Receptor-mediated Internalization Research Articles

Chemically engineered polyethylenimine conjugates for mannose receptor-mediated gene delivery with potential antimicrobial and anticancer activity

Polyethylenimines exhibit unique intrinsic properties, which make them potential gene delivery agents. However, nonspecific interactions and cytotoxicity in the cells/tissues have significantly hampered their use in clinical applications. To subside these concerns, plant-based cyclitols, shikimic and quinic acids, have been tethered to polyethylenimine (PEI) to fabricate shikimoyl-PEI (SP) and quinoyl-PEI (QP) conjugates under mild coupling conditions. On interactions with plasmid DNA, the size and zeta potential of SP and QP complexes were found in the range of ∼245–158 nm and ∼ +22–10 mV, respectively. Subsequent assessment of these complexes revealed that cytocompatibility (∼90%–94%) and transfection efficiency (∼90% cells transfected) were significantly higher as compared to the native PEI/pDNA and Lipofectamine/pDNA complexes. Further, a higher degree of pDNA release was also obtained from SP/DNA and QP/DNA complexes. A decrease in GFP expression was observed in a competitive assay on RAW 264.7 cells, which reflected receptor-mediated internalization of SP and QP/DNA complexes. Besides, both SP and QP conjugates also displayed efficient antimicrobial activity against differential pathogenic strains of bacteria. Of these conjugates, QP conjugates exhibited promising anticancer activity against variable cell lines such as RAW 264.7, MCF-7, HepG2 cells. Altogether, these results advocate the promising potential of SP and QP conjugates as efficient gene delivery agents having effective antimicrobial and anticancer competence.

Transferrin-conjugated UiO-66 metal organic frameworks loaded with doxorubicin and indocyanine green: A multimodal nanoplatform for chemo-photothermal-photodynamic approach in cancer management

Stimuli-responsive nanoplatforms have been popular in controlled drug delivery research because of their ability to differentiate the tumor microenvironment from the normal tissue environment in a spatiotemporally controllable manner. The synergistic therapeutic approach of combining cancer chemotherapy with photothermal tumor ablation has improved the therapeutic efficacy of cancer therapeutics. In this study, a UiO-66 metal organic framework (MOF)-based system loaded with doxorubicin (DOX), surface decorated with the photothermal agents indocyanine green (ICG) and polydopamine (PDA), and conjugated with transferrin (TF) was successfully designed to operate as a responsive system to pH changes, featuring photothermal capabilities and target specificity for the purpose of treating breast cancer. The synthesized nanoplatform benefits from its uniform size, excellent DOX encapsulation efficiency (91.66 %), and efficient pH/NIR-mediated controlled release of the drug. In vitro photothermal studies indicate excellent photothermal stability of the formulation even after 6 on–off cycles of NIR irradiation. The in vitro cytotoxicity assessment using an NIR laser (808 nm) revealed that the DOX-loaded functionalized UiO-66 nanocarriers had outstanding inhibitory effects on 4T1 cells because of synergistic chemo-photo therapies, with no substantial toxicity by the carriers. In addition, cellular uptake evaluations revealed that UiO-DOX-ICG@PDA-TF could specifically target 4T1 cells on the basis of receptor-mediated internalization of transferrin receptors. Additionally, in vivo toxicity studies in Wistar rats indicated no signs of significant toxicity. The UiO-based nanoformulations effectively inhibited and destroyed cancer cells under 808 nm laser irradiation because of their minimal toxicity, strong biocompatibility, and outstanding synergistic chemo/photothermal/photodynamic treatment.

T cell receptor-directed antibody-drug conjugates for the treatment of T cell-derived cancers

T cell-derived cancers are hallmarked by heterogeneity, aggressiveness, and poor clinical outcomes. Available targeted therapies are severely limited due to a lack of target antigens that allow discrimination of malignant from healthy T cells. Here we report a novel approach for the treatment of T cell diseases based on targeting the clonally rearranged T cell receptor displayed by the cancerous T cell population. As a proof of concept, we identified an antibody with unique specificity towards a distinct TCR and developed antibody-drug conjugates, precisely recognizing and eliminating target T cells while preserving overall T cell repertoire integrity and cellular immunity. Our anti-TCR antibody-drug conjugates demonstrated effective receptor-mediated cell internalization, associated with induction of cancer cell death with strong signs of apoptosis. Furthermore, cell proliferation-inhibiting bystander effects observed on target-negative cells may contribute to the molecules’ anti-tumor properties precluding potential tumor escape mechanisms. To our knowledge, this represents the first anti-TCR antibody-drug conjugate designed as custom-tailored immunotherapy for T cell-driven pathologies.

