Targeted therapies prime oncogene-driven lung cancers for macrophage-mediated destruction.

Macrophage immune checkpoint inhibitors, such as anti-CD47 antibodies, show promise in clinical trials for solid and hematologic malignancies. However, the best strategies to use these therapies remain unknown, and ongoing studies suggest they may be most effective when used in combination with other anticancer agents. Here, we developed an unbiased, high-throughput screening platform to identify drugs that render lung cancer cells more vulnerable to macrophage attack, and we found that therapeutic synergy exists between genotype-directed therapies and anti-CD47 antibodies. In validation studies, we found that the combination of genotype-directed therapies and CD47 blockade elicited robust phagocytosis and eliminated persister cells in vitro and maximized antitumor responses in vivo. Importantly, these findings broadly applied to lung cancers with various RTK/MAPK pathway alterations - including EGFR mutations, ALK fusions, or KRASG12C mutations. We observed downregulation of ß2-microglobulin and CD73 as molecular mechanisms contributing to enhanced sensitivity to macrophage attack. Our findings demonstrate that dual inhibition of the RTK/MAPK pathway and the CD47/SIRPa axis is a promising immunotherapeutic strategy. Our study provides strong rationale for testing this therapeutic combination in patients with lung cancers bearing driver mutations.

MMD collaborates with ACSL4 and MBOAT7 to promote polyunsaturated phosphatidylinositol remodeling and susceptibility to ferroptosis.

Ferroptosis is a form of regulated cell death with roles in degenerative diseases and cancer. Excessive iron-catalyzed peroxidation of membrane phospholipids, especially those containing the polyunsaturated fatty acid arachidonic acid (AA), is central in driving ferroptosis. Here, we reveal that an understudied Golgi-resident scaffold protein, MMD, promotes susceptibility to ferroptosis in ovarian and renal carcinoma cells in an ACSL4- and MBOAT7-dependent manner. Mechanistically, MMD physically interacts with both ACSL4 and MBOAT7, two enzymes that catalyze sequential steps to incorporate AA in phosphatidylinositol (PI) lipids. Thus, MMD increases the flux of AA into PI, resulting in heightened cellular levels of AA-PI and other AA-containing phospholipid species. This molecular mechanism points to a pro-ferroptotic role for MBOAT7 and AA-PI, with potential therapeutic implications, and reveals that MMD is an important regulator of cellular lipid metabolism.

Targeted therapies prime oncogene-driven lung cancers for macrophage-mediated destruction.

Macrophage immune checkpoint inhibitors, such as anti-CD47 antibodies, show promise in clinical trials for solid and hematologic malignancies. However, the best strategies to use these therapies remain unknown and ongoing studies suggest they may be most effective when used in combination with other anticancer agents. Here, we developed a novel screening platform to identify drugs that render lung cancer cells more vulnerable to macrophage attack, and we identified therapeutic synergy exists between genotype-directed therapies and anti-CD47 antibodies. In validation studies, we found the combination of genotype-directed therapies and CD47 blockade elicited robust phagocytosis and eliminated persister cells in vitro and maximized anti-tumor responses in vivo. Importantly, these findings broadly applied to lung cancers with various RTK/MAPK pathway alterations-including EGFR mutations, ALK fusions, or KRASG12C mutations. We observed downregulation of ß2-microglobulin and CD73 as molecular mechanisms contributing to enhanced sensitivity to macrophage attack. Our findings demonstrate that dual inhibition of the RTK/MAPK pathway and the CD47/SIRPa axis is a promising immunotherapeutic strategy. Our study provides strong rationale for testing this therapeutic combination in patients with lung cancers bearing driver mutations.

Evolutionarily conserved resistance to phagocytosis observed in melanoma cells is insensitive to upregulation of pro-phagocytic signals and to CD47 blockade.

