199 Enhanced immune responses in human breast and colon cancer following checkpoint therapy in a CD34⁺stem cell humanized NCG (HuCD34NCG) mouse model

Abstract

BackgroundBreast and colon cancer rank second and third, respectively, in world-wide prevalence of malignancies and present a large unmet medical need. The correlation between lymphocyte infiltration into the tumor microenvironment and efficacy of anti-cancer immunotherapies has been established. Therefore, relevant and cost-saving pre-clinical models are needed for developing new treatment approaches to predominant human tumor types. HuCD34NCG mice facilitate studying human immune responses in vivo elicited by experimental therapeutic antibodies. We characterized growth kinetics and human immune responses to checkpoint blockade in human breast and colon tumor-bearing HuCD34NCG mice. Aging, non tumor-bearing HuCD34NCG mice were also monitored for indicators of spontaneous hematopoietic cancer formation.MethodsHSC engraftment was quality controlled prior to inoculating HuCD34NCG mice with either colon adenocarcinoma (COLO 205) or triple negative breast cancer (MDA-MB-436) cells (both purchased from American Type Culture Collection, Manassas, VA). Mice were randomized into treatment groups based on tumor size, and checkpoint inhibitor antibodies were dosed twice weekly (anti-human PD-1, BioXcell clone: RMP1-14 or Keytruda; anti-human CTLA-4, BioXcell clone: BN13; and combination therapy). Body weights, general health status and survival were monitored. Peripheral blood (PB) and selected tissues were analyzed for the presence and composition of human immune cells by acoustic focusing flow cytometry. Non tumor-bearing aged HuCD34NCG mice (27 weeks post-engraftment) were sampled biweekly over ten weeks for lymphoma immunophenotyping.ResultsBoth tumor-bearing models showed significant anti-hPD-1 and anti-hCTLA-4 responses, but combination therapy only enhanced growth reduction significantly in MDA-MB-436 tumors. Flow cytometric analysis identified viable human leukocytes in tumor and spleen at study termination. These tumor-infiltrating lymphocytes (TIL) and splenocytes from surviving COLO 205 and MDA-MB-436 mice consisted of a total T-cell phenotype (CD3+) with proliferating (Ki67+), CD4+, CD8+ and Treg subsets. Additionally, myeloid cells (CD11b+, CD11c+) and M1/M2 macrophages were detected within these infiltrates. Splenic and tumor-infiltrating T-cells readily secreted human cytokines (IFN-γ, IL-2, TNF-α) and granzyme B upon ex vivo activation exhibiting polyfunctional and cytotoxic capabilities in all treatment groups. Baseline murine and human cytokine levels were distinguished in plasma from aging, non tumor-bearing HuCD34NCGs. Their phenotypes also showed no conclusive indicators of abnormal blood cells developing or graft failure.ConclusionsBreast and colon tumor cell-line derived models were established in HuCD34NCG mice. Standard checkpoint inhibitor treatment promoted human T-cell infiltration into tumor microenvironments inhibiting growth. These results demonstrate that HuCD34NCG are a robust and relevant host for various human cell xenotransplants to advance preclinical immuno-oncology drug development.Ethics ApprovalAnimal studies were executed in compliance with local Charles River IACUC guidelines, IACUC number I-033.

SARS-CoV-2 Omicron (B.1.1.529) shows minimal neurotropism in a double-humanized mouse model

“…One advantage to the use of mice for modeling of SARS-CoV-2 infection and COVID-19 is the availability of immunodeficient, human immune-reconstituted, and transgenic strains, making it possible to assess the importance of both viral and host factors in infection and disease development ( Kenney et al, 2022 ; Fu et al, 2021 ; Shang et al, 2021 ; Wahl et al, 2021 ; Sefik et al, 2021 , 2022 ; Mao et al, 2021 ). In the present study, we employed human ACE2 knock-in (NOD/ShiLtJGpt- Prkdc em26Cd52 IL2rg em26Cd22 Ace2 em1Cin(hACE2) /GptCRL) or mouse ACE2 (NOD- Prkdc em26Cd52 Il2rg em26Cd22 /NjuCrl) triple-immunodeficient (NCG) mice, which lack functional mouse T-, B-, and natural killer (NK) cells, and have reduced numbers of dendritic cells and macrophages ( Eberle et al, 2020 ; Tarpinian et al, 2020 ). Myeloablation of NCG mice followed by reconstitution with human umbilical cord blood-derived CD34 + stem cells generates double-humanized huCD34 + -hACE2-NCG or huCD34 + -mACE2-NCG mice, which have a broad range of circulating and tissue-associated human immune cells ( Shultz et al, 2007 ).…”

“…Myeloablation of NCG mice followed by reconstitution with human umbilical cord blood-derived CD34 + stem cells generates double-humanized huCD34 + -hACE2-NCG or huCD34 + -mACE2-NCG mice, which have a broad range of circulating and tissue-associated human immune cells ( Shultz et al, 2007 ). Previous studies with NCG mice have demonstrated that human immune cells take residence in mouse tissues, including the lungs, for months after stem cell engraftment ( Eberle et al, 2020 ). Importantly, apheresis prior to engraftment ensures that no anti-SARS-CoV-2 antibodies present in cord blood are passively transferred with the graft .…”

Non-Clinical Cell Therapy Development Using the NCG Mouse Model as a Test System

“…Importantly, previous work demonstrated that NCG mice were a stable host for human immune cells and tumors in various disease contexts including bladder, colon, and breast cancer. [12][13][14][15] The present study consisted of two phases, the first phase optimized total body irradiation dose for myeloablation in NCG mice, while the second phase characterized the model for toxicity, engraftment, biodistribution, and tumorigenicity endpoints with different conditioning regimens. In Phase 1, the optimized irradiation dose was established to be 200 cGy.…”