From humans to dogs: harnessing CAR-T therapies to cure B cell lymphomas in canines
Updated: Feb 6, 2020
This post was inspired by LEAH Laboratories- a company that is working to cure cancer in dogs. Last week they reached a benchmark of $100,000 through the equity crowdfunding website Wefunder. If you are interested in investing and owning a stake in their mission - check out the website!
The One Health Initiative brings together experts in a variety of fields- researchers, clinicians, veterinarians- to help solve biomedical questions related to different disease states in humans and animals. Each realm of expertise can bring something different to the table, which ultimately makes for a rich collaborative experience.
In biomedical research, it is critical to test therapeutics in animal models to verify safety and efficacy prior to clinical trials. Testing in animal models is known as preclinical testing. The preclinical trial phase allows researchers to pinpoint the mechanisms for how drugs and other therapeutics work, as well as to address any safety concerns before drugs can reach human hands.
While most preclinical research is specifically aimed at finding cures for humans, there are groups working towards therapies for our canine friends as well.
Cancer in dogs
There are approximately 90 million dogs across 60,000,000 households around the United States. Unfortunately, as many of us may know, 1/3 of all dogs will develop cancer in their lives. The most common types of cancer in dogs are mast cell tumors, melanoma, lymphoma, and osteosarcoma (bone cancer). Solid tumor types have historically been difficult to maintain and cure, however there has been substantial progress made in curing liquid tumors- that is, lymphomas and leukemias.
One type of cancer that dogs can develop is called large B cell lymphoma, which is a clinical analog of human non-Hodgkin’s lymphoma . Approximately 250,000 dogs die per year due to this cancer. The typical treatment for these dogs is chemotherapy. Unfortunately, animals that are treated on a 5-month regimen will relapse and pass due to their cancer within a year of the original diagnosis. Better treatment options are needed to help these dogs.
Genome Editing- CRISPR/Cas9
We have all probably heard some form of research being done with CRISPR/Cas9; I will give a quick summary of the system here. CRISPR- or clustered regulatory interspaced short palindromic repeats- are DNA sequences in prokaryotic organisms that function in part of the cell’s “immune system”. Upon infection of the prokaryotic cell with a virus, the viral DNA will be cleaved and integrated adjacent to CRISPR sequences. Once integrated, CRISPR RNA containing CRISPR and viral sequence is transcribed, and then binds to new viral DNA invading the cell. Once the CRISPR RNA is bound to incoming viral DNA, Cas9 protein is recruited to the area and cleaves the viral DNA, preventing the cell from being further infected.
The significance of this system is that the Cas9 cleaving protein is recruited to a specific sequence of DNA which is designated by the CRISPR RNA sequence- called guide RNA (gRNA) in the laboratory. For example, if you wanted to knock out a gene IL2RG in pigs, you could create a gRNA containing a sequence complimentary to IL2RG along with a CRISPR sequence . The CRISPR RNA contains a protospacer adjacent motif (PAM) sequence that helps to guide the Cas9 to the proper site of cleavage.
To learn more about CRISPR/Cas9 in a little more detail check out these reviews:  
CAR-T (chimeric antigen receptor) cells utilize signaling components found on T and B cells. Simply speaking, the extracellular domain of the receptor is derived from a variable region of an antibody (the part of the antibody that binds to an antigen- scFv), while the intracellular regions are derived from signaling components of stimulatory T cell receptors (CD3ζ, 41BB, CD28).
The idea of a CAR-T therapy is that the scFv can be designed such that it will bind to a cellular component that is highly expressed on cancerous cells. For example, for B cell lymphoma, the CAR target could be against CD19. To produce the CAR-T cells, T cells are removed from patients and then genetically modified to express a CAR that recognizes CD19 (autologous therapy). The CAR-T will then bind to the CD19 on cancerous cells and the CAR-T cell will become activated and will kill the target cancer cell. KYMRIAH (https://www.hcp.novartis.com/products/kymriah/acute-lymphoblastic-leukemia-children/) and YESCARTA (https://www.yescartahcp.com/efficacy) both target CD19 and are the only FDA approved CAR-T cell therapies for human B cell malignancies.
