Monitoring changing patterns in HER2 addiction by liquid biopsy in advanced breast cancer patients.

BACKGROUND: During targeted treatment, HER2-positive breast cancers invariably lose HER2 DNA amplification. In contrast, and interestingly, HER2 proteins may be either lost or gained. To longitudinally and systematically appreciate complex/discordant changes in HER2 DNA/protein stoichiometry, HER2 DNA copy numbers and soluble blood proteins (aHER2/sHER2) were tested in parallel, non-invasively (by liquid biopsy), and in two-dimensions, hence HER2-2D. METHODS: aHER2 and sHER2 were assessed by digital PCR and ELISA before and after standard-of-care treatment of advanced HER2-positive breast cancer patients (n=37) with the antibody-drug conjugate (ADC) Trastuzumab-emtansine (T-DM1). RESULTS: As expected, aHER2 was invariably suppressed by T-DM1, but this loss was surprisingly mirrored by sHER2 gain, sometimes of considerable entity, in most (30/37; 81%) patients. This unorthodox split in HER2 oncogenic dosage was supported by reciprocal aHER2/sHER2 kinetics in two representative cases, and an immunohistochemistry-high status despite copy-number-neutrality in 4/5 available post-T-DM1 tumor re-biopsies from sHER2-gain patients. Moreover, sHER2 was preferentially released by dying breast cancer cell lines treated in vitro by T-DM1. Finally, sHER2 gain was associated with a longer PFS than sHER2 loss (mean PFS 282 vs 133 days, 95% CI [210-354] vs [56-209], log-rank test p=0.047), particularly when cases (n=11) developing circulating HER2-bypass alterations during T-DM1 treatment were excluded (mean PFS 349 vs 139 days, 95% CI [255-444] vs [45-232], log-rank test p=0.009). CONCLUSIONS: HER2 gain is adaptively selected in tumor tissues and recapitulated in blood by sHER2 gain. Possibly, an increased oncogenic dosage is beneficial to the tumor during anti-HER2 treatment with naked antibodies, but favorable to the host during treatment with a strongly cytotoxic ADC such as T-DM1. In the latter case, HER2-gain tumors may be kept transiently in check until alternative oncogenic drivers, revealed by liquid biopsy, bypass HER2. Whichever the interpretation, HER2-2D might help to tailor/prioritize anti-HER2 treatments, particularly ADCs active on aHER2-low/sHER2-low tumors. TRIAL REGISTRATION: NCT05735392 retrospectively registered on January 31, 2023 https://www. CLINICALTRIALS: gov/search?term=NCT05735392.

Trastuzumab, a monoclonal anti-HER2 antibody modulates cytotoxicity against cholangiocarcinoma via multiple mechanisms.

Cholangiocarcinoma (CCA) is an aggressive and fatal cancer. The prognosis is very poor and no optimal chemotherapy has been established. Human epidermal growth factor receptor 2 (HER2, neu, and erbB2) is highly-expressed in breast cancer and is expressed in many other tumors but poorly expressed in CCA. The anti-HER2 antibody, trastuzumab, has been used for the treatment of HER2-positive breast and gastric cancer. In this study, we examined the surface expression of HER2 on seven Thai liver-fluke-associated CCA cell lines by flow cytometry, and found all of these CCA cells were weakly positive for HER2. MTT assay revealed that trastuzumab directly suppressed the growth of CCA. By using FcR-bearing recombinant Jurkat T-cell-expressing firefly luciferase gene under the control of NFAT response elements, we defined the activities of antibody-dependent cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). ADCC was confirmed by using expanded NK cells. ADCP was confirmed by using mouse peritoneal macrophages and human monocyte-derived macrophages as effector cells. Rabbit serum was administered to test the complement-dependent cytotoxicity (CDC) activity of trastuzumab. Finally, we evaluated the efficacy of trastuzumab in in vivo patient-derived cell xenograft and patient-derived xenograft (PDX) models. Our results showed that a distinct population of CCA (liver-fluke-associated CCA) expressed HER2. Trastuzumab demonstrated a potent inhibitory effect on even HER2 weakly positive CCA both in vitro and in vivo via multiple mechanisms. Thus, HER2 is a promising target in anti-CCA therapy, and trastuzumab can be considered a promising antibody immunotherapy agent for the treatment of CCA.

