Assuntos
Antígeno CD47 , Neoplasias , Receptores Imunológicos , Humanos , Antígeno CD47/metabolismo , Antígeno CD47/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Receptores Imunológicos/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Animais , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos/uso terapêutico , Terapia de Alvo Molecular/métodos , Antígenos de DiferenciaçãoRESUMO
The antitumor strategies based on innate immunity activation have become favored by researchers in recent years. In particular, strategies targeting antiphagocytic signaling blockade to enhance phagocytosis have been widely reported. For example, the addition of prophagocytic signals such as calreticulin could make the strategy significantly more effective. In this study, an antitumor strategy that combines photodynamic therapy (PDT) with CD47 blockade has been reported. This approach promotes the maturation of dendritic cells and the presentation of tumor antigens by PDT-mediated tumor immunogenic cell death, as well as the enhancement of cytotoxic T lymphocyte infiltration in tumor areas and the phagocytic activity of phagocytes. Furthermore, the downregulation and blockage of CD47 protein could further promote phagocytic activity, strengthen the innate immune system, and ultimately elevate the antitumor efficacy and inhibit tumor metastasis.
Assuntos
Antígeno CD47 , Células Dendríticas , Fagocitose , Fotoquimioterapia , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/metabolismo , Fotoquimioterapia/métodos , Animais , Camundongos , Fagocitose/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Linhagem Celular Tumoral , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/patologia , Camundongos Endogâmicos C57BL , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Humanos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Camundongos Endogâmicos BALB C , FemininoRESUMO
Background: CD38 and CD47 are expressed in many hematologic malignancies, including multiple myeloma (MM), B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia (ALL), and B-cell chronic lymphocytic leukemia (CLL). Here, we evaluated the antitumor activities of CD38/CD47 bispecific antibodies (BsAbs). Methods: Five suitable anti-CD38 antibodies for co-targeting CD47 and CD38 BsAb were developed using a 2 + 2 "mAb-trap" platform. The activity characteristics of the CD38/CD47 BsAbs were evaluated using in vitro and in vivo systems. Results: Using hybridoma screening technology, we obtained nine suitable anti-CD38 antibodies. All anti-CD38 antibodies bind to CD38+ tumor cells and kill tumor cells via antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Five anti-CD38 antibodies (4A8, 12C10, 26B4, 35G5, and 65A7) were selected for designing CD38/CD47 BsAbs (IMM5605) using a "mAb-trap" platform. BsAbs had higher affinity and binding activity to the CD38 target than those to the CD47 target, decreasing the potential on-target potential and off-tumor effects. The CD38/CD47 BsAbs did not bind to RBCs and did not induce RBC agglutination; thus, BsAbs had much lower blood toxicity. The CD38/CD47 BsAbs had a greater ability to block the CD47/SIRPα signal in CD38+/CD47+ tumor cells than IMM01 (SIRPα Fc fusion protein). Through Fc domain engineering, CD38/CD47 BsAbs were shown to kill tumors more effectively by inducing ADCC and ADCP. IMM5605-26B4 had the strongest inhibitory effect on cellular CD38 enzymatic activity. IMM5605-12C10 had the strongest ability to directly induce the apoptosis of tumor cells. The anti-CD38 antibody 26B4 combined with the SIRPα-Fc fusion proteins showed strong antitumor effects, which were better than any of the mono-therapeutic agents used alone in the NCI-H929 cell xenograft model. The CD38/CD47 BsAbs exhibited strong antitumor effects; specifically, IMM5605-12C10 efficiently eradicated all established tumors in all mice. Conclusion: A panel of BsAbs targeting CD38 and CD47 developed based on the "mAb-tarp" platform showed potent tumor-killing ability in vitro and in vivo. As BsAbs had lower affinity for binding to CD47, higher affinity for binding to CD38, no affinity for binding to RBCs, and did not induce RBC agglutination, we concluded that CD38/CD47 BsAbs are safe and have a satisfactory tolerability profile.
