Sensing antibody functions with a novel CCR8-responsive engineered cell.

Human chemokine receptor 8 (CCR8) is a promising drug target for immunotherapy of cancer and autoimmune diseases. Monoclonal antibody-based CCR8 targeted treatment shows significant inhibition in tumor growth. The inhibition of CCR8 results in the improvement of antitumor immunity and patient survival rates by regulating tumor-resident regulatory T cells. Recently monoclonal antibody drug development targeting CCR8 has become a research hotspot, which also promotes the advancement of antibody evaluation methods. Therefore, we constructed a novel engineered customized cell line HEK293-cAMP-biosensor-CCR8 combined with CCR8 and a cAMP-biosensor reporter. It can be used for the detection of anti-CCR8 antibody functions like specificity and biological activity, in addition to the detection of antibody-dependent cell-mediated cytotoxicity and antibody-dependent-cellular-phagocytosis. We obtained a new CCR8 mAb 22H9 and successfully verified its biological activities with HEK293-cAMP-biosensor-CCR8. Our reporter cell line has high sensitivity and specificity, and also offers a rapid kinetic detection platform for evaluating anti-CCR8 antibody functions.

Immunoglobulins and serum proteins impair anti-tumor NK cell effector functions in malignant ascites.

Introduction: Malignant ascites indicates ovarian cancer progression and predicts poor clinical outcome. Various ascites components induce an immunosuppressive crosstalk between tumor and immune cells, which is poorly understood. In our previous study, imbalanced electrolytes, particularly high sodium content in malignant ascites, have been identified as a main immunosuppressive mechanism that impaired NK and T-cell activity. Methods: In the present study, we explored the role of high concentrations of ascites proteins and immunoglobulins on antitumoral NK effector functions. To this end, a coculture system consisting of healthy donor NK cells and ovarian cancer cells was used. The anti-EGFR antibody Cetuximab was added to induce antibody-dependent cellular cytotoxicity (ADCC). NK activity was assessed in the presence of different patient ascites samples and immunoglobulins that were isolated from ascites. Results: Overall high protein concentration in ascites impaired NK cell degranulation, conjugation to tumor cells, and intracellular calcium signaling. Immunoglobulins isolated from ascites samples competitively interfered with NK ADCC and inhibited the conjugation to target cells. Furthermore, downregulation of regulatory surface markers CD16 and DNAM-1 on NK cells was prevented by ascites-derived immunoglobulins during NK cell activation. Conclusion: Our data show that high protein concentrations in biological fluids are able to suppress antitumoral activity of NK cells independent from the mechanism mediated by imbalanced electrolytes. The competitive interference between immunoglobulins of ascites and specific therapeutic antibodies could diminish the efficacy of antibody-based therapies and should be considered in antibody-based immunotherapies.

SARS-CoV-2 membrane protein-specific antibodies from critically ill SARS-CoV-2-infected individuals interact with Fc receptor-expressing cells but do not neutralize the virus.

The membrane (M) glycoprotein of SARS-CoV-2 is one of the key viral proteins regulating virion assembly and morphogenesis. Immunologically, the M protein is a major source of peptide antigens driving T cell responses, and most individuals who have been infected with SARS-CoV-2 make antibodies to the N-terminal, surface-exposed peptide of the M protein. We now report that although the M protein is abundant in the viral particle, antibodies to the surface-exposed N-terminal epitope of M do not appear to neutralize the virus. M protein-specific antibodies do, however, activate antibody-dependent cell-mediated cytotoxicity and cytokine secretion by primary human natural killer cells. Interestingly, while patients with severe or mild disease make comparable levels of M antigen-binding antibodies, M-specific antibodies from the serum of critically ill patients are significantly more potent activators of antibody-dependent cell-mediated cytotoxicity than antibodies found in individuals with mild or asymptomatic infection.

Enhanced Immunomodulatory Effect of Intravenous Immunoglobulin by Fc Galactosylation and Nonfucosylation.

