抗CD47抗体在实体瘤中的安全性特征及管理策略（如IgG4型的magrolimab/Hu5F9）

近年来,以免疫检查点相关信号通路为靶点的新型单抗药物在肿瘤治疗领域显示出了强大潜力。新近研发的Hu5F9-G4、IBI188、Daratumumab均是分别以CD47、CD38为治疗靶点制备的单抗类药物,这些药物在治疗淋巴瘤、多发性骨髓瘤或其他恶性肿瘤方面,显示出了特异性高、临床疗效好等优点。但是CD38和CD47分子不仅表达在肿瘤细胞上,在人红细胞上也有表达,因此经抗-CD38和抗-CD47单抗药物治疗的患者,会因为血液中存在抗-CD38或抗-CD47抗体与红细胞特异性结合,而导致血型鉴定、交叉配血和不规则抗体等输血相容性检测出现假阳性,威胁输血安全。本文阐述了目前抗-CD38单抗和抗-CD47单抗药物对输血相容性检测存在的干扰问题和现有排除策略,为最大限度降低抗-CD38单抗和抗-CD47单抗药物对输血安全造成的影响提供参考。

目的 分析探讨CD47单抗对输血相容性检测的干扰和处理。 方法 血型鉴定采用盐水法和微柱凝胶法；盐水法、间接抗人球蛋白法(IAT法)、凝聚胺法、酸放散处理法进行抗体筛选和鉴定；分别用木瓜蛋白酶、二硫苏糖醇(DTT)处理抗筛细胞，观察能否消除抗CD47抗体的干扰；用O型献血者红细胞与患者血浆进行吸收试验。 结果 CD47单抗干扰ABO血型反定型，且患者抗体筛选和鉴定试验在所有介质中均出现凝集，患者红细胞酸放散液与谱细胞反应均为阳性。酶及DTT不能消除CD47单抗对抗筛试验的影响。异体红细胞吸收4次患者体内抗体效价为2 187。 结论 抗CD47抗体会干扰输血前检测，建议治疗前检测患者的血型并进行抗体筛选等，输注同型匹配性血液，以保障安全输血。

在肿瘤发生发展过程中，肿瘤细胞逃逸了免疫系统的监视；免疫逃逸机制对于研发新的抗肿瘤治疗方案具有重要的意义。免疫治疗旨在激活患者自身的免疫系统，是肿瘤治疗相关研究的前沿，具有广阔的应用前景。CD47是一种广泛表达的细胞表面分子，肿瘤细胞可能借此“别吃我”信号，逃避了肿瘤免疫；肿瘤干细胞中CD47的表达水平甚至比肿瘤细胞更高。通过使用抗CD47抗体阻断CD47-SIRPα通路，从而介导细胞吞噬作用，能够靶向性杀伤肿瘤细胞。现在多项关于CD47靶向治疗的临床试验正在进行中，包括两种单克隆抗体和一种融合蛋白。然而由于使用动物模型不一，可能高估了这些药物的临床疗效预测。现将CD47抗肿瘤治疗相关研究的背景及潜在问题加以综述，并对该治疗的未来应用前景予以展望。

目的 1）探索不同Rh表型细胞与抗CD47的IgG4单克隆抗体TJC4结合的血清学特征;2）明确TJC4对输血相容性试验干扰消除的处理策略。 方法 抽取EDTA-K 2 抗凝非肿瘤且无急慢性基础疾病A型Rh表型分别为CCDee、CcDEe、CcDee、ccDEE、ccDEe、ccdee、Ccdee、CCdee患者静脉血各5 mL,运用微柱凝胶法、盐水法、聚凝胺法分别做DAT（直接抗球蛋白试验）、IAT（间接抗球蛋白试验）、交叉配血等输血相容性试验,检测TJC4体外致敏、独特型抗体ID-ab中和、Immucor AHG干扰去除情况。 结果 1）该研究纳入所有Rh表型细胞被TJC4致敏后盐水、聚凝胺、微柱凝胶三种方法DAT、IAT、交叉配血试验均呈不同凝集强度的阳性反应,且该干扰均能够通过Immucor AHG LISS增强剂法消除;2）独特型抗体ID-ab中和试验去除CD47单抗对ccDEe和ccdee表型血清学实验的干扰需加大剂量。 结论 1）不同Rh表型细胞与TJC4结合后不同输血相容性试验方法呈现不同的血清学差异;2）独特型抗体ID-ab中和试验及缺乏IgG4亚型检测功能的Immucor AHG LISS增强剂法均可消除CD47单抗所致的血清学干扰,但不同Rh表型细胞前者存在剂量差异。

CD47作为“don't eat me”信号在髓系肿瘤表面表达上调，可与巨噬细胞上信号调节蛋白α相结合抑制巨噬细胞吞噬功能，在肿瘤细胞的免疫逃避中起作用。有研究表明，CD47高表达与骨髓增生异常综合征的进展及急性髓系白血病的不良预后相关。CD47作为固有免疫中备受瞩目的免疫检查点，靶向CD47治疗髓系肿瘤的多种药物目前已经进入临床试验阶段，有望成为治疗髓系肿瘤的强有力手段。

肿瘤细胞可以通过多种途径逃避机体免疫系统的识别和清除，如诱导免疫抑制的肿瘤微环境，降低肿瘤细胞的免疫原性等；其中，肿瘤细胞逃避天然免疫系统如巨噬细胞等吞噬细胞清除的机制之一是上调细胞表面“别吃我”（don't eat me）信号的表达。整合素相关蛋白（IAP，即CD47）便是一种重要的自我信号，它通过与巨噬细胞上的配体信号调节蛋白α（SIRPα）结合，进而抑制巨噬细胞对肿瘤细胞的吞噬；此外，在天然免疫细胞如树突状细胞等向适应性免疫T细胞提呈抗原的过程中，CD47也发挥着抑制作用。因此，CD47在肿瘤免疫中发挥着重要的调节作用，靶向CD47是一个潜在的抗肿瘤方向。本文对CD47的抗肿瘤作用进行了详述，包括分子结构、信号转导、调节吞噬与促凋亡作用及成药性考虑等。