Receptor-mediated internalisation of the long-acting growth hormone analogue somapacitan in comparison with recombinant human growth hormone and pegvisomant

Receptor-mediated internalisation of the long-acting growth hormone analogue somapacitan in comparison with recombinant human growth hormone and pegvisomant

Abstract PO3-18-04: MBRC-101: a novel antibody-drug conjugate (ADC) targeting the membrane-associated tyrosine kinase receptor EphA5 in breast cancer

Abstract Objectives: ADCs have emerged as a major advance in contemporary medical oncology. The receptor tyrosine kinase (RTK) EphA5 possesses the classical receptor kinase topology with an extracellular-binding domain, a single-pass transmembrane domain, and a cytoplasmic kinase domain. We have previously shown that EphA5 is highly expressed in non-small cell lung cancer and contributes to DNA-damage repair and radiation therapy resistance. Others have reported EphA5 expression in gastric, ovarian, and pancreatic cancers. Here, we investigated the expression of EphA5 in breast cancer (BC) and assessed EphA5 as a potential cancer-specific target using a novel ADC designated MBRC-101. Methods: MBRC-101 is composed of a humanized anti-EphA5 IgG1 kappa monoclonal antibody conjugated to the small molecule microtubule-disrupting agent monomethyl auristatin E (MMAE) via a protease cleavable valine-citrulline (vc) ThioBridge linker (ThioBridge™-Glu-[Val-Cit-PAB-MMAE] PEG [24u]). MBRC-101 averages a drug-to-antibody ratio (DAR)=4. The specificity profile of the anti-EphA5 monoclonal antibody was characterized using a Membrane Proteome Array (MPA). Receptor-mediated antibody internalization was determined by real-time live-cell analysis; cell killing assays used both live-cell analysis of cell confluence and the CellTiter-Glo® luminescent cell viability assay. Expression of EphA5 in archival human breast tumor tissue sections was evaluated by immunohistochemistry (IHC) using a commercial polyclonal antibody against human EphA5. In vivo efficacy studies used patient-derived xenograft (PDX) models of BC. Results: IHC demonstrated robust and selective EphA5 expression in 87% (20 of 23) of triple negative breast cancer (TNBC) and 88% (23 of 26) of hormone receptor-positive (HR+) patient tumors tested. EphA5 expression was not detected in adjacent, non-malignant breast tissue. Antibody-specificity profile testing showed that the anti-EphA5 antibody bound exclusively to EphA5 and did not cross-react with other members of the Eph receptor/ephrin ligand family. Binding of the anti-EphA5 antibody to EphA5 on the cell surface triggered rapid internalization and processing of the EphA5/antibody complex through the endosomal-lysosomal system. Consistently, MBRC-101 was cytotoxic to EphA5-expressing cells and its activity was concentration-dependent and commensurate to the levels of EphA5 on the cell surface. In PDX murine models of TNBC, once weekly administrations of intravenous (IV) MBRC-101 showed dose-dependent, robust, and reproducible anti-tumor activity in vivo. Partial tumor responses were observed at doses of 2.5 mg/Kg IV and complete and nearly complete tumor responses starting at 5 mg/Kg IV. Doses up to 10 mg/Kg IV were well-tolerated in tumor-bearing mice with no observable weight loss. Pre-clinical safety confirmed MBRC-101 is also well tolerated in rats and in cynomolgus monkeys. Conclusions: These results support EphA5 as a new and promising membrane-associated molecular target for the design and development of ADC-based therapies against hormone-positive breast cancer and TNBC and support the conduct of a Phase 1/1b study of MBRC-101 in patients with breast cancer. Citation Format: Fernanda Staquicini, Fenny Tang, Vanessa de Oliveira, Daniela Staquicini, Yongjian Wu, Kirstin Barnhart, J Kellogg Parsons, Wadih Arap, Isan Chen, Renata Pasqualini. MBRC-101: a novel antibody-drug conjugate (ADC) targeting the membrane-associated tyrosine kinase receptor EphA5 in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-18-04.