Assuntos
ADP-Ribosil Ciclase 1 , Antígeno CD47 , Neoplasias Hematológicas , Antígeno CD47/imunologia , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/metabolismo , ADP-Ribosil Ciclase 1/antagonistas & inibidores , ADP-Ribosil Ciclase 1/imunologia , ADP-Ribosil Ciclase 1/metabolismo , Humanos , Animais , Camundongos , Neoplasias Hematológicas/terapia , Neoplasias Hematológicas/imunologia , Linhagem Celular Tumoral , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/antagonistas & inibidores , Citotoxicidade Celular Dependente de Anticorpos , Feminino , Antineoplásicos Imunológicos/farmacologiaRESUMO
Signal regulatory protein alpha (SIRPα) is an immune inhibitory receptor on myeloid cells including macrophages and dendritic cells, which binds to CD47, a ubiquitous self-associated molecule. SIRPα-CD47 interaction is exploited by cancer cells to suppress anti-tumor activity of myeloid cells, therefore emerging as a novel immune checkpoint for cancer immunotherapy. In blood cancer, several SIRPα-CD47 blockers have shown encouraging monotherapy activity. However, the anti-tumor activity of SIRPα-CD47 blockers in solid tumors seems limited, suggesting the need for combination therapies to fully exploit the myeloid immune checkpoint in solid tumors. Here we tested whether combination of SIRPα-CD47 blocker with antibody-drug conjugate bearing a topoisomerase I inhibitor DXd (DXd-ADC) would enhance anti-tumor activity in solid tumors. To this end, DS-1103a, a newly developed anti-human SIRPα antibody (Ab), was assessed for the potential combination benefit with datopotamab deruxtecan (Dato-DXd) and trastuzumab deruxtecan (T-DXd), DXd-ADCs targeting human trophoblast cell-surface antigen 2 and human epidermal growth factor receptor 2, respectively. DS-1103a inhibited SIRPα-CD47 interaction and enhanced antibody-dependent cellular phagocytosis of Dato-DXd and T-DXd against human cancer cells. In a whole cancer cell vaccination model, vaccination with DXd-treated cancer cells led to activation of tumor-specific T cells when combined with an anti-mouse SIRPα (anti-mSIRPα) Ab, implying the benefit of combining DXd-ADCs with anti-SIRPα Ab on anti-tumor immunity. Furthermore, in syngeneic mouse models, both Dato-DXd and T-DXd combination with anti-mSIRPα Ab showed stronger anti-tumor activity over the monotherapies. Taken together, this study provides a preclinical rationale of novel therapies for solid tumors combining SIRPα-CD47 blockers with DXd-ADCs.
Assuntos
Antígenos de Diferenciação , Antígeno CD47 , Imunoconjugados , Receptores Imunológicos , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/imunologia , Animais , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Receptores Imunológicos/imunologia , Humanos , Camundongos , Imunoconjugados/farmacologia , Antígenos de Diferenciação/imunologia , Linhagem Celular Tumoral , Feminino , Trastuzumab/farmacologia , Inibidores da Topoisomerase I/farmacologia , Imunoterapia/métodos , Camundongos Endogâmicos BALB CRESUMO
The use of immune checkpoint inhibitors, which activate T cells, is a paradigm shift in the treatment of non-small cell lung cancer. However, the overall response remains low. To address this limitation, here we describe a novel platform, termed antibody-conjugated drug-loaded nanotherapeutics (ADN), which combines immunotherapy and molecularly targeted therapy. An ADN was designed with an anti-CD47 and anti-programmed death ligand 1 (PDL1) antibody pair on the surface of the nanoparticle and a molecularly targeted inhibitor of the PI3K (phosphatidylinositol 3-kinase)/AKT/mTOR (mammalian target of rapamycin) pathway, PI103, entrapped in the nanoparticle. The anti-CD47-PDL1-ADN exhibited greater antitumor efficacy than current treatment options with a PDL1 inhibitor in vivo in an aggressive lung cancer immunocompetent mouse model. Dual antibody-drug-loaded nanotherapeutics can emerge as an attractive platform to improve outcomes with cancer immunotherapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Imunoterapia , Neoplasias Pulmonares , Nanopartículas , Carcinoma Pulmonar de Células não Pequenas/terapia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Animais , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/imunologia , Imunoterapia/métodos , Humanos , Camundongos , Nanopartículas/química , Linhagem Celular Tumoral , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Imunoconjugados/química , Ensaios Antitumorais Modelo de Xenoenxerto , Modelos Animais de Doenças , Antígeno CD47/imunologia , Antígeno CD47/antagonistas & inibidores , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêuticoRESUMO
Certain therapies that target CD markers on some blood cells can affect pretransfusion testing. Key examples are anti-CD38, CD47 monoclonal antibody (mAb) therapies such as daratumumab (DARA) and magrolimab, which have presented a challenge for transfusion medicine laboratories around the globe. Scientists have been faced with not only introducing a protocol to provide safe blood to patients but also investigating the most effective method to remove the pretransfusion pan-agglutinating interference caused. A number of papers in the last 5 years have reported on various methods to remove pretransfusion interference; however, most of these studies have been conducted only in a few countries. Most recent reviews on this topic have focused on techniques and reagents to remove pretransfusion interference, and dithiothreitol is currently the gold standard for removing DARA interference. However, it was clear from this review that while many laboratories have developed processes for addressing interference in pretransfusion testing, and DARA interference may not be a major issue, there are still laboratories around the world, that may not have adequately addressed this issue. In addition, the impact of mAb interference on widely used techniques such as flow cytometry is unclear.
Assuntos
ADP-Ribosil Ciclase 1 , Anticorpos Monoclonais , Humanos , Anticorpos Monoclonais/uso terapêutico , ADP-Ribosil Ciclase 1/antagonistas & inibidores , ADP-Ribosil Ciclase 1/imunologia , Transfusão de Sangue , Glicoproteínas de Membrana/imunologia , Antígeno CD47/antagonistas & inibidores , Citometria de Fluxo/métodosRESUMO
Targeting tumor-associated macrophages (TAMs) is an emerging approach being tested in multiple clinical trials. TAMs, depending on their differentiation state, can exhibit pro- or antitumorigenic functions. For example, the M2-like phenotype represents a protumoral state that can stimulate tumor growth, angiogenesis, metastasis, therapy resistance, and immune evasion by expressing immune checkpoint proteins. In this issue of the JCI, Vaccaro and colleagues utilized an innovative drug screen approach to demonstrate that targeting driver oncogenic signaling pathways concurrently with anti-CD47 sensitizes tumor cells, causing them to undergo macrophage-induced phagocytosis. The combination treatment altered expression of molecules on the tumor cells that typically limit phagocytosis. It also reprogrammed macrophages to an M1-like antitumor state. Moreover, the approach was generalizable to tumor cells with different oncogenic pathways, opening the door to precision oncology-based rationale combination therapies that have the potential to improve outcomes for patients with oncogene-driven lung cancers and likely other cancer types.
Assuntos
Antígeno CD47 , Macrófagos Associados a Tumor , Humanos , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/efeitos dos fármacos , Macrófagos Associados a Tumor/imunologia , Antígeno CD47/metabolismo , Antígeno CD47/antagonistas & inibidores , Animais , Fagocitose/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/metabolismo , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismoRESUMO
BACKGROUND: Approximately half of the neuroblastoma patients develop high-risk neuroblastoma. Current treatment involves a multimodal strategy, including immunotherapy with dinutuximab (IgG ch14.18) targeting GD2. Despite achieving promising results, the recurrence rate remains high and poor survival persists. The therapeutic efficacy of dinutuximab is compromised by suboptimal activation of neutrophils and severe neuropathic pain, partially induced by complement activation. METHODS: To enhance neutrophil cytotoxicity, IgG ch14.18 was converted to the IgA isotype, resulting in potent neutrophil-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), without complement activation. However, myeloid checkpoint molecules hamper neutrophil cytotoxicity, for example through CD47 that is overexpressed on neuroblastomas and orchestrates an immunosuppressive environment upon ligation to signal regulatory protein alpha (SIRPα) expressed on neutrophils. In this study, we combined IgA therapy with CD47 blockade. RESULTS: In vitro killing assays showed enhanced IgA-mediated ADCC by neutrophils targeting neuroblastoma cell lines and organoids in comparison to IgG. Notably, when combined with CD47 blockade, both IgG and IgA therapy were enhanced, though the combination with IgA resulted in the greatest improvement of ADCC. Furthermore, in a neuroblastoma xenograft model, we systemically blocked CD47 with a SIRPα fusion protein containing an ablated IgG1 Fc, and compared IgA therapy to IgG therapy. Only IgA therapy combined with CD47 blockade increased neutrophil influx to the tumor microenvironment. Moreover, the IgA combination strategy hampered tumor outgrowth most effectively and prolonged tumor-specific survival. CONCLUSION: These promising results highlight the potential to enhance immunotherapy efficacy against high-risk neuroblastoma through improved neutrophil cytotoxicity by combining IgA therapy with CD47 blockade.