Therapeutic activation of macrophage phagocytosis has the ability to restrain tumour growth through phagocytic clearance of tumour cells and activation of the adaptive immune response. Our objective for this study was to evaluate the effects of modulating pro- and anti-phagocytic pathways in malignant melanoma. In order to identify evolutionarily conserved mechanisms of resistance that may be important for melanoma cell survival, we utilized a multi-species approach and examined the phagocytosis of human, mouse, and dog melanoma cells. We observed that melanoma cells from all three species displayed unexpected resistance to phagocytosis that could not be fully mitigated by blockade of the 'don't eat me' signal CD47 or by chemotherapeutic enhancement of known 'eat me' signals. Additionally, CD47 blockade failed to promote anti-melanoma immune responses or tumour regression in vivo. This melanoma resistance to phagocytosis was not mediated by soluble factors, and it was unaffected by siRNA-mediated knockdown of 47 prospective 'don't eat me' signals or by CRISPR-Cas-mediated CD47 knockout. Unexpectedly, CD47 knockout also did not enhance phagocytosis of lymphoma cells, but it eliminated the pro-phagocytic effect of CD47 blockade, suggesting that the pro-phagocytic effects of CD47 blockade are due in part to Fc receptor engagement. From this study, we conclude that melanoma cells possess an evolutionarily conserved resistance to macrophage phagocytosis. Further investigation will be needed to overcome the mechanisms that mediate melanoma cell resistance to innate immunity.

Engagement of MHC class I by the inhibitory receptor LILRB1 suppresses macrophages and is a target of cancer immunotherapy.

Exciting progress in the field of cancer immunotherapy has renewed the urgency of the need for basic studies of immunoregulation in both adaptive cell lineages and innate cell lineages. Here we found a central role for major histocompatibility complex (MHC) class I in controlling the phagocytic function of macrophages. Our results demonstrated that expression of the common MHC class I component ß2-microglobulin (ß2M) by cancer cells directly protected them from phagocytosis. We further showed that this protection was mediated by the inhibitory receptor LILRB1, whose expression was upregulated on the surface of macrophages, including tumor-associated macrophages. Disruption of either MHC class I or LILRB1 potentiated phagocytosis of tumor cells both in vitro and in vivo, which defines the MHC class I-LILRB1 signaling axis as an important regulator of the effector function of innate immune cells, a potential biomarker for therapeutic response to agents directed against the signal-regulatory protein CD47 and a potential target of anti-cancer immunotherapy.

Anti-SIRPα antibody immunotherapy enhances neutrophil and macrophage antitumor activity.

Cancer immunotherapy has emerged as a promising therapeutic intervention. However, complete and durable responses are only seen in a fraction of patients who have cancer. A key factor that limits therapeutic success is the infiltration of tumors by cells of the myeloid lineage. The inhibitory receptor signal regulatory protein-α (SIRPα) is a myeloid-specific immune checkpoint that engages the "don't eat me" signal CD47 expressed on tumors and normal tissues. We therefore developed the monoclonal antibody KWAR23, which binds human SIRPα with high affinity and disrupts its binding to CD47. Administered by itself, KWAR23 is inert, but given in combination with tumor-opsonizing monoclonal antibodies, KWAR23 greatly augments myeloid cell-dependent killing of a collection of hematopoietic and nonhematopoietic human tumor-derived cell lines. Following KWAR23 antibody treatment in a human SIRPA knockin mouse model, both neutrophils and macrophages infiltrate a human Burkitt's lymphoma xenograft and inhibit tumor growth, generating complete responses in the majority of treated animals. We further demonstrate that a bispecific anti-CD70/SIRPα antibody outperforms individually delivered antibodies in specific types of cancers. These studies demonstrate that SIRPα blockade induces potent antitumor activity by targeting multiple myeloid cell subsets that frequently infiltrate tumors. Thus, KWAR23 represents a promising candidate for combination therapy.

Delivery of monocyte lineage cells in a biomimetic scaffold enhances tissue repair.

The monocyte lineage is essential to normal wound healing. Macrophage inhibition or knockout in mice results in impaired wound healing through reduced neovascularization, granulation tissue formation, and reepithelialization. Numerous studies have either depleted macrophages or reduced their activity in the context of wound healing. Here, we demonstrate that by increasing the number of macrophages or monocytes in the wound site above physiologic levels via pullulan-collagen composite dermal hydrogel scaffold delivery, the rate of wound healing can be significantly accelerated in both wild-type and diabetic mice, with no adverse effect on the quality of repair. Macrophages transplanted onto wounds differentiate into M1 and M2 phenotypes of different proportions at various time points, ultimately increasing angiogenesis. Given that monocytes can be readily isolated from peripheral blood without in vitro manipulation, these findings hold promise for translational medicine aimed at accelerating wound healing across a broad spectrum of diseases.

