Abstract B027: Antitumor effects of the programmed death receptor-1 and the programmed death-ligand 1 blockade in human colorectal carcinoma in a humanized orthotopic mouse model
Authors: Li Li , Zhang Xin , Maresh Grace , Hellmers Linh , Patel Avi , Moret Ravan , Cohen Sarah , Margolin David 
Published: February 2019

Abstract
Background: The immune system plays an important role in tumor immune surveillance and progression. Immune checkpoint blockade is a new approach for cancer immunotherapy. The programmed death receptor-1 (PD-1) and the programmed death-ligand 1 (PD-L1) are immune checkpoint molecules and their expression results in negative regulation of T-cells primarily within the tumor microenvironment by preventing the killing of cancer cells by cytotoxic T-lymphocytes. Antibody blocking of the PD-1/PD-L1 signal exhibits promising therapeutic effects in non-small cell lung cancer and melanoma in patients. Due to its early success, more trials have been conducted to evaluate their efficacy for different tumors. We have developed a patient-derived orthotopic xenograft mouse model for colorectal carcinoma (CRC). Here, we investigate the potential efficacy of PD-1/PD-L1 blockade in our humanized orthotopic mouse models for human CRC. Methods: All studies were conducted under approved guidelines of the Institutional Animal Care and Use Committee and the Investigative Review Board of Ochsner Clinic Foundation. Humanized mice were established by intraperitoneal injection of donor human peripheral blood mononuclear cells (PBMCs) into recombinase activating gene 2 (Rag2) and common cytokine receptor gamma chain gene (IL2Rγ) double knockout (Rag2-/-/IL2Rγ-/-) Rag2 mice. Luciferase-tagged CRC cells were injected intra-rectally into Rag2 mice. One group of mice received a combination of anti-PD-1 and anti-PD-L1 antibodies (nivolumab, 200 µg/mouse and atezolizumab, 200 µg/mouse, intravenous injection) once a week for 3 weeks. Tumor growth was measured weekly by bioluminescent imaging (BLI). At necropsy, the CRC tumor weights were measured. Human CD45+ hematopoietic cells, CD4+ and CD8+ T-cells, and CD20+ B cells were detected in peritoneal lavage, blood, and tumor by flow cytometry. The presence of human immune cells (humanization) and tumor-infiltrating human lymphocytes was further confirmed by immunohistochemistry staining on paraffin-embedded tissue slides of mouse spleen and tumor, respectively. Results: Blood from mice receiving human PBMCs contained on average 31.4% CD45+ human cells (n=9) when tested by FACS analysis. Smaller tumor growth was observed in mice given PBMCs. This may be due to alloreactivity based on the recognition of MHC alloantigens in the transplanted tumor cells. However, mice further treated with the combination therapy of anti-PD-1 and anti-PD-L1 antibodies exhibited better antitumor response as well as less development of lung metastases compared to untreated controls. On average, tumor weight was reduced by 36% and lung metastases measured by ex-vivo BLI was reduced by 82% (n=5). In addition, circulating CD326+ tumor cells were reduced by 21% and CD3, CD4, CD8 and CD20 positive human lymphocytes were also reduced in the combination treatment group. Conclusion: Our study provides preclinical evidence of establishment of humanized Rag2 mice to be used as an orthotopic xenograft model for CRC; and treatment benefit for CRC through targeting immune checkpoint molecules PD-1 and PD-L1. The effect of immune checkpoint blockade in combination with conventional chemotherapy using CRC patient-derived specimens and PBMCs from MHC matched donors or autologous PBMCs will be further investigated.
Citation Format: Li Li, Xin Zhang, Grace Maresh, Linh Hellmers, Avi Patel, Ravan Moret, Sarah Cohen, David Margolin. Antitumor effects of the programmed death receptor-1 and the programmed death-ligand 1 blockade in human colorectal carcinoma in a humanized orthotopic mouse model [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B027.