Intravenous immunoglobulin (IVIG) is used as an immunomodulatory agent in the treatment of various autoimmune/inflammatory diseases although its mechanism of action remains elusive. Recently, nonfucosylated IgG has been shown to be preferentially bound to Fcγ receptor IIIa (FcγRIIIa) on circulating natural killer cells; therefore, we hypothesized that nonfucosylated IVIG may modulate immune responses through FcγRIIIa blockade. Here, homogeneous fucosylated or nonfucosylated glycoforms of normal polyclonal IgG bearing sialylated, galactosylated or nongalactosylated Fc oligosaccharides were generated by chemoenzymatic glycoengineering to investigate whether the IgG glycoforms can inhibit antibody-dependent cellular cytotoxicity (ADCC).　Among the six IgG glycoforms, galactosylated, nonfucosylated IgG [(G2)2] had the highest affinity to FcγRIIIa and 20 times higher potency to inhibit ADCC than native IgG. A pilot study of IVIG treatment in mice with collagen antibody-induced arthritis highlighted the low-dose (G2)2 glycoform of IVIG (0.1 g/kg) as an effective immunomodulatory agent as the 10-fold higher dose of native IVIG. These preliminary results suggest that the anti-inflammatory activity of IVIG is in part mediated via activating FcγR blockade by galactosylated, nonfucosylated IgG and that such nonfucosylated IgG glycoforms bound to FcγRs on immune cells play immunomodulatory roles in health and disease. This study provides insights into improved therapeutic strategies for autoimmune/inflammatory diseases using glycoengineered IVIG and recombinant Fc.

Phagocytosis by an HIV antibody is associated with reduced viremia irrespective of enhanced complement lysis.

Increasingly, antibodies are being used to treat and prevent viral infections. In the context of HIV, efficacy is primarily attributed to dose-dependent neutralization potency and to a lesser extent Fc-mediated effector functions. It remains unclear whether augmenting effector functions of broadly neutralizing antibodies (bNAbs) may improve their clinical potential. Here, we use bNAb 10E8v4 targeting the membrane external proximal region (MPER) to examine the role of antibody-mediated effector and complement (C') activity when administered prophylactically against SHIV challenge in rhesus macaques. With sub-protective dosing, we find a 78-88% reduction in post-acute viremia that is associated with 10E8v4-mediated phagocytosis acting at the time of challenge. Neither plasma nor tissue viremic outcomes in vivo is improved with an Fc-modified variant of 10E8v4 enhanced for C' functions as determined in vitro. These results suggest that effector functions inherent to unmodified 10E8v4 contribute to efficacy against SHIVSF162P3 in the absence of plasma neutralizing titers, while C' functions are dispensable in this setting, informing design of bNAb modifications for improving protective efficacy.

Engineered human antibodies for the opsonization and killing of Staphylococcus aureus.

Gram-positive organisms with their thick envelope cannot be lysed by complement alone. Nonetheless, antibody-binding on the surface can recruit complement and mark these invaders for uptake and killing by phagocytes, a process known as opsonophagocytosis. The crystallizable fragment of immunoglobulins (Fcγ) is key for complement recruitment. The cell surface of S. aureus is coated with Staphylococcal protein A (SpA). SpA captures the Fcγ domain of IgG and interferes with opsonization by anti-S. aureus antibodies. In principle, the Fcγ domain of therapeutic antibodies could be engineered to avoid the inhibitory activity of SpA. However, the SpA-binding site on Fcγ overlaps with that of the neonatal Fc receptor (FcRn), an interaction that is critical for prolonging the half-life of serum IgG. This evolutionary adaptation poses a challenge for the exploration of Fcγ mutants that can both weaken SpA-IgG interactions and retain stability. Here, we use both wild-type and transgenic human FcRn mice to identify antibodies with enhanced half-life and increased opsonophagocytic killing in models of S. aureus infection and demonstrate that antibody-based immunotherapy can be improved by modifying Fcγ. Our experiments also show that by competing for FcRn-binding, staphylococci effectively reduce the half-life of antibodies during infection. These observations may have profound impact in treating cancer, autoimmune, and asthma patients colonized or infected with S. aureus and undergoing monoclonal antibody treatment.

SIRPα-specific monoclonal antibody enables antibody-dependent phagocytosis of neuroblastoma cells.

Immunotherapy with anti-GD2 monoclonal antibodies (mAbs) provides some benefits for patients with neuroblastoma (NB). However, the therapeutic efficacy remains limited, and treatment is associated with significant neuropathic pain. Targeting O-acetylated GD2 (OAcGD2) by 8B6 mAb has been proposed to avoid pain by more selective tumor cell targeting. Thorough understanding of its mode of action is necessary to optimize this treatment strategy. Here, we found that 8B6-mediated antibody-dependent cellular phagocytosis (ADCP) performed by macrophages is a key effector mechanism. But efficacy is limited by upregulation of CD47 expression on neuroblastoma cells in response to OAcGD2 mAb targeting, inhibiting 8B6-mediated ADCP. Antibody specific for the CD47 receptor SIRPα on macrophages restored 8B6-induced ADCP of CD47-expressing NB cells and improved the antitumor activity of 8B6 mAb therapy. These results identify ADCP as a critical mechanism for tumor cytolysis by anti-disialoganglioside mAb and support a combination with SIRPα blocking agents for effective neuroblastoma therapy.

