Targeted Radionuclide Therapy (TRT) enables selective delivery of radionuclides for cancer treatment. Alpha particle emitters such as 212Pb are emerging as potential gamechangers, representing highly potent payloads for precision therapy and refractory cancer treatment. While small-molecule carriers are widely explored due to favorable pharmacokinetics, nanoparticle-based TRT remains less studied due to perceived non-ideal pharmacokinetics. We report a unique nanoparticle-TRT pretargeting approach using bispecific antibodies (BsAbs) which "prime" tumor surfaces, enhancing tumor-specific delivery and minimizing off-target deposition of 212Pb. We developed a poly(ethylene glycol) (PEG)-based nanomedicine platform to carry 212Pb, and employed in-house designed and manufactured BsAb (α-epidermal growth factor receptor (EGFR)/α-PEG) to prime tumors to receive the nanocarrier. Sequential administration of each component to EGFR-expressing cells produced enhanced receptor-mediated internalization of the BsAb upon nanomedicine binding, resulting in improved radionuclide delivery and efficacy in a series of in vitro assays. The pretargeting approach more than tripled tumor retention of the 212Pb-nanomedicine compared to the untargeted nanomaterial in a murine EGFR+ breast cancer xenograft model, evidenced by single-photon emission computed tomography (SPECT) imaging of 212Pb-loaded nanomaterials and gamma analysis of the excised organs. Therapeutic studies demonstrated the 212Pb-nanomedicine to produce well-tolerated and statistically-enhanced therapeutic outcomes for the pretargeting versus conventional 212Pb-nanomedicine, with no observed long term hematological effects. This work establishes a modular strategy for targeted TRT nanomedicine delivery. The platform has potential for broad applicability, including simultaneous delivery of diverse or synergistic payloads. These findings represent an important advance toward precision nanomedicine approaches in radionuclide therapy.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an emerging target in cancer immunotherapy, recognized for its consistent and elevated expression across several epithelial tumors, including triple-negative breast cancer (TNBC). TNBC is an aggressive and difficult-to-treat cancer, with limited effective therapeutic options currently available. Therapeutic approaches centered on targeting ROR1 have therefore become increasingly popular, with ROR1 chimeric antigen receptor (CAR) T cells currently in clinical trials to treat TNBC patients. While ROR1-targeting therapies have shown promising preclinical results, single arm treatment has often shown low efficacy as well as off-target toxicity. Natural killer (NK) cell-based immunotherapies, such as antibody-dependent cell cytotoxicity-inducing monoclonal antibodies and CAR NK cells, have also been shown to induce cancer cell cytotoxicity; however, with less toxicity compared with CAR T cells. Here, we developed and characterized a phage-derived single-chain fragment variable (scFv) against a highly specific ROR1 region and generated scFv-derived chimeric monoclonal antibodies and anti-ROR1-CAR NK cells, which show anti-cancer efficacy against TNBC cells. Additionally, we found TGF-β inhibition using either small-molecule inhibitors or CRISPR-Cas9-edited NK cells could further enhance ROR1-targeting therapy persistence and efficacy in controlling TNBC tumor growth.

Anti-cancer monoclonal antibodies often fail to provide therapeutic benefit in receptor-positive patients due to rapid endocytosis of antibody-bound cell surface receptors. High dose co-administration of prochlorperazine (PCZ) inhibits endocytosis and sensitises tumours to mAbs by inhibiting dynamin II but can also introduce neurological side effects. We examined the potential to use PEGylated liposomal formulations of PCZ (LPCZ) to retain the anti-cancer effects of PCZ, but limit brain uptake. Uncharged liposomes showed complete drug encapsulation and pH-dependent drug release, but cationic liposomes showed limited drug encapsulation and lacked pH-dependent drug release. Uncharged LPCZ showed comparable inhibition of EGFR internalisation to free PCZ in KJD cells. After IV administration to rats, LPCZ reduced the plasma clearance and brain uptake of PCZ compared to IV PCZ. The results suggest that LPCZ may offer some benefit over PCZ as an adjunct therapy in cancer patients receiving mAb treatment.