A High Immune-Related Index with the Suppression of cGAS-STING Pathway is a Key Determinant to Herceptin Resistance in HER2+ Breast Cancer.

Resistance to HER2-targeted therapy is the major cause of treatment failure in patients with HER2+ breast cancer (BC). Given the key role of immune microenvironment in tumor development, there is a lack of an ideal prognostic model that fully accounts for immune infiltration. In this study, WGCNA analysis was performed to discover the relationship between immune-related signaling and prognosis of HER2+ BC. After Herceptin-resistant BC cell lines established, transcriptional profiles of resistant cell line and RNA-sequencing data from GSE76360 cohort were analyzed for candidate genes. 85 samples of HER2+ BC from TCGA database were analyzed by the Cox regression, XGBoost and Lasso algorithm to generalize a credible immune-related prognostic index (IRPI). Correlations between the IRPI signature and tumor microenvironment were further analyzed by multiple algorithms, including single-cell RNA sequencing data analysis. Patients with high IRPI had suppressive tumor immune microenvironment and worse prognosis. The suppression of type I interferon signaling indicated by the IRPI in Herceptin-resistant HER2+ BC was validated. And we elucidated that the suppression of cGAS-STING pathway is the key determinant underlying immune escape in Herceptin-resistant BC with high IRPI. A combination of STING agonist and DS-8201 could serve as a new strategy for Herceptin-resistant HER2+ BC.

Proteomic Characterization of a 3D HER2+ Breast Cancer Model Reveals the Role of Mitochondrial Complex I in Acquired Resistance to Trastuzumab.

HER2-targeted therapies, such as Trastuzumab (Tz), have significantly improved the clinical outcomes for patients with HER2+ breast cancer (BC). However, treatment resistance remains a major obstacle. To elucidate functional and metabolic changes associated with acquired resistance, we characterized protein profiles of BC Tz-responder spheroids (RSs) and non-responder spheroids (nRSs) by a proteomic approach. Three-dimensional cultures were generated from the HER2+ human mammary adenocarcinoma cell line BT-474 and a derived resistant cell line. Before and after a 15-day Tz treatment, samples of each condition were collected and analyzed by liquid chromatography-mass spectrometry. The analysis of differentially expressed proteins exhibited the deregulation of energetic metabolism and mitochondrial pathways. A down-regulation of carbohydrate metabolism and up-regulation of mitochondria organization proteins, the tricarboxylic acid cycle, and oxidative phosphorylation, were observed in nRSs. Of note, Complex I-related proteins were increased in this condition and the inhibition by metformin highlighted that their activity is necessary for nRS survival. Furthermore, a correlation analysis showed that overexpression of Complex I proteins NDUFA10 and NDUFS2 was associated with high clinical risk and worse survival for HER2+ BC patients. In conclusion, the non-responder phenotype identified here provides a signature of proteins and related pathways that could lead to therapeutic biomarker investigation.

Trastuzumab-deruxtecan in solid tumors with HER2 alterations: from early phase development to the first agnostic approval of an antibody-drug conjugate.

INTRODUCTION: Antibody-drug conjugates (ADCs) represent a revolutionary approach in the systemic treatment for both solid and hematologic tumors. Constituted by an antibody, a cytotoxic payload, and a linker, ADCs aim to selectively deliver cytotoxic agents to tumors while sparing normal tissues. Various ADCs have been tested and approved for multiple solid tumors so far, but if there is one that had a major impact on clinical practice, this is Trastuzumab-deruxtecan (T-DXd). Notably, T-DXd was approved for HER2-positive and HER2-low metastatic breast cancer (MBC), HER2-positive gastric cancer (GC), HER2-mutant non-small cell lung cancer (NSCLC) and HER2 3+ solid tumors. Moreover, it received Breakthrough Therapy Designation for HER2-positive colorectal cancer (CRC). AREAS COVERED: We review preclinical and clinical data of T-DXd, focusing on early-phase ongoing trials exploring combination therapies to enhance the activity of T-DXd in HER2-expressing solid tumors. EXPERT OPINION: The clinical use of T-DXd still raises questions about selection of patients, treatment duration, prioritization over other approved ADCs, and management of resistance. Concerns regarding the toxicity of T-DXd remain, particularly with combinations involving potentially toxic drugs. Advancements in biomarker identification and combination therapies offer promising avenues to enhance efficacy and overcome resistance to T-DXd, ultimately improving outcomes for patients with cancer.