T cells, particularly activated CD4+ cells, maintain anti-CD20-mediated NK cell viability and antibody dependent cellular cytotoxicity.

Anti-CD20 monoclonal antibody (mAb) therapy is a mainstay of therapy for B cell malignancies, however many patients fail to respond or eventually develop resistance. The current understanding of mechanisms responsible for this resistance is limited. When peripheral blood mononuclear cells of healthy donors were cultured with Raji cells for 7 days, rituximab (RTX) induced NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC), enhanced NK cell viability and increased or maintained NK expression of CD56, CD16, CD57 and KIR. T cells, mainly CD4+, mediated these changes in a contact-dependent manner, with local T cell production of IL2 playing a central role. Similar findings were found when autologous B cells were used as target cells demonstrating the need for T cell help was not due to allogenic reaction. Results with other anti-CD20 and anti-EGFR antibodies were consistent. Small numbers of T cells activated by anti-CD3/CD28 beads or bispecific antibody enhanced RTX-mediated NK cell ADCC, viability and phenotypical changes. Pathway analysis of bulk NK cell mRNA sequencing after activation by RTX with and without T cells was consistent with T cells maintaining the viability of the activated NK cells. These findings suggest T cell help, mediated in large part by local production of IL2, contributes to NK cell ADCC and viability, and that activating T cells in the tumor microenvironment, such as through the use of anti-CD3 based bispecific antibodies, could enhance the efficacy of anti-CD20 and other mAb therapies where NK-mediated ADCC is a primary mechanism of action.

Effect of ibrutinib on CCR7 expression and functionality in chronic lymphocytic leukemia and its implication for the activity of CAP-100, a novel therapeutic anti-CCR7 antibody.

CAP-100 is a novel therapeutic antibody directed against the ligand binding site of human CCR7. This chemokine receptor is overexpressed in chronic lymphocytic leukemia (CLL) and orchestrates the homing of CLL cells into the lymph node. Previous studies, on a very limited number of samples, hypothesized that the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib might induce loss of surface CCR7 levels in CLL cells. CAP-100 will be evaluated in clinical trials as a therapy for relapse/refractory CLL patients, who have received at least two systemic therapies (NCT04704323). As nowadays many relapse/refractory CLL patients will have received ibrutinib as a prior therapy, we aimed to investigate in a large cohort of CLL patients the impact of this BTKi on CCR7 expression and functionality as well as on the therapeutic activity of CAP-100. Our data confirm that ibrutinib moderately down-regulates the very high expression of CCR7 in CLL cells but has no apparent effect on CCR7-induced chemotaxis. Moreover, CLL cells are perfectly targetable by CAP-100 which led to a complete inhibition of CCR7-mediated migration and induced strong target cell killing through antibody-dependent cell-mediated cytotoxicity, irrespective of previous or contemporary ibrutinib administration. Together, these results validate the therapeutic utility of CAP-100 as a next-line single-agent therapy for CLL patients who failed to ibrutinib and confirm that CAP-100 and ibrutinib have complementary non-overlapping mechanisms of action, potentially allowing for combination therapy.

Combined strategies for effective cancer immunotherapy with a novel anti-CD47 monoclonal antibody.

CD47 is a widely expressed cell-surface protein that regulates phagocytosis mediated by cells of the innate immune system, such as macrophages and dendritic cells. CD47 serves as the ligand for a receptor on these innate immune cells, signal regulatory protein (SIRP)-α, which in turn inhibits phagocytosis. Several targeted CD47 therapeutic antibodies have been investigated clinically; however, how to improve its therapeutic efficacy remains unclear. Herein, we developed a CD47 blocking antibody, named IBI188, that could specifically block the CD47-SIRP-α axis, which transduces the "don't eat me" signal to macrophages. In vitro phagocytosis assays demonstrated the pro-phagocytosis ability of IBI188. Furthermore, several in vivo models were chosen to evaluate the anti-tumor efficacy of IBI188. IBI188 treatment upregulated cell movement- and inflammation-related genes in macrophages. Synergism was observed when combined with an anti-CD20 therapeutic antibody, whose function depends on antibody-dependent cellular cytotoxicity/phagocytosis (ADCC/ADCP). CD47 expression was evaluated following azacytidine (AZA) treatment, a standard-of-care for patients with multiple myeloma; enhanced anti-tumor efficacy was observed in the combination group in AML xenograft models. Notably, IBI188 treatment increased vascular endothelial growth factor-A (VEGF-A) levels in a solid tumor model, and combined treatment with an anti-VEGF-A antibody and IBI188 resulted in an enhanced anti-tumor effect. These data indicate that IBI188 is a therapeutic anti-CD47 antibody with anti-tumor potency, which can be enhanced when used in combination with standard-of-care drugs for cancer treatment.