Assuntos
Antígeno CD47 , Imunoglobulina A , Neuroblastoma , Neutrófilos , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/metabolismo , Antígeno CD47/imunologia , Humanos , Neuroblastoma/imunologia , Neuroblastoma/tratamento farmacológico , Neutrófilos/imunologia , Neutrófilos/metabolismo , Animais , Camundongos , Imunoglobulina A/imunologia , Imunoglobulina A/farmacologia , Imunoglobulina A/metabolismo , Linhagem Celular Tumoral , Citotoxicidade Celular Dependente de Anticorpos , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Imunoterapia/métodos , Feminino , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêuticoRESUMO
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.
Assuntos
Anticorpos Biespecíficos , Antígeno CD47 , Antígeno Carcinoembrionário , Animais , Feminino , Humanos , Camundongos , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Antígeno Carcinoembrionário/imunologia , Antígeno CD47/imunologia , Antígeno CD47/antagonistas & inibidores , Linhagem Celular Tumoral , Proteínas Ligadas por GPI , Macaca fascicularis , Neoplasias/tratamento farmacológico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Immune checkpoint inhibitors remained the standard-of-care treatment for advanced non-small cell lung cancer (NSCLC) for the past decade. In unselected patients, anti-PD-(L)1 monotherapy achieved an overall response rate of about 20%. In this analysis, we developed a pharmacokinetic and pharmacodynamic module for our previously calibrated quantitative systems pharmacology model (QSP) to simulate the effectiveness of macrophage-targeted therapies in combination with PD-L1 inhibition in advanced NSCLC. By conducting in silico clinical trials, the model confirmed that anti-CD47 treatment is not an optimal option of second- and later-line treatment for advanced NSCLC resistant to PD-(L)1 blockade. Furthermore, the model predicted that inhibition of macrophage recruitment, such as using CCR2 inhibitors, can potentially improve tumor size reduction when combined with anti-PD-(L)1 therapy, especially in patients who are likely to respond to anti-PD-(L)1 monotherapy and those with a high level of tumor-associated macrophages. Here, we demonstrate the application of the QSP platform on predicting the effectiveness of novel drug combinations involving immune checkpoint inhibitors based on preclinical or early-stage clinical trial data.
Assuntos
Antígeno B7-H1 , Carcinoma Pulmonar de Células não Pequenas , Inibidores de Checkpoint Imunológico , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/imunologia , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/administração & dosagem , Inibidores de Checkpoint Imunológico/farmacocinética , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/metabolismo , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Receptores CCR2/antagonistas & inibidores , Receptores CCR2/metabolismo , Farmacologia em Rede/métodos , Simulação por Computador , Modelos Biológicos , Macrófagos Associados a Tumor/efeitos dos fármacos , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/metabolismoRESUMO
Antibody-based CD47 blockade aims to activate macrophage phagocytosis of tumor cells. However, macrophages possess a high degree of phenotype heterogeneity that likely influences phagocytic capacity. In murine models, proinflammatory (M1) activation increases macrophage phagocytosis of tumor cells, but in human models, results have been conflicting. Here, we investigated the effects of proinflammatory polarization on the phagocytic response of human monocyte-derived macrophages in an in vitro model. Using both flow cytometry-based and fluorescence live-cell imaging-based phagocytosis assays, we observed that mouse monoclonal anti-CD47 antibody (B6H12) induced monocyte-derived macrophage phagocytosis of cancer cells in vitro. Proinflammatory (M1) macrophage polarization with IFN-γ+LPS resulted in a severe reduction in phagocytic response to CD47 blockade. This reduction coincided with increased expression of the antiphagocytic membrane proteins LILRB1 and Siglec-10 but was not rescued by combination blockade of the corresponding ligands. However, matrix metalloproteinase inhibitors (TAPI-0 or GM6001) partly restored response to CD47 blockade in a dose-dependent manner. In summary, these data suggest that proinflammatory (M1) activation reduces phagocytic response to CD47 blockade in human monocyte-derived macrophages.
