SPR) to more closely interrogate the ability of our isolated antibody fragments to neutralize the activity of VEGF (i.e. inhibit the ability of VEGF to interact with a key VEGF receptor). Disappointingly, the results of these experiments have shown that the most abundant antibody fragment that we isolated from our library does not appear to affect the interaction of VEGF with its receptor suggesting that this clone would not inhibit the biological effects of VEGF on tumor blood vessels. However, we are now testing the remaining 2 isolated scFv using these advanced techniques to determine whether they can neutralize VEGF.
In summary, this work has 1) demonstrated that canine derived antibody fragments that bind to VEGF can be isolated from libraries using simple panning techniques; and 2) optimized sophisticated techniques (surface plasmon resonance) that we can now use to determine whether antibody fragments interact with molecules of interest and can inhibit particular molecular interactions in vitro prior to their translation into patients with diseases such as hemangiosarcoma. We will continue to utilize these techniques to evaluate the remaining 2 scFv. If we find that one or both of these scFv neutralize VEGF activity then we intend to identify commercial partners that will assist in generating canine monoclonal antibodies based on these scFv that could then enter clinical trials in patients with hemangiosarcoma or other tumor types that are associated with excess production of VEGF.
Grant Progress Report: 1484 MY2 FINAL Summary.pdf
Grant Title: 01609: Probiotic VSL# 3 Reduces Enteritis in Dogs with Inflammatory Bowel Disease
Principal Investigator: Dr. Albert E. Jergens, DVM, PhD
Research Institution: Iowa State University
Start - End Dates: 1/1/2012 - 12/31/2014
Original Project Description:
Idiopathic inflammatory bowel disease (IBD) is a common cause of chronic gastrointestinal disease in dogs. Accumulating evidence in human IBD and animal models suggests that imbalances in composition of the intestinal microbiota contribute to the pathogenesis of chronic intestinal inflammation. Recent studies have also shown that dogs with IBD have distinctly different duodenal microbial communities compared to healthy dogs. Current treatments for IBD include the administration of nonspecific anti-inflammatory drugs which may confer serious side effects and do not address the underlying basis for disease, namely, altered microbial composition. Use of probiotics (viable, non-pathogenic bacteria that exert health benefits beyond basic nutrition) offers an attractive, physiologic, and non-toxic alternative to shift the balance to protective species and treat IBD. The aim of the proposed study is to investigate the clinical, microbiologic, and anti-inflammatory effects of probiotic VSL#3 in the treatment of canine IBD. We hypothesis that VSL#3 used as an adjunct to standard therapy (i.e., elimination diet and prednisone) will induce a beneficial alteration of enteric bacteria leading to induction and maintenance of remission in dogs with IBD. A randomized, controlled clinical trial of 8 weeks duration will assess the efficacy of standard therapy + probiotic versus standard therapy alone. There is a need for additional data to be generated to provide proof of efficacy in probiotic therapy before these agents can be applied to widespread clinical use. These studies will also provide highly relevant insight into the anti-inflammatory effects of probiotics for treatment of human and canine IBD.
To determine the clinical, microbiologic, and anti-inflammatory effects of probiotic VSL #3 in the treatment of canine IBD.
Report to Grant Sponsor from Investigator:
Idiopathic inflammatory bowel disease (IBD) is a common cause of chronic gastrointestinal disease in dogs. Accumulating evidence in human IBD and animal models suggests that imbalances in composition of the intestinal microbiota contribute to the pathogenesis of chronic intestinal inflammation. Recent studies in dogs with IBD have shown that they have distinctly different duodenal microbial communities compared to healthy dogs. Current treatments for IBD include the administration of nonspecific anti-inflammatory drugs which may confer serious side effects and do not address the underlying basis for disease, namely, altered microbial composition. Use of probiotics (viable, non-pathogenic bacteria that exert health benefits beyond basic nutrition) offers an attractive, physiologic, and non-toxic alternative to shift the balance to protective species and treat IBD. The aim of the proposed study is to investigate the clinical, microbiologic, and anti-inflammatory effects of probiotic VSL#3 in the treatment of canine IBD. We hypothesize that VSL#3 used as an adjunct to standard therapy (i.e., elimination diet and prednisone) will induce a beneficial alteration of enteric bacteria leading to induction and maintenance of remission in dogs with IBD. A randomized, controlled clinical trial of 8 weeks duration is presently being performed to assess the efficacy of standard therapy + probiotic versus standard therapy alone.
Our data to date suggests that dogs treated with VSL#3 do respond favorably to the probiotic as evidenced by a reduction in their clinical disease severity. What remains unclear (ie, the clinicians are blinded as to whether a dog receives VSL#3 or placebo) is whether remission occurs more quickly in VSL#3-treated dogs vs placebo-medicated dogs. There is a need for additional data to be generated to provide proof of efficacy in probiotic therapy before these agents can be applied to widespread clinical use. These studies will also provide highly relevant insight into the anti-inflammatory effects of probiotics for treatment of human and canine IBD.
We are actively seeking additional dogs for inclusion into the trial at this time, and would particularly welcome purebred dogs at increased risk for IBD including GSD, boxer, and French Bull dog breeds.
Grant Abstract: 1609 EY1 Summary.pdf
Grant Title: Investigation of the Genetic Mutations Underlying Progressive Retinal Atrophy
Principal Investigator: Simon Petersen-Jones, PhD
Research Institution: Michigan State University
Purpose: To identify the gene mutation that causes PRA in the Old English Sheepdog and develop a screening test to identify carriers and affected PRA dogs. Such a test could enable the eradication of PRA in the breed.
You Can Participate: If you own a PRA diagnosed dog, help researchers by collecting and submitting DNA samples from the PRA dog and immediate relatives.
If you have DNA stored on a dog who was diagnosed with PRA, contact Dr. Petersen-Jones. Stored blood can also contain valuable information.
Over 30 breeds have a screening test for PRA.
Download the submission form.