Utilizing CAR-T cells to cure B cell malignancies in dogs
Bringing all of these components leads to the question: can we cure canine B cell lymphoma with CAR-T cell therapies?
With better gene editing technology being discovered and described, improved CAR-T therapies can be produced. Genetic modification is required to produce CAR-T cell. Historically, CAR-T cells have been produced by viral (retroviral or lentiviral), transposon mediated gene transfer, or through mRNA based systems. Viral integration can be an expensive methodology for engineering CAR-T therapy, and this FDA regulated step in CAR-T manufacturing can cost around $40,000. Additionally, viral and transposon methodologies randomly insert DNA within the genome, resulting in two major implications: (1) the insertion could be mutagenic and (2) every CAR-T cell could have a different insertion site with different expression patterns (heterogenous population).
Utilizing a CRISPR mediated gene targeting methodology called GeneWeld , the issues mentioned above can be avoided. As CRISPR gene editing is precise and can be localized to a specific area of the genome, the CAR-T gene insertion can be directed to a specific area in the genome. This directed insertion would significantly reduce incidences of mutagenic insertions, and all CAR-T cells would have the same gene insertion. The main hypothesis is that the homogeneity of the CAR-T population would increase therapeutic efficacy. In addition, with GeneWeld methodology, research can be performed on knocking out cellular components that could be recognized as “foreign” by an allogeneic recipient (immunologically dissimilar from the donor), which could increase efficacy of the CAR-T therapy. Use of an allogeneic donor compared to an autologous (self) can decrease price of the therapy, as well as provide more treatment options for the dogs.
The field of immuno-oncology in canine cancer is relatively new. With these new innovative technologies, researchers can make strides towards curing B cell malignancies in dogs. These studies also pave the way for curing other types of cancers in our four-legged friends as well.
And of course, check out these references:
1. Aresu L. Canine Lymphoma, More Than a Morphological Diagnosis: What We Have Learned about Diffuse Large B-Cell Lymphoma. Front Vet Sci. 2016;3: 77. doi:10.3389/fvets.2016.00077
2. Boettcher AN, Li Y, Ahrens AP, Kiupel M, Byrne KA, Loving CL, et al. Novel engraftment and T cell differentiation of human hematopoietic cells in -/- SCID pigs. bioRxiv. 2019; 614404. doi:10.1101/614404
3. Thurtle-Schmidt DM, Lo T-W. Molecular biology at the cutting edge: A review on CRISPR/CAS9 gene editing for undergraduates. Biochem Mol Biol Educ. 2018/01/30. 2018;46: 195–205. doi:10.1002/bmb.21108
4. Adli M. The CRISPR tool kit for genome editing and beyond. Nat Commun. 2018;9: 1911. doi:10.1038/s41467-018-04252-2
5. Mochel JP, Ekker SC, Johannes CM, Jergens AE, Allenspach K, Bourgois-Mochel A, et al. CAR T Cell Immunotherapy in Human and Veterinary Oncology: Changing the Odds Against Hematological Malignancies. AAPS J. 2019;21: 50. doi:10.1208/s12248-019-0322-1
6. Wierson WA, Welker JM, Almeida MP, Mann CM, Webster DA, Torrie ME, et al. GeneWeld: a method for efficient targeted integration directed by short homology. bioRxiv. 2019; 431627. doi:10.1101/431627
7. Duroux-Richard I, Giovannangeli C, Apparailly F. CRISPR-Cas9: A revolution in genome editing in rheumatic diseases. Joint, bone, spine : revue du rhumatisme. France; 2017. pp. 1–4. doi:10.1016/j.jbspin.2016.09.012
8. Klampatsa A, Haas AR, Moon EK, Albelda SM. Chimeric Antigen Receptor (CAR) T Cell Therapy for Malignant Pleural Mesothelioma (MPM). Cancers (Basel). 2017;9. doi:10.3390/cancers9090115