Assuntos
Antígeno CD47 , Macrófagos , Fagocitose , Humanos , Antígeno CD47/imunologia , Antígeno CD47/metabolismo , Antígeno CD47/antagonistas & inibidores , Macrófagos/imunologia , Macrófagos/metabolismo , Fagocitose/imunologia , Ativação de Macrófagos/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Inflamação/imunologia , Anticorpos Monoclonais/farmacologia , Camundongos , Animais , Linhagem Celular Tumoral , Neoplasias/imunologia , Interferon gama/metabolismo , Interferon gama/imunologiaRESUMO
BACKGROUND: Myocardial ischemia-reperfusion injury (MIRI) is an important cause of early dysfunction and exacerbation of immune rejection in transplanted hearts. The integrin-related protein CD47 exacerbates myocardial ischemia-reperfusion injury by inhibiting the nitric oxide signaling pathway through interaction with thrombospondin-1 (TSP-1). In addition, the preservation quality of the donor hearts is a key determinant of transplant success. Preservation duration beyond four hours is associated with primary graft dysfunction. We hypothesized that blocking the CD47-TSP-1 system would attenuate ischemia-reperfusion injury in the transplanted heart and, thus, improve the preservation of donor hearts. METHODS: We utilized a syngeneic mouse heart transplant model to assess the effect of CD47 monoclonal antibody (CD47mAb) to treat MIRI. Donor hearts were perfused with CD47mAb or an isotype-matched control immunoglobulin (IgG2a) and were implanted into the abdominal cavity of the recipients after being stored in histidine-tryptophan-ketoglutarate (HTK) solution at 4 °C for 4 h or 8 h. RESULTS: At both the 4-h and 8-h preservation time points, mice in the experimental group perfused with CD47mAb exhibited prolonged survival in the transplanted heart, reduced inflammatory response and oxidative stress, significantly decreased inflammatory cell infiltration, and fewer apoptosis-related biomarkers. CONCLUSION: The application of CD47mAb for the blocking of CD47 attenuates MIRI as well as improves the preservation and prognosis of the transplanted heart in a murine heart transplant model.