Cancer immunotherapy targeting the CD47/SIRPα axis.

The success of cancer immunotherapy has generated tremendous interest in identifying new immunotherapeutic targets. To date, the majority of therapies have focussed on stimulating the adaptive immune system to attack cancer, including agents targeting CTLA-4 and the PD-1/PD-L1 axis. However, macrophages and other myeloid immune cells offer much promise as effectors of cancer immunotherapy. The CD47/signal regulatory protein alpha (SIRPα) axis is a critical regulator of myeloid cell activation and serves a broader role as a myeloid-specific immune checkpoint. CD47 is highly expressed on many different types of cancer, and it transduces inhibitory signals through SIRPα on macrophages and other myeloid cells. In a diverse range of preclinical models, therapies that block the CD47/SIRPα axis stimulate phagocytosis of cancer cells in vitro and anti-tumour immune responses in vivo. A number of therapeutics that target the CD47/SIRPα axis are under preclinical and clinical investigation. These include anti-CD47 antibodies, engineered receptor decoys, anti-SIRPα antibodies and bispecific agents. These therapeutics differ in their pharmacodynamic, pharmacokinetic and toxicological properties. Clinical trials are underway for both solid and haematologic malignancies using anti-CD47 antibodies and recombinant SIRPα proteins. Since the CD47/SIRPα axis also limits the efficacy of tumour-opsonising antibodies, additional trials will examine their potential synergy with agents such as rituximab, cetuximab and trastuzumab. Phagocytosis in response to CD47/SIRPα-blocking agents results in antigen uptake and presentation, thereby linking the innate and adaptive immune systems. CD47/SIRPα blocking therapies may therefore synergise with immune checkpoint inhibitors that target the adaptive immune system. As a critical regulator of macrophage phagocytosis and activation, the potential applications of CD47/SIRPα blocking therapies extend beyond human cancer. They may be useful for the treatment of infectious disease, conditioning for stem cell transplant, and many other clinical indications.

Eradication of Canine Diffuse Large B-Cell Lymphoma in a Murine Xenograft Model with CD47 Blockade and Anti-CD20.

Cancer immunotherapies hold much promise, but their potential in veterinary settings has not yet been fully appreciated. Canine lymphomas are among the most common tumors of dogs and bear remarkable similarity to human disease. In this study, we examined the combination of CD47 blockade with anti-CD20 passive immunotherapy for canine lymphoma. The CD47/SIRPα axis is an immune checkpoint that regulates macrophage activation. In humans, CD47 is expressed on cancer cells and enables evasion from phagocytosis. CD47-blocking therapies are now under investigation in clinical trials for a variety of human cancers. We found the canine CD47/SIRPα axis to be conserved biochemically and functionally. We identified high-affinity SIRPα variants that antagonize canine CD47 and stimulate phagocytosis of canine cancer cells in vitro When tested as Fc fusion proteins, these therapeutic agents exhibited single-agent efficacy in a mouse xenograft model of canine lymphoma. As robust synergy between CD47 blockade and tumor-specific antibodies has been demonstrated for human cancer, we evaluated the combination of CD47 blockade with 1E4-cIgGB, a canine-specific antibody to CD20. 1E4-cIgGB could elicit a therapeutic response against canine lymphoma in vivo as a single agent. However, augmented responses were observed when combined with CD47-blocking therapies, resulting in synergy in vitro and in vivo and eliciting cures in 100% of mice bearing canine lymphoma. Our findings support further testing of CD47-blocking therapies alone and in combination with CD20 antibodies in the veterinary setting. Cancer Immunol Res; 4(12); 1072-87. ©2016 AACR.

Myeloid Cell Origins, Differentiation, and Clinical Implications.

