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Anti-CD47 Monoclonal Antibody-Drug Conjugate: A Targeted Therapy to Treat Triple-Negative Breast Cancers.

ABSTRACT: Triple-negative breast cancers (TNBCs) are frequently recurrent due to the development of drug resistance post chemotherapy. Both the existing literature and our study found that surface receptor CD47 (cluster of differentiation 47) was upregulated in chemotherapy-treated TNBC cells. The goal of this study was to develop a monoclonal antibody (mAb)-based targeting strategy to treat TNBC after standard treatment. Specifically, a new mAb that targets the extracellular domain of receptor CD47 was developed using hybridoma technology and produced in fed-batch culture. Flow cytometry, confocal microscopy, and in vivo imaging system (IVIS) showed that the anti-CD47 mAb effectively targeted human and mouse TNBC cells and xenograft models with high specificity. The antibody-drug conjugate (ADC) carrying mertansine was constructed and demonstrated higher potency with reduced IC₅₀ in TNBC cells than did the free drug and significantly inhibited tumor growth post gemcitabine treatment in MDA-MB-231 xenograft NSG model. Finally, whole blood analysis indicated that the anti-CD47 mAb had no general immune toxicity, flow cytometry analysis of lymph nodes revealed an increase of CD69⁺ NK, CD11c⁺ DC, and CD4⁺ T cells, and IHC staining showed tumoral infiltration of macrophage in the 4T1 xenograft BALB/cJ model. This study demonstrated that targeting CD47 with ADC has great potential to treat TNBCs as a targeted therapy.

SUBMITTER: Si Y

PROVIDER: S-EPMC8402537 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Intercellular communication is critical for integrating complex signals in multicellular eukaryotes. Vascular endothelial cells and T lymphocytes closely interact during the recirculation and trans-endothelial migration of T cells. In addition to direct cell-cell contact, we show that T cell derived extracellular vesicles can interact with endothelial cells and modulate their cellular functions. Thrombospondin-1 and its receptor CD47 are expressed on exosomes/ectosomes derived from T cells, and these extracellular vesicles are internalized and modulate signaling in both T cells and endothelial cells. Extracellular vesicles released from cells expressing or lacking CD47 differentially regulate activation of T cells induced by engaging the T cell receptor. Similarly, T cell-derived extracellular vesicles modulate endothelial cell responses to vascular endothelial growth factor and tube formation in a CD47-dependent manner. Uptake of T cell derived extracellular vesicles by recipient endothelial cells globally alters gene expression in a CD47-dependent manner. CD47 also regulates the mRNA content of extracellular vesicles in a manner consistent with some of the resulting alterations in target endothelial cell gene expression. Therefore, the thrombospondin-1 receptor CD47 directly or indirectly regulates intercellular communication mediated by the transfer of extracellular vesicles between vascular cells. Treatment with B6H12 antibody inhibited co-immunoprecipitation of EGFR with CD47 and inhibited EGF-induced EGFR tyrosine phosphorylation. B6H12 treatment of bCSC also suppressed asymmetric cell division and cell proliferation and up-regulated caspase 3/7 activity. Correspondingly, caspase-7 cleavage in human breast cancers correlated with CD47 expression. Our data shows that B6H12 specifically targets bCSCs but not differentiated cancer cells, and this CD47 signaling is independent of SIRPÎ±. Three replicates of each condition were generated. Three replicates of each MDA-231 attached cells (differentiated), MDA-231 in suspension cells (bCSC), MDA-231 in suspension cells (bCSC) treated with Control Antibody and MDA-231 in suspension cells (bCSC) treated with B6H12 Antibody.

Project description:PurposeAnti-EGFR antibodies show limited response in breast cancer, partly due to activation of compensatory pathways. Furthermore, despite clinical success of CDK4/6 inhibitors in hormone receptor-positive tumors, aggressive triple-negative breast cancers (TNBCs) are largely resistant due to CDK2/cyclin E expression, while free CDK2 inhibitors display normal tissue toxicity, limiting their therapeutic application. A cetuximab-based antibody drug conjugate (ADC) carrying a CDK inhibitor selected based on oncogene dysregulation, alongside patient subgroup stratification, may provide EGFR-targeted delivery.Experimental designExpression of G1/S-phase cell cycle regulators were evaluated alongside EGFR in breast cancer. We conjugated cetuximab with CDK inhibitor SNS-032, for specific delivery to EGFR-expressing cells. We assessed ADC internalization, and its anti-tumor functions in vitro and in orthotopically-grown basal-like/TNBC xenografts.ResultsTranscriptomic (6173 primary, 27 baseline and matched post-chemotherapy residual tumors), scRNA-seq (150290 cells, 27 treatment-naïve tumors) and spatial transcriptomic (43 tumor sections, 22 TNBCs) analyses confirmed expression of CDK2 and its cyclin partners in basal-like/TNBCs, associated with EGFR. Spatiotemporal live-cell imaging and super-resolution confocal microscopy demonstrated ADC colocalization with late lysosomal clusters. The ADC inhibited cell cycle progression, induced cytotoxicity against high EGFR-expressing tumor cells and bystander killing of neighboring EGFR-low tumor cells, but minimal effects on immune cells. Despite carrying a small fraction of the drug, the ADC restricted EGFR-expressing spheroid and cell line/patient-derived xenograft tumor growth.ConclusionsExploiting EGFR overexpression, and dysregulated cell cycle in aggressive and treatment-refractory tumors, a cetuximab-CDK inhibitor ADC may provide selective and efficacious delivery of cell cycle-targeted agents to basal-like/TNBCs, including chemotherapy-resistant residual disease.

Project description:BackgroundAnti-CD47 monoclonal antibodies have increasing clinical applications in the treatment of cancer. However, anti-CD47 monoclonal antibodies interfere with immunohematology testing in patients who require blood transfusion. As the current approaches to removing any interferences have technical problems, new methods need to be developed to resolve anti-CD47 interference in immunohematology testing.Materials and methodsWe evaluated the Daudi cell line for the adsorption of free anti-CD47 monoclonal antibodies from patients' plasma to facilitate immunohematology testing in patients treated with anti-CD47 monoclonal antibody. CD47 expression was identified on the Daudi cells using flow cytometry and confocal microscopy. Next, we tested the ability of intact Daudi cells mixed with simulating plasma and clinical samples to achieve efficient removal of interfering anti-CD47 monoclonal antibodies. The indirect antiglobulin test was used to verify whether interference from anti-CD47 monoclonal antibodies in plasma was eliminated and whether the detection of other irregular antibodies was affected. The effect of eliminating interference was also investigated in relation to the time that the Daudi cells were stored after having been fixed with paraformaldehyde.ResultsCD47 expression was higher on Daudi cells than on red blood cells. Analysis of the indirect antiglobulin test results revealed that anti-CD47 monoclonal antibody-treated patients' plasma absorbed by Daudi cells for 15 min at 37°C could completely prevent the interference of anti-CD47 monoclonal antibodies in immunohematology testing while the detection of the tested antibodies, including anti-D and anti-K, was unaffected.DiscussionBy decreasing the incubation time, we discovered that interferences in samples with agglutination strengths below 2+ could be eliminated after incubation for 5 min. Of importance, Daudi cells can be preserved with 4% paraformaldehyde for 14 days as short-term storage reagents. This is the first study in which Daudi cells were used to effectively resolve the interference of anti-CD47 monoclonal antibodies in pretransfusion tests.

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Anti-CD47 Monoclonal Antibody-Drug Conjugate: A Targeted Therapy to Treat Triple-Negative Breast Cancers.

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