CD47 Studies Take Immunotherapy in New Direction

During the past several decades, it has become increasingly clear that there is a complicated relationship between a tumor and the patient’s immune system. Although the genetic and epigenetic changes that fuel cancer development create foreign antigens that should trigger an immune response, one of the hallmarks of cancer is its ability to evade this immune recognition.

The immuno-oncology field has blossomed as researchers have gotten a better grasp of the mechanisms that cancer cells use to suppress the antitumor immune response.

The dominant strategy thus far has been to activate T cells, the central mediators of the adaptive immune response. This approach is antigen specific, involves memory recall, and could in theory have long-lasting therapeutic benefit.

Yet there are 2 arms to the immune system that act in concert with one another and evidence is building that the second arm—the innate immune response—is also likely to be important.

As a result, researchers and pharmaceutical companies, seeking to carve out new niches in the immuno-oncology field, are looking to the major player in the innate immune response: the macrophages.

A particularly promising therapeutic target in this area is the CD47 protein, dubbed the “don’t eat me” signal for its role in inhibiting the phagocytic activity of macrophages and seemingly exploited by cancer cells to make them appear as normal cells to the innate immune response.

Exploration of CD47-targeting antibodies in preclinical and early clinical trials is beginning to scratch at the surface of the hidden complexities of this nearly ubiquitously expressed protein. It has also revealed the likely complementary role of CD47-targeted therapies to both other immunotherapies and targeted therapies, which could prove beneficial in rationally designed combination therapy.

The Other Arm of the Immune Response

Tumors clearly engage the cells of the innate immune response. Macrophages are among the most abundant normal cells found in the tumor microenvironment and evidence suggests that they are manipulated by cancer cells to become protumoral.

Furthermore, there is crosstalk between the innate and adaptive arms since macrophages and other phagocytic cells can present foreign antigens to T cells, triggering an adaptive immune response.

It is becoming increasingly clear that cancer cells have to sneak past the effector cells of both the adaptive and innate immune response to avoid destruction and that the innate immune response plays an important role—possibly, a therapeutically complementary role—in cancer development and progression.

Macrophages are the key effectors of the innate immune response and their primary function is to patrol the body in search of damaged, old, or foreign cells, and clear them from the body via phagocytosis in order to maintain cellular homeostasis.

Phagocytosis appears to be regulated by a delicate balance in the expression of cell-surface proteins that act as either “eat me” or “don’t eat me” signals to phagocytic effectors. Similar to T-cell activation, switching on macrophages also appears to be a 2-step process that requires both an upregulation of the “eat me” signals and a downregulation of the “don’t eat me” signals.

“Don’t Eat Me”

That is where CD47, the cluster of differentiation 47 protein (also known as integrin-associated protein), comes into focus.CD47 is a membrane-spanning protein that belongs to the immunoglobulin superfamily and is expressed on almost every cell in the body. It has become best known for its pivotal role in regulating phagocytosis, thus earning it the moniker of being the “don’t eat me” signal.

In its phagocytic role, CD47 binds to signal regulatory protein alpha (SIRPα), another member of the immunoglobulin superfamily that is expressed on the surface of macrophages, as well as other phagocytic cells. The SIRPα protein consists of 3 immunoglobulin—like domains that protrude outside the cell, a portion that crosses the cell membrane, and a tail section that resides inside the cell and contains 4 immunoreceptor tyrosine-based inhibitory motifs (ITIMs).

By binding to one of SIRPα’s extracellular domains, CD47 triggers phosphorylation of the intracellular ITIMs, transmitting the signal through the cell membrane of the macrophage. This drives the recruitment and activation of 2 phosphatase enzymes, SHP1 and SHP2, and one of the downstream effects of this is the inhibition of myosin IIA accumulation at the phagocytic synapse, which ultimately suppresses phagocytosis.

CD47 as an Anticancer Target

In this way, CD47 acts as a marker of self for the innate immune system; cells that are old or nonself express little or no CD47 and therefore do not engage SIRPα on the surface of macrophages, tipping the balance in favor of the “eat me” signals that they express and stimulating phagocytosis. Meanwhile, young, healthy cells express higher levels of CD47 and essentially switch off any macrophages they encounter.High levels of the CD47 protein were first observed on ovarian cancer cells. Since then, it has been shown to be overexpressed on a variety of different tumor types including such hematologic malignancies such as acute myeloid leukemia (AML) and non-Hodgkin lymphoma, and solid tumors such as breast, bladder, and head and neck cancers.

Furthermore, increased CD47 expression appears to correlate with poor prognosis in many cancers. In addition, it has been noted that CD47 expression is increased on tumor-infiltrating cells and cells of the tumor microenvironment, and could play a critical role in establishing an immunosuppressive niche.

The current thinking is that cancer cells ramp up their expression of CD47 to hijack the “don’t eat me” signal, which then acts as a kind of invisibility cloak, enabling them to avoid phagocytic clearance by the innate immune response.