针对CTLA-4/B7和PD-1/PD-L1的免疫检查点抑制剂现已广泛应用于临床，其他检查点抑制剂也正在开发中。CD47是广泛表达于正常细胞表面的蛋白质，在肿瘤细胞中呈过表达状态，其受体为髓系抑制性免疫受体SIRPα。阻断CD47与SIRPα之间的相互作用可增强巨噬细胞和中性粒细胞的吞噬作用进而消灭肿瘤细胞。此外，越来越多的证据表明阻断CD47-SIRPα轴还可以增强抗原呈递细胞的功能，从而刺激T细胞介导的抗癌免疫。随着该领域研究的发展，CD47-SIRPα轴的临床应用将会是未来肿瘤免疫治疗的热点之一。

目的 比较靶向CD47的免疫治疗对于免疫健全小鼠口腔-肠道菌群的影响。 方法 构建免疫健全小鼠的结肠癌腹腔转移模型，经腹腔实验组给予抗CD47单克隆抗体或对照组给予PBS溶液进行治疗，用小动物活体成像技术记录肿瘤生长，采用16S rRNA基因测序技术进行口腔-肠道微生物群落组成及多样性分析。 结果 抗CD47单克隆抗体治疗组小鼠口腔微生物Alpha多样性降低，差异有统计学意义（P<0.05）；肠道微生物Alpha多样性则无显著改变；差异物种分析显示，接受治疗小鼠口腔葡萄球菌属（Staphylococcus）、Jeotgalicoccus、球菌属（Sporosarcina）较对照组丰度显著降低，肠道菌群拟杆菌属（Bacteroides）在接受靶向CD47免疫治疗组丰度显著增高。 结论 靶向CD47的免疫治疗对小鼠口腔菌群多样性有着较大的影响，而对肠道菌群物种多样性无显著影响。

PURPOSE Magrolimab is a first-in-class humanized monoclonal antibody against cluster of differentiation 47, an antiphagocytic signal used by cancer cells to evade phagocytosis. Azacitidine upregulates prophagocytic signals on AML cells, further increasing phagocytosis when combined with magrolimab. We report final phase Ib data for magrolimab with azacitidine in patients with untreated AML ineligible for intensive chemotherapy (ClinicalTrials.gov identifier: NCT03248479). PATIENTS AND METHODS Patients with previously untreated AML, including TP53-mutant AML, received magrolimab intravenously as an initial dose (1 mg/kg, days 1 and 4), followed by 15 mg/kg once on day 8 and 30 mg/kg once weekly or every 2 weeks as maintenance. Azacitidine 75 mg/m2 was administered intravenously/subcutaneously once daily on days 1-7 of each 28-day cycle. Primary end points were safety/tolerability and proportion with complete remission (CR). RESULTS Eighty-seven patients were enrolled and treated; 72 (82.8%) had TP53 mutations with a median variant allele frequency of 61% (range, 9.8-98.7). Fifty-seven (79.2%) of TP53-mutant patients had European LeukemiaNet 2017 adverse-risk cytogenetics. Patients received a median of 4 (range, 1-39) cycles of treatment. The most common treatment-emergent adverse events included constipation (49.4%), nausea (49.4%), and diarrhea (48.3%). Thirty (34.5%) experienced anemia, and the median hemoglobin change from baseline to first postdose assessment was –0.9 g/dL (range, –3.6 to 2.5 g/dL). Twenty-eight (32.2%) patients achieved CR, including 23 (31.9%) patients with TP53 mutations. The median overall survival in TP53-mutant and wild-type patients were 9.8 months and 18.9 months, respectively. CONCLUSION Magrolimab with azacitidine was relatively well tolerated with promising efficacy in patients with AML ineligible for intensive induction chemotherapy, including those with TP53 mutations, warranting further evaluation of magrolimab with azacitidine in AML. The phase III randomized ENHANCE-2 (ClinicalTrials.gov identifier: NCT04778397) and ENHANCE-3 (ClinicalTrials.gov identifier: NCT05079230) studies are recruiting frontline patients with AML.

… trial of magrolimab combined with azacitidine for treatment of AML is ongoing.Magrolimab-… Here, we review the interference of magrolimab in RBC typing and serological testing and …

Objective Magrolimab (ONO-7913) is an anti-cluster of differentiation 47 (CD47) monoclonal antibody. This was a phase 1, open-label, uncontrolled, dose-escalation study that assessed the tolerability and safety of intravenous magrolimab (priming dose: 1 mg/kg; maintenance dose: 20 mg/kg [Cohort 1] or 30 mg/kg [Cohort 2]; 28-day treatment cycle) in Japanese adult patients with histologically or cytologically confirmed advanced or metastatic solid tumors, ≥ 1 measurable lesion, an Eastern Cooperative Oncology Group performance score of 0–1, and an expected survival ≥ 3 months. Results Seven patients were enrolled and received magrolimab (Cohort 1, n = 4; Cohort 2, n = 3) with the median follow-up of 170.0 (34–491) days. All 7 patients discontinued the magrolimab monotherapy, and 4 (57.1%) patients completed the study as per the protocol. No dose-limiting toxicities were observed in both cohorts, and the maximum tolerated dose was not reached. All patients experienced treatment-emergent adverse events (TEAEs) but not any serious adverse events, TEAEs leading to treatment discontinuation or interruption, or TEAE-related deaths. No complete or partial responses occurred, while the disease control rate was 42.9% (best overall response: stable disease, n = 3; disease progression, n = 3; not evaluable, n = 1). Magrolimab was well tolerated in Japanese patients with advanced or metastatic solid tumors. Trial registration: NCT04403308, submitted on May 21, 2020. Supplementary Information The online version contains supplementary material available at 10.1186/s13104-026-07787-6.

