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We have created the immunodeficient SRG rat, a Sprague-Dawley Rag2/Il2rg double knockout that lacks mature B cells, T cells, and circulating NK cells. This model has been tested and validated for use in oncology (SRG OncoRat®). The SRG rat demonstrates efficient tumor take rates and growth kinetics with different human cancer cell lines and PDXs. Although multiple immunodeficient rodent strains are available, some important human cancer cell lines exhibit poor tumor growth and high variability in those models. The VCaP prostate cancer model is one such cell line that engrafts unreliably and grows irregularly in existing models but displays over 90% engraftment rate in the SRG rat with uniform growth kinetics. Since rats can support much larger tumors than mice, the SRG rat is an attractive host for PDX establishment. Surgically resected NSCLC tissue from nine patients were implanted in SRG rats, seven of which engrafted and grew for an overall success rate of 78%. These developed into a large tumor volume, over 20,000 mm3 in the first passage, which would provide an ample source of tissue for characterization and/or subsequent passage into NSG mice for drug efficacy studies. Molecular characterization and histological analyses were performed for three PDX lines and showed high concordance between passages 1, 2 and 3 (P1, P2, P3), and the original patient sample. Our data suggest the SRG OncoRat is a valuable tool for establishing PDX banks and thus serves as an alternative to current PDX mouse models hindered by low engraftment rates, slow tumor growth kinetics, and multiple passages to develop adequate tissue banks.

Human tumor xenografts are a staple tool for understanding tumor biology, growth kinetics, and therapeutic efficacy. While these studies are most commonly done in immunocompromised mice, we have created a Sprague Dawley Rag2 null, Il2rgamma null SRGTM rat that is an excellent host for human xenografts (OncoRat®). Lacking B, T, and NK cells, the SRG rat readily supports the growth of multiple human cancer cell lines, including lines that do not engraft well or grow consistently in existing mouse models. The tumor microenvironment (TME) is a critical factor for supporting xenograft tumors, and the microenvironment of a human tumor grown in the rat has yet to be fully characterized. In this study, a collaborative effort between research institutions discovered that the tumor microenvironment in the SRG rat is more robust, involved, and more supportive of human tumor growth than in NSG mice. To characterize the aforementioned differences in rat and mouse TME, human prostate cancer cell lines LNCaP and VCaP were grown in NSG mice and SRG rats. Formalin fixed paraffin embedded sections were stained via immunohistochemistry (IHC) for both rat and mouse tumor microenvironment markers. Collagen marker CD29, endothelial cell marker CD31, macrophage marker CD45, smooth muscle actin, and stromal markers CD54 and vimentin were analyzed in both animal hosts. When applicable, staining was quantified via counting positive cells per high powered field of view. When VCaP and LNCaP xenograft tumors are hosted by SRG rats, the host TME is significantly more involved within the human tumor, and readily supports tumor growth. Comparing the same markers in SRG rat and NSG mouse hosts revealed a stark difference - the SRG rat TME is more prevalent than the mouse. Results show significantly increased stromal cells per high powered field in the SRG rat when compared to tumors of the same cell line grown in the NSG mouse. There is heavily increased endothelial and stromal cell infiltration from the host into the human tumor, and higher heterogeneity within the cell population in tumors hosted by SRG rats. These data show that there are more human tumor epithelial cell interactions within the TME of the SRG rat than in NSG mouse. Increased stromal involvement more accurately recapitulates a human TME and may help explain the better take rates and faster growth rates of xenografts in SRG rats versus NSG mice. It is well known that recapitulating the tumor cell population heterogeneity is a study limitation when using animal models. Utilization of the SRG rat TME has great value in nonclinical research by more accurately translating into human disease, while remaining in a readily available immunodeficient animal model (i.e., OncoRat). Citation Format: Diane Begemann, Bisoye Towobola Adedeji, Valeriya Steffey, Sam Moody, Goutham Narla, Fallon Noto. Sprague-Dawley Rag2 null Il2rgamma null SRG rat (OncoRat®) has enhanced tumor microenvironment in human prostate cancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2949.