Assuntos
Antígeno CD47 , Transplante de Coração , Camundongos Endogâmicos C57BL , Animais , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/metabolismo , Antígeno CD47/imunologia , Camundongos , Masculino , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Preservação de Órgãos/métodos , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/imunologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Trombospondina 1/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Modelos Animais de Doenças , Apoptose/efeitos dos fármacosRESUMO
Introduction: Chemotherapy remains the mainstay treatment for triple-negative breast cancer (TNBC) due to the lack of specific targets. Given a modest response of immune checkpoint inhibitors in TNBC patients, improving immunotherapy is an urgent and crucial task in this field. CD73 has emerged as a novel immunotherapeutic target, given its elevated expression on tumor, stromal, and specific immune cells, and its established role in inhibiting anti-cancer immunity. CD73-generated adenosine suppresses immunity by attenuating tumor-infiltrating T- and NK-cell activation, while amplifying regulatory T cell activation. Chemotherapy often leads to increased CD73 expression and activity, further suppressing anti-tumor immunity. While debulking the tumor mass, chemotherapy also enriches heterogenous cancer stem cells (CSC), potentially leading to tumor relapse. Therefore, drugs targeting both CD73, and CSCs hold promise for enhancing chemotherapy efficacy, overcoming treatment resistance, and improving clinical outcomes. However, safe and effective inhibitors of CD73 have not been developed as of now. Methods: We used in silico docking to screen compounds that may be repurposed for inhibiting CD73. The efficacy of these compounds was investigated through flow cytometry, RT-qPCR, CD73 activity, cell viability, tumorsphere formation, and other in vitro functional assays. For assessment of clinical translatability, TNBC patient-derived xenograft organotypic cultures were utilized. We also employed the ovalbumin-expressing AT3 TNBC mouse model to evaluate tumor-specific lymphocyte responses. Results: We identified quercetin and luteolin, currently used as over-the-counter supplements, to have high in silico complementarity with CD73. When quercetin and luteolin were combined with the chemotherapeutic paclitaxel in a triple-drug regimen, we found an effective downregulation in paclitaxel-enhanced CD73 and CSC-promoting pathways YAP and Wnt. We found that CD73 expression was required for the maintenance of CD44highCD24low CSCs, and co-targeting CD73, YAP, and Wnt effectively suppressed the growth of human TNBC cell lines and patient-derived xenograft organotypic cultures. Furthermore, triple-drug combination inhibited paclitaxel-enriched CSCs and simultaneously improved lymphocyte infiltration in syngeneic TNBC mouse tumors. Discussion: Conclusively, our findings elucidate the significance of CSCs in impairing anti-tumor immunity. The high efficacy of our triple-drug regimen in clinically relevant platforms not only underscores the importance for further mechanistic investigations but also paves the way for potential development of new, safe, and cost-effective therapeutic strategies for TNBC.
Assuntos
Antígeno CD47 , Neoplasias de Mama Triplo Negativas , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Flavonoides/farmacologia , Luteolina/metabolismo , Células-Tronco Neoplásicas/metabolismo , Paclitaxel/uso terapêutico , Quercetina/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Antígeno CD47/antagonistas & inibidoresRESUMO
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.
Assuntos
Antígeno CD47 , Neoplasias Pulmonares , Macrófagos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Humanos , Antígeno CD47/genética , Antígeno CD47/metabolismo , Antígeno CD47/imunologia , Antígeno CD47/antagonistas & inibidores , Camundongos , Animais , Macrófagos/metabolismo , Macrófagos/imunologia , Macrófagos/patologia , Linhagem Celular Tumoral , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Terapia de Alvo Molecular , Receptores ErbB/genética , Receptores ErbB/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/imunologia , Sistema de Sinalização das MAP Quinases/genética , Fagocitose , FemininoAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Compostos Bicíclicos Heterocíclicos com Pontes , Antígeno CD47 , Sulfonamidas , Humanos , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Sulfonamidas/uso terapêutico , Sulfonamidas/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Resultado do Tratamento , Antígeno CD47/antagonistas & inibidores , Masculino , Resistencia a Medicamentos Antineoplásicos , Idoso , Pessoa de Meia-Idade , Feminino , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Adulto , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/diagnósticoRESUMO
The treatment for trastuzumab-resistant breast cancer (BC) remains a challenge in clinical settings. It was known that CD47 is preferentially upregulated in HER2+ BC cells, which is correlated with drug resistance to trastuzumab. Here, we developed a novel anti-CD47/HER2 bispecific antibody (BsAb) against trastuzumab-resistant BC, named IMM2902. IMM2902 demonstrated high binding affinity, blocking activity, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and internalization degradation effects against both trastuzumab-sensitive and trastuzumab-resistant BC cells in vitro. The in vivo experimental data indicated that IMM2902 was more effective than their respective controls in inhibiting tumor growth in a trastuzumab-sensitive BT474 mouse model, a trastuzumab-resistant HCC1954 mouse model, two trastuzumab-resistant patient-derived xenograft (PDX) mouse models and a cord blood (CB)-humanized HCC1954 mouse model. Through spatial transcriptome assays, multiplex immunofluorescence (mIFC) and in vitro assays, our findings provided evidence that IMM2902 effectively stimulates macrophages to generate C-X-C motif chemokine ligand (CXCL) 9 and CXCL10, thereby facilitating the recruitment of T cells and NK cells to the tumor site. Moreover, IMM2902 demonstrated a high safety profile regarding anemia and non-specific cytokines release. Collectively, our results highlighted a novel therapeutic approach for the treatment of HER2+ BCs and this approach exhibits significant anti-tumor efficacy without causing off-target toxicity in trastuzumab-resistant BC cells.