Abstract LB131: CBO421, a novel drug Fc-conjugate, inhibits the enzymatic activity of CD73 and triggers CD73 internalization

Abstract Background: CD73 (5’-nucleotidase or NT5E) is a cell surface enzyme responsible for the rate limiting step in adenosine production; the conversion of adenosine monophosphate (AMP) into adenosine. Immunosupressive adenosine contributes to immune evasion in solid tumors by accumulating within the tumor microenvironment and thereby inhibiting immune effector cells (e.g., CD8+ T cells). CD73 expression levels correlate with a worse prognosis in specific cancer types, including breast cancer. Adenosine production via CD73 can be therapeutically inhibited by two independent mechanisms: (1) enzyme inhibition, and (2) receptor internalization and subsequent degradation. CBO421, a drug Fc-conjugate (DFC), comprised of a novel small molecule CD73 inhibitor stably conjugated to an immune-silent human IgG1 Fc, achieves both mechanisms, combining the strengths of small molecule inhibitors and CD73-targeting monoclonal antibodies (mAbs) with potential best-in-class efficacy. Methods: The activities of CBO421, a commercially sourced CD73 small molecule inhibitor, quemliclustat (AB680), and biosimilar anti-CD73 mAb, oleclumab (MEDI9447), were investigated. Enzymatic inhibition of soluble CD73 and membrane-bound CD73 on MDA-MB-231 cells was determined. Receptor-mediated internalization of CD73 was assessed using the human triple-negative breast cancer cell line (TNBC), MDA-MB-231, by flow cytometry. Cells were treated with CBO421 or oleclumab. CD73 internalization was measured as the decrease in CD73 surface receptor signal over a 6 h time-course using an anti-human CD73 detection antibody with non-overlapping epitopes to CBO421 or oleclumab. Results: CD73 inhibition by CBO421 of soluble CD73 or CD73 expressed on the surface of the MDA-MB-231 cell line was comparable or superior to AB680 or oleclumab. Receptor internalization activities of CBO421 and oleclumab were compared. CBO421 and oleclumab internalization percentages were determined at 1 h (52.3% vs 26.7%), 4 h (80.8% vs 38.0%), and 6 h (84.8% vs 42.9%) respectively, with CBO421 demonstrating superior maximal receptor internalization. The unconjugated hIgG1 Fc negative control did not exhibit any receptor-mediated internalization of CD73, as expected. IC50 values were also calculated for CBO421 and oleclumab at 1 h (0.20 nM vs 0.88 nM), 4 h (0.07 nM vs 0.27 nM), and 6 h (0.08 nM vs 0.14 nM), respectively. Conclusions: CBO421 demonstrated potent and complete CD73 enzyme inhibition and robust CD73 receptor internalization in CD73+ cancer cells that was superior to oleclumab, thereby further differentiating CBO421 from existing CD73-targeting therapeutics currently in clinical development. Based on these compelling results and additional emerging data, CBO421 is progressing as a potential best-in-class clinical development candidate for the treatment of solid cancers. Citation Format: Elizabeth Abelovski, Nicholas Dedeic, Simon Döhrmann, James Levin, Amanda Almaguer, Doug Zuill, Joanne Fortier, Qiping Zhao, Maria Hernandez, Karin Amundson, Madison Moniz, Hongyuan Chen, Dhanya Panickar, Thanh Lam, Tom Brady, Allen Borchardt, Grayson Hough, Jeffrey B. Locke, Jason N. Cole, Leslie W. Tari. CBO421, a novel drug Fc-conjugate, inhibits the enzymatic activity of CD73 and triggers CD73 internalization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB131.

Abstract 2577: Analyzing specific and nonspecific tissue uptake of antibodies by SPECT imaging and non-linear compartmental modeling

