Macrophage engineering to initiate anti-tumor immunity

Introduction:: Macrophages and monocytes have understudied roles in acquired immunity. These cells are familiar to bioengineers as being recruited into foreign body responses to implants and also in clearance of synthetic particles and invasive microbes. Solid tumors often have many macrophages, but they arise from 'Self' and are not attacked. They are also not typically viewed as helping with immune rejection of the tumor. To address these issues, our goals have been to engineer macrophages to not only initiate elimination of 'cold' solid tumors that are a challenge with Tcell pathways but also to show that macrophages can make use of anti-tumor antibodies to help eliminate recurring tumors.

Materials and Methods:: We focus on deep suppression of the macrophage 'Self' checkpoint CD47-SIRPα in combination with an otherwise ineffective tumor-opsonizing monoclonal antibody. Macrophage engineering entailed deleting SIRPα in conditionally immortalized macrophage (CIM) progenitors. B16 melanoma is widely used for cancer immunotherapy development and does not respond in vivo to T cell checkpoint blockade nor to CD47 disruption and therefore represents a large number of patients thus far unresponsive to immunotherapy. Moreover, because monotherapy with CD47 blockade shows no clinical efficacy against solid tumors it was important to focus on a cancer line such as B16 that is anticipated to be similarly unaffected by CD47 disruption, rather than study lines already known to respond to CD47 disruption alone (e.g. MC38 colon cancer). Melanin granule synthesis generally relies on Tyrp1 so that anti-Tyrp1 is relatively specific to melanocytes and can be used to help drive phagocytosis via Fc-Receptors (FcR). However, anti-Tyrp1 is ineffective in the clinic against melanoma and shows little to no effect on B16 tumors established in mice. Engineered SIRPα-KO CIM readily phagocytosed target B16s with anti-Tyrp1 opsonization of immuno-tumoroids in vitro, and tail-vein injections of SIRPα-KO CIM’s plus anti-Tyrp1 was the key approach in vivo with fully immunocompetent mice.

Results, Conclusions, and Discussions:: With immuno-tumoroids, more than 80% of engulfment events showed phagocytosis of an entire cell including its nucleus and cytoplasm with just a minority of engulfment events suggesting 'trogocytosis' of cytoplasmic fragments. The latter could conceivably contribute to tumour cell elimination and perhaps to cell death. Importantly, only for the maximal phagocytosis condition of CD47-SIRPa suppression plus anti-Tyrp1 opsonization is the exponential growth of tumoroids inverted to tumoroids eradication. In terms of a effector:target cell effect, adding 3:1 macrophages suffices to suppress growth and fit the calculus of cell proliferation with n ~2 phagocytes working together to pull apart and eliminate the tumour.
Systemic delivery of our engineered macrophages leads to ≥60% of mice showing complete tumour elimination. Our method with these invasive immune cells potentially overcomes challenges for solid tumor therapies that include limited permeation of antibodies. Furthermore, induction of anti-cancer IgG prove both tumor-specific and multi-epitope beyond Tyrp1, and can drive phagocytosis via FcR activation. Given that solid tumors remain challenging for immunotherapies, durable anti-tumor responses here illustrate unexpected advantages in maximizing net phagocytic activity.
Our studies are timely in at least two ways. First, anti-cancer Ab's are being reported as functionally important in various cancers including lung cancer (e.g. Ng et al., Nature 2023). However, determining which immune cells are effector cells and which immune checkpoints are relevant and can be optimized remain unknown. Secondly, cancer vaccines such as with mRNA are likely to produce anti-tumour Ab's, and attention to whether such Ab's bind and opsonize the tumour cell surface for phagocytosis is a potential opportunity and consideration.


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