Anti-disialoganglioside antibody internalization by neuroblastoma cells as a mechanism of immunotherapy resistance.

Neuroblastoma (NBL) accounts for a disproportionate number of deaths among childhood malignancies despite intensive multimodal therapy that includes antibody targeting disialoganglioside GD2, a NBL antigen. Unfortunately, resistance to anti-GD2 immunotherapy is frequent and we aimed to investigate mechanisms of resistance in NBL. GD2 expression was quantified by flow cytometry and anti-GD2 antibody internalization was measured using real-time microscopy in 20 human NBL cell lines. Neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) assays were performed on a subset of the cell lines (n = 12), and results were correlated with GD2 expression and antibody internalization. GD2 was expressed on 19 of 20 NBL cell lines at variable levels, and neutrophil-mediated ADCC was observed only in GD2-expressing cell lines. We found no correlation between level of GD2 expression and sensitivity to neutrophil-mediated ADCC, suggesting that GD2 expression of many cell lines was above a threshold required for maximal ADCC, such that expression level could not be used to predict subsequent cytotoxicity. Instead, anti-GD2 antibody internalization, a process that occurred universally but differentially across GD2-expressing NBL cell lines, was inversely correlated with ADCC. Treatment with endocytosis inhibitors EIPA, chlorpromazine, MBCD, and cytochalasin-D showed potential to inhibit antibody internalization; however, only MBCD resulted in significantly increased sensitivity to neutrophil-mediated ADCC in 4 of 4 cell lines in vitro. Our data suggest that antibody internalization may represent a novel mechanism of immunotherapy escape by NBL and provide proof-of-principle that targeting pathways involved in antibody internalization may improve the efficacy of anti-GD2 immunotherapies.

Chemical Synthesis of Antibody-Hapten Conjugates Capable of Recruiting the Endogenous Antibody to Magnify the Fc Effector Immunity of Antibody for Cancer Immunotherapy.

Monoclonal antibodies (mAbs) with enhanced effector functions in cancer immunotherapy, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), could improve the clinical performance. Here, we develop an mAb-hapten conjugate strategy to augment the mAb effector functions with the engagement of endogenous antibodies. An "off-the-shelf" mAb, rituximab, is site-specifically conjugated with the rhamnose (Rha) hapten to generate rituximab-Rha conjugates. The octopus-like conjugates could recruit anti-Rha antibodies onto the cancer cell surface and further form an immune complex that is able to provide multivalent Fc domains to interact with immune cells or complement protein C1q, leading to magnified ADCC and CDC simultaneously. One optimal conjugate R2 with PEG2 as a linker exhibits the most potent in vitro cancer cell killing activity and significant in vivo antitumor efficacy in a xenograft model. This is a general and cost-effective approach to generate mAb with improved effector functions that may have broad applications.

Fc Binding by FcγRIIa Is Essential for Cellular Activation by the Anti-FcγRIIa mAbs 8.26 and 8.2.

FcγR activity underpins the role of antibodies in both protective immunity and auto-immunity and importantly, the therapeutic activity of many monoclonal antibody therapies. Some monoclonal anti-FcγR antibodies activate their receptors, but the properties required for cell activation are not well defined. Here we examined activation of the most widely expressed human FcγR; FcγRIIa, by two non-blocking, mAbs, 8.26 and 8.2. Crosslinking of FcγRIIa by the mAb F(ab')2 regions alone was insufficient for activation, indicating activation also required receptor engagement by the Fc region. Similarly, when mutant receptors were inactivated in the Fc binding site, so that intact mAb was only able to engage receptors via its two Fab regions, again activation did not occur. Mutation of FcγRIIa in the epitope recognized by the agonist mAbs, completely abrogated the activity of mAb 8.26, but mAb 8.2 activity was only partially inhibited indicating differences in receptor recognition by these mAbs. FcγRIIa inactivated in the Fc binding site was next co-expressed with the FcγRIIa mutated in the epitope recognized by the Fab so that each mAb 8.26 molecule can contribute only three interactions, each with separate receptors, one via the Fc and two via the Fab regions. When the Fab and Fc binding were thus segregated onto different receptor molecules receptor activation by intact mAb did not occur. Thus, receptor activation requires mAb 8.26 Fab and Fc interaction simultaneously with the same receptor molecules. Establishing the molecular nature of FcγR engagement required for cell activation may inform the optimal design of therapeutic mAbs.