FDA approval summary: fam-trastuzumab deruxtecan-nxki for unresectable or metastatic non-small cell lung cancer with activating HER2 mutations.

On August 11, 2022, FDA granted accelerated approval to fam-trastuzumab deruxtecan-nxki (DS-8201a, T-DXd, ENHERTU, Daiichi Sankyo) for adult patients with unresectable or metastatic non-small cell lung cancer (NSCLC) whose tumors have activating human epidermal growth factor receptor 2 (HER2) mutations, as detected by an FDA-approved test, and who have received a prior systemic therapy. The approval was based on a prespecified interim analysis of DESTINY-Lung02 (Study U206), a multi-center, randomized, dose-optimization trial in patients with NSCLC harboring activating HER2-mutations. At the approved dose of 5.4 mg/kg given intravenously every 3 weeks, the overall response rate (ORR) was 58% (95% confidence interval [CI]: 43, 71). The median duration of response was 8.7 months (95% CI: 7.1, not estimable). These results were consistent with response rates observed at the 6.4 mg/kg dose level. The most common (≥ 20%) adverse reactions were nausea, constipation, decreased appetite, vomiting, fatigue, and alopecia. The rate of interstitial lung disease (ILD) or pneumonitis was 6% at the 5.4 mg/kg dose level and 14% at the 6.4 mg/kg dose level. In the setting of similar efficacy and reduced toxicity, approval was granted for the 5.4 mg/kg dose level. The applicant conducted a randomized, dose-optimization study with guidance from the FDA Oncology Center of Excellence's Project Optimus. This is the first approval of a targeted therapy for HER2-mutated NSCLC.

Establishment of a murine model of breast cancer expressing human epidermal growth factor receptor 2 (4T1-HER2).

AIM: Although people with HER2-positive breast cancer benefit from approved HER2-targeted therapy, acquiring resistance to the therapies occurs. Animal models can play a part in gaining a deep understanding of such a process and addressing questions concerning developing and improving immunotherapy approaches. MATERIALS AND METHODS: To develop such a model, we transfected murine 4T1 cells with the pCMV6-Neo-HER2 construct and evaluated HER2 expression and its effects on the established cell line behavior in vitro and in vivo. RESULTS: Data illustrated that human HER2 protein was expressed on isolated 4T1-HER2 clones in vitro and in vivo. Except for proliferation over 48 hours, such expression did not change 4T1-HER2 characteristics compared to 4T1 in vitro. Notwithstanding the reduction in proliferation, the rate of tumorigenicity was 90% in challenged mice and Herceptin therapy significantly decreased tumors' growth and metastasis compared to the control group. CONCLUSION: We describe a murine model for HER2-positive breast cancer not only helping shed light on the mechanisms by which the tumor evades antitumor immunity but also playing a key role in making breast cancer more sensitive to novel immunotherapy modalities.

Blockade of SIRPα-CD47 axis by anti-SIRPα antibody enhances anti-tumor activity of DXd antibody-drug conjugates.

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.

Trastuzumab-functionalized bionic pyrotinib liposomes for targeted therapy of HER2-positive breast cancer.

In this study, we prepared a bionic nanosystem of trastuzumab-functionalized SK-BR-3 cell membrane hybrid liposome-coated pyrotinib (Ptb-M-Lip-Her) for the treatment of HER2-positive breast cancer. Transmission electron microscopy, dynamic light scattering, polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were used to verify the successful preparation of Ptb-M-Lip-Her. In vitro drug release experiments proved that Ptb-M-Lip-Her had a sustained release effect. Cell uptake experiments and in vivo imaging experiments proved that Ptb-M-Lip-Her had good targeting ability to homologous tumor cells (SK-BR-3). The results of cell experiments such as MTT, flow cytometry, immunofluorescence staining and in vivo antitumor experiments showed that Ptb-M-Lip-Her could significantly promote apoptosis and inhibit the proliferation of SK-BR-3 cells. These results clearly indicated that Ptb-M-Lip-Her may be a promising biomimetic nanosystem for targeted therapy of HER2-positive breast cancer.