Assuntos
Anticorpos Biespecíficos , Neoplasias da Mama , Antígeno CD47 , Resistencia a Medicamentos Antineoplásicos , Imunoterapia , Receptor ErbB-2 , Trastuzumab , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Feminino , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Camundongos , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/imunologia , Imunoterapia/métodos , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Linhagem Celular Tumoral , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Fagocitose/efeitos dos fármacosRESUMO
CD47-SIRPa is a myeloid check point pathway that promotes phagocytosis of cells lacking markers for self-recognition. Tumor cells can overexpress CD47 and bind to SIRPa on macrophages, preventing phagocytosis. CD47 expression is enhanced and correlated with a negative prognosis in acute myeloid leukemia (AML), with its blockade leading to cell clearance. ALX90 is an engineered fusion protein with high affinity for CD47. Composed of the N-terminal D1 domain of SIRPα genetically linked to an inactive Fc domain from human immunoglobulin (Ig) G, ALX90 is designed to avoid potential toxicity of CD47-expressing red blood cells. Venetoclax (VEN) is a specific B-cell lymphoma-2 (BCL-2) inhibitor that can restore apoptosis in malignant cells. In AML, VEN is combined with azanucleosides to induce superior remission rates, however treatment for refractory/relapse is an unmet need. We questioned whether the anti-tumor activity of a VENbased regimen can be augmented through CD47 inhibition (CD47i) in AML and how this triplet may be enhanced. Human AML cell lines were sensitive to ALX90 and its addition increased efficacy of a VEN plus azacitidin (VEN+AZA) regimen in vivo. However, CD47i failed to clear bone marrow tumor burden in PDX models. We hypothesized that the loss of resident macrophages in the bone marrow in AML reduced efficiency of CD47i. Therefore, we attempted to enhance this medullary macrophage population with agonism of TLR3 via polyinosinic:polycytidylic acid (poly(I:C)), which led to expansion and activation of medullary macrophages in in vivo AML PDX models and potentiated CD47i. In summary, the addition of poly(I:C) can enhance medullary macrophage populations to potentiate the phagocytosis merited by therapeutic inhibition of CD47.
Assuntos
Antígeno CD47 , Leucemia Mieloide Aguda , Antígeno CD47/metabolismo , Antígeno CD47/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Humanos , Animais , Camundongos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Sulfonamidas/farmacologia , Receptores Imunológicos/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Antígenos de Diferenciação/metabolismo , Fagocitose/efeitos dos fármacos , Poli I-C/farmacologiaRESUMO
While anti-CD47 antibodies hold promise for cancer immunotherapy, early-phase clinical trials have shown limited clinical benefit, suggesting that CD47 blockade alone might be insufficient for effective tumor control. Here, we investigate the contributions of the Fc domain of anti-CD47 antibodies required for optimal in vivo antitumor activity across multiple species-matched models, providing insights into the mechanisms behind the efficacy of this emerging class of therapeutic antibodies. Using a mouse model humanized for CD47, SIRPα, and FcγRs, we demonstrate that local administration of Fc-engineered anti-CD47 antibodies with enhanced binding to activating FcγRs promotes tumor infiltration of macrophages and antigen-specific T cells, while depleting regulatory T cells. These effects result in improved long-term systemic antitumor immunity and minimal on-target off-tumor toxicity. Our results highlight the importance of Fc optimization in the development of effective anti-CD47 therapies and provide an attractive strategy to enhance the activity of this promising immunotherapy.