HIV-1 Envelope Glycoproteins Proteolytic Cleavage Protects Infected Cells from ADCC Mediated by Plasma from Infected Individuals.

The HIV-1 envelope glycoprotein (Env) is synthesized in the endoplasmic reticulum as a trimeric gp160 precursor, which requires proteolytic cleavage by a cellular furin protease to mediate virus-cell fusion. Env is conformationally flexible but controls its transition from the unbound "closed" conformation (State 1) to downstream CD4-bound conformations (States 2/3), which are required for fusion. In particular, HIV-1 has evolved several mechanisms that reduce the premature "opening" of Env which exposes highly conserved epitopes recognized by non-neutralizing antibodies (nnAbs) capable of mediating antibody-dependent cellular cytotoxicity (ADCC). Env cleavage decreases its conformational transitions favoring the adoption of the "closed" conformation. Here we altered the gp160 furin cleavage site to impair Env cleavage and to examine its impact on ADCC responses mediated by plasma from HIV-1-infected individuals. We found that infected primary CD4+ T cells expressing uncleaved, but not wildtype, Env are efficiently recognized by nnAbs and become highly susceptible to ADCC responses mediated by plasma from HIV-1-infected individuals. Thus, HIV-1 limits the exposure of uncleaved Env at the surface of HIV-1-infected cells at least in part to escape ADCC responses.

Cancer-Cell-Derived IgG and Its Potential Role in Tumor Development.

Human immunoglobulin G (IgG) is the primary component of the human serum antibody fraction, representing about 75% of the immunoglobulins and 10-20% of the total circulating plasma proteins. Generally, IgG sequences are highly conserved, yet the four subclasses, IgG1, IgG2, IgG3, and IgG4, differ in their physiological effector functions by binding to different IgG-Fc receptors (FcγR). Thus, despite a similarity of about 90% on the amino acid level, each subclass possesses a unique manner of antigen binding and immune complex formation. Triggering FcγR-expressing cells results in a wide range of responses, including phagocytosis, antibody-dependent cell-mediated cytotoxicity, and complement activation. Textbook knowledge implies that only B lymphocytes are capable of producing antibodies, which recognize specific antigenic structures derived from pathogens and infected endogenous or tumorigenic cells. Here, we review recent discoveries, including our own observations, about misplaced IgG expression in tumor cells. Various studies described the presence of IgG in tumor cells using immunohistology and established correlations between high antibody levels and promotion of cancer cell proliferation, invasion, and poor clinical prognosis for the respective tumor patients. Furthermore, blocking tumor-cell-derived IgG inhibited tumor cells. Tumor-cell-derived IgG might impede antigen-dependent cellular cytotoxicity by binding antigens while, at the same time, lacking the capacity for complement activation. These findings recommend tumor-cell-derived IgG as a potential therapeutic target. The observed uniqueness of Ig heavy chains expressed by tumor cells, using PCR with V(D)J rearrangement specific primers, suggests that this specific part of IgG may additionally play a role as a potential tumor marker and, thus, also qualify for the neoantigen category.

NK Cell-Dependent Antibody-Mediated Immunotherapy Is Improved In Vitro and In Vivo When Combined with Agonists for Toll-like Receptor 2 in Head and Neck Cancer Models.

