Cancer immunotherapies enhance the body's own immune system to control and eliminate tumors. Most clinical-stage immunotherapies target T cells of the adaptive immune system, especially cytotoxic CD8+ T cells. Myeloid cells have risen to prominence as worthwhile targets for immunomodulation. Within the immune landscape of the tumor microenvironment (TME), tumor-associated macrophages (TAMs) significantly influence the activities of T cells and the outcome of T cell-targeting therapies. TAMs are broadly categorized based on macrophage polarization and fall within one of two major subsets describing their activity:
- Classically activated, pro-inflammatory M1 macrophages, which can directly kill tumor cells and stimulate adaptive immune responses against tumors
- Alternatively activated, anti-inflammatory M2 macrophages, which suppress T cell-mediated anti-tumor responses and remodel tissues promoting angiogenesis and metastasis.
Figure 1. Schematic of proposed mechanism of action driving Dectin-2-mediated anti-tumor activity, as shown in a poster presented at the SITC 2022 Annual Meeting. (Kenkel, J.A. et al. BDC-3042: A Dectin-2 agonistic antibody for tumor-associated macrophage-directed immunotherapy. Annual meeting for the Society of Immunotherapy of Cancer, abstract 1348 (2022).)
Humanized Animal Models for Macrophage-targeted Immunotherapy
Super immunodeficient mice can be engrafted with a humanized immune system (HIS) and human tumors, generating powerful models of the TME. These enable direct assessment of immune-targeting therapeutics within a humanized immune/malignancy milieu. As our understanding of the complexity within the TME evolves, so too do rodent models to support increasingly complex systems required for drug development and clinical translation.The NOG-EXL mouse is on the leading edge of biomedical research models. It is transgenic for two human signaling molecules, interleukin-3 and granulocyte-macrophage colony-stimulating factor, which support differentiation of myeloid lineages from engrafted hematopoietic stem cells. Within a humanized huNOG-EXL mouse, macrophages develop robustly alongside T cells and can infiltrate the TME of a cancer xenograft1.
At last year's annual meetings of the American Association for Cancer Research and the Society for Immunotherapy of Cancer, a preclinical research team from Bolt Biotherapeutics presented posters (1, 2) outlining a compelling case study of macrophage reprogramming within the TME of the huNOG-EXL mouse2,3. Their work strongly supports the model's utility for establishing a translational TME as a relevant model for evaluating TAM-directed immunotherapies.
Targeting Pattern Recognition Receptors in the TME
Bolt Biotherapeutics was spun out of Dr. Edgar Engleman's lab at Stanford University to commercialize the research team's pioneering work in myeloid biology. Recently, the poster's lead author, Justin Kenkel, PhD, a principal scientist at Bolt Biotherapeutics, and Shelley Ackerman, PhD, Director, met with Taconic Biosciences to discuss the findings and significance of the poster presentations. Their research represents a translational extension of a project originally pursued by Dr. Kenkel while he was a postdoctoral fellow in the Engleman Lab4.The therapeutic described in the posters, BDC-3042, is an agonistic antibody for the C-type lectin receptor Dectin 2. This is a pattern recognition receptor (PRR) that senses glycan structures found within fungi and other microorganisms5. Macrophages and other innate immune cells are activated by characteristic molecular motifs from microbial and viral pathogens via an assortment of PRRs that trigger acute microbicidal and inflammatory responses required to contain and eliminate infection. Upon ligation, Dectin 2 mediates phagocytosis, antigen processing and presentation, and secretion of pro-inflammatory cytokines and chemokines—inducing a classically activated M1 functional profile within the triggered macrophages. In the lead up to in vivo studies, Bolt Biotherapeutics demonstrated that BDC-3042 can activate human macrophages in a series of in vitro and ex vivo assays, eliciting secretion of an array of pro-inflammatory cytokines and chemokines.