Elimination of radiation-induced senescent cancer cells and stromal cells in vitro by near-infrared photoimmunotherapy.

INTRODUCTION: Therapy-induced senescent cancer and stromal cells secrete cytokines and growth factors to promote tumor progression. Therefore, senescent cells may be novel targets for tumor treatment. Near-infrared photoimmunotherapy (NIR-PIT) is a highly tumor-selective therapy that employs conjugates of a molecular-targeting antibody and photoabsorber. Thus, NIR-PIT has the potential to be applied as a novel senolytic therapy. This study aims to investigate the efficacy of NIR-PIT treatment on senescent cancer and stromal cells. METHODS: Two cancer cell lines (human lung adenocarcinoma A549 cells and human pancreatic cancer MIA PaCa-2 cells) and two normal cell lines (mouse fibroblast transfected with human epidermal growth factor receptor 2 [HER2] cells and human fibroblast WI38 cells) were used. The cytotoxicity of NIR-PIT was evaluated using anti-epidermal growth factor receptor (EGFR) antibody panitumumab and anti-HER2 antibody transtuzumab. RESULTS: Cellular senescence was induced in A549 and MIA PaCa-2 cells by 10 Gy γ-irradiation. The up-regulation of cellular senescence markers and characteristic morphological changes in senescent cells, including enlargement, flattening, and multinucleation, were observed in cancer cells after 5 days of γ-irradiation. Then, NIR-PIT targeting EGFR was performed on these senescent cancer cells. The NIR-PIT induced morphological changes, including bleb formation, swelling, and the inflow of extracellular fluid, and induced a significant decrease in cellular viability. These results suggested that NIR-PIT may induce cytotoxicity using the same mechanism in senescent cancer cells. In addition, similar morphological changes were also induced in radiation-induced senescent 3T3-HER2 fibroblasts by NIR-PIT targeting human epidermal growth factor receptor 2. CONCLUSION: NIR-PIT eliminates both senescent cancer and stromal cells in vitro suggesting it may be a novel strategy for tumor treatment.

HER-2 positive gastric cancer: Current targeted treatments.

Gastric cancer (GC) is highly metastatic and characterized by HER2 amplification. Aberrant HER2 expression drives metastasis, therapy resistance, and tumor recurrence. HER2 amplification contributes to drug resistance by upregulating DNA repair enzymes and drug afflux proteins, reducing drug efficacy. HER2 modulates transcription factors critical for cancer stem cell properties, further impacting drug resistance. HER2 activity is influenced by HER-family ligands, promoting oncogenic signaling. These features point to HER2 as a targetable driver in GC. This review outlines recent advances in HER2-mediated mechanisms and their upstream and downstream signaling pathways in GC. Additionally, it discusses preclinical research investigation that comprehends trastuzumab-sensitizing phytochemicals, chemotherapeutics, and nanoparticles as adjunct therapies. These developments hold promise for improving outcomes and enhancing the management of HER2-positive GC.

3D layered co-culture model enhances Trastuzumab Deruxtecan sensitivity and reveals the combined effect with G007-LK in HER2-positive non-small cell lung cancer.

Human epidermal growth factor receptor 2 (HER2) aberrations are observed in various cancers. In non-small cell lung cancer, genetic alterations activating HER2, mostly exon 20 insertion mutations, occur in approximately 2-4% of cases. Trastuzumab deruxtecan (T-DXd), a HER2-targeted antibody-drug conjugate has been approved as the first HER2-targeted drug for HER2-mutant lung cancer. However, some cases are not responsive to T-DXd and the primary resistant mechanism remains unclear. In this study, we assessed sensitivity to T-DXd in JFCR-007, a patient-derived HER2-mutant lung cancer cell line. Although JFCR-007 was sensitive to HER2 tyrosine kinase inhibitors, it showed resistance to T-DXd in attachment or spheroid conditions. Accordingly, we established a three-dimensional (3D) layered co-culture model of JFCR-007, where it exhibited a lumen-like structure and became sensitive to T-DXd. In addition, an in-house inhibitor library screening revealed that G007-LK, a tankyrase inhibitor, was effective when combined with T-DXd. G007-LK increased the cytotoxicity of topoisomerase-I inhibitor, DXd, a payload of T-DXd and SN-38. This combined effect was also observed in H2170, an HER2-amplified lung cancer cell line. These results suggest that the proposed 3D co-culture system may help in evaluating the efficacy of T-DXd and may recapitulate the tumor microenvironment.

