Intratumoral Fc-optimized agonistic CD40 antibody induces tumor rejection and systemic antitumor immunity in patients with metastatic cancer.

While CD40 agonism is an attractive approach for activating antigen-presenting cells and initiating antitumor responses, previous attempts have encountered limited clinical efficacy coupled with toxicity. We previously demonstrated that interactions between the antibody Fc domain and the inhibitory receptor FcγRIIB are critical for enhanced antitumor activity. Here, we present the results of a phase 1 study on intratumoral administration of an anti-CD40 agonistic antibody (2141-V11) Fc-engineered to enhance FcγRIIB binding. Primary endpoints included safety, maximum tolerated dose (MTD), and recommended phase 2 dose. Secondary objectives included preliminary clinical activity and correlative studies from biospecimens. 2141-V11 was well-tolerated without dose-limiting toxicities and MTD was not reached. In ten evaluable patients with metastatic cancer, the overall response rate was 20%, with complete responses in two patients (melanoma and breast carcinoma) and stable disease in six patients. 2141-V11 induced tumor regression in injected and non-injected lesions, with increased leukocyte infiltration and tertiary lymphoid structures (TLS) formation in post-treatment biopsies. In a humanized mouse model for CD40 and FcγRs, 2141-V11 induced TLS formation in mice bearing orthotopic breast carcinoma, correlating with local and abscopal antitumor effects, systemic immune activation, and immune memory. These findings support the safety and efficacy of 2141-V11, warranting phase 2 studies and suggesting a unique mechanism of action for this Fc-enhanced immunotherapy (NCT04059588).

Siglecs-7/9 function as inhibitory immune checkpoints in vivo and can be targeted to enhance therapeutic antitumor immunity.

Given the role of myeloid cells in T cell activation and in the antitumor response, targeting checkpoint molecules expressed on this population represents a promising strategy to augment antitumor immunity. However, myeloid checkpoints that can be effectively used as immunotherapy targets are still lacking. Here, we demonstrate the therapeutic potential of targeting the myeloid receptors Siglec-7 and Siglec-9 in vivo. By using a humanized immunocompetent murine model, we demonstrate that human Siglec-7 and Siglec-9, in addition to the murine homolog Siglec-E, inhibit the endogenous antitumor immune response, as well as the response to tumor-targeting and immune checkpoint inhibiting antibodies in vivo. The impact of these Siglecs on tumor progression is highly dependent on the anatomical distribution of the tumor and, as a consequence, the local tumor microenvironment, as tumors with a more immune-suppressive tumor microenvironment are less sensitive to Siglec perturbation. Finally, to assess the potential of these two receptors as targets for immunotherapy, we developed Fc engineered blocking antibodies to Siglec-7 and Siglec-9 and demonstrate that Siglec-7 and Siglec-9 blockade can significantly reduce tumor burden in vivo, demonstrating the therapeutic potential of targeting these two receptors.

Antibodies targeting sialyl Lewis A mediate tumor clearance through distinct effector pathways.

Sialyl Lewis A (sLeA, also known as CA19-9), a tetrasaccharide selectively and highly expressed on advanced adenocarcinomas including colon, stomach, and pancreatic cancers, has long been considered as an attractive target for active and passive vaccination. While progress in antibodies targeting tumor-associated protein antigens resulted in an impressive array of therapeutics for cancer treatment, similar progress in exploiting tumor-associated carbohydrate antigens, such as sLeA, has been hampered by the lack of a detailed understanding of the singular characteristics of these antigens. We have addressed this issue by analyzing antibodies derived from patients immunized with an sLeA/KLH vaccine. These antibodies were engineered to mediate tumor clearance in vivo in preclinical models through Fc-FcγR interactions. However, in contrast to protein antigens in which hFcγRIIIA engagement was both necessary and sufficient to mediate tumor clearance in both preclinical and clinical settings, a similar selective dependence was not seen for anti-sLeA antibodies. Thus, re-engineering the Fc portion of sLeA-targeting antibodies to broadly enhance their affinity for activating FcγRs led to an enhanced therapeutic effect. These findings will facilitate the development of more efficient anticancer therapies and further advance this promising class of therapeutic antibodies into clinical use.

Microenvironmental networks promote tumor heterogeneity and enrich for metastatic cancer stem-like cells in Luminal-A breast tumor cells.