Assuntos
Anticorpos , Antígeno CD47 , Neoplasias , Fagocitose , Humanos , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/imunologia , Imunoterapia/métodos , Macrófagos , Neoplasias/tratamento farmacológico , Receptores de IgG/metabolismo , Anticorpos/uso terapêutico , Animais , CamundongosRESUMO
Cluster of differentiation 47 (CD47) plays an important role in the pathophysiology of various diseases including atherosclerosis but its role in neointimal hyperplasia which contributes to restenosis has not been studied. Using molecular approaches in combination with a mouse vascular endothelial denudation model, we studied the role of CD47 in injury-induced neointimal hyperplasia. We determined that thrombin-induced CD47 expression both in human aortic smooth muscle cells (HASMCs) and mouse aortic smooth muscle cells. In exploring the mechanisms, we found that the protease-activated receptor 1-Gα protein q/11 (Gαq/11)-phospholipase Cß3-nuclear factor of activated T cells c1 signaling axis regulates thrombin-induced CD47 expression in HASMCs. Depletion of CD47 levels using its siRNA or interference of its function by its blocking antibody (bAb) blunted thrombin-induced migration and proliferation of HASMCs and mouse aortic smooth muscle cells. In addition, we found that thrombin-induced HASMC migration requires CD47 interaction with integrin ß3. On the other hand, thrombin-induced HASMC proliferation was dependent on CD47's role in nuclear export and degradation of cyclin-dependent kinase-interacting protein 1. In addition, suppression of CD47 function by its bAb rescued HASMC efferocytosis from inhibition by thrombin. We also found that vascular injury induces CD47 expression in intimal SMCs and that inhibition of CD47 function by its bAb, while alleviating injury-induced inhibition of SMC efferocytosis, attenuated SMC migration, and proliferation resulting in reduced neointima formation. Thus, these findings reveal a pathological role for CD47 in neointimal hyperplasia.
Assuntos
Antígeno CD47 , Reestenose Coronária , Miócitos de Músculo Liso , Animais , Humanos , Camundongos , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/genética , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Hiperplasia/metabolismo , Hiperplasia/fisiopatologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Neointima/metabolismo , Neointima/fisiopatologia , Trombina/metabolismo , Lesões do Sistema Vascular/fisiopatologia , Regulação da Expressão Gênica/genética , Reestenose Coronária/fisiopatologiaRESUMO
BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has been successfully applied in treating lymphoma malignancies, but not in solid tumors. CD47 is highly expressed on tumor cells and its overexpression is believed to inhibit phagocytosis by macrophages and dendritic cells. Given the antitumor activity against preclinical model of CD47-blocking to induce the innate and adaptive immune system in the tumor microenvironment, here we developed a CAR-T cell secreting CD47 blocker signal regulatory protein α (SIRPα)-Fc fusion protein (Sirf CAR-T) to boost CAR-T cell therapeutic effect in solid tumor therapy. METHODS: Murine T cells were transduced to express a conventional anti-Trop2 CAR and Sirf CAR. The expression of SIRPα-Fc fusion protein in the supernatant of CAR-T cells and its effect on macrophage phagocytosis were tested in vitro. In vivo antitumor efficacy of CAR-T cells was evaluated in immunocompetent mice and analysis of the tumor microenvironment in the tumor-bearing mice was performed. RESULTS: We found that Sirf CAR-T cells dramatically decreased tumor burden and significantly prolonged survival in several syngeneic immunocompetent tumor models. Furthermore, we found that Sirf CAR-T cells induced more central memory T cells (TCM) and improved the persistence of CAR-T cells in tumor tissue, as well as decreased PD-1 expression on the CAR-T cell surface. In addition, we demonstrated that Sirf CAR-T cells could modulate the tumor microenvironment by decreasing myeloid-derived stem cells as well as increasing CD11c+ dendritic cells and M1-type macrophages in tumor tissue. CONCLUSIONS: In summary, our findings indicate that CD47 blocker SIRPα-Fc enhances the antitumor efficacy of CAR-T cells and propose to block CD47/SIRPα signaling effect on CAR-T cells function, which could provide a new strategy for successful cancer immunotherapy by rationalizing combination of CD47 blocker and CAR-T cell therapy.