A metabolic inhibitor blocks cellular fucosylation and enables production of afucosylated antibodies.

The fucosylation of glycoproteins regulates diverse physiological processes. Inhibitors that can control cellular levels of protein fucosylation have consequently emerged as being of high interest. One area where inhibitors of fucosylation have gained significant attention is in the production of afucosylated antibodies, which exhibit superior antibody-dependent cell cytotoxicity as compared to their fucosylated counterparts. Here, we describe ß-carbafucose, a fucose derivative in which the endocyclic ring oxygen is replaced by a methylene group, and show that it acts as a potent metabolic inhibitor within cells to antagonize protein fucosylation. ß-carbafucose is assimilated by the fucose salvage pathway to form GDP-carbafucose which, due to its being unable to form the oxocarbenium ion-like transition states used by fucosyltransferases, is an incompetent substrate for these enzymes. ß-carbafucose treatment of a CHO cell line used for high-level production of the therapeutic antibody Herceptin leads to dose-dependent reductions in core fucosylation without affecting cell growth or antibody production. Mass spectrometry analyses of the intact antibody and N-glycans show that ß-carbafucose is not incorporated into the antibody N-glycans at detectable levels. We expect that ß-carbafucose will serve as a useful research tool for the community and may find immediate application for the rapid production of afucosylated antibodies for therapeutic purposes.

Polyglycerol-Shelled Reduction-Sensitive Polymersome for DM1 Delivery to HER-2-Positive Breast Cancer.

Supramolecular delivery systems with the prolonged circulation, the potential for diverse functionalization, and few toxin-related limitations have been extensively studied. For the present study, we constructed a linear polyglycerol-shelled polymersome attached with the anti-HER-2-antibody trastuzumab. We then covalently loaded the anticancer drug DM1 in the polymersome via dynamic disulfide bonding. The resulted trastuzumab-polymersome-DM1 (Tra-PS-DM1) exhibits a mean size of 95.3 nm and remarkable drug loading efficiency % of 99.3%. In addition to its superior stability, we observed the rapid release of DM1 in a controlled manner under reductive conditions. Compared to the native polymersomes, Tra-PS-DM1 has shown greatly improved cellular uptake and significantly reduced IC50 up to 17-fold among HER-2-positive cancer cells. Moreover, Tra-PS-DM1 demonstrated superb growth inhibition of HER-2-positive tumoroids; specifically, BT474 tumoroids shrunk up to 62% after 12 h treatment. With exceptional stability and targetability, the PG-shelled Tra-PS-DM1 appears as an attractive approach for HER-2-positive tumor treatment.

Cubosomes functionalized with antibodies as a potential strategy for the treatment of HER2-positive breast cancer.

Breast cancers that overexpress human epidermal growth factor receptor 2 (HER2) have poor prognosis. Moreover, available chemotherapies cause numerous side effects due to poor selectivity. To advance more effective and safer therapies for HER2-positive breast cancer, we explored the fusion of drug delivery technology and immunotherapy. Our research led to the design of immunocubosomes loaded with panobinostat and functionalized with trastuzumab antibodies, enabling precise targeting of breast cancer cells that overexpress HER2. We characterised the nanostructure of cubosomes using small-angle X-ray scattering (SAXS), cryo-transmission electron microscopy (cryo-TEM), and dynamic light scattering (DLS). Moreover, we confirmed the integrity of the trastuzumab antibodies on the immunocubosomes by Fourier-transform infrared spectroscopy (FTIR) and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, we found that panobinostat-loaded immunocubosomes were more cytotoxic, and in an uptake-dependant manner, towards a HER2-positive breast cancer cell line (SKBR3) compared to a cell line representing healthy cells (L929). These results support that the functionalization of cubosomes with antibodies enhances both the effectiveness of the loaded drug and its selectivity for targeting HER2-positive breast cancer cells.

Photoimmunotherapy of HER2-expressing Breast Cancer Cells.