The roles of the tumor microenvironment (TME) in generating intra-tumoral diversity within each specific breast cancer subtype are far from being fully elucidated. In this study, we exposed Luminal-A breast cancer cells in culture to combined "TME Stimulation", representing three typical arms of the breast TME: hormonal (estrogen), inflammatory (tumor necrosis factor α) and growth-promoting (epidermal growth factor). In addition to enriching the tumor cell population with CD44+/ß1+ cells (as we previously published), TME Stimulation selected for CD44+/CD24low/- stem-like cells, that were further enriched by doxorubicin treatment and demonstrated high plasticity in vitro and in vivo. Knock-down experiments revealed that CD44 and Zeb1 regulated CD24 and ß1 expression and controlled differently cell spreading and formation of cellular protrusions. TME-enriched CD44+/CD24low/- stem-like cells promoted dissemination to bones and lymph nodes, whereas CD44+/ß1+ cells had a low metastatic potential. Mixed co-injections of TME-enriched CD44+/CD24low/- and CD44+/ß1+ sub-populations generated metastases populated mostly by CD44+/CD24low/--derived cells. Thus, combined activities of several TME factors select for CD44+/CD24low/- stem-like cells that dictate the metastatic phenotype of Luminal-A breast tumor cells, suggesting that therapeutic modalities targeting the TME could be introduced as a potential strategy of inhibiting the detrimental stem-like sub-population in this disease subtype.

Chemokine axes in breast cancer: factors of the tumor microenvironment reshape the CCR7-driven metastatic spread of luminal-A breast tumors.

Chemokine axes have been shown to mediate site-specific metastasis in breast cancer, but their relevance to different subtypes has been hardly addressed. Here, with the focus on the CCR7-CCL21 axis, patient datasets demonstrated that luminal-A tumors express relatively low CCR7 levels compared with more aggressive disease subtypes. Furthermore, lymph node metastasis was not associated with high CCR7 levels in luminal-A patients. The metastatic pattern of luminal-A breast tumors may be influenced by the way luminal-A tumor cells interpret signals provided by factors of the primary tumor microenvironment. Thus, CCR7-expressing human luminal-A cells were stimulated simultaneously by factors representing 3 tumor microenvironment arms typical of luminal-A tumors, hormonal, inflammatory, and growth stimulating: estrogen + TNF-α + epidermal growth factor. Such tumor microenvironment stimulation down-regulated the migration of CCR7-expressing tumor cells toward CCL21 and inhibited the formation of directional protrusions toward CCL21 in a novel 3-dimensional hydrogel system. CCL21-induced migration of CCR7-expressing tumor cells depended on PI3K and MAPK activation; however, when CCR7-expressing cancer cells were prestimulated by tumor microenvironment factors, CCL21 could not effectively activate these signaling pathways. In vivo, pre-exposure of the tumor cells to tumor microenvironment factors has put restraints on CCL21-mediated lymph node-homing cues and shifted the metastatic pattern of CCR7-expressing cells to the aggressive phenotype of dissemination to bones. Several of the aspects were also studied in the CXCR4-CXCL12 system, demonstrating similar patient and in vitro findings. Thus, we provide novel evidence to subtype-specific regulation of the CCR7-CCL21 axis, with more general implications to chemokine-dependent patterns of metastatic spread, revealing differential regulation in the luminal-A subtype.

The chemokine system, and its CCR5 and CXCR4 receptors, as potential targets for personalized therapy in cancer.

Chemokines and their receptors regulate the trafficking of leukocytes in hematopoiesis and inflammation, and thus are fundamental to the immune integrity of the host. In parallel, members of the chemokine system exert a large variety of functions that dictate processes of cancer development and progression. Chemokines can act as pro-tumoral or anti-tumoral regulators of malignancy by affecting cells of the tumor microenvironment (leukocytes, endothelial cells, fibroblasts) and the tumor cells themselves (migration, invasion, proliferation, resistance to chemotherapy). Several of the chemokines are generally skewed towards the cancer-promoting direction, including primarily the CCR5-CCL5 (RANTES) and the CXCR4-CXCL12 (SDF-1) axes. This review provides a general view of chemokines and chemokine receptors as regulators of malignancy, describing their multi-faceted activities in cancer. The tumor-promoting activities of the CCR5-CCL5 and CXCR4-CXCL12 pathways are enlightened, emphasizing their potential use as targets for personalized therapy. Indeed, novel blockers of chemokines and their receptors are constantly emerging, and two chemokine receptor inhibitors were recently approved for clinical use: Maraviroc for CCR5 and Plerixafor for CXCR4. The review addresses ongoing pre-clinical and clinical trials using these modalities and others in cancer. Then, challenges and opportunities of personalized therapy directed against chemokines and their receptors in malignancy are discussed, demonstrating that such novel personalized cancer therapies hold many challenges, but also offer hope for cancer patients.

Progression of luminal breast tumors is promoted by ménage à trois between the inflammatory cytokine TNFα and the hormonal and growth-supporting arms of the tumor microenvironment.