&lt;div&gt;Abstract&lt;p&gt;The CD47/SIRPα axis serves as a “do not eat me” signal, protecting normal cells from phagocytosis, but in the meantime, enabling immune evasion by tumor cells. Whereas substantial progress has been made in developing CD47 antagonists, achieving a balance between hematotoxicity and antitumor efficacy remains a critical challenge. In this study, we demonstrated that the bivalent anti-CD47 antibody, Hu5F9, caused severe anemia in both human CD47 knock-in mice lacking the CD47/SIRPα signal and human CD47/SIRPα double knock-in mice with complete signal, suggesting the CD47/SIRPα signal is not essential. Moreover, the single-arm CD47 antibody Hu5F9/gp120 exhibited only mild red blood cell (RBC) destruction &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt;. These findings reveal that RBC toxicity induced by anti-CD47 antibodies is determined by the bivalency of the antibody rather than the CD47/SIRPα signal engagement. Based on this, we engineered TJH2201, a novel anti-CD47 antibody that avoids RBC agglutination while retaining high-affinity CD47 binding, robust signaling blockade, and enhanced pro-phagocytosis activity &lt;i&gt;in vitro&lt;/i&gt;. In xenograft models with Raji and MV-4-11 cells, TJH2201 demonstrated potent antitumor activity without inducing body weight loss. These results suggest that TJH2201 is a promising CD47 antagonist that balances antitumor efficacy and hematologic safety, providing a new therapeutic approach for CD47-expressing malignancies.&lt;/p&gt;&lt;/div&gt;

CD47 is a surface glycoprotein expressed by host cells to impede phagocytosis upon binding to macrophage SIRPα, thereby represents an immune checkpoint known as the “don't-eat-me” signal. However, accumulating evidence shows that solid and hematologic tumor cells overexpress CD47 to escape immune surveillance. Thus, targeting the CD47-SIRPa axis by limiting the activity of this checkpoint has emerged as a key area of research. In this review, we will provide an update on the landscape of CD47-targeting antibodies for hematological malignancies, including monoclonal and bi-specific antibodies, with a special emphasis on agents in clinical trials and novel approaches to overcome toxicity.

Extensive clinical and experimental evidence suggests that macrophages play a crucial role in cancer immunotherapy. Cluster of differentiation (CD) 47, which is found on both healthy and malignant cells, regulates macrophage-mediated phagocytosis by sending a "don't eat me" signal to the signal regulatory protein alpha (SIRPα) receptor. Increasing evidence demonstrates that blocking CD47 interaction with SIRPα can enhance cancer cell clearance by macrophages. Additionally, inhibition of CD47/SIRPα interaction can increase antigen cross-presentation, leading to T-cell priming and an activated adaptive antitumor immune response. Therefore, inhibiting CD47/SIRPα axis has a significant impact on tumor immunotherapy. Studies on CD47 monoclonal antibodies are at the forefront of research, and impressive results have been obtained. Nevertheless, hematotoxicity, especially anemia, has become the most common adverse effect of the CD47 monoclonal antibody. More specific targeted drugs ( i.e. , bispecific antibodies, SIRPα/Fc fusion protein antibodies, and small-molecule inhibitors) have been developed to reduce hematotoxicity. Here, we review the present usage of CD47 antagonists for the treatment of lymphomas and hematologic neoplasms from the perspectives of structure, function, and clinical trials, including a comprehensive overview of the drugs in development.

Abstract Background The CD47‐signal regulatory protein alpha (SIRPα) ‘don't eat me’ signalling axis is perhaps the most prominent innate immune checkpoint to date. However, from initial clinical trials, it is evident that monotherapy with CD47‐SIRPα blocking has a limited therapeutic effect at the maximum tolerated dose. Furthermore, treatment is associated with severe side effects, most notably anaemia, that are attributable to the ubiquitous expression of CD47. Nevertheless, promising clinical responses have been reported upon combination with the tumour‐targeting antibody rituximab or azacytidine, although toxicity issues still hamper clinical application. Main body Here, we discuss the current state of CD47‐SIRPα blocking therapy with a focus on limitations of current strategies, such as depletion of red blood cells. Subsequently, we focus on innovations designed to overcome these limitations. These include novel antibody formats designed to selectively target CD47 on tumour cells as well as tumour‐targeted bispecific antibodies with improved selectivity. In addition, the rationale and outcome of combinatorial approaches to improve the therapeutic effect of CD47 blockade are discussed. Such combinations include those with tumour‐targeted opsonizing antibodies, systemic therapy, epigenetic drugs, other immunomodulatory T‐cell‐targeted therapeutics or dual immunomodulatory CD47 bispecific antibodies. Conclusion With these advances in the design of CD47‐SIRPα‐targeting therapeutic strategies and increasing insight into the mechanism of action of this innate checkpoint, including the role of adaptive immunity, further advances in the clinical application of this checkpoint can be anticipated.

… A triplet regimen of magrolimab + azacitidine + venetoclax has shown promising activity in patients with AML. We aim to evaluate the efficacy, safety, and tolerability of magrolimab + …

Monoclonal antibody (MoAb) therapy has been increasingly used in recent years for hematologic malignancies. The MoAbs anti-CD38 and anti-CD47 are immunoglobulins directed against epitopes that are highly expressed not only on cancer cells, but also on red blood cells (RBCs), as well as platelets. Additionally, producing an off-target effect interferes in pre-transfusion testing, having the potential to unchain hemolytic anemia. Blood banks must assure the availability and safety of blood products for patients in need. Thus, MoAbs have become a challenge for blood banks, since methods to overcome interferences must be adopted. Several strategies have been proposed to mitigate pan-reactivity in pre-transfusion indirect antiglobulin tests, such as the treatment of reagent RBCs with enzymes or reducing agents, allogeneic RBC adsorptions, and drug-specific neutralization assays. All of these have some kind of limitation. This review summarizes the interferences of MoAbs in pre-transfusion testing, focusing on the available strategies to mitigate them in order to provide a safe transfusion.