CD47 Beyond Phagocytosis

Blocking that CD47 signal with targeted therapies could allow researchers to reinstate the phagocytic signal to destroy tumor cells, as well as repurpose the tumor-associated macrophages that form such an essential part of the tumor microenvironment, switching them from a protumoral state back to a tumoricidal state.Interestingly, cancer stem cells (CSCs)—cells within the tumor population with the capacity for self-renewal and differentiation—have also been reported to express elevated CD47 levels. As yet, the role of CD47 in regulating CSC function has not been widely examined, although some studies have suggested that it regulates self-renewal.

In a preclinical study using a breast cancer model, it was shown that the function of CD47 in breast CSCs may be independent of SIRPα signaling and instead may have to do with its effects on the epidermal growth factor receptor (EGFR). The use of a CD47 antibody appeared to target breast CSCs by downregulating cell-surface expression of EGFR.

While undoubtedly highlighting the potential for therapeutic targeting of CSCs using CD47-directed drugs, this also leads to an important point: that CD47 has cellular activities beyond phagocytosis, which may also be important in the context of cancer.

In addition to SIRPα, another important binding partner for CD47 is the thrombospondin-1 (TSP-1) protein, a glycoprotein that is expressed by a wide variety of different cell types, including platelets, monocytes, and macrophages, as well as several non-hematopoietic cell types including smooth muscle and endothelial cells. TSP-1 is a multifunctional protein, impacting such processes as aggregation of platelets, cell-to-cell adhesion, and angiogenesis.

Contrary to the CD47/SIRPα axis, which likely promotes tumor growth, CD47/TSP-1 signaling appears to be an important tumor suppressor pathway in cancer. Best characterized are the inhibitory effects of this pathway on angiogenesis. CD47 acts as a receptor for TSP-1 and mediates its antiangiogenic impact through its effects on endothelial cell adhesion, migration, and proliferation.

Pharmaceutical Companies Take Aim

The therapeutic potential of CD47, reinforced by promising data from preclinical trials, has attracted the attention of several pharmaceutical companies that are developing monoclonal antibodies targeting this protein. Thus far, disrupting the CD47/SIRPα interaction and therefore blocking the ability of cancer cells to disrupt phagocytosis has been at the heart of these efforts.

Among the leaders in the field is the immuno-oncology company Forty Seven Inc, founded by several of the Stanford University researchers who were among the early pioneers of CD47 research. The company name is partly a play on their lead compound, Hu5F9-G4, a humanized monoclonal antibody targeting CD47.

This antibody is currently being evaluated in phase I clinical trials in patients with relapsed or refractory solid tumors or AML.

Preclinical studies suggested that Hu5F9-G4 caused a transient anemia that could be managed by the use of a single, low priming dose followed by subsequent higher maintenance doses. Thus, this clinical trial is a 2-part study evaluating the optimal priming dose (part A) and the optimal maintenance dose (part B).

At the time of reporting, 19 patients had been enrolled (11 in part A and 8 in part B). A 1-mg/ kg dose was selected as the optimal priming dose since the 0.1-, 0.3- and 1-mg/kg doses were well tolerated, but the 3 mg/kg dose led to grade 3 dose-limiting toxicities.

In part B, a 1-mg/kg priming dose followed by a 3-mg/kg maintenance dose appears well tolerated thus far. Adverse events have included anemia, hyperbilirubinemia, headache, nausea and retinal toxicity, but were mostly associated with the priming dose and were reversible.

Also investing in CD47-targeted therapies is Celgene, which is developing CC-90002, a humanized monoclonal antibody the company licensed from Inhibrx. Phase I trials are underway in both solid tumors and hematologic malignancies with a focus on AML.

Meanwhile, Trillium Therapeutics is developing TTI-621, a soluble recombinant fusion protein that is created by joining the CD47 binding region of SIRPα to the fragment crystallizable domain of human immunoglobulin G1. Two phase I clinical trials are ongoing in hematologic malignancies and solid tumors.

Preclinical testing of CD47 antibodies has reinforced the complex mechanism of action of these drugs, and it is still not entirely clear how they are exerting their antitumor effects. It has long been suspected that targeting CD47 could activate both the innate and adaptive immune responses. It was hypothesized that CD47 blockade would reinstate the phagocytic activity of macrophages and that these cells could then present the digested cancer cell antigens to the T cells to activate an adaptiveimmuneresponse.

The results of a study published last year in Nature Medicine suggested that this may not be the case and that macrophages may not play as vital a role in the antitumor activity of CD47 blockade as first thought. CD47 antibodies may instead stimulate dendritic cells to activate T cells and thus their adaptive immune-mediated effects.

Preclinical studies have also strongly hinted at the synergistic efficacy of CD47 antibodies with immunotherapies targeting the adaptive immune response, such as checkpoint inhibitors, and with targeted therapies, including EGFR- and HER2-targeting drugs.

Although many open questions remain to be addressed as the clinical development of these antibodies continues, the progress made thus far bears testament to the potential of this therapeutic strategy.

Jane de Lartigue, PhD, is a freelance medical writer and editor based in New Haven, Connecticut.

KEY RESEARCH

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