A Dual-Payload Antibody-Drug Conjugate Targeting CD276/B7-H3 Elicits Cytotoxicity and Immune Activation in Triple-Negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous disease that often relapses following treatment with standard radiotherapies and cytotoxic chemotherapies. Combination therapies have potential for treating refractory metastatic TNBC. In this study, we aimed to develop an antibody-drug conjugate with dual payloads (DualADC) as a chemoimmunotherapy for TNBC. The overexpression of an immune checkpoint transmembrane CD276 (also known as B7-H3) was associated with angiogenesis, metastasis, and immune tolerance in more than 60% of patients with TNBC. Development of a mAb capable of targeting the extracellular domain of surface CD276 enabled delivery of payloads to tumors, and a platform was established for concurrent conjugation of a traditional cytotoxic payload and an immunoregulating Toll-like receptor 7/8 agonist to the CD276 mAb. The DualADC effectively killed multiple TNBC subtypes, significantly enhanced immune functions in the tumor microenvironment, and reduced tumor burden by up to 90% to 100% in animal studies. Single-cell RNA sequencing, multiplex cytokine analysis, and histology elucidated the impact of treatment on tumor cells and the immune landscape. This study suggests that the developed DualADC could represent a promising targeted chemoimmunotherapy for TNBC. Significance: An anti-CD276 monoclonal antibody conjugated with both a cytotoxic drug and an immune boosting reagent effectively targets triple-negative breast cancer by inducing tumor cell death and stimulating immune cell infiltration.

Graphical abstract

Figure 1.

Evaluations of CD276 and characterization of humanized CD276 mAb in TNBCs. A, IHC staining of TMA with anti-CD276 antibody. B, Representative images of low, medium, and high CD276 expressions. C, TNBC surface binding by engineered CD276 mAbs. D, TNBC MDA-MB-468 cell surface binding and internalization of humanized CD276 mAb (Hu276 mAb) labeled with Cy5.5 fluorescent dye (red). E, Live-animal and ex vivo IVIS to confirm TNBC-specific targeting by Hu276 mAb-Cy5.5 at 24 hours after tail vein injection. F, IHC staining of 33 human normal organs with our humanized CD276 mAb.

Figure 2.

Construction of DualADC via cysteine and lysine. A, Structure of DualADC CD276 mAb–MMAF/IMQ. B, Structure of DualADC. C, HPLC confirmed the conjugation of single payload and dual payloads with chimeric anti-CD276 mAb. D, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometer to validate the molecular weight of mAb, single ADC (mAb–MMAF, mAb–IMQ), and DualADC (mAb–MMAF/IMQ). E, SDS-PAGE of ADCs. M, marker; 1, CD276 mAb; 2, mAb–MMAF; 3, mAb–IMQ; 4, mAb–MMAF/IMQ.

Figure 3.

In vitro evaluations of DualADC using humanized CD276 mAb. The TNBC MDA-MB-231, MDA-MB-468, and 4T1 cells were used in cytotoxicity studies with free drugs and single-payload ADC as controls. A, Anti-TNBC cytotoxicity and IC₅₀ of free DM1 and MMAF drugs. B, Cytotoxicity and IC₅₀ of IMQ. C, EC₅₀ of IMQ on human and murine TLR8⁺ HEK cells. D, Anti-TNBC cytotoxicity and IC₅₀ of single-payload ADC (mAb–MMAF). E, Cytotoxicity and IC₅₀ of single-payload (mAb–IMQ). F, Cytotoxicity and IC₅₀ of DualADC mAb–MMAF/IMQ. n = 3.

Figure 4.

Antitumor efficacy of DualADC in the TNBC PDX xenograft model. A, Tumor volume changes after treatment with humanized CD276 mAb–derived ADC following the schedule of Q7Dx3 as indicated by the black arrow. Data were presented as mean ± SEM, n = 5–7. Saline (○), 16 mg/kg of mAb–MMAF (▲), and 16 mg/kg of mAb–MMAF/IMQ (●). *, P < 0.05 vs. saline using ANOVA followed by Dunnett t test. B, Body weight. C, White light images at 14 days after treatment stopped. D, IHC staining of TNBC PDX tumor tissue. Scale bar, 20 μm.

Figure 5.

Anti-TNBC efficacy of 276 mAb–MMAF/IMQ in immunocompetent models. The mouse TNBC 4T1-FLuc xenografted female BALB/cJ mice were treated with DualADC (8, 16, 24 mg/kg mAb–MMAF/IMQ), mAb–MMAF, mAb–IMQ, or saline (controls) via i.v. injection through tail vein. n = 5–8. A, Tumor volume after treatment following the schedule of Q7Dx4 as indicated by black arrow. Data were presented as mean ± SEM. *, P < 0.05 vs. saline using ANOVA followed by Dunnett t test. B, Weight of terminal wet tumors treated with 16 mg/kg of DualADCs using chimeric CD276 mAb and humanized 276 mAb. *, P < 0.05 vs. saline using ANOVA followed by Dunnett t test. C, H&E staining of major organs. Scale bar, 70 μm. D, IHC staining of harvested tumors using markers of cell proliferation (Ki67), apoptosis (CCasp3), immune checkpoint inhibition (PD-1), and filtration and activation of CD8⁺ T, NK and macrophage cells (CD8, CD45, F4/80). Scale bar, 20 μm. E, H&E staining to analyze TNBC cell death in the treatment group. Scale bar, 40 μm.

Figure 6.

Analysis of tumoral cytokines and general toxicity after treatment of DualADC. The same mice as in Fig. 5 were used here. A, Luminex assay identified several enhanced cytokines and downregulated PD-1 in the TME. B, The complete blood cell counts. 1, Saline (control); 2, 8 mg/kg mAb–MMAF (control); 3, 8 mg/kg mAb–IMQ (control); 4, 8 mg/kg mAb–MMAF/IMQ; 5, 16 mg/kg mAb–MMAF/IMQ; 6, 24 mg/kg mAb–MMAF/IMQ. Data were presented as mean ± SEM, n = 4. *, P < 0.05 vs. saline using ANOVA followed by Dunnett t test. BA, basophil; EO, eosinophil; Hb, hemoglobin; HCT, hematocrit; LY, lymphocyte; MCH, hexachlorocyclohexane; MCHC, mean corpuscular hemoglobin concentration; MO, monocytes; MCV, mean corpuscular volume; MPV, mean platelet volume; NE, neutrophils; PDW, platelet distribution width; PTL, platelet; PCT, procalcitonin; RBC, red blood cell; RDW, red cell distribution width; WBC, white blood cell.

Figure 7.

Analysis of immune cell infiltration and immune functions in the TME using scRNA-seq. The tumor tissues were harvested from the same animal study in Fig. 5. A, Overview of all cell types in TNBC tumors. B, Immune functions in the TME. C, Immune responses of macrophage. D, Analysis of mitotic activities. The adjusted P value is 0.05. UMAP, Uniform Manifold Approximation and Projection.