Drugs such as daratumumab (Darzalex, anti‐CD38) and Hu5F9‐G4 (magrolimab, anti‐CD47) may interfere with red blood cell compatibility testing as CD38 and CD47 are expressed on red blood cells.

Acute myeloid leukemia (AML), a heterogeneous and aggressive hematologic malignancy, remains challenging to treat due to high relapse rates, chemotherapy resistance, and the immunosuppressive tumor microenvironment (TME). While traditional therapies like chemotherapy and hematopoietic stem cell transplantation have improved outcomes, their efficacy is often limited by toxicity and disease recurrence. Recent advancements in antibody engineering have revolutionized AML immunotherapy, offering precision-targeted strategies to overcome these barriers. This narrative review explores the transformative role of monoclonal antibodies (mAbs), antibody–drug conjugates (ADCs), and bispecific antibodies (bsAbs) in redirecting immune effector cells, blocking immune checkpoints, and eradicating leukemic stem cells (LSCs). Key innovations include CD33-targeted gemtuzumab ozogamicin, CD123-directed bispecific engagers, and anti-CD47 agents that disrupt “don’t eat me” signals. We highlight breakthroughs in antibody design—such as Fc optimization, trispecific constructs, and conditionally active biologics—that enhance specificity while minimizing on-target off-tumor toxicity. Clinical trials demonstrate promising results, including improved remission rates and survival in refractory/relapsed AML when combining antibodies with hypomethylating agents, venetoclax, or checkpoint inhibitors. However, challenges persist, including AML’s genetic heterogeneity, adaptive immune evasion, and cytokine release syndrome (CRS) risks. Emerging strategies such as biomarker-driven personalization, TME modulation, and engineered NK-cell engagers are poised to address these limitations. By integrating preclinical insights with clinical data, this review underscores the potential of antibody-based combinatorial regimens to redefine AML therapy, offering durable responses and bridging the gap to curative approaches.

Monoclonal antibody (mAb) therapy targeting CD38 and CD47 antigens expressed on cancer cells has transformed therapy options for patients with multiple myeloma as well as other haematological and non-haematological malignancies. While the on target effects of these new drugs highlight the promise of precision cancer therapeutics, the unintended, off target binding of drugs to red blood cells (RBCs) and platelets has required transfusion service laboratories (TSL) and immunohaematology reference laboratories (IRL) to innovate and rapidly set up processes and testing protocols to overcome the significant interference in routine pre-transfusion tests caused by these agents. Binding of anti-CD38 and anti-CD47 drugs to reagent RBCs leads to false positive pan-agglutination during the antihuman globulin phase of testing, making it difficult to rule out underlying alloantibodies, and leading to delays in setting up compatible units for RBC transfusion. Anti-CD47 agents can also interfere with ABO/Rh typing studies. Several methods to successfully mitigate interference have been described, such as treatment of reagent RBCs with reducing agents or enzymes, allogeneic RBC adsorption studies and drug specific neutralisation assays; all methods have limitations. TSLs should select an approach that best fits their workflow and expertise and takes into consideration their level of access to specialised outside testing, local blood supplier capabilities, and the type of patient population served. For platelet refractory patients, samples should be tested by platelet antibody assays that are known to be unaffected by drug therapy. RBC transfusion support for multiple myeloma patients receiving anti-CD38 or anti-CD47 drugs can be optimised by establishing good communication between the clinical teams and TSLs, building electronic notification processes, and ensuring timely completion of baseline pre-transfusion testing and RBC phenotype/genotype prior to starting therapy. Staff education, standardisation of laboratory mitigation measures, and implementation of testing algorithms that consider mAb-induced interference when working up a pan-agglutinin help to significantly decrease delays that would otherwise result if standard methods were employed to complete antibody identification studies.

Immune evasion is a hallmark of cancer and there is mounting evidence that the tumor microenvironment (TME) plays a role in the pathogenesis of haematologic malignancies as well as treatment resistance. Macrophages play a central role in anti-tumor immunity, and dysregulation of macrophage mediated phagocytosis has recently emerged as a key player in blood cancers. The integrin associated protein CD47 is expressed in a variety of cancers and interacts with its ligand, signal regulatory protein α (SIRPα) expressed on macrophages, resulting in down regulation of macrophage-mediated phagocytosis. CD47 is highly expressed in various cancers including multiple myeloma (MM). It is therefore postulated that blockade of the CD47-SIRPα immune checkpoint has the potential to ‘re-awaken’ macrophage mediated phagocytosis of MM plasma cells. In this review, we provide our perspective on the key pre-clinical data supporting the CD47-SIRPα axis as a therapeutic target in MM. We subsequently discuss the ongoing clinical trials which may provide the basis for future clinical translation of these agents. We also highlight key gaps in our knowledge of macrophage biology in MM which need to be addressed by future research. Finally, we present potential future directions for translational research and personalized application of macrophage-based immunotherapy in MM.

Highlights • High M2d macrophage infiltration correlates with poor prognosis in colorectal cancer (CRC).• Hu5F9-G4 promotes M2d-to-M1 macrophage polarization, inhibits CRC cell proliferation and migration, and enhances autophagy in macrophages.• In vivo studies show that Hu5F9-G4 reduces tumor growth and modulates immune cell populations in CRC-bearing mice.• Integrative analyses identify CDKN1A and MAP1LC3B as potential therapeutic targets associated with the autophagy pathway.• Hu5F9-G4 has therapeutic potential in CRC by modulating macrophage polarization, tumor progression, and autophagy-related pathways.