Abstract Understanding the mechanisms driving specific target engagement versus off-target nonspecific cellular uptake of antibodies can help inform design and/or delivery strategies that maximize therapeutic indices. Receptor binding kinetics, rates of receptor-mediated internalization and nonspecific pinocytosis are often measured through in vitro cell culture models, but there have been fewer efforts to collectively measure these cellular parameters in vivo. Single photon emission computed tomography (SPECT) imaging is a powerful method of estimating these parameters by quantifying both free/bound (intact) and internalized/catabolized antibody using non-residualizing halogen labels and residualizing radiometal-chelates, respectively. Modeling longitudinal in vivo imaging data of non-residualizing antibody can inform receptor occupancy (RO), while residualizing tracer kinetics can provide additional insights into catabolism due to pinocytosis and receptor-mediated internalization in the intracellular spaces. Here, we describe SPECT imaging of tumor-targeting trastuzumab (anti-HER2) and non-targeting anti-glycoprotein D (anti-gD) antibody, each labeled with non-residualizing ([125I]SIB) and residualizing radiometal-chelate (111In-DOTA) radionuclides in HER2+ xenograft-bearing mice. A non-linear compartmental model was fitted to SPECT imaging data to characterize RO of 10 mg/kg of anti-HER2 out to 7 days post dosing as well as to estimate rate constants of pinocytosis and receptor-mediated internalization of anti-HER2 in tumor. Longitudinal SPECT imaging demonstrated statistically significant differences in tumor drug exposure between 125I- and 111In-labeled anti-HER2 relative to anti-gD (p < 0.01). Tracer kinetics of anti-HER2 and associated RO simulations in tumor showed rapid extravasation from blood to tumor with peak RO of 25% at 13 hours post administration followed by a reduction in RO, reaching ~10% by day 7. Simulations for free, bound and catabolized tracer kinetics from our fitted model showed that anti-HER2 in the tumor interstitium was primarily receptor-bound with minimal pinocytosis of unbound anti-HER2 as well as gradual receptor-mediated catabolism, reaching a peak concentration of 113 µg/mL 7 days post dosing. Rate constants of nonspecific pinocytosis and HER2-mediated receptor internalization were estimated to be 0.0145 and 0.036 hours−1, respectively. Our model allowed us to successfully capture anti-HER2 antibody receptor binding and specific and nonspecific internalization over time in vivo. These data and analyses demonstrate the power of SPECT imaging using both non-residualizing and residualizing radiolabels combined with compartmental modeling to better characterize the different biological states (free, bound, and catabolized) of antibody within interstitial and intracellular compartments. Citation Format: Nicholas Cho, Phillip Spinosa, Danielle Mandikian, Geoffrey Del Rosario, Shang-Fan Yu, Jason Ho, Greg Ferl, Charles A. Boswell. Analyzing specific and nonspecific tissue uptake of antibodies by SPECT imaging and non-linear compartmental modeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2577.

Abstract 718: CCR8 antibody drug conjugates: Targeting regulatory T cells for tumor sensitization

Abstract Background: Regulatory T (Treg) cells in the tumor microenvironment have an immune suppressive function, and their presence is associated with poor prognosis. Depletion of Treg cells may be a mechanism to sensitize tumors and represents a novel strategy for cancer immunotherapy. High expression of chemokine receptor CCR8 has been found in Treg cells in microenvironments of numerous tumors but not on Treg cells in systemic lymphoid tissues. The ability to selectively kill Treg cells in tumor environments may provide an opportunity for therapy that leaves Treg function intact elsewhere in the body where they normally mediate tolerance to self-antigen. Methods: We sought to identify CCR8 antibodies for therapeutics and designed an antibody discovery strategy to address the challenges of this target. CCR8 is a member of the G protein-coupled receptor (GPCR) family. Like many GPCRs, CCR8 is a valuable therapeutic antibody target but challenging to access due to its poor expression, membrane-dependent structure, and poor immunogenicity due to sequence conservation. We developed an antibody discovery platform (MPS) that specifically addresses each of these challenges through use of advanced immunization techniques and evolutionarily divergent host species (chickens) for robust immune responses against conserved targets. We extensively engineered CCR8 antigen to enable high expression for immunization. When sufficient antibody diversity was not obtained using our standard immunization techniques, we developed mRNA immunization techniques for this program. Antibody binding was measured using flow cytometry on transfected cells and human Treg cells. The ability of antibodies to inhibit CCR8 function was measured using intracellular calcium influx assays. Antibody internalization was measured using dye uptake assays and flow cytometry. Antibodies were conjugated with cytotoxic payloads and tested for the ability to mediate killing. Results: Using these approaches, we identified a panel of high-affinity antibodies that bound in the nanomolar or subnanomolar range with high specificity, and we selected a lead molecule to progress. We will present data from our antibody discovery campaign including mRNA immunization, binding profiles, GPCR functional data, receptor-mediated internalization, and the ability of antibody-drug conjugates (ADC) to kill CCR8+ cells, including human Treg cells, in vitro. Conclusion: Treg cells are a primary driver of the immunosuppressive microenvironment in many solid tumors. Targeting immune-suppressive CCR8+ Treg cells using ADC has potential to expand the treatment of solid tumors Citation Format: Lewis J. Stafford, Hayley Roth, Sarah Campbell, Samantha Brady, Valerie Fiers, FeiFan Yu, Andrew Tsourkas, Riley Payne, Meghan Pitts, Kyle Guldner, Tim Phillips, Breanna Tyrell, Colleen Ulke, Allison Snyder, Marianne Assogba, Holden Ohl, Ileine Sanchez, Benjamin Doranz, Joseph Rucker, Ross Chambers. CCR8 antibody drug conjugates: Targeting regulatory T cells for tumor sensitization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 718.