Cold atmospheric plasma (CAP) holds promise as a cancer-specific treatment that selectively kills various types of malignant cells. We used CAP-activated media (PAM) to utilize a range of the generated short- and long-lived reactive species. Specific antibodies, small molecule inhibitors and CRISPR/Cas9 gene-editing approaches showed an essential role for receptor tyrosine kinases, especially epidermal growth factor (EGF) receptor, in mediating triple negative breast cancer (TNBC) cell responses to PAM. EGF also dramatically enhanced the sensitivity and specificity of PAM against TNBC cells. Site-specific phospho-EGFR analysis, signal transduction inhibitors and reconstitution of EGFR-depleted cells with EGFR-mutants confirmed the role of phospho-tyrosines 992/1173 and phospholipase C gamma signaling in up-regulating levels of reactive oxygen species above the apoptotic threshold. EGF-triggered EGFR activation enhanced the sensitivity and selectivity of PAM effects on TNBC cells. The proposed approach based on the synergy of CAP and EGFR-targeted therapy may provide new opportunities to improve the clinical management of TNBC.

OPINION STATEMENT:Monoclonal antibody (mAb) therapy is now considered a main component of cancer therapy in Australia. Although traditionally thought of as pure signalling inhibitors, a large proponent of these medications function through antibody-dependent cell-mediated cytotoxicity (ADCC). Currently, most protocols and institutional guidelines for ADCC-mediated mAbs promote the use of corticosteroids as premedication: this is implemented to reduce infusion-related reactions (IRRs) and antiemesis prophylaxis and combat concurrently administered chemotherapy-related syndromes. Concerningly, the inhibitory effects of ADCC by corticosteroids are well documented; henceforth, it is possible the current standard of care is misaligned to the literature surrounding ADCC. Subsequently, clinicians' decisions to act in contrast to this literature may be reducing the efficacy of mAbs. The literature suggests that the redundant use of corticosteroids should be cautioned against when used in conjunction with ADCC-mediated mAbs-this is due to the consequent reduction in anti-tumour activity. Owing to the fact IRRs typically occur upon initial infusion, the authors advocate for individual clinicians and institutional protocols to considering augmenting their practice to corticosteroid premedication at the first dose only, unless clinically indicated. Additionally, product information (PI) and consumer medicine information (CMI) documents distributed by Australian and international regulatory agencies should consider disclosing the risk of concurrent steroids with these medications. Moreover, the authors suggest considering alternative medications for the management of side effects.

Breast cancer exhibits the second-highest incidence and fourth-highest mortality rates worldwide based on current global cancer statistics.1 Significant advances such as surgical resection,radiation therapy,chemotherapy,endocrine therapy,and the emergence of targeted therapy and immunotherapy have notably enhanced survival outcomes for in-dividuals with early and moderately advanced breast cancer.Metastasis,the process by which an original primary tumor develops in a distal second organ,lacks efficacious treatment modalities for patients with advanced breast cancer.There is an imminent need to investigate novel approaches to treat patients with gastrointestinal metastasis of breast cancer.

IntroductionAbout 50% of cutaneous melanoma (CM) patients present activating BRAF mutations that can be effectively targeted by BRAF inhibitors (BRAFi). However, 20% of CM patients exhibit intrinsic drug resistance to BRAFi, while most of the others develop adaptive resistance over time. The mechanisms involved in BRAFi resistance are disparate and globally seem to rewire the cellular signaling profile by up-regulating different receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR). RTKs inhibitors have not clearly demonstrated anti-tumor activity in BRAFi resistant models. To overcome this issue, we wondered whether the shared up-regulated RTK phenotype associated with BRAFi resistance could be exploited by using immune weapons as the antibody-dependent cell cytotoxicity (ADCC)-mediated effect of anti-RTKs antibodies, and kill tumor cells independently from the mechanistic roots.Methods and resultsBy using an in vitro model of BRAFi resistance, we detected increased membrane expression of EGFR, both at mRNA and protein level in 4 out of 9 BRAFi-resistant (VR) CM cultures as compared to their parental sensitive cells. Increased EGFR phosphorylation and AKT activation were observed in the VR CM cultures. EGFR signaling appeared dispensable for maintaining resistance, since small molecule-, antibody- and CRISPR-targeting of EGFR did not restore sensitivity of VR cells to BRAFi. Importantly, immune-targeting of EGFR by the anti-EGFR antibody cetuximab efficiently and specifically killed EGFR-expressing VR CM cells, both in vitro and in humanized mouse models in vivo, triggering ADCC by healthy donors’ and patients’ peripheral blood cells. ConclusionOur data demonstrate the efficacy of immune targeting of RTKs expressed by CM relapsing on BRAFi, providing the proof-of-concept supporting the assessment of anti-RTK antibodies in combination therapies in this setting. This strategy might be expected to concomitantly trigger the crosstalk of adaptive immune response leading to a complementing T cell immune rejection of tumors.

