February 2022 Cancer Research Lab Update

As development of our current bacterially delivered anticancer immune therapy continues toward human clinical trials, we are working to determine the strain of bacteria that will be used in those trials. Our most cutting-edge candidate has been genetically engineered to secrete seven different immune stimulating proteins directly into tumors. Off-target immune stimulation has limited the amount and number of immune-stimulating proteins that current anticancer therapy can deliver. We have engineered a regulatory switch into our bacteria that senses their presence in the tumor environment and then turns on the molecular machinery to manufacture and secrete the anticancer proteins. This avoids over-stimulation of the immune system elsewhere in the body. Experiments are proceeding to optimize the amount of each protein secreted into tumors by adjusting DNA sequences in the bacteria. In addition, we continue to work to determine the optimal dosing of this therapy in combination with drugs that increase the ability of our bacteria to colonize tumors. Once these optimization experiments are completed, this therapy will be moved from our laboratory to the clinic. Thus, we are working to complete development of a therapy to eliminate tumors and establish an immune memory to guard against return of the cancer, without the debilitating toxic side-effects of current cancer therapy.

August 2021 Cancer Research Lab Update

All of the experiments being performed in our laboratory are designed to increase the ability of our genetically engineered bacteria to induce the immune system to eliminate cancer and guard against its return, without the debilitating side effects of current cancer therapy. These experiments include: 1) optimizing the secretion of immune modulating proteins from bacteria into tumors; 2) determining conditions for multiple dosing of the bacteria; 3) analysis of the molecular and cellular changes in tumors in response to treatment with bacterial strains secreting various immune modulating proteins; 4) monitoring increased survival of tumor-burdened mice due to genetic changes we introduce into the bacteria.

Initially, our bacteria were engineered to secrete proteins through a membrane pore system normally used by pathogenic bacteria to secrete toxins. While this was successful and resulted in a degree of anticancer efficacy, we are testing a more robust system to secrete more immune modulating protein through a molecular apparatus normally used by the bacteria to secrete proteins to elongate their flagella. Secreting more immune modulating proteins should result in a stronger anticancer immune response.

Cancer therapy is usually given in multiple doses over time to ensure tumors are completely destroyed and residual disease is eliminated. However, once the immune system encounters our bacteria, it is better able to eliminate additional doses of the bacteria before they can reach tumors and secrete immune modulating proteins. Therefore, we are working to engineer the ability of our bacteria to remain stealthy upon multiple dosing. Two mechanisms are being tested. First, we have engineered a strain of bacteria to encapsulate itself in a sugar coat while being grown prior to dosing. This coat will eliminate the ability of the immune system to detect any bacterial surface proteins it became aware of during the first dosing with the uncoated bacteria. Second, we are wrapping our bacteria in a coat of fat that will also protect it from interacting with previously established immune surveillance. These methods of increased bacterial stealth will allow multiple dosing and an extended anticancer immune response.

Analyzing the molecular and cellular changes in the tumor microenvironment in response to the various immune modulating proteins secreted from bacterial strains will allow selection of the most efficacious strains to be included in the therapy based on anticancer immune mechanisms. Using mass spectrometry to analyze the concentration of thousands of proteins in the fluid surrounding cancer cells in treated tumors, we are examining changes in the molecular immune state in response to treatment. Similarly, using flow cytometry to analyze the concentration and state of immune cells within tumors also allows for mechanistic determination of the effect of each bacterial strain on the antitumor immune response.

These experiments are allowing continual optimization of our nontoxic anticancer bacterial immune therapy and will soon allow translation into human clinical trials.

Fall 2020 Cancer Research Lab Update

Although we have accomplished reduction of tumor growth and extension of life in mice with cancer, we are working to improve this therapy before embarking on clinical trials in humans. This involves focusing our investigation on half a dozen topics.

Making the bacteria less visible to the immune system, allowing for increased and multiple dosing. We have recently deleted a gene for a protein on the surface of the bacteria that stimulates a toxic response to the bacteria while they are in blood vessels prior to reaching tumors. We have inserted genes into the bacteria that provide a capsule that also interferes with toxic recognition. We are also investigating wrapping the bacteria in a coat of fat that decreases its visibility while traversing blood vessels.

Increased secretion of proteins that stimulate an anticancer immune response in tumors. We are at the final step of engineering the bacteria to secrete proteins through an apparatus that may be 100 times more efficient than the current secretion apparatus.

As knowledge of cancer immunology advances we are engineering our bacteria to secrete additional molecules that have been identified to enhance destruction of cancer cells by the immune system.

We are working to optimize the amounts and timing of administration of each element of our therapy to achieve the greatest possible anticancer effect. Once we have completed the work described above, we will change the proteins secreted by our bacteria from mouse to human and proceed to clinical trials.

Fall 2019 Cancer Research Lab Update

The main goal of our work this year has been to engineer bacteria that colonize tumors consistently, comprehensively and without toxicity. These bacteria have been modified to secrete molecules into the tumor that stimulate the immune system to destroy the cancer and guard against its return.