The development of Novel cancer immunotherapies has relied on the use of immune humanized mice. The field of study can be enhanced using an immune humanized rat, which will provide several advantages over the currently available humanized mouse models. The SRG OncoRat®; A Rag2/Il2rg double-knockout rat supports the growth of larger tumors for serial fine needle biopsies to assess immune infiltration and serial blood draws for assessing human immune development and tumor biomarkers in real-time throughout an efficacy study. Using this rat, we developed a novel autologous human skin and immune cells-humanized rat model by co-engrafting full-thickness human-fetal skin and autologous fetal lymphoid organoids under the kidney capsule along with intravenous injection of autologous fetal-liver derived hematopoietic stem cells, thus termed, human skin-immune system humanized rat model (hSIS-humanized rat). We demonstrated the development of adult-like, full-thickness human skin and human lymphoid organoids along with human immune cells. Methicillin-resistant Staphylococcus aureus inoculation in the human skin results in infection and skin pathology, thus recapitulating clinical outcomes. This model will enable in vivo mechanistic studies for development and evaluation of novel therapeutics for skin infectious disease and may also provide a model for establishing skin grafts of patient-derived melanoma tumors to investigate melanoma metastasis and response to therapies. In addition, engrafting the rat with human lymphoid organs and human immune cells may provide a similar platform to the BLT mouse for immunotherapy studies. Furthermore, we demonstrated the presence of Human CD45+, CD3+, and CD20+ cells in peripheral blood, spleen, and bone marrow of the SRG OncoRat® engrafted with human PBMCs, hence humanizing the rat's immune system. These immune humanized rat models may be beneficial for evaluating immunotherapies in human cancer models, including assessment of immune cell infiltration through fine needle biopsies. Citation Format: Bisoye Towobola Adedeji, Fallon K. Noto, Tseten Yeshi Jamling, Yash Argawal, Cole Jamison Beatty, Sara Grace Ho, Antu Das, Rajeev Kishore Salunke, Moses Turkle Bility. Humanized rat model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5621.

The use of the most predictive model is essential in transferring scientific knowledge from bench to bedside. In In vivo cancer research, immunodeficient mice models are being used, but have been found with limitations. They fall short in tumor take rates, growth kinetics, and their ability to support the growth of some human Cancer cell lines. To address these limitations, we produced an immunodeficient rat, a Sprague-Dawley Rag2/Il2rg double knockout (SRG OncoRat) rat that lacks mature B cells, T cells, and circulating NK cells. Using a variety of human cell lines (VCaP, LNCaP, 22RV1) which have poor or highly variable growth kinetics in commercially available immunodeficient mouse models, we demonstrated that the SRG rat has superior tumor take rates and growth kinetics, providing a more efficient model for drug efficacy studies. Enhanced take rates enable the use of fewer animals for studies, allows for faster timelines to drug efficacy data, and overall, may provide a better platform for reproducible drug efficacy results. In addition, the SRG rat developed larger tumor volumes, allowing for serial fine needle aspirate biopsy for PK, PD and molecular analysis in the same animal throughout the course of the study without significantly affecting normal tumor growth. The SRG OncoRat is a valuable addition to current available rodent xenograft models for evaluating novel therapies. Citation Format: Bisoye Towobola Adedeji, Noto K. Fallon, Sam Moody, Valeriya Steffey, Chris Brenzel, Jack Crawford, Tseten Yeshi Jamling, Goutham Narla. Rats support cancer studies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6133.