The hematopoietic stem cell (HSC) is a multipotent stem cell that resides in the bone marrow and has the ability to form all of the cells of the blood and immune system. Since its first purification in 1988, additional studies have refined the phenotype and functionality of HSCs and characterized all of their downstream progeny. The hematopoietic lineage is divided into two main branches: the myeloid and lymphoid arms. The myeloid arm is characterized by the common myeloid progenitor and all of its resulting cell types. The stages of hematopoiesis have been defined in both mice and humans. During embryological development, the earliest hematopoiesis takes place in yolk sac blood islands and then migrates to the fetal liver and hematopoietic organs. Some adult myeloid populations develop directly from yolk sac progenitors without apparent bone marrow intermediates, such as tissue-resident macrophages. Hematopoiesis also changes over time, with a bias of the dominating HSCs toward myeloid development as animals age. Defects in myelopoiesis contribute to many hematologic disorders, and some of these can be overcome with therapies that target the aberrant stage of development. Furthermore, insights into myeloid development have informed us of mechanisms of programmed cell removal. The CD47/SIRPα axis, a myeloid-specific immune checkpoint, limits macrophage removal of HSCs but can be exploited by hematologic and solid malignancies. Therapeutics targeting CD47 represent a new strategy for treating cancer. Overall, an understanding of hematopoiesis and myeloid cell development has implications for regenerative medicine, hematopoietic cell transplantation, malignancy, and many other diseases.

Hematopoietic stem cell transplantation in immunocompetent hosts without radiation or chemotherapy.

Hematopoietic stem cell (HSC) transplantation can cure diverse diseases of the blood system, including hematologic malignancies, anemias, and autoimmune disorders. However, patients must undergo toxic conditioning regimens that use chemotherapy and/or radiation to eliminate host HSCs and enable donor HSC engraftment. Previous studies have shown that anti-c-Kit monoclonal antibodies deplete HSCs from bone marrow niches, allowing donor HSC engraftment in immunodeficient mice. We show that host HSC clearance is dependent on Fc-mediated antibody effector functions, and enhancing effector activity through blockade of CD47, a myeloid-specific immune checkpoint, extends anti-c-Kit conditioning to fully immunocompetent mice. The combined treatment leads to elimination of >99% of host HSCs and robust multilineage blood reconstitution after HSC transplantation. This targeted conditioning regimen that uses only biologic agents has the potential to transform the practice of HSC transplantation and enable its use in a wider spectrum of patients.

Antibody Therapy Targeting CD47 and CD271 Effectively Suppresses Melanoma Metastasis in Patient-Derived Xenografts.

The high rate of metastasis and recurrence among melanoma patients indicates the existence of cells within melanoma that have the ability to both initiate metastatic programs and bypass immune recognition. Here, we identify CD47 as a regulator of melanoma tumor metastasis and immune evasion. Protein and gene expression analysis of clinical melanoma samples reveals that CD47, an anti-phagocytic signal, correlates with melanoma metastasis. Antibody-mediated blockade of CD47 coupled with targeting of CD271(+) melanoma cells strongly inhibits tumor metastasis in patient-derived xenografts. This therapeutic effect is mediated by drastic changes in the tumor and metastatic site immune microenvironments, both of whichwhich exhibit greatly increased density of differentiated macrophages and significantly fewer inflammatory monocytes, pro-metastatic macrophages (CCR2(+)/VEGFR1(+)), and neutrophils, all of which are associated with disease progression. Thus, antibody therapy that activates the innate immune response in combination with selective targeting of CD271(+) melanoma cells represents a powerful therapeutic approach against metastatic melanoma.

CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer.

Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers.

Targeting lymphoma with precision using semisynthetic anti-idiotype peptibodies.

B-cell lymphomas express a functionally active and truly tumor-specific cell-surface product, the variable region of the B-cell receptor (BCR), otherwise known as idiotype. The tumor idiotype differs, however, from patient to patient, making it a technical challenge to exploit for therapy. We have developed a method of targeting idiotype by using a semisynthetic personalized therapeutic that is more practical to produce on a patient-by-patient basis than monoclonal antibodies. In this method, a small peptide with affinity for a tumor idiotype is identified by screening a library, chemically synthesized, and then affixed to the amino terminus of a premade IgG Fc protein. We demonstrate that the resultant semisynthetic anti-idiotype peptibodies kill tumor cells in vitro with specificity, trigger tumor cell phagocytosis by macrophages, and efficiently clear human lymphoma in a murine xenograft model. This method could be used to target tumor with true precision on a personalized basis.

SIRPα-Antibody Fusion Proteins Selectively Bind and Eliminate Dual Antigen-Expressing Tumor Cells.