TPS584 Background: Patients with solid tumors who progress on standard chemotherapy and/or immune checkpoint inhibitors have limited efficacy with existing standard-of-care chemotherapy options (objective response rates [ORRs] ̃10%). These patients have a significant unmet medical need. Novel agents that can safely enhance treatment efficacy are urgently needed. Magrolimab is a first-in-class monoclonal antibody that blocks the macrophage inhibitory immune checkpoint CD47, a “do not eat me” signal overexpressed on tumor cells. Preclinical studies provide compelling evidence that magrolimab triggers phagocytosis and eliminates cancer cells from human solid tumors and hematologic malignancies. Magrolimab has demonstrated clinical activity in both hematologic and solid tumor malignancies. Chemotherapeutic agents, including taxanes, enhance prophagocytic signals on tumor cells, leading to synergistic antitumor activity when combined with magrolimab. This study (NCT04827576) is evaluating the safety, tolerability, and efficacy of magrolimab with docetaxel in relapsed/refractory (R/R) metastatic urothelial cancer (mUC), non-small-cell lung cancer (mNSCLC), and small-cell lung cancer (mSCLC). Methods: This Phase 2, open-label, multi-arm study (NCT04827576) consists of a safety run-in cohort and a Phase 2 cohort. Eligible patients are ≥18 years old with chemotherapy- and/or immunotherapy-refractory mUC, mNSCLC, or mSCLC. Magrolimab is administered intravenously (IV) with an initial 1-mg/kg priming dose to mitigate on-target anemia, followed by a 30-mg/kg dose during cycle 1 (cycles are 21 days) in the safety run-in to identify any dose-limiting toxicities (DLTs) and determine a recommended Phase 2 dose (RP2D). De-escalation may occur for DLTs per protocol. In Phase 2, following the priming dose on day 1, the highest acceptable dose of magrolimab will be administered on days 8 and 15 of cycle 1; days 1, 8, and 15 of cycle 2; and day 1 for cycles 3 and beyond. Docetaxel 75 mg/m2 is administered IV on day 1 of each cycle for all study participants. Patients may continue treatment until unacceptable toxicity, progressive disease by RECIST 1.1, or patient/investigator choice to discontinue. The primary endpoints are incidence of adverse events (safety and Phase 2 cohorts) and ORR (Phase 2). Secondary endpoints (Phase 2) are progression-free survival, duration of response, and overall survival. Exploratory endpoints are to evaluate the pharmacodynamic, mechanism of action, and/or therapeutic response of biomarkers in blood and tumor biopsy samples and to explore biomarkers that may predict response to therapy. Enrollment began in August 2021. Planned enrollment is approximately 116 patients, and as of October 1, 2021 recruitment is ongoing. Clinical trial information: NCT04827576.

2516 Background: AO-176 is a humanized IgG2 antibody that specifically targets CD47. Expressed by multiple tumor types, CD47 binds to signal regulatory protein a (SIRPa) on phagocytes, including macrophages and dendritic cells. The CD47-SIRPa complex results in a “don’t eat me” signal that allows the tumor to escape removal by the innate immune system, disabling the generation of an adaptive immune response. The differentiated mechanisms of action of AO-176 include promotion of phagocytosis, direct tumor cell killing through programmed cell death type III and induction of damage associated molecular patterns/immunogenic cell death, preferentially binding to tumor cells vs. normal cells, and enhanced binding at an acidic pH as found in tumor microenvironments. AO-176 has negligible binding to RBCs. Methods: In a phase 1/2 first-in-human study (NCT03834948) of AO-176, pts with advanced solid tumors associated with high CD47 expression and an ECOG PS of 0-1 were enrolled into escalating dose cohorts of AO-176 given IV every 7 days. Objectives included evaluation of safety, dose-limiting toxicity (DLT) and recommended phase 2 dose (RP2D), antitumor activity, pharmacokinetic (PK) parameters and exploratory biomarkers. Results: As of 4 Jan 2021, 27 pts were enrolled (median age 64 years; 67% female; 67% ECOG PS 1; median [range] of 4 [1-7] prior therapies for metastatic disease). Dose levels of 1, 3, 10, 20 and 20 (using step-up dosing) mg/kg were evaluated in >250 infusions. Most common (>10%) treatment-related adverse events (TRAEs) of any grade were thrombocytopenia and infusion-related reaction (IRR) (33% each), anemia (22%) with no evidence of hemolysis, nausea (19%), and fatigue (15%). The only G3+ TRAE occurring in >10% of pts was asymptomatic, brief thrombocytopenia (22%). No platelet transfusions were given. DLTs included IRRs in 2 pts dosed at 20 mg/kg, and asymptomatic thrombocytopenia and a cerebrovascular accident in 1 pt each in the 20 mg/kg step-up cohort. The RP2D was 10 mg/kg. Implementation of additional pre-medication and a 6-hr infusion duration in cycle 1 eliminated subsequent IRRs. Dexamethasone tapering and shortening of the infusion duration to 2 hrs was successful in all pts after cycle 1. Interim PK analysis of AO-176 demonstrated consistent exposure with linear PK. The T1/2 was ̃5 days. One pt with endometrial carcinoma who had not responded to any of 4 prior systemic regimens had a confirmed PR and remains on study for >1 year. 7 pts had SD as a best response, with 2 pts (endometrial carcinoma, gastric cancer) on study for >6 mos. Conclusions: AO-176 is a well-tolerated, differentiated anti-CD47 therapeutic. Durable anti-tumor activity was observed. Evaluations of AO-176 in combination with paclitaxel in pts with select solid tumors (NCT03834948) and as a single-agent in pts with multiple myeloma (NCT04445701) are ongoing. Clinical trial information: NCT03834948.