Current cell line-derived cancer and PDX mouse models are hindered by low engraftment rates and slow tumor growth kinetics. Furthermore, serial passaging of patient derived tissue results in changes to the genomic landscape such that the transplanted tumor no longer reflects the primary tumor. To address these limitations, we produced an immunodeficient rat, a Sprague-Dawley Rag2/Il2rg double knockout (SRG) rat that lacked mature B cells, T cells, and circulating NK cells. We demonstrated that the SRG rat has improved tumor take rates and growth kinetics using a variety of human cell lines and PDXs. Cell lines tested include VCaP and LNCaP, which have poor or highly variable growth kinetics in commercially available immunodeficient mouse models. We have demonstrated that both of these cell lines have superior take rate and growth kinetics in the SRG rat, providing a more efficient model for drug efficacy studies. In addition, the rat can accommodate a larger tumor volume, allowing for serial fine needle aspirate biopsy for PK/PD analysis in the same animal throughout the course of the study without significantly affecting normal tumor growth. The PDXs in the SRG developed larger tumor volume, over 20,000 mm3 in the first passage, which provided an ample source of tissue for characterization and/or subsequent passage into SRG for drug efficacy studies. Larger tumor volumes enabled fewer animals to be needed for a study, allowed for faster timelines to drug efficacy data, and reduced the need for serial passaging to generate enough tissue, which limited genomic divergence from the parental tumor tissue. As proof of principle, we used next generation sequencing based genomic analysis to direct a precision medicine strategy to guide in vivo efficacy studies. Specifically, using this approach we found a novel mutation in the MET pathway in a primary patient derived sample and tested therapies that were predicted to target this mutation and compared efficacy to standard of care agents in this unique SRG rat derived PDX model. Collectively, this data suggests that this novel rat model could serve as a patient avatar to better predict outcomes to genomically-directed therapies. In addition, we tested the ability of the SRG rat to support the growth of patient-derived tissue that was cryopreserved directly from patient harvested and had not previously been xenografted into an animal model. This concept may provide a means for establishing PDX models from cryopreserved samples when animals are not immediately available for xenografting. Citation Format: Fallon K Noto, Bisoye Towobola Adedeji, Sam Moody, Chris Brenzel, Jack Crawford, Goutham Narla, B. Mark Evers, Tseten Yeshi Jamling. A Rag2/Il2rg double-knockout rat (SRG OncoRat) enables precision-medicine based cancer studies with cell line- and patient-derived xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B067. doi:10.1158/1535-7163.TARG-19-B067

Immune humanized mice have been valuable in the development of novel cancer immunotherapies and have demonstrated stronger efficacy when combined with standard of care chemotherapy. An immune humanized rat could provide several advantages over the currently available humanized mouse models, including supporting the growth of larger tumors for serial fine needle biopsies to assess immune infiltration and serial blood draws for assessing human immune development and tumor biomarkers in real-time throughout an efficacy study. We developed a novel autologous human skin and immune cells-humanized rat model by co-engrafting full-thickness human-fetal skin and autologous fetal lymphoid organoids under the kidney capsule along with intravenous injection of autologous fetal-liver derived hematopoietic stem cells, thus termed, human skin-immune system humanized rat model (hSIS-humanized rat). hSIS-humanized rat support development of adult-like, full-thickness human skin and human lymphoid organoids along with human immune cells. Methicillin-resistant Staphylococcus aureus inoculation in the human skin results in infection and skin pathology, thus recapitulating clinical outcomes. This model will enable in vivo mechanistic studies for development and evaluation of novel therapeutics for skin infectious disease and may also provide a model for establishing skin grafts of patient-derived melanoma tumors to investigate melanoma metastasis and response to therapies. In addition, engrafting the rat with human lymphoid organs and human immune cells may provide a similar platform to the BLT mouse for immunotherapy studies. Finally, we have demonstrated humanization of the rat immune system using human PBMCs. Human CD45+, CD3+, and CD20+ cells can be found in the peripheral blood, spleen, and bone marrow of engrafted rats. These immune humanized rat models may be beneficial for evaluating immunotherapies in human cancer models, including assessment of immune cell infiltration through fine needle biopsies. Citation Format: Fallon K Noto, Bisoye Towobola Adedeji, Yash Agarwal, Cole Beatty, Sara Ho, Antu Das, Rajeev Salunke, Moses Bility, Tseten Yeshi Jamling. A Rag2/Il2rg double-knockout rat supports engraftment of human immune system for immunotherapy-based cancer efficacy studies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A007. doi:10.1158/1535-7163.TARG-19-A007