Endocytosis is a complex process whereby cell surface proteins, lipids and fluid from the extracellular environment are packaged, sorted and internalized into cells. Endocytosis is also a mechanism of drug internalization into cells. There are multiple routes of endocytosis that determine the fate of molecules, from degradation in the lysosomes to recycling back to the plasma membrane. The overall rates of endocytosis and temporal regulation of molecules transiting through endocytic pathways are also intricately linked with signalling outcomes. This process relies on an array of factors, such as intrinsic amino acid motifs and post-translational modifications. Endocytosis is frequently disrupted in cancer. These disruptions lead to inappropriate retention of receptor tyrosine kinases on the tumour cell membrane, changes in the recycling of oncogenic molecules, defective signalling feedback loops and loss of cell polarity. In the past decade, endocytosis has emerged as a pivotal regulator of nutrient scavenging, response to and regulation of immune surveillance and tumour immune evasion, tumour metastasis and therapeutic drug delivery. This Review summarizes and integrates these advances into the understanding of endocytosis in cancer. The potential to regulate these pathways in the clinic to improve cancer therapy is also discussed.

Figure S1. Generation of E2F7 and E2F8 deficient murine keratinocytes and the validation of E2F1 levels in E2F1KO mice.

Dynamin II (dynII) plays a significant role in the internalization pathways of endocytic cells, by allowing membrane invaginations to "bud off". An important class of dynII inhibitors that are used clinically are phenothiazines, such as prochlorperazine (PCZ). PCZ is an antipsychotic drug but is also currently in clinical trials at higher concentrations as an adjuvant in cancer patients that increases the efficacy of monoclonal antibodies at high intravenous doses. It is unknown, however, whether high-dose dynII inhibitors have the potential to alter the pharmacokinetics of co-administered chemotherapeutic nanomedicines that are largely cleared via the mononuclear phagocyte system. This work therefore sought to investigate the impact of clinically relevant concentrations of phenothiazines, PCZ and thioridazine, on in vitro liposome endocytosis and in vivo liposome pharmacokinetics after PCZ infusion in rats. The uptake of fluorescently labeled PEGylated liposomes into differentiated and undifferentiated THP-1 and RAW246.7 cells, and primary human peripheral white blood cells, was investigated via flow cytometry after co-incubation with dynII inhibitors. The IV pharmacokinetics of PEGylated liposomes were also investigated in rats after a 20 min infusion with PCZ. Phenothiazines and dyngo4a reduced the uptake of PEGylated liposomes by THP-1 and RAW264.7 cells in a concentration-dependent manner in vitro. However, dynII inhibitors did not alter the mean uptake of liposomes by human peripheral white blood cells, but endocytic white cells from some donors exhibited sensitivity to phenothiazine exposure. When a clinically relevant dose of PCZ was co-administered with PEGylated liposomal doxorubicin (Caelyx/Doxil) in rats, the pharmacokinetics and biodistribution of liposomes were unaltered. These data suggest that while clinically relevant doses of dynII inhibitors can inhibit the uptake of liposomes by endocytic cells in vitro, they are unlikely to significantly affect the pharmacokinetics of long-circulating, co-administered liposomes.

Figure S2. Adenovirus infection of murine keratinocytes does not alter normal cell responses.

Figure S3. Cytotoxic responses to doxorubicin selectively enhanced in E2F7-deficient murine keratinocytes.

Supplementary Table S1: Differentially expressed genes (List A and List B combined) identified as E2F7-dependent cytotoxic sensitivity genes

Figure S5. SK1-I did not enhance doxorubicin sensitivity in KJDSV40 cells in vitro.