The immunosuppressive character of head and neck cancers may explain the relatively low response rates to antibody therapy targeting a tumor antigen, such as cetuximab, and anti-PD-1 checkpoint inhibition. Immunostimulatory agents that overcome tumor-derived inhibitory signals could augment therapeutic efficacy, thereby enhancing tumor elimination and improving patient survival. Here, we demonstrate that cetuximab treatment combined with immunostimulatory agonists for Toll-like receptor (TLR) 2 induces profound immune responses. Natural killer (NK) cells, isolated from healthy individuals or patients with head and neck cancer, harbored enhanced cytotoxic capacity and increased tumor-killing potential in vitro. Additionally, combination treatment increased the release of several pro-inflammatory cytokines and chemokines by NK cells. Tumor-bearing mice that received cetuximab and the TLR2 ligand Pam3CSK4 showed increased infiltration of immune cells into the tumors compared to mice that received cetuximab monotherapy, resulting in a significant delay in tumor growth or even complete tumor regression. Moreover, combination treatment resulted in improved overall survival in vivo. In conclusion, combining tumor-targeting antibody-based immunotherapy with TLR stimulation represents a promising treatment strategy to improve the clinical outcomes of cancer patients. This treatment could well be applied together with other therapeutic strategies such as anti-PD-(L)1 checkpoint inhibition to further overcome immunosuppression.

Histopathologic Features of Antibody Mediated Rejection: The Banff Classification and Beyond.

Antibody mediated rejection (ABMR) in the kidney can show a wide range of clinical presentations and histopathologic patterns. The Banff 2019 classification currently recognizes four diagnostic categories: 1. Active ABMR, 2. Chronic active ABMR, 3. Chronic (inactive) ABMR, and 4. C4d staining without evidence of rejection. This categorization is limited in that it does not adequately represent the spectrum of antibody associated injury in allograft, it is based on biopsy findings without incorporating clinical features (e.g., time post-transplant, de novo versus preformed DSA, protocol versus indication biopsy, complement inhibitor drugs), the scoring is not adequately reproducible, and the terminology is confusing. These limitations are particularly relevant in patients undergoing desensitization or positive crossmatch kidney transplantation. In this article, I discuss Banff criteria for these ABMR categories, with a focus on patients with pre-transplant DSA, and offer a framework for considering the continuum of allograft injury associated with donor specific antibody in these patients.

Human Cytomegalovirus Infection Promotes Expansion of a Functionally Superior Cytoplasmic CD3+ NK Cell Subset with a Bcl11b-Regulated T Cell Signature.

Human CMV (HCMV) is a ubiquitous pathogen that indelibly shapes the NK cell repertoire. Using transcriptomic, epigenomic, and proteomic approaches to evaluate peripheral blood NK cells from healthy human volunteers, we find that prior HCMV infection promotes NK cells with a T cell-like gene profile, including the canonical markers CD3ε, CD5, and CD8ß, as well as the T cell lineage-commitment transcription factor Bcl11b. Although Bcl11b expression is upregulated during NK maturation from CD56bright to CD56dim, we find a Bcl11b-mediated signature at the protein level for FcεRIγ, PLZF, IL-2Rß, CD3γ, CD3δ, and CD3ε in later-stage, HCMV-induced NK cells. BCL11B is targeted by Notch signaling in T cell development, and culture of NK cells with Notch ligand increases cytoplasmic CD3ε expression. The Bcl11b-mediated gain of CD3ε, physically associated with CD16 signaling molecules Lck and CD247 in NK cells is correlated with increased Ab-dependent effector function, including against HCMV-infected cells, identifying a potential mechanism for their prevalence in HCMV-infected individuals and their prospective clinical use in Ab-based therapies.

Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis.

Monoclonal antibody therapies targeting tumour antigens drive cancer cell elimination in large part by triggering macrophage phagocytosis of cancer cells1-7. However, cancer cells evade phagocytosis using mechanisms that are incompletely understood. Here we develop a platform for unbiased identification of factors that impede antibody-dependent cellular phagocytosis (ADCP) using complementary genome-wide CRISPR knockout and overexpression screens in both cancer cells and macrophages. In cancer cells, beyond known factors such as CD47, we identify many regulators of susceptibility to ADCP, including the poorly characterized enzyme adipocyte plasma membrane-associated protein (APMAP). We find that loss of APMAP synergizes with tumour antigen-targeting monoclonal antibodies and/or CD47-blocking monoclonal antibodies to drive markedly increased phagocytosis across a wide range of cancer cell types, including those that are otherwise resistant to ADCP. Additionally, we show that APMAP loss synergizes with several different tumour-targeting monoclonal antibodies to inhibit tumour growth in mice. Using genome-wide counterscreens in macrophages, we find that the G-protein-coupled receptor GPR84 mediates enhanced phagocytosis of APMAP-deficient cancer cells. This work reveals a cancer-intrinsic regulator of susceptibility to antibody-driven phagocytosis and, more broadly, expands our knowledge of the mechanisms governing cancer resistance to macrophage phagocytosis.