Strategies for targeting CD47 are becoming a hot spot of cancer immunotherapy. However the ubiquitous expression of CD47, especially on the RBC, makes the targeted therapy facing safety risk issues. So, how to balance the safety and efficacy during CD47 inhibition is currently a major question. We had reported an anti-CD47 antibody ZF1 with potent anti-tumor effect. In this study, we further developed and assessed a novel fully human anti-CD47 antibody, AMMS4-G4, derived from ZF1 using affinity maturation. AMMS4-G4 exhibited equivalent anticancer effects with Hu5F9-G4, a humanized anti-CD47 antibody in clinical trial, on the potential of inducing significant phagocytosis of tumor cells in vitro and prolonging the survival of leukemia xenografted mice. Additionally, AMMS4-G4 significantly inhibited the growth of grafted solid tumors by enhancing macrophage infiltration and modestly enhanced the anti-tumor activity of opsonizing antibody and antiangiogenic therapy. In cynomolgus monkeys, AMMS4-G4 was safely administered, was well tolerated at doses of 30 and 60 mg/kg, and did not produce serious adverse events, except for the reversible anemia, which was observed after 3 days and started to recover from 9 days later. Remarkably, it was proved by in vitro assay that Hu5F9-G4 induced RBC hemagglutination which wasn't observed in AMMS4-G4. On the whole, AMMS4-G4 was demonstrated to be a promising candidate with great potential and safe profile for cancer immunotherapy.

Rationale: Many cancers have evolved different mechanisms to evade immune surveillance. Macrophages, the innate defense of the immune system, are limited in their phagocytosis by CD47 anti-phagocytic signaling expressed on the surface of tumor cells. Although the CD47 monoclonal antibody (aCD47) strategy has been extensively studied in clinical trials, the depletion of aCD47 by red blood cells (RBCs) and the resulting hematotoxicity have impeded their application in tumor treatment. Methods: Here, we reported an injectable hydrogel scaffold that allowed for local delivery of small-molecule inhibitor PQ912. The biodegradable hydrogel scaffold (PQ/PB-Gel) was formed by rapid cross-linking of tetra-armed PEG succinimidyl succinate (Tetra-PEG-SS) solution and alkalescent bovine serum albumin (BSA) solution through ammonolysis reaction. Results: PQ/PB-Gel had excellent effect on inhibiting local recurrence of two kinds of tumors. The hydrogel system inhibited the generation of “don't eat me” signals during the treatment cycle by inhibiting the expression of newly generated neoplastic CD47. Thus, it avoided adverse reactions such as erythrocytopenia after the use of aCD47 in terms of safety. After the “don't eat me” signal was blocked the clearance and recognition of cancer cells by macrophages and antigen-presenting cells were enhanced, sequentially systemic immune response was activated and further memory T lymphocyte (T cell) formation was induced. Conclusions: PQ/PB-Gel had a simple preparation and administration method, low production cost, excellent efficacy and low toxicity, so it had good practicability. This might provide a safe alternative strategy for aCD47 for inhibit local tumor recurrence and distal metastasis in postoperative immunotherapy.

Background CD47 is a widely expressed transmembrane glycoprotein that delivers an antiphagocytic signal on macrophages through its interaction with SIRPα. CD47 is highly expressed in cancer cells and its overexpression is correlated with poor prognosis. CD47 blocking antibodies are actively being developed worldwide for cancer therapy, and the most challenging concern is associated with hematotoxicity. Ligufalimab (AK117) is a novel humanized IgG4 anti-CD47 antibody without hemagglutination effect. Blockade of CD47-SIRPα pathway by AK117 leads to a promising therapeutic strategy for cancer treatment with unique safety features. Methods AK117 was discovered through a screening hierarchy excluding hemagglutination. AK117 was characterized by detecting CD47-SIRPα blocking potential. Its effect on human red blood cells was examined and the mechanism of its binding with erythrocytes was studied. The abilities of AK117 and its combination with various opsonizing antibodies to promote macrophage-dependent phagocytosis of multiple human tumor cells were determined using fluorescence microscopy and flow cytometry. In vivo, the antitumor efficacy of AK117 monotherapy and combination with AK112 (an anti-PD-1/VEGF-A bispecific antibody) was assessed in a variety of xenograft models. Toxicologic studies were evaluated in non-human primates. Results AK117 bound to CD47 with high affinity and blocked the CD47-SIRPα interaction. AK117 did not induce hemagglutination and showed significantly lower degree of erythrophagocytosis compared with Hu5F9-G4, and this mechanism of hemagglutination resistance might be related to the binding conformation. AK117 enhanced macrophage-mediated phagocytosis in both hematologic cancer and solid tumor cell lines as a single agent or in combination with cetuximab and rituximab in vitro, respectively. The antitumor effects of AK117 as a single agent or in combination with AK112 were also encouraging in various xenograft models. In non-human primates, AK117 showed less hematotoxicity compared with Hu5F9-G4. Conclusions AK117 eliminated hemagglutination and also enabled to maintain full effectiveness of CD47 blockade on tumor cells, which resulted in excellent antitumor efficacy and favorable safety profile of AK117. A series of clinical trials of AK117 as a therapeutic agent in combination with various agents such as AK112 are in progress for the treatment of multiple hematologic malignancies and solid tumors.

