Poster CC18 - SIRPa deleted Myeloid Progenitors Eliminate Solid Tumors, Induce Anti-Tumor IgG, and Provide a Platform For Multi-Targeting with IgGs and CARs

Introduction: Immune checkpoint blockade therapies that are focused on acquired immune cells have shown clinical success against some solid tumors, but many tumors remain a challenge, including some cases of melanoma. In liquid (blood) cancers, trials with antibody-based blockade of the macrophage checkpoint Sirpα-CD47 has raised safety issues and achieved limited success as part of combination therapies, but efficacy against solid tumors remains a challenge. Involvement of myeloid cells including macrophages is nonetheless attractive given the possibilities of acquired immunity against cancer. Here, we show that combining SIRPa knockout (SKO) myeloid progenitors with an anti-Tyrp1 monoclonal antibody can successfully cure immunocompetent mice with B16 melanoma tumors. Survivors possess anti-B16 IgG against multiple epitopes that can also promote engulfment. This provides opportunities to synergize with Chimeric Antigen Receptors (CARs) against known targets on the B16 cells. Multi-targeting provides important advantages for highly heterogeneous solid tumors, where different cells express different antigens at different levels.

Materials and

Methods: Conditionally immortalized macrophages (CIMs) lacking SIRPa (SKO) were created using CRISPR/Cas9 gene editing and subsequently maintained in RPMI growth media and maintained with the progenitor state using β-estrogen and GM-CSF. To generate tumors, 2x105 B16 WT cells were injected subcutaneously into 6-week-old C57 mice. Each mouse was treated intravenously with 250 µg anti-Tyrp1 antibody on days 4, and 7, and treated with one dose of 4x106 SKO CIMs on day 4. Tumors were measured with digital calipers every 2 days. Blood was collected retro orbitally every 2 weeks. Animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania.

Results, Conclusions, and Discussions: Results and

Discussion: A fraction of mice with WT B16 tumors survived when treated with i.v. anti-Tyrp1 and SKO CIMs compared to no long-term survival among untreated mice or in a solely antibody treated cohort of mice with WT B16 tumors. Some treated mice showed a partial response characterized by slower tumor growth compared to non-responding and control mice. We hypothesize that such partial responders would benefit from increased numbers of phagocytic macrophages. Long-term survivors from the treated cohort resisted re-challenge, suggesting an acquired immune response. We identified anti-B16 IgG antibodies in the sera of survivors and showed this is functional in in vitro phagocytosis assays and when pre-bound to WT B16 cells prior to injection in mice. Furthermore, comparison of monoclonal antibodies to CAR macrophages shows increased phagocytic capability of CAR macrophages compared to mAb opsonization. More interestingly, opsonization of tumor cells with monoclonal antibodies in tandem with CAR macrophage show an on average higher phagocytic capability of hinting at encouraging results of dual target immunotherapies. To further investigate this trend, ongoing studies on longer timescales will probe the importance of antigen heterogeneity and minor populations in solid tumors to elucidate the therapeutic benefit of dual targets.


Conclusions: We show that myeloid progenitors lacking SIRPα can initiate in vivo clearance of tumors when paired with an opsonizing monoclonal antibody, and new anti-tumor IgG develop. CAR expression in progenitor-derived macrophages also drives phagocytosis, and enables comparisons of synergy against cancer cells with the various IgG over hours, days, and weeks in vitro and in vivo. A key and intriguing aspect of the overall approach will be the broad activation of adaptive immunity, especially anti-cancer IgG.

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