BACKGROUND/AIM: Breast cancer (BC) is the most common malignant disease worldwide. Localized stages of BC can be successfully treated by surgery. However, local recurrence occurs in about 4-10% of patients, requiring systemic treatments that impair the patients' quality of life and shortens life expectancy. Therefore, new therapeutic options are needed, which can be used intraoperatively and contribute to the complete removal of residual tumor cells in the surgical area. In the present study, we describe a cysteine-modified variant of the anti-HER2 antibody trastuzumab, that was coupled to the silicon phthalocyanine photosensitizer dye WB692-CB1 for the photoimmunotherapy (PIT) of BC. MATERIALS AND METHODS: The cysteine modified trastuzumab variant was cloned and expressed in Expi293F cells. After purification via immobilized affinity chromatography, the antibody was coupled to the dye. Cell binding of the antibody and the antibody dye conjugate was measured by flow cytometry. After incubation of BC cells with the conjugate and activation of the dye by irradiation with red light, cell viability was determined. RESULTS: The antibody and the conjugate showed specific binding to HER2-expressing BC cells. Treatment of the HER2high BC cell line SK-BR-3 with the conjugate followed by irradiation with a red light dose of 32 J/cm2 led to complete cell killing within 24 h. CONCLUSION: Our novel antibody dye conjugate represents a promising candidate for intraoperative treatment of localized BC, aiming to eliminate residual tumor cells in the surgical area and potentially reduce local recurrence, thereby improving recovery prospects for BC patients.

In Vivo PET Imaging of 89Zr-Labeled Natural Killer Cells and the Modulating Effects of a Therapeutic Antibody.

Natural killer (NK) cells can kill cancer cells via antibody-dependent cell-mediated cytotoxicity (ADCC): a tumor-associated IgG antibody binds to the Fcγ receptor CD16 on NK cells via the antibody Fc region and activates the cytotoxic functions of the NK cell. Here, we used PET imaging to assess NK cell migration to human epidermal growth factor receptor 2 (HER2)-positive HCC1954 breast tumors, examining the influence of HER2-targeted trastuzumab antibody treatment on NK cell tumor accumulation. Methods: Human NK cells from healthy donors were expanded ex vivo and labeled with [89Zr]Zr-oxine. In vitro experiments compared the phenotypic markers, viability, proliferation, migration, degranulation, and ADCC behaviors of both labeled (89Zr-NK) and unlabeled NK cells. Female mice bearing orthotopic human breast HCC1954 tumors were administered 89Zr-NK cells alongside trastuzumab treatment or a sham treatment and then scanned using PET/CT imaging over 7 d. Flow cytometry and γ-counting were used to analyze the presence of 89Zr-NK cells in liver and spleen tissues. Results: 89Zr cell radiolabeling yields measured 42.2% ± 8.0%. At an average specific activity of 16.7 ± 4.7 kBq/106 cells, 89Zr-NK cells retained phenotypic and functional characteristics including CD56 and CD16 expression, viability, migration, degranulation, and ADCC capabilities. In vivo PET/CT studies indicated predominant accumulation of 89Zr-NK cells in the liver and spleen. Ex vivo analyses of liver and spleen tissues indicated that the administered human 89Zr-NK cells retained their radioactivity in vivo and that 89Zr did not transfer to cells of murine soft tissues, thus validating this 89Zr PET method for NK cell tracking. Notably, 89Zr-NK cells migrated to HER2-positive tumors, both with and without trastuzumab treatment. Trastuzumab treatment was associated with an increased 89Zr-NK cell signal at days 1 and 3 after injection. Conclusion: In vitro, 89Zr-NK cells maintained key cellular and cytotoxic functions. In vivo, 89Zr-NK cells trafficked to HER2-postive tumors, with trastuzumab treatment correlating with enhanced 89Zr-NK infiltration. This study demonstrates the feasibility of using PET to image 89Zr-NK cell infiltration into solid tumors.

An optimised patient-derived explant platform for breast cancer reflects clinical responses to chemotherapy and antibody-directed therapy.