Researchers at Bolt Biotherapeutics engrafted huNOG-EXL mice with a triple-negative breast cancer cell line that they found supports macrophage infiltration into the TME. The tumors subsequently grew in vivo and were infiltrated by human immune cells, including Dectin-2-expressing TAMs. Mice were then treated with BDC-3042 and benchmarked against a standard-of-care drug, pembrolizumab, which targets an immune checkpoint pathway in T cells. Treatment with BDC-3042 was shown to inhibit tumor growth to a significantly greater extent than pembrolizumab when comparing tumor volumes from huNOG-EXL cohorts humanized with HSCs across nine different donors. This crucial proof of concept work supports further clinical evaluation of the drug candidate.
Figure 2. huNOG-EXL mice were implanted with triple-negative breast cancer cell line and treated with control antibody, pembrolizumab, or BDC-3042. (Left) Tumor growth inhibition (TGI) across 9 HSC donor cohorts for Pembrolizumab or BDC-3042 compared to an isotype control antibody. (Right) Tumor growth curves for mice from a representative donor treated with BDC-3042 (1 mg/kg), isotype control mAb (5 mg/kg), or pembrolizumab (5 mg/kg). (Kenkel, J.A. et al. BDC-3042: A Dectin-2 agonistic antibody for tumor-associated macrophage-directed immunotherapy. Annual meeting for the Society of Immunotherapy of Cancer, abstract 1348 (2022).)
What attracted Bolt scientists to Dectin-2, instead of more well-known PRR family members? Dr. Kenkel explained that Dectin 2 expression is largely restricted to monocytes and macrophages within the myeloid compartment, as compared to other PRRs that are often more broadly expressed. This selectivity may mean treatment is accompanied by a lower risk of triggering systemic inflammatory responses.Identification of Dectin-2 expression within multiple tumor types affirms it is a broadly applicable target preferentially localized within the TME6. Furthermore, its expression and druggability can be modeled within the huNOG-EXL mouse. Data presented at the meetings last year demonstrated that Dectin-2 is most highly expressed by macrophages residing in the TME in the humanized mouse model (Figure 3). Correspondingly, BDC-3042 bound preferentially to macrophages found within the TME while showing minimal binding to macrophages in other tissues (Figure 4).
Figure 3. Immune landscape and Dectin-2 expression in tumor-bearing humanized mice, as shown in a poster presentation at the AACR 2022 Annual Meeting. (Left) The tumor immune compartment was assessed in huNOG-EXL mice from four unique HSC donors (n=3 mice/donor). Quantification of human immune cell subsets is shown as the percentage of total live cells, and total human immune cells (CD45+) are marked as "Total". (Right) Dectin-2 expression on human monocytes and macrophages recovered from the indicated tissues in tumor-bearing huNOG-EXL mice was assessed using a commercially available anti-Dectin-2 antibody.
Figure 4. Human macrophage distribution and binding of BDC-3042 in tumor-bearing humanized mice, as shown in a poster presented at the 2022 annual meeting of the Society for Immunotherapy of Cancer. (Left) Human macrophage frequency was assessed in the indicated tissues from tumor-bearing huNOG-EXL mice generated using 5 unique HSC donors (n = 4-5 mice/donor) and expressed as a percentage of total live cells. (Right) BDC-3042 binding to human macrophages recovered from the indicated tissues in tumor-bearing huNOG-EXL mice is shown.
Additional Considerations
The Bolt poster presentations offer insight into other important attributes of the model. A key consideration for using HIS mice is that they are most typically engrafted with immune cells from a healthy, but otherwise random, donor. Among lots of animals, the researcher should be prepared to encounter a degree of variability in immune composition and responses. This is evident in the poster's depiction of the immune richness within the TME of mice grouped by donor. Some are richly infiltrated; others are sparsely infiltrated. The ratios of macrophages to cytotoxic T cells to regulatory T cells further define the relative "hotness or coldness" of the tumor.At Bolt Biotherapeutics, this is viewed as an informative representation of heterogeneity rather than a hurdle of the model. Dr. Ackerman explains that patients in the clinic are heterogeneous in terms of TME immune composition and responsiveness to immune-targeting agents. Therefore, when good study design comprises a representative sampling of HIS donors—three or more, as a best practice for Bolt Biotherapeutics—clinical heterogeneity may be better predicted. Many published studies do not address this variability.
Interested in learning more about humanized immune system (HIS) mouse models for immuno-oncology research? Watch the Related Taconic Biosciences' Webinar:
