Author: Ginger Ding
On September 4, 2020, I-Mab Biopharma announced a collaboration with AbbVie on the global development and commercialization of CD47 monoclonal antibody TJC4. The US$2.9 billion collaboration signifies the largest out-licensing in the history of Chinese biotech companies.
Less than a week later, on September 8, another CD47 company, Trillium Therapeutics, announced the sale of 2,297,794 common shares to Pfizer at a price of $10.88 per share, totaling $25 million. That same day, the Hong Kong Stock Exchange announced that SciClone Pharmaceuticals had submitted the IPO application. SciClone Pharmaceuticals had introduced RRx-001, a small molecule immunotherapy targeting CD47-SIRPα, from EpicentRx in July this year for US$120 million.
At present, more than 30 companies are developing drugs against CD47 or its ligand SIRPα, including monoclonal antibodies, bispecific antibodies, fusion proteins, and small molecules. More than 20 of them have entered the clinical research stage. From the perspective of the geographical distribution of CD47 projects, China has become the most competitive arena in the post-PD-1 era.
Figure 1: List of Global CD47 Projects
In fact, there are endless opportunities for CD47 projects around the world. In March of this year, Gilead acquired Forty Seven, a company that specializes in CD47 antibodies, for US$4.9 billion. Investment analysts also predict that Trillium Therapeutics is the next to be acquired. Pfizer might be one of the prospective buyers.
Figure 2: CD47 project deals
Although there has frequently been good news about CD47 projects in 2020, reviewing development history has not all been smooth sailing: From 2017 to 2018 due to toxic side effects, Tioma Therapeutics (later renamed Arch Oncology) and Celgene (CC-90002) terminated all CD47 monoclonal antibody clinical trial tests.
Will CD47 become the next hot target for immunotherapy after PD(L)1? What are the differentiated advantages of the products under development? How to avoid the failed tragetory of Celegen’s CD47 monoclonal antibody? What are the challenges and opportunities for the commercialization of CD47 antibodies? This article will discuss the above issues.
Introduction of CD47
CD47 is a glycoprotein that was first identified as a tumor antigen of human ovarian cancer in the 1980s. It was later found to be overexpressed in a variety of tumors, and its expression level was related to the prognosis of tumors. After CD47 binds to the receptor SIRPɑ on the surface of macrophages, it will release a “don’t eat me” signal to help cancer cells shield the innate immune system and induce immune suppression.
Figure 3： D47 mRNA expression level is related to the prognosis of solid tumors
Image source: PNAS 2012;109:17:6662-6667
Using specific antibodies and other drugs to block the CD47/SIRPα signaling pathway can restore the phagocytosis of tumor cells by macrophages and suppress the “don’t eat me” signal. Based on this discovery, in recent years, CD47 has been regarded by the industry as one of the most important targets in the field of immunotherapy in the post-PD-(L)1 era.
“We think CD47 is the most promising next-generation immuno checkpoint besides PD-(L)1.”
—Tian Wenzhi, Chairman of Immune Onco
Therapies targeting the CD47-SIRPα signaling pathway have a variety of mechanisms of action, including:
- Blocking CD47 and SIRPα interactions, disintegrating the “don’t eat me” signal, and promoting phagocytosis.
- CD47 antibody can stimulate the anti-tumor adaptive response by promoting the phagocytosis of tumor cells by DC cells, and then presenting the antigen to T cells.
- CD47 antibody kills tumor cells through ADCC and CDC.
- CD47 antibody can activate tumor cell apoptosis pathway and directly induce tumor cell death.
Figure 4: The four mechanisms of targeting CD47.
Image source: Crit Rev Oncol Hematol. 2020 Aug;152:103014
How to effectively avoid off-target effects?
Given the ubiquitous expression of CD47, potential problems with anti-CD47 therapy include off-target effects. For example, CD47 is widely expressed on the surface of red blood cells to protect itself from phagocytosis. This means that while CD47 drugs target and kill tumor cells, they will likely also injure red blood cells.
Severe anemia is the main obstacle to the clinical development of CD47 antibody drugs. At least two projects have been terminated because of the decline in the number of red blood cells and platelets of grade 3 and above in early clinical trials, including Celgene’s CC-90002 .
The above situation is very common in the development of innovative drugs. Efficacy and safety issues are solved through experiences of the first-generation drug molecules and multiple rounds of trying-out second-generation products. The new generation of CD47 antibodies proposes solutions from four perspectives:
- Antibody fusion protein, such as Trilium Therapeutics’ TTI-621 and Sumgen Bio’s SG404 or nano antibody, such as Novamab.
- Prime dose combined with therapeutic dose, such as Forty Seven’s Hu5F9-G4.
- Reduce the binding of the CD47 antibody and red blood cell CD47 through structural optimization, such as TJC4 of I-Mab Biopharma and ALX148 of ALX Oncology
- Combine with drug delivery carriers such as nanoparticles.
In theory, CD47 on the surface of red blood cells and CD47 on the surface of tumor cells may have different conformations. Since the shape and membrane fluidity of red blood cells are also different from spherical tumor cells, so it is possible to discover antibody that binds to tumor cells instead of red blood cell.
Beginning in 2017, I-Mab Biopharma began the development of CD47 antibodies. Using human antibody libraries and phage display technology, they took a new path through reverse screening and finally obtained CD47 monoclonal antibody TJC4 that does not bind to red blood cells and has anti-tumor activity. Unlike other CD47 antibodies, TJC4 can recognize a unique CD47 epitope, which cannot be fully exposed due to glycosylation modification on red blood cells, resulting in weak binding of TJC4 to red blood cells.
The preliminary results of the Phase I TJC4 clinical study in the United States show that it has a differentiated advantage in terms of safety and better pharmacokinetic characteristics with a single injection of TJC4 is administered from 1 mg/kg to 30; no dose-limiting toxicity or serious hematological adverse events were observed in patients.
Similar to TJC4, Arch Oncology’s AO-176 can also selectively bind to tumor cells but not red blood cells.
Figure 5: Combination of AO-176 and different cells
Image source: Arch Oncology official website
Hu5F9-G4（Forty Seven/Gilead Sciences）
Irving Weissman, one of the founders of Forty Seven, discovered the mechanism of CD47. Forty Seven’s Hu5F9-G4 uses an improved dosing strategy in its clinical design. First, the patient is given a 1 mg/kg priming dose to trigger the elimination of aging red blood cells in the body. Although it will cause temporary mild anemia, at the same time it can stimulate the maturation and differentiation of reticulocytes to produce fresh young red blood cells.
These new red blood cells express a low “eat me” signal on the surface and are less sensitive to CD47 antibody-mediated phagocytosis. Thus, they can withstand the subsequent higher therapeutic dose of Hu5F9 30 mg/kg and achieve sufficient CD47 receptor occupancy and have an anti-tumor effect.
The Phase Ib clinical trial data released by Forty Seven at the ASH conference last year showed that Hu5F9-G4 combined with azacitidine showed promising afficacy for patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) effect. Among 24 MDS patients, the overall response rate (ORR) reached 92%, with 50% (12 cases) achieving complete response (CR). In 22 AML patients, ORR is 64 % and CR is 41% (9 cases).
TTI-621 is an antibody fusion protein composed of the N-terminal V domain of human SIRPα and the Fc region of human IgG1. It has been engineered to minimize the binding to human red blood cells, thereby minimizing the risk of anemia. It can be administered at low therapeutic doses while still maintaining sufficient receptor binding.
Figure 6: Combination of TTI-621/622 and red blood cells
Image source: Trillum Therapeutics’ official website
It is worth noting that significant dose-limiting thrombocytopenia was observed when TTI-621 was administered to patients, which may be related to the use of activated IgG1. Trillium Therapeutics’ other CD47 inhibitor TTI-622 is conjugated to IgG4, which is weaker in binding to Fc receptors on immune cells, which also enhances the protection of CD47-expressing non-tumor cells.
Figure 7: Comparison of CD47 drug candidates
Image source: Trillum Therapeutics’ official website
Nanobodies are a new type of single domain antibody fragments derived from naturally occurring heavy chain IgG antibodies. Due to their small size (about 12 kDa), high affinity and high stability, they are considered an ideal candidate for the development of new antibody.
In order to reduce the adverse effects of blocking the CD47-SIRPα interaction, Novamab is developing a CD47 nanobody HuNb1-IgG4 with low affinity to red blood cells.
Preclinical trials have shown that HuNb1-IgG4 enhances macrophage-mediated phagocytosis and shows strong anti-tumor activity in vivo. More importantly, in a test on monkeys, HuNb1-IgG4 did not cause platelet aggregation (Figure 8), showing high safety. In order to further improve the safety and effectiveness of HuNb1-IgG4, Novamab has also established an anti-CD47/CD20 bispecific antibody composed of HuNb1 and rituximab.
Figure 8: The effect of HuNb1-IgG4 on red blood cells in monkeys
Image source: J Nanobiotechnology. 2020; 18: 12.
Drug delivery methods
New drug delivery methods, such as modified nanoparticles, can also reduce the off-target effects of CD47 antibodies.
Multifunctional iron oxide magnetic nanoparticles have been developed as carriers for selective treatment of pancreatic cancer, including the simultaneous delivery of gemcitabine and anti-CD47 antibodies. Compared with free antibodies, treatment by nanoparticles has no additional cytotoxicity, and nanoparticle-mediated delivery of CD47 antibody to tumor cells effectively leads to PDAC cell apoptosis.
Combination therapy strategies
With reference to the development track of PD-(L)1, the combination of CD47 antibodies will undoubtedly be the next research focus.
In fact, a clinical trial of Hu5F9-G4 combined with azacitidine has been mentioned above. When AbbVie introduced TJC4, they also mentioned that they would consider BCL-2 inhibitor (Venclexta) as a potential drug candidate for the pairing.
In terms of scientific rationality, which strategies are solid?
- T cell checkpoint inhibitors: After blocking macrophage-mediated phagocytosis of cancer cells through CD47, these phagocytes can present tumor antigens to T cells, thereby inducing anti-cancer T cell responses. Therefore, combined use with T cell checkpoint inhibitor (PD-(L)1) can further enhance T cell response and enhance efficacy. A number of preclinical studies have shown that CD47 and PD-(L)1 inhibitors can enhance anti-tumor activity. 
Figure 9: Survival rate of mice; HAC: PD-L1 inhibitor
Image source: Nature 545, 495–499 (2017)
It can be seen from table 1 that among the 19 bispecific antibodies, the combination with PD-(L)1 is the the most selected. Existing clinical trials have also carried out the exploration of CD47 antibodies and PD(L)1, such as NCT03922477 and NCT02518958.
The purpose of the NCT02518958 clinical trial is to determine the safety and afficacy of RRx-001 (CD47 antibody) and nivolumab. The objective response rate at week 12 was 25%, and the disease control rate was 67%.
- Other immunotherapies: Such methods can increase tumor specificity and reduce the targeted toxicity of healthy cells expressing CD47. In a mouse model of non-Hodgkin’s lymphoma, the synergy of CD47 antibody and rituximab (CD20 antibody) promoted phagocytosis and eradicated non-Hodgkin’s lymphoma. Another bispecific antibody against CD47 and CD19 (NI-1701) is also designed for B-cell lymphoma and refractory leukemia.
Figure 10: Survival rate of mice
Image source: Cell. 2010 Sep 3; 142(5): 699–713.
- Phagocytosis-promoting drugs: Since CD47 is an anti-phagocytic signal, increasing the tumor-promoting phagocytic signal can play a good synergistic effect with CD47 inhibition. Enhancing the phagocytic signal includes chemotherapy (NCT04313881 and NCT02953509), cytotoxic agents, and drugs that induce apoptosis (NCT04435691).
- CD24: In addition to CD47, Professor Irving Weissman also discovered the second “don’t eat me” signal CD24. Inhibiting CD47 and CD24 at the same time is equivalent to double insurance.
Other challenges and opportunities
- Antigen Sink: The ubiquitous expression of CD47 means that the drug may require large doses or frequent administration to achieve effective therapeutic blockage of CD47. Preclinical studies have shown that induction of phagocytosis requires a receptor occupancy rate of 40-60%. The high therapeutic dose used in the clinical trials of Hu5F9-G4 is based on this consideration.
- Targeting SIRPα: In addition to CD47, many existing pipelines target SIRPα. Compared with CD47, the tissue distribution of SIRPα is more restricted, as a target potentially with reduced toxicity. However, SIRPα is highly expressed in myeloid cells and cells of the central nervous system and peripheral nervous system. Therefore, SIRPα therapy should consider potential nervous system side effects. In addition, due to their sequence similarity, other SIRP family members (SIRPβ and SIRPγ) may cross-react.
- Animal models: When evaluating early stage CD47 project, the selection of animal models is very important. It has been shown that the binding affinity of human CD47 and SirpαNOD model SIRPα is about 10 times that of human SIRPα, so the data is difficult to replicate in human clinical trials. Moreover, SirpαNOD is from an immunocompromised mouse model and cannot restore important mechanisms such as ADCC. The ideal xenograft model should be that SIRPα has a lower affinity for CD47 than NOD but higher than C57BL/6. BALB/c may be such a candidate. 
Biomarkers: Judging from the development experience of PD-(L)1 inhibitors, biomarkers play a key role in precision treatment and also play an important role in the optimization of patient recruitment in clinical trials
 Crit Rev Oncol Hematol. 2020 Aug;152:103014
 MAbs. Feb/Mar 2018;10(2):315-324
 Nature 545, 495–499 (2017)
 Exp Hematol. 2014 Mar;42(3):163-171.e1.
About the author:
Dr. Ginger is the Director of Investment Analysis at MyBioGate and an advisor for the Texas Medical Center and Columbia University Consulting Club. She has worked at the Howard Hughes Medical Institute and MD Anderson Cancer Center. During the period, she participated in the preclinical research of tumor brain metastasis which received funding from the National Institutes of Health RO1, Susan G. Komen, and Japan’s Taiho Pharmaceutical.
Chen Lixing is an investment analyst at MyBioGate. He has a double master’s degree from KGI Graduate School and Drucker School of Management. As a graduate consultant, he provided strategic consulting services for Samumed and SomaLogic.