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WEBINAR REVIEW

In vivo models of human immunity in human tumor-bearing mice are frequently used to study mechanisms of targeted immunotherapies, safety, and efficacy. In a recent webinar Jenny Rowe, PhD, Scientist III at Charles River Laboratories (CRL), overviews the advantages of using humanized mice and details characterization data on the novel HuCD34-NCG strain.

Some immunodeficient strains are defective in T and B cell functions but retain varying amounts of NK cell activity and innate immunity. These severely immunodeficient animals are required to support human cell engraftment to generate multiple differentiated lineages from human hematopoietic stem cells (HSCs).

NCG mice (NOD SCID-like CRISPR Il2R Gamma) are made on the NOD background; these mice carry the SIRP-alpha gene similar to humans that allows the engraftment of donor cells. CRL created a triple-immunodeficient mouse by CRISPR/Cas9 editing of the Prkdc and IL2r-gamma loci.

Humanized NCG mice have severely impaired murine T and B cells or innate immunity provided by macrophages and NK cells. They robustly support the engraftment of human cells, and develop functional human immune, systems making them ideal for studying immune-related therapies in a vast array of applications.

A broadly used technique for humanizing mice, termed Hu-HSC, involves the transfer of HSCs isolated from a variety of sources. These models display a robust and broad-spectrum human hematopoiesis, are suitable for large cohort generation, and are thoroughly standardized for reconstitution kinetics. Hu-HSC models have utility for long-term studies up to several months, especially those involving tumors or patient-derived xenografts (PDXs).

CRL’s CD34+ humanized NCG model, HuCD34-NCG, injected with umbilical cord blood–derived CD34+ cells shows robust engraftment of major human immune cells and demonstrates infiltration of T cells into tissues and xenograft tumors.

During the initial development of the HuCD34-NCG mouse, peripheral blood samples were collected at various timepoints, along with periodic tissue sampling, to verify engraftment and sustainability.

A QC screening flow cytometry panel and a more extensive T cell and myeloid panel identify the presence of human cell populations and evaluate the model’s functionality. These baseline data are used for comparison to other humanized models.

Flow cytometry analysis of peripheral blood at 12 and 16 weeks post injection verified engraftment of human cell subpopulations. Levels of total CD3+ T cells and CD4+ and CD8+ subsets in addition to other cell populations, such as CD19+ and CD16/56+, were evaluated as well as cells from spleen, liver, bone marrow, lung, and lysed whole blood. Viable human CD45+ cells were detected in all samples.

HuCD34-NCG mice sustain humanization through day 236 post engraftment. Human immune cells repopulate and maintain lymphoid and partial myeloid differentiation; polyfunctional responses demonstrate that T lymphocytes are activatable and not exhausted.

In pilot studies, CRL evaluated the antitumor effects of immune checkpoint inhibitors (CPIs) on colon epithelial carcinoma (RKO) and basal lung cell adenocarcinoma (A549) cell lines in the HuCD34-NCG model and showed significant immunomodulatory antitumor response. Anti-PD1 (Pembrolizumab [Keytruda] or CD279, BioXCell) monotherapy significantly inhibited RKO and A549 tumor growth whereas anti-CTLA-4 (CD152, BioXCell) dosing, but not combined blockade of CTLA-4 and PD-1, increased intratumoral CD8+ T lymphocytes in A549 tumor-bearing mice.

Human T cell infiltration was observed in both carcinomas with the majority of live T cells responsive post infiltration. Intracellular cytokine staining showed release of human IFN-γ, IL-2, and TNF-α by the activated tumor-infiltrating lymphocytes (TILs).

Another study assessed the sensitivity of six NSCLC (non-small cell lung cancer) PDX models toward the same two CPIs and the combinations thereof. A distinct sensitivity profile was seen, ranging from partial remission to progression. The TIL analysis revealed a pronounced rise in the CD3+ subset and a modulation of CD4+/CD8+ cells.

These results demonstrate that the HuCD34-NCG model is a fully functional drug development tool. CRL offers scientists access to an exploratory trial program to determine if a Charles River Hu-Mouse model, including the new HuCD34-NCG mouse, meets research requirements.

Learn more about the Charles River Humanized NCG Exploratory Program to determine if a no-cost trial program is the right fit for your study needs.
www.criver.com/goinghuman

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