Antibody-mediated phagocytosis in cancer immunotherapy

Targeted therapy, a goal first popularized by Paul Erlich in the 19th century,¹ has the potential to increase the efficacy and decrease toxicity of treatment for malignancies. Capability to manufacture monoclonal antibodies (mAbs) in the 1970s followed by genetic engineering to develop chimeric, humanized and then fully human mAb constructs to overcome the development of neutralizing antibodies by patients resulted in the ability to make targeted mAb.^{2, 3} These mAb can bind target cells to activate immune cytotoxicity, induce apoptosis, block ligation of cell surface receptors or sequestrate their ligands. The prototypic mAbs rituximab (chimeric mouse/human IgG1) and alemtuzumab (humanized rat/human IgG1) that activated innate immune cytotoxicity were clinically effective and tolerable. Rituximab was approved by the FDA for treatment of B-cell non-Hodgkin lymphomas in 1997 and alemtuzumab for treatment of chronic lymphocytic leukemia in 2001. Rituximab, alemtuzumab and next generation mAbs have significantly improved treatment outcomes for several human malignancies by mechanisms that include mAb-induced antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC). To date, the best studied mAb for which ADCP is a major mechanism of action has been in B-cell malignancies, which will be the clinical focus of this review.

The two main forms of cellular phagocytosis are efferocytosis (the engulfment of dead or dying cells) and ADCP.^4-8 Despite decades of research into the mechanisms of cellular phagocytosis, this innate immune effector mechanism is still underutilized as means for the targeted killing of malignant cells. This is due in part to our lack of information on how ADCP is carried out in vivo and a poor understanding of the factors that control the efficacy of mAbs in vivo. In recent years, important studies in mice have revealed key new insights into how ADCP-inducing mAbs traffic in the body and how phagocytes recognize and clear mAb-opsonized target cells. At the same time, advances in therapeutic antibody design, including glyco-engineering and the development of hexameric and mAbs, have opened new avenues to leverage the power of ADCP for better clearance of malignant cells in a wide range of cancers. Here, we will first review our current understanding of the cellular and molecular mechanisms of ADCP and the current state of ADCP-inducing mAbs used in human cancer. Then we will discuss key factors that control ADCP in the context of mAb cancer immunotherapy and to provide perspective on some of the most important and outstanding questions in this field.

Therapeutic mAb-mediated cytotoxicity can be induced by direct cytototoxic effects, receptor blockade, and activation of innate immune cytotoxicity by ADCP, ADCC, and CDC.^{47, 48} The mechanisms of action of mAbs that activate of innate immune cytotoxicity have not yet been fully elucidated. Examples of B cell targeting mAbs that can activate ADCP and are effective therapy for B-cell malignancies are listed in Table 1. This section will review how ADCP contributes to the efficacy of these mAbs with a focus on those targeting CD20.

The prototypic therapeutic anti-CD20 mAb rituximab (chimeric mouse/human IgG1) was registered by the FDA for treatment of B-cell non-Hodgkin lymphomas in 1997. Rituximab targets CD20, a B-cell-specific, homodimeric cell-surface protein expressed from the late pre-B cell through memory B cells and by most B-cell lymphomas.⁴⁹ CD20, a transmembrane calcium channel implicated in B-cell activation, proliferation, and differentiation was selected as a target for mAb therapy because of both its B-cell specificity and limited endocytosis after ligation by mAb.⁵⁰

Monotherapy with rituximab is an effective but non-curative therapy for B-cell lymphomas.⁵¹ Next generation anti-CD20 mAbs were subsequently developed in efforts to improve efficacy by increasing complement activation (ofatumumab) and cellular cytotoxicity (obinutuzumab) with modest clinical success.⁵² In contrast, use of rituximab in combination therapy has been very successful. Addition of rituximab to chemotherapy (chemoimmunotherapy) significantly increases the efficacy of treatment of many B-cell lymphomas with improved response rates, progression free survival, and overall survival. Addition of rituximab to the potentially curative combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) significantly improved outcomes for patients with diffuse large B-cell lymphoma.⁵³ Addition of rituximab to the combination of fludarabine and cyclophosphamide (FCR) for treatment of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) significantly improved response duration and overall survival but is not curative.⁵⁴

Combining anti-CD20 mAb with other targeted drugs is a promising treatment option that reduces the potential genetic toxicity of chemotherapy agents. In treatment of CLL, targeted therapies inhibiting B-cell receptor initiated signaling mobilize malignant B cells into the circulation where they are more susceptible to mAb-mediated cytotoxicity.⁵⁵ Inhibition of B-cell receptor signaling by the Bruton tyrosine kinase (BTK) inhibitors ibrutinib results in rapid and prolonged mobilization of CLL cells into the circulation.⁵⁶ However, addition of rituximab to the first generation BTK inhibitors ibrutinib did not increase clinical efficacy.⁵⁷ Several studies suggested that this could be a result of inhibition of ADCP by off target effects of ibrutinib.^58-60 Subsequent clinical trials using rituximab or obinutuzumab with acalabrutinib, a second generation more targeted BTK inhibitors, suggest that these regimens could be more effective.^61-63 Combination of rituximab or obinutuzumab with the BCL2 inhibitor (BCL2i) venetoclax has also been shown to be effective and tolerable in the treatment of CLL.^{64, 65} Results from Phase II trials suggest that three drug combinations of anti-CD20 mAb with BTK inhibitors and BCL2i could be more effective and are also tolerable.^{65, 66} These efforts to improve treatment by combination target therapy are ongoing.

The reasons why anti-CD20 mAb monotherapy has limited efficacy are not well understood.^{49, 51} Potential reasons include pharmacokinetic constraints, limited immune cytotoxic capacity, loss of target cell antigen, and intrinsic cell resistance. We will further discuss the factors influencing ADCP in Section 5.

As we now appreciate the great potential of ADCP as a cytotoxic mechanism for mAb therapies, it is clear that new approaches are needed to optimize and improve the efficacy of these therapeutics to treat malignancies. There are now multiple approaches being explored to increase ADCP efficacy.

Since its discovery by Elie Metchnikoff in the late 19th century, phagocytosis has been studied extensively at the cellular and molecular levels.^{110, 111} Historically, however, ADCP has been studied largely in the context of host pathogen defense. Now, though, as the number of clinically approved mAbs have grown exponentially since the introduction of rituximab, we are gaining an impressive amount of insight into how ADCP can be leveraged as a cytotoxic process in the modern era of immunotherapy. Clearly, there are substantial barriers to achieve optimal efficacy in vivo, and it is also very clear that in vitro studies of mAb mechanisms of action do not exactly align with in vivo mechanisms. To this end, we believe that the development of better in vivo models and techniques, and in vitro models that recapitulate the tissue environment, will provide a path to develop new, clinically useful strategies that leverage the impressive cell killing capacity of macrophage phagocytosis to achieve better patient outcomes and possible cures for cancer and many other diseases.

All authors contributed to the writing, review, and editing of the manuscript.

No conflicts to declare.