Current treatment options for advanced castration-resistant prostate cancers (CRPC) are effective for a brief period before becoming refractory. It is important to use relevant in vivo models for candidate selection in the development of next-generation mechanistically-distinct drugs to treat castration- and drug- resistant prostate cancers. Here, we describe a series of AR pan-antagonists (selective AR degraders (SARDs)) that degrade the AR and AR splice variants. SARDs inhibit the wild-type and LBD mutant ARs comparably and inhibit the in vitro proliferation and in vivo growth of enzalutamide-sensitive and resistant prostate cancer xenografts. Xenograft studies conducted in immune-compromised SRG rats (Sprague Dawley Rag2-/- Il2rg -/-; Hera Biolabs) with three lead SARDs demonstrated regression of enzalutamide-sensitive and -resistant VCaP tumors both in castrated and in intact rats. SRG rats provide the benefit of abundant blood samples for weekly evaluation of rising PSA, which also indicated that the SARDs inhibit the rising PSA, while enzalutamide was inactive in enzalutamide-resistant model. Drug metabolism and pharmacokinetic (DMPK) studies, also conducted with SRG and wild-type rats and in combination with efficacy, indicate that the molecules possess all the necessary drug-like properties. The molecules exhibit a broad safety margin with no cross-reactivity with G-Protein Coupled Receptor, kinase, and nuclear receptor family members. Collectively, the SARDs exhibit the properties necessary for a next-generation prostate cancer drug and that use of SRG rats facilitated thorough characterization of their in vivo properties. Citation Format: Suriyan Udhayasuriyan Ponnusamy, Fallon K Noto, Bisoye Towobola Adedeji, Yali He, Dong-Jin Hwang, Sam Moody, Thirumagal Thiyagarajan, Tseten Yeshi Jamling, Duane D Miller, Ramesh Narayanan. Potential next-generation androgen receptor-targeted therapeutic for enzalutamide-resistant prostate cancer; In vivo characterization in immune-compromised SRG rats [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B037. doi:10.1158/1535-7163.TARG-19-B037

Human cancer xenografts in rodents can provide predictive data on the success of candidate drugs in clinical trials and have been a pivotal tool in moving new drugs from the bench to the clinic. However, currently available immunodeficient mouse models have shown some limitation and variability in tumor take rates and growth kinetics in cancer cell lines. In addition, commercially available human cancer cell lines aren’t representative of the genomic and molecular diversity of cancers found in patients. Patient Derived Xenograft (PDX), in which tumor tissue is transplanted directly into rodents after biopsy from the patient, better represents that molecular signature, heterogeneity, and pathology of the original tumor. Therefore, in vivo efficacy studies with PDX models could be highly predictive for treatment sensitivity. Despite the many advantages of PDXs for preclinical research, PDX mouse models are hindered by low engraftment rates and slow tumor growth kinetics. The loss of patient tumor heterogeneity and stromal cells as the PDX is passaged multiple times to generate sufficient tumor tissue to inoculate a cohort of animals for efficacy studies is also a disadvantage in the immunodeficient mouse models. To address these limitations, we have introduced the OncoRat®; built on the SRGTM Platform, a Sprague-Dawley Rag2/Il2rg double knockout rat that lacks mature B cells, T cells, and circulating NK cells. We have demonstrated that the OncoRat has improved tumor take rate and growth kinetics for non-small cell lung cancer (NSCLC) PDXs. The NSLSC PDXs in the OncoRat have a much larger tumor volume, over 20,000 mm3 in the first passage (P0) in the rat, which provides an ample source of tissue for characterization and/or subsequent passage (P1) into OncoRat for drug efficacy studies. This leads to fewer animals used for study and faster timelines to drug efficacy data, resulting in a reduction in cost. In addition, we have used genomic analysis for guidance in planning in vivo efficacy studies. One of our NSCLC PDX models harbors a novel mutation in the MET pathway, suggesting this tumor would not be responsive to standard of care treatment. An efficacy study we performed in the OncoRat suggests that this particular tumor would respond well to Type II MET inhibitors, such as Cabonzantinib. This proof of concept study demonstrates that genomic and molecular analysis can provide insight into treatment outcomes and that PDX models in the OncoRat could serve as patient avatars for predicting treatment outcomes. Citation Format: Fallon K. Noto, Bisoye Towobola Adedeji, Sam Moody, Chris Brenzel, Jack Crawford, Goutham Narla, Tseten Yeshi Jamling. A case study: OncoRat is a viable patient avatar for a NSCLC patient with a Y1248H Met activating mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1059.