Background Blocking the CD47 “don’t eat me”-signal on tumor cells with monoclonal antibodies or fusion proteins has shown limited clinical activity in hematologic malignancies and solid tumors thus far. Main side effects are associated with non-tumor targeted binding to CD47 particularly on blood cells. Methods We present here the generation and preclinical development of NILK-2401, a CEACAM5×CD47 bispecific antibody (BsAb) composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format). Results NILK-2401 is a fully human BsAb binding the CEACAM5 N-terminal domain on tumor cells by its lambda light chain arm with an affinity of ≈4 nM and CD47 with its kappa chain arm with an intendedly low affinity of ≈500 nM to enabling tumor-specific blockade of the CD47-SIRPα interaction. For increased activity, NILK-2401 features a functional IgG1 Fc-part. NILK-2401 eliminates CEACAM5-positive tumor cell lines (3/3 colorectal, 2/2 gastric, 2/2 lung) with EC50 for antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity ranging from 0.38 to 25.84 nM and 0.04 to 0.25 nM, respectively. NILK-2401 binds neither CD47-positive/CEACAM5-negative cell lines nor primary epithelial cells. No erythrophagocytosis or platelet activation is observed. Quantification of the pre-existing NILK-2401-reactive T-cell repertoire in the blood of 14 healthy donors with diverse HLA molecules shows a low immunogenic potential. In vivo, NILK-2401 significantly delayed tumor growth in a NOD-SCID colon cancer model and a syngeneic mouse model using human CD47/human SIRPα transgenic mice and prolonged survival. In cynomolgus monkeys, single doses of 0.5 and 20 mg/kg were well tolerated; PK linked to anti-CD47 and Fc-binding seemed to be more than dose-proportional for Cmax and AUC0-inf. Data were validated in human FcRn TG32 mice. Combination of a CEACAM5-targeting T-cell engager (NILK-2301) with NILK-2401 can either boost NILK-2301 activity (Emax) up to 2.5-fold or allows reaching equal NILK-2301 activity at >600-fold (LS174T) to >3,000-fold (MKN-45) lower doses. Conclusion NILK-2401 combines promising preclinical activity with limited potential side effects due to the tumor-targeted blockade of CD47 and low immunogenicity and is planned to enter clinical testing.

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Recently, many immunosuppressive checkpoints such as PD-L1, CTLA-4 and CD47, were identified in succession and serve as potential immunotherapy targets in human cancers. Among them, CD47, a 'marker-of-self' protein that is overexpressed broadly across tumor types, is emerging as a novel potent macrophage immune checkpoint for cancer immunotherapy. In this review, we highlight the prominent role of CD47 as a 'don't-eat-me' signal that inhibits macrophage phagocytosis for immune evasion of a tumor and presents the opportunities and challenges for CD47 inhibitors both as monotherapy and in combination treatments for hematological cancers and solid tumors; some of these agents are currently in clinical trials.

CD47 is a widely expressed cell surface protein that functions as a regulator of 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, SIRP-alpha, which in turn delivers an inhibitory signal for phagocytosis. We previously found increased expression of CD47 on primary human acute myeloid leukemia (AML) stem cells, and demonstrated that blocking monoclonal antibodies directed against CD47 enabled the phagocytosis and elimination of AML, non-Hodgkin’s lymphoma (NHL), and many solid tumors in xenograft models. Here, we report the development of a humanized anti-CD47 antibody with potent efficacy and favorable toxicokinetic properties as a candidate therapeutic. A novel monoclonal anti-human CD47 antibody, 5F9, was generated, and antibody humanization was carried out by grafting its complementarity determining regions (CDRs) onto a human IgG4 format. The resulting humanized 5F9 antibody (Hu5F9-G4) bound monomeric human CD47 with an 8 nM affinity. Hu5F9-G4 induced potent macrophage-mediated phagocytosis of primary human AML cells in vitro and completely eradicated human AML in vivo, leading to long-term disease-free survival of patient-derived xenografts. Moreover, Hu5F9-G4 synergized with rituximab to eliminate NHL engraftment and cure xenografted mice. Finally, toxicokinetic studies in non-human primates showed that Hu5F9-G4 could be safely administered intravenously at doses able to achieve potentially therapeutic serum levels. Thus, Hu5F9-G4 is actively being developed for and has been entered into clinical trials in patients with AML and solid tumors (ClinicalTrials.gov identifier: NCT02216409).

Cluster of differentiation 47 (CD47) (also known as integrin-associated protein) is a ubiquitously expressed glycoprotein of the immunoglobulin superfamily that plays a critical role in self-recognition. Various solid and hematologic cancers exploit CD47 expression in order to evade immunological eradication, and its overexpression is clinically correlated with poor prognoses. One essential mechanism behind CD47-mediated immune evasion is that it can interact with signal regulatory protein-alpha (SIRPα) expressed on myeloid cells, causing phosphorylation of the SIRPα cytoplasmic immunoreceptor tyrosine-based inhibition motifs and recruitment of Src homology 2 domain-containing tyrosine phosphatases to ultimately result in delivering an anti-phagocytic—“don’t eat me”—signal. Given its essential role as a negative checkpoint for innate immunity and subsequent adaptive immunity, CD47-SIRPα axis has been explored as a new target for cancer immunotherapy and its disruption has demonstrated great therapeutic promise. Indeed, CD47 blocking antibodies have been found to decrease primary tumor size and/or metastasis in various pre-clinical models. In this review, we highlight the various functions of CD47, discuss anti-tumor responses generated by both the innate and adaptive immune systems as a consequence of administering anti-CD47 blocking antibody, and finally elaborate on the clinical potential of CD47 blockade. We argue that CD47 is a checkpoint molecule for both innate and adaptive immunity for tumor evasion and is thus a promising target for cancer immunotherapy.