PURPOSE: CD47 is highly expressed on a variety of tumor cells. The interaction of CD47 with signal regulatory protein alpha (SIRPα), a protein on phagocytic cells, transmits a "don't eat me" signal that negatively regulates phagocytosis. CD47-SIRPα antagonists enable phagocytosis by disrupting the inhibitory signal and can synergize with Fc-mediated pro-phagocytic signals for potent elimination of tumor cells. A potential limitation of therapeutic CD47-SIRPα antagonists is that expression of CD47 on normal cells may create sites of toxicity or an "antigen sink." To overcome these limitations and address selective tumor targeting, we developed SIRPabodies to improve the therapeutic benefits of CD47-SIRPα blockade specifically toward tumor. EXPERIMENTAL DESIGN: SIRPabodies were generated by grafting the wild-type SIRPα either to the N-terminus or to the C-terminus of the heavy chain of rituximab. Selective tumor binding was tested using CFSE-labeled human primary CLL cells in the presence of 20-fold excess of human RBCs. NSG mice were transplanted with Raji-luciferase cells and were assigned to controls versus SIRPabody treatment. Cynomolgus nonhuman primates were administered a single intravenous infusion of SIRPabody at 3, 10, or 30 mg/kg. RESULTS: SIRPabodies selectively bound to dual antigen-expressing tumor cells in the presence of a large antigen sink. SIRPabody reduced tumor burden and extended survival in mouse xenograft lymphoma models. SIRPabody caused no significant toxicity in nonhuman primates. CONCLUSIONS: These findings establish SIRPabodies as a promising approach to deliver the therapeutic benefit of CD47-SIRPα blockade specifically toward tumor cells. SIRPabodies may be applied to additional cancer types by grafting SIRPα onto other tumor-specific therapeutic antibodies. Clin Cancer Res; 22(20); 5109-19. ©2016 AACR.

Identification of tumorigenic cells and therapeutic targets in pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (PanNETs) are a type of pancreatic cancer with limited therapeutic options. Consequently, most patients with advanced disease die from tumor progression. Current evidence indicates that a subset of cancer cells is responsible for tumor development, metastasis, and recurrence, and targeting these tumor-initiating cells is necessary to eradicate tumors. However, tumor-initiating cells and the biological processes that promote pathogenesis remain largely uncharacterized in PanNETs. Here we profile primary and metastatic tumors from an index patient and demonstrate that MET proto-oncogene activation is important for tumor growth in PanNET xenograft models. We identify a highly tumorigenic cell population within several independent surgically acquired PanNETs characterized by increased cell-surface protein CD90 expression and aldehyde dehydrogenase A1 (ALDHA1) activity, and provide in vitro and in vivo evidence for their stem-like properties. We performed proteomic profiling of 332 antigens in two cell lines and four primary tumors, and showed that CD47, a cell-surface protein that acts as a "don't eat me" signal co-opted by cancers to evade innate immune surveillance, is ubiquitously expressed. Moreover, CD47 coexpresses with MET and is enriched in CD90(hi)cells. Furthermore, blocking CD47 signaling promotes engulfment of tumor cells by macrophages in vitro and inhibits xenograft tumor growth, prevents metastases, and prolongs survival in vivo.

A bispecific antibody targeting CD47 and CD20 selectively binds and eliminates dual antigen expressing lymphoma cells.

Agents that block the anti-phagocytic signal CD47 can synergize with pro-phagocytic anti-tumor antigen antibodies to potently eliminate tumors. While CD47 is overexpressed on cancer cells, its expression in many normal tissues may create an 'antigen sink' that could minimize the therapeutic efficacy of CD47 blocking agents. Here, we report development of bispecific antibodies (BsAbs) that co-target CD47 and CD20, a therapeutic target for non-Hodgkin lymphoma (NHL), that have reduced affinity for CD47 relative to the parental antibody, but retain strong binding to CD20. These characteristics facilitate selective binding of BsAbs to tumor cells, leading to phagocytosis. Treatment of human NHL-engrafted mice with BsAbs reduced lymphoma burden and extended survival while recapitulating the synergistic efficacy of anti-CD47 and anti-CD20 combination therapy. These findings serve as proof of principle for BsAb targeting of CD47 with tumor-associated antigens as a viable strategy to induce selective phagocytosis of tumor cells and recapitulate the synergy of combination antibody therapy. This approach may be broadly applied to cancer to add a CD47 blocking component to existing antibody therapies.