This year we have:

  • Completed engineering of bacteria that allow delivery of anticancer molecules to tumors throughout the body without therapy-limiting toxicity. We are now working to determine optimal dosing of these bacteria to eliminate tumors.
  • Obtained a library of DNA to be screened for human-specific molecules to replace the mouse-specific molecules currently secreted into tumors by our bacteria, which will be necessary before proceeding to clinical trials. This library also allows us to screen for additional cancer-killing molecules.
  • Worked with additional strains of bacteria to accomplish oral delivery of the therapy in addition to the current intravenous delivery method.
  • Expanded our mouse colony so that we now have mice with tumors that can be used for weekly experiments.

We are grateful for your support and excited about the anticancer immune therapy being developed with that support.

November 2018 Cancer Research Lab Update

1. We are using a mouse colony that mimics human cancer, called the autochthonous approach, where the cancer starts from a single cell and develops just as human cancer progresses. This system is much more efficient than what drug companies use, where the cancer is injected under the skin. Our system allows a natural progression of stimulating the immune system in proper, realistic fashion.

2. We have instituted antibodies against checkpoint inhibitors PDL-1 and CTLA-4. The 2018 Nobel Prize for Medicine was awarded this year to the two groups (Japan and Texas) for the discovery of these immune modulators. The biggest drawback to the use of the checkpoint immune inhibitors is the toxic side effects. We have been able to use our innovative Salmonella Delivery System to deliver both the PDL-1 and CTLA-4 proteins without any toxicity whatsoever.

3. We are working at present to develop a method to alter the blood supply to the solid tumor. By changing how blood flows to a tumor, we can cause necrosis, or disintegration of the tumor. This changes the tumor microenvironment to make it more attractive of our Salmonella to invade and colonize these solid cancers. Thus, we are able to use Salmonella to act as an in situ factory to produce a cocktail of immune modulating proteins directly within the tumor itself and eliminate any toxic side effects.

4. Also, we have added promoter genes to our genetic systems that also enhance the effect of all the killing power of the tumor with our genetic engineering. Hopefully, with your continued support and our recent progress, we expect to not only kill the tumor, but also with the application of our system, prevent later metastatic spread.

5. In addition to the genetic engineering advance above, we also added an experiment with chemotherapy from 0-100% to helpbreak down part of the tumor. We are very excited to report that a 75% reduction in the dose of chemotherapy coupled with our genetic engineering construct kills as much tumor as 100% chemotherapy. This advance also gives quality of life to patients without the grave side effects of 100% chemotherapy itself.

Thank you for your generosity that allows us to continue this most exciting work. Our lab was chosen to help run a consensus conference generated by the National Cancer Institute and the National Institute of Health last year. This gave great exposure to our lab and system as we presented our approach and helped lead the seminar. This is a complicated approach but we are making significant progress and again, thank you for your help.

2019 Lab Personnel:

The principle investigator of our cancer therapy development, Dr. Daniel A. Saltzman, has studied the use of attenuated Salmonella as a tumor-targeted vehicle to carry immune modulating proteins to solid tumors for over 20 years. His Ph.D. thesis was in this field. He wrote and published one of the very first manuscripts highlighting the use of Salmonella as a potential cancer treatment and has been sought out as an expert in this field throughout the world. Despite his clinical duties as an active pediatric surgeon, basic science research with a focus on translating that research to the clinic has always been his passion. His dedication to the field of microbial based cancer therapy was recently recognized when he was invited to co-chair a session in a recent NCI sponsored consensus conference on microbial based cancer therapy. Dr. Saltzman’s expertise, experience, training, leadership, and tenacity to successfully carry out the proposed research inspires our team.

As a co-investigator, Dr. Janet L. Schottel provides a wealth of knowledge in microbiology, biochemistry and molecular biology, and extensive experience in bacterial physiology. During her career as a professor she has mentored numerous graduate and undergraduate students on projects concerning transcriptional and translational regulation of gene expression, mechanisms of mRNA degradation, secondary metabolite production, plant-pathogen interactions, biological control of disease, biosensor development, and mechanisms of desiccation tolerance. In addition to providing insight into the design of experiments and analysis of data, Dr. Schottel supervises staff and students involved in the development of our cancer therapy in her laboratory in the Department of Biochemistry, Molecular Biology & Biophysics at the University of Minnesota.

Our laboratory director, Dr. Lance B. Augustin, began his career in life science research over thirty years ago as a graduate student in the University of Minnesota Medical School Department of Biochemistry. During the ensuing years he developed skills in microbial engineering and molecular biology. He has expertise in engineering mammalian cells and extensive experience with mouse husbandry and surgery. During his work as a member of cancer research teams he has accumulated the knowledge of cancer biology and immunology needed for his current position directing the efforts of a laboratory developing bacterially delivered anticancer immunotherapy. Dr. Augustin has authored several publications contributing new knowledge to our understanding of cancer.

Additional full-time staff in our laboratory include Dr. Liming Milbauer with over 20 years of experience in: biochemical assays, nucleic acid manipulation, bacterial strain construction, tissue culture, and flow cytometry. Dr. Milbauer is responsible for data collection and analysis and she provides daily oversight of all animal experiments.

Ms. Sara Hastings carries out daily experimentation and manages our mouse breeding colony. Ms. Hastings joined our team in 2017 after completing her B.S. degree at Carleton College.

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