HER2Pro: A Phase 1b dose de-escalation study of high dose prochlorperazine added to paclitaxel, trastuzumab and pertuzumab in patients with previously untreated HER2-positive metastatic breast cancer Authors Teesha Downton1,2, Emma Karlsen1, Katharine Cuff3,4, Euan Walpole3,4, Fiona Simpson3,4, Elgene Lim1,2. Affiliations 1Garvan Institute of Medical Research, Darlinghurst NSW, Australia; 2School of Clinical Medicine, St Vincent’s Healthcare Clinical Campus, Faculty of Medicine and Health, University of New South Wales Sydney, Australia; 3Diamantina Institute, University of Queensland, Woolloongabba QLD, Australia; 4Princess Alexandra Hospital, Brisbane QLD, Australia Disclosures T. Downton: None. E. Karlsen: None. K. Cuff: None. E. Walpole: None. F. Simpson: None. E. Lim. Advisory Board for Pfizer, Astra Zeneca, Lilly, Roche, Novartis, Gilead Australia. Research Funding from Pfizer, Novartis, Bayer. Abstract Background: The anti-emetic prochlorperazine reversibly inhibits dynamin-mediated endocytosis. Preclinical studies have demonstrated that through this mechanism, high dose prochlorperazine can temporarily increase tumor cell antigen presentation, enhance interaction of tumor antigens with therapeutic monoclonal antibodies, and improve antibody-dependent cellular cytotoxicity (Chew H et al. Cell 2020). High dose prochlorperazine has been well tolerated and proof of mechanism demonstrated in a pilot study (ACTRN12619001051134), and in a phase Ib trial (ACTRN12619001527156) of prochlorperazine with cetuximab in patients with EGFR-expressing advanced head and neck squamous cell carcinoma or breast cancer. Prochlorperazine potentially offers a novel approach to improve the efficacy of a range of therapeutic antibodies. This study HER2Pro (ACTRN12622000016730) aims to evaluate the feasibility and safety of high dose prochlorperazine in combination with paclitaxel, trastuzumab, and pertuzumab in patients with HER2-positive metastatic breast cancer. Trial Design: In this phase Ib single-arm trial, patients receive standard of care trastuzumab and pertuzumab 3-weekly and paclitaxel weekly. From cycle 2, patients in addition receive de-escalating doses of prochlorperazine weekly for 6 weeks, though an additional 6 weeks may be given if there are no treatment associated serious adverse events. Dose de-escalation will be evaluated using a 3+3 design, commencing at a prochlorperazine intravenous dose of 0.8mg/kg weekly. Eligibility: Eligible patients must have previously untreated HER2-positive metastatic breast cancer, Eastern Cooperative Oncology Group performance status 0-1, and baseline left ventricular ejection fraction 50%. Key exclusion criteria include current daily treatment with corticosteroids at a dose >10mg prednisolone or equivalent, blood pressure < 90/50 mmHg, prolonged QT interval, and Parkinson’s disease. Objectives: The primary objective is to determine the recommended phase 2 dose. Secondary objectives include determining the frequency and severity of adverse events, rates of cardiotoxicity, objective response rate, duration of response and progression free survival. Tertiary objectives include analysis of receptor trafficking and immune system activation on paired tumor biopsies obtained before and after first prochlorperazine dose. Accrual: 6-12 patients will be enrolled across 2 Australian sites. Enrollment for this trial is expected to commence late 2022. Citation Format: Teesha Downton, Emma Karlsen, Katharine Cuff, Euan Walpole, Fiona Simpson, Elgene Lim. HER2Pro: A Phase 1b dose de-escalation study of high dose prochlorperazine added to paclitaxel, trastuzumab and pertuzumab in patients with previously untreated HER2-positive metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-10-05.

Figure S4. Validation of siRNA directed against E2F7 and E2F1 mRNA expression level in SCC25 cells in which E2F7 had been silenced by siRNA.

Figure S6. Inhibition of Sphk1 sensitizes FaDu cells to the cytotoxic actions of doxorubicin in vitro and in vivo.

Enhancing natural killer (NK) cell-based innate immunity has become a promis-ing strategy for immunotherapy against hard-to-cure solid cancers. Monoclonal antibody (mAb) therapy has been used to activate NK-cell-mediated antibody-dependent cellular cytotoxicity (ADCC) towards solid cancers. Cancer cells, however, can subvert immunosurveillance using multiple immunosuppressive mechanisms, which may hamper NK cell ADCC. Mechanisms to safely enhance ADCC by NK cells, such as utilizing temporary inhibition of receptor endocytosis to increase antibody presentation from target to effector cells can now be used to enhance NK-cell-mediated ADCC against solid tumors. This review summa-rizes and discusses the recent advances in the field and highlights current and potential future use of immunotherapies to maximize the therapeutic efficacy of innate anticancer immunity.