Breast cancer progression is strongly linked to inflammatory processes, aggravating disease course. The impacts of the inflammatory cytokine TNF α on breast malignancy are not fully substantiated, and they may be affected by cooperativity between TNF α and other protumoral mediators. Here, we show that together with representatives of other important arms of the tumor microenvironment, estrogen (hormonal) and EGF (growth-supporting), TNF α potently induced metastasis-related properties and functions in luminal breast tumor cells, representing the most common type of breast cancer. Jointly, TNFα + Estrogen + EGF had a stronger effect on breast cancer cells than each element alone, leading to the following: (1) extensive cell spreading and formation of FAK/paxillin-enriched cellular protrusions; (2) elevated proportion of tumor cells coexpressing high levels of CD44 and ß 1 and VLA6; (3) EMT and cell migration; (4) resistance to chemotherapy; (5) release of protumoral factors (CXCL8, CCL2, MMPs). Importantly, the tumor cells used in this study are known to be nonmetastatic under all conditions; nevertheless, they have acquired high metastasizing abilities in vivo in mice, following a brief stimulation by TNFα + Estrogen + EGF. These dramatic findings indicate that TNF α can turn into a strong prometastatic factor, suggesting a paradigm shift in which clinically approved inhibitors of TNFα would be applied in breast cancer therapy.

Inflammatory mediators in breast cancer: coordinated expression of TNFα & IL-1ß with CCL2 & CCL5 and effects on epithelial-to-mesenchymal transition.

BACKGROUND: The inflammatory chemokines CCL2 (MCP-1) & CCL5 (RANTES) and the inflammatory cytokines TNFα & IL-1ß were shown to contribute to breast cancer development and metastasis. In this study, we wished to determine whether there are associations between these factors along stages of breast cancer progression, and to identify the possible implications of these factors to disease course. METHODS: The expression of CCL2, CCL5, TNFα and IL-1ß was determined by immunohistochemistry in patients diagnosed with: (1) Benign breast disorders (=healthy individuals); (2) Ductal Carcinoma In Situ (DCIS); (3) Invasive Ducal Carcinoma without relapse (IDC-no-relapse); (4) IDC-with-relapse. Based on the results obtained, breast tumor cells were stimulated by the inflammatory cytokines, and epithelial-to-mesenchymal transition (EMT) was determined by flow cytometry, confocal analyses and adhesion, migration and invasion experiments. RESULTS: CCL2, CCL5, TNFα and IL-1ß were expressed at very low incidence in normal breast epithelial cells, but their incidence was significantly elevated in tumor cells of the three groups of cancer patients. Significant associations were found between CCL2 & CCL5 and TNFα & IL-1ß in the tumor cells in DCIS and IDC-no-relapse patients. In the IDC-with-relapse group, the expression of CCL2 & CCL5 was accompanied by further elevated incidence of TNFα & IL-1ß expression. These results suggest progression-related roles for TNFα and IL-1ß in breast cancer, as indeed indicated by the following: (1) Tumors of the IDC-with-relapse group had significantly higher persistence of TNFα and IL-1ß compared to tumors of DCIS or IDC-no-relapse; (2) Continuous stimulation of the tumor cells by TNFα (and to some extent IL-1ß) has led to EMT in the tumor cells; (3) Combined analyses with relevant clinical parameters suggested that IL-1ß acts jointly with other pro-malignancy factors to promote disease relapse. CONCLUSIONS: Our findings suggest that the coordinated expression of CCL2 & CCL5 and TNFα & IL-1ß may be important for disease course, and that TNFα & IL-1ß may promote disease relapse. Further in vitro and in vivo studies are needed for determination of the joint powers of the four factors in breast cancer, as well as analyses of their combined targeting in breast cancer.

Epidermal growth factor and estrogen act by independent pathways to additively promote the release of the angiogenic chemokine CXCL8 by breast tumor cells.

The tumor microenvironment contains multiple cancer-supporting factors, whose joint activities promote malignancy. Here, we show that epidermal growth factor (EGF) and estrogen upregulate in an additive manner the transcription and the secretion of the angiogenic chemokine CXCL8 (interleukin 8 [IL-8]) in breast tumor cells. In view of published findings on cross-regulatory interactions between EGF receptors and estrogen receptors in breast tumor cells, we asked whether the additive effects of EGF and estrogen were due to their ability to (1) induce intracellular cross talk and amplify shared regulatory pathways or (2) act in independent mechanisms, which complement each other. We found that stimulation by EGF alone induced the release of CXCL8 through signaling pathways involving ErbB2, ErbB1, Erk, and phosphoinositide 3-kinase (PI3K). ErbB2 and Erk were also involved in estrogen activities on CXCL8 but to a lower extent than with EGF. However, in the joint stimulatory setup, the addition of estrogen to EGF has led to partial (ErbB2, ErbB1, Erk) or complete (PI3K) shutoff of the involvement of these activation pathways in CXCL8 up-regulation. Furthermore, when costimulation by EGF + estrogen was applied, the effects of estrogen were channeled to regulation of CXCL8 at the transcription level, acting through the transcription factor estrogen receptor α (ERα). In parallel, in the joint stimulation, EGF acted independently at the transcription level through AP-1, to upregulate CXCL8 expression. The independent activities of EGF and estrogen on CXCL8 transcription reinforce the need to introduce simultaneous targeting of ErbBs and ERα to achieve effective therapy in breast cancer.