… range of patient experiences and adverse events. When traditional … Anti-CD47 combinations may also work particularly well in … suited to treating solid tumours due to its potency profile. A …

CD47 is an antiphagocytic molecule that acts via ligation to signal regulatory protein alpha on phagocytes; its enhanced expression and therapeutic targeting have recently been reported for several malignancies. However, CD47 expression in gastric cancer is not well documented. Immunohistochemical expression of CD47 in surgical specimens was investigated. Expression of CD47 and CD44, a known gastric cancer stem cell marker, were investigated in gastric cancer cell lines by flow cytometry. MKN45 and MKN74 gastric cancer cells were sorted by fluorescence‐activated cell sorting according to CD44 and CD47 expression levels, and their in vitro proliferation, spheroid‐forming capacity, and in vivo tumorigenicity were studied. In vitro phagocytosis of cancer cells by human macrophages in the presence of a CD47 blocking monoclonal antibody (B6H12) and the survival of immunodeficient mice intraperitoneally engrafted with MKN45 cells and B6H12 were compared to experiments using control antibodies. Immunohistochemistry of the clinical specimens indicated that CD47 was positive in 57 out of 115 cases, and its positivity was an independent adverse prognostic factor. Approximately 90% of the MKN45 and MKN74 cells expressed CD47 and CD44. CD47hi gastric cancer cells showed significantly higher proliferation and spheroid colony formation than CD47lo, and CD44hiCD47hi cells showed the highest proliferation in vitro and tumorigenicity in vivo. B6H12 significantly enhanced in vitro phagocytosis of cancer cells by human macrophages and prolonged the survival of intraperitoneal cancer dissemination in mice compared to control antibodies. In conclusion, CD47 is an adverse prognostic factor and promising therapeutic target in gastric cancer.

Magrolimab (Hu5F9‐G4) is a first‐in‐class anti‐CD47 IgG4 monoclonal antibody, with potential applications in several malignancies including myelodysplastic syndrome. CD47 blockade in malignancy has been shown to promote antitumour effects. However, the ubiquity of CD47 on red blood cells can result in interference in pretransfusion immunohaematology investigations and hinder timely provision of red blood cell units, with potential to mask clinically significant alloantibodies. We reviewed the literature for pretransfusion interference seen with magrolimab and methods to circumvent potential issues, and sought to provide clinical and laboratory recommendations for safe local transfusion practices. These recommendations are based on expert opinion and available literature, including the Victorian Senior Transfusion Scientist working group and professional societies and organisations (Australian & New Zealand Society of Blood Transfusion and Lifeblood representatives), to establish consensus recommendations. Interference in the ABO group and antibody screen can occur, and baseline immunohaematology testing prior to magrolimab therapy is critical. Antibody screening using an antihuman globulin reagent that does not detect human IgG4 subclass may distinguish magrolimab interference from an underlying alloantibody in patient plasma. Clear and consistent protocols for laboratories and close communication with clinicians are paramount to facilitate timely and safe transfusion support for patients receiving magrolimab therapy. As local transfusion laboratories gain experience with magrolimab, this will assist in our understanding and comfort in managing these patients.

… adverse reactions under these specific study conditions. … target not just for hematologic cancers and solid tumors,1 but also for viral … Anti-CD47 antibody and rituximab efficacy in a B-cell …

CD47, serving as an intrinsic immune checkpoint, has demonstrated efficacy as an anti-tumor target in hematologic malignancies. Nevertheless, the clinical relevance of CD47 in gastric cancer and its potential as a therapeutic target remains unclear. The expression of CD47 in clinical gastric cancer tissues was assessed using immunohistochemistry and Western blot. Patient-derived cells were obtained from gastric cancer tissues and co-cultured with macrophages derived from human peripheral blood mononuclear cells. Flow cytometry analyses were employed to evaluate the rate of phagocytosis. Humanized patient-derived xenografts (Hu-PDXs) models were established to assess the efficacy of anti-CD47 immunotherapy or the combination of anti-CD47 and anti-VEGF therapy in treating gastric cancer. The infiltrated immune cells in the xenograft were analyzed by immunohistochemistry. In this study, we have substantiated the high expression of CD47 in gastric cancer tissues, establishing a strong association with unfavorable prognosis. Through the utilization of SIRPα-Fc to target CD47, we have effectively enhanced macrophage phagocytosis of PDCs in vitro and impeded the growth of Hu-PDXs. It is noteworthy that anti-CD47 immunotherapy has been observed to sustain tumor angiogenic vasculature, with a positive correlation between the expression of VEGF and CD47 in gastric cancer. Furthermore, the successful implementation of anti-angiogenic treatment has further augmented the anti-tumor efficacy of anti-CD47 therapy. In addition, the potent suppression of tumor growth, prevention of cancer recurrence after surgery, and significant prolongation of overall survival in Hu-PDX models can be achieved through the simultaneous targeting of CD47 and VEGF using the bispecific fusion protein SIRPα-VEGFR1 or by combining the two single-targeted agents. Our preclinical studies collectively offer substantiation that CD47 holds promise as a prospective target for gastric cancer, while also highlighting the potential of anti-angiogenic therapy to enhance tumor responsiveness to anti-CD47 immunotherapy.

Accumulating evidence indicates that a small subset of cancer cells, termed the tumor-initiating cells or cancer stem cells (CSCs), construct a reservoir of self-sustaining cancer cells with the characteristic ability to self-renew and maintain the tumor mass. The CSCs play an important role in the tumor initiation, development, relapse, metastasis, and the ineffectiveness of conventional cancer therapies. CD47 is a ligand for signal-regulatory protein-α expressed on phagocytic cells and functions to inhibit phagocytosis. This study was to explore if the expression of CD47 is the mechanism used by lung cancer cells, especially CSCs, to escape phagocytosis in vitro and in vivo. Here, we selected CD133 as the marker for lung CSCs according to previous reports. We analyzed lung cancer and matched adjacent normal (non-tumor) tissue and revealed that CD47 is overexpressed on lung cancer cells, especially on lung CSCs. The mRNA expression levels of CD47 and CD133 correlated with a decreased probability of survival for multiple types of lung cancer. Blocking CD47 function with anti-CD47 antibodies enabled macrophage phagocytosis of lung cancer cells and lung CSCs. Anti-CD47 antibodies inhibited tumor growth in immunodeficient mouse xenotransplantation models established with lung cancer cells or lung CSCs and improved survival in tumor-bearing animals. These data indicate that CD47 is a valid target for cancer therapies, especially for anti-CSC therapies.