Breast Cancer is the most common cancer among women globally. Despite significant improvements in overall survival, many tumours are refractory to therapy and so novel approaches are required to improve patient outcomes. We have evaluated patient-derived explants (PDEs) as a novel preclinical platform for breast cancer (BC) and implemented cutting-edge digital pathology and multi-immunofluorescent approaches for investigating biomarker changes in both tumour and stromal areas at endpoint. Short-term culture of intact fragments of BCs as PDEs retained an intact immune microenvironment, and tumour architecture was augmented by the inclusion of autologous serum in the culture media. Cell death/proliferation responses to FET chemotherapy in BC-PDEs correlated significantly with BC patient progression-free survival (p = 0.012 and p = 0.0041, respectively) and cell death responses to the HER2 antibody therapy trastuzumab correlated significantly with HER2 status (p = 0.018). These studies show that the PDE platform combined with digital pathology is a robust preclinical approach for informing clinical responses to chemotherapy and antibody-directed therapies in breast cancer. Furthermore, since BC-PDEs retain an intact tumour architecture over the short-term, they facilitate the preclinical testing of anti-cancer agents targeting the tumour microenvironment.

Synthesis and evaluation of antibody-drug conjugates with high drug-to-antibody ratio using dimaleimide-DM1 as a linker- payload.

The notable characteristics of recently emerged Antibody-Drug Conjugates (ADCs) encompass the targeting of Human Epidermal growth factor Receptor 2 (HER2) through monoclonal antibodies (mAbs) and a high ratio of drug to antibody (DAR). The achievements of Kadcyla® (T-DM1) and Enhertu® (T-Dxd) have demonstrated that HER2-targeting antibodies, such as trastuzumab, have shown to be competitive in terms of efficacy and price for development. Furthermore, with the arrival of T-Dxd and Trodelvy®, high-DAR (7-8) ADCs, which differ from the moderate DAR (3-4) ADCs that were formerly regarded as conventional, are being acknowledged for their worth. Following this trend of drug development, we endeavored to develop a high-DAR ADC using a straightforward approach involving the utilization of DM1, a highly potent substance, in combination with the widely recognized trastuzumab. To achieve a high DAR, DM1 was conjugated to reduced cysteine through the simple design and synthesis of various dimaleimide linkers with differing lengths. Using LC and MS analysis, we have demonstrated that our synthesis methodology is uncomplicated and efficacious, yielding trastuzumab-based ADCs that exhibit a remarkable degree of uniformity. These ADCs have been experimentally substantiated to exert an inhibitory effect on cancer cells in vitro, thus affirming their value as noteworthy additions to the realm of ADCs.

Phase Ib dose-escalation trial of taselisib (GDC-0032) in combination with HER2-directed therapies in patients with advanced HER2+ breast cancer.

BACKGROUND: In most patients with advanced human epidermal growth factor receptor-2-positive (HER2+) breast cancer, anti-HER2 therapies fail due to the development of acquired resistance, potentially mediated through phosphoinositide-3-kinase (PI3K) signaling. We investigated adding taselisib, an α-selective potent oral inhibitor of PI3K, to different HER2-directed regimens in order to improve disease control. PATIENTS AND METHODS: Patients (n = 68) with advanced HER2+ breast cancer were enrolled to this open-label, dose-escalation phase Ib study. The primary endpoint was defining the maximal tolerated dose (MTD) for the various taselisib-containing combinations. The secondary endpoint was safety. Exploratory endpoints included circulating tumor DNA analysis. The study included four cohorts: (A) taselisib + trastuzumab emtansine (T-DM1), (C) taselisib + trastuzumab and pertuzumab (TP), (D) taselisib + TP + paclitaxel, and (E) taselisib + TP + fulvestrant. RESULTS: Following dose escalation, the taselisib MTD was defined as 4 mg once daily. Treatment was associated with significant toxicities, as 34 out of 68 patients experienced grade ≥3 adverse events (AEs) attributed to taselisib, the most common all-grade AEs being diarrhea, fatigue, and oral mucositis. At a median follow-up of 43.8 months, median progression-free survival (PFS) for the MTD-treated population in cohorts A, C, and E was 6.3 [95% confidence interval (CI) 3.2-not applicable (NA)] months, 1.7 (95% CI 1.4-NA) months, and 10.6 (95% CI 8.3-NA) months, respectively. The median PFS for patients in cohort A with prior T-DM1 use was 10.4 (95% CI 2.7-NA) months. CONCLUSIONS: PIK3CA targeting with taselisib in combination with HER2-targeted therapies was associated with both promising efficacy and substantial toxicities.