Abstract 738: MBRC-101: a novel antibody-drug conjugate (ADC) targeting the tyrosine kinase receptor EphA5

Abstract Introduction: The receptor tyrosine kinase EphA5 has the classical receptor kinase topology with an extracellular-binding domain, a single-pass transmembrane domain, and a cytoplasmic kinase domain. Previously, EphA5 has been shown to contribute to DNA-damage repair and radiation therapy resistance in lung cancer. We investigated EphA5 expression in lung cancer as well as breast, pancreatic, gastric, and colorectal cancer and assessed its potential as a therapeutic target using a novel ADC, MBRC-101. Methods: MBRC-101 is composed of a humanized anti-EphA5 IgG1 monoclonal antibody conjugated to monomethyl auristatin E (MMAE) via a cleavable valine-citrulline linker using site-specific ThioBridge™ technology. The specificity profile of the anti-EphA5 antibody was characterized using a membrane proteome array. Receptor-mediated antibody internalization was determined by real-time live-cell analysis, and cell killing assays were performed using analysis of cell confluence and luminescent-based viability. Expression of EphA5 in archival human tissue sections was evaluated by immunohistochemistry (IHC) using a commercial antibody. Efficacy studies used patient-derived xenograft (PDX) models of lung adenocarcinoma, squamous cell carcinoma of both lung and head and neck, and breast cancer. Results: IHC demonstrated selective EphA5 membrane expression in 78% (18/23) of triple negative and 88% (23/26) of hormone receptor-positive breast cancer tumors tested. EphA5 membrane expression was also detected in 85% (29/34), 73% (16/22), 70% (7/10), 70% (15/22), and 50% (6/12) of lung squamous cell carcinoma, lung adenocarcinoma, gastric, colorectal, and pancreatic patient tumors tested, respectively. EphA5 expression was not detected in adjacent, non-malignant tissue in any of the tumors examined. The anti-EphA5 antibody bound exclusively to EphA5 and did not cross-react with other members of the Eph receptor/ephrin ligand family. Binding of the anti-EphA5 antibody on the cell surface triggered rapid internalization and processing through the endosomal-lysosomal system. MBRC-101 was cytotoxic to EphA5-expressing cells and its activity was concentration-dependent and commensurate to levels of EphA5 on the cell surface. In PDX models, once weekly administrations of intravenous MBRC-101 showed dose-dependent, robust, and reproducible anti-tumor activity. Partial tumor responses were observed at doses of 2.5 mg/Kg and complete tumor responses starting at 5 mg/Kg. Toxicologic findings in Sprague-Dawley rats and cynomolgus monkeys were attributed to the MMAE payload and not target-related. Based on an HNSTD of 10 mg/kg in monkeys, the starting human dose was 0.5 mg/kg with ~15-fold safety margin. Conclusions: EphA5 is a new and promising molecular target for the development of ADC-based therapies against many human cancers. A Phase 1/1b study of MBRC-101 is currently recruiting (NCT06014658). Citation Format: Fernanda I. Staquicini, Fenny H. Tang, Vanessa de Oliveira, Daniela I. Staquicini, Yongjian Wu, Kirstin F. Barnhart, J Kellogg Parsons, Wadih Arap, Isan Chen, Renata Pasqualini. MBRC-101: a novel antibody-drug conjugate (ADC) targeting the tyrosine kinase receptor EphA5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 738.

Abstract 6573: Targeting DLK1, a Notch ligand, with an antibody-drug conjugate in adrenocortical carcinoma

Abstract Adrenocortical carcinoma (ACC) is an aggressive and rare endocrine malignancy with poor prognosis. First-line therapy (adrenolytic agent mitotane in combination with chemotherapy) for recurrent and metastatic ACC has limited efficacy and there are no approved second-line therapies. Antibody-drug conjugates (ADCs) are an emerging immunotherapeutic class in which a cytotoxic payload is directed to tumor cells via antibodies to cell surface proteins. As Notch ligands, such as DLL3, are ADC targets in neuroendocrine cancers, we screened for expression of Notch ligands in ACC. Among Notch ligands (DLL1, DLL3, DLK1, JAG1, JAG2), DLK1 (delta-like non-canonical Notch ligand 1) was the most highly expressed. Moreover, ACC had the near highest expression of DLK1 across all TCGA tumors likely because this ligand is of adrenal origin (with limited expression in other normal tissues apart from the pituitary gland and ovaries). By immunohistochemistry, we verified DLK1 to be expressed, at variable levels, in 97% of ACC tumors (n=30/31). We then tested a DLK1-directed antibody-drug conjugate (ADCT-701; DLK1-PBD), consisting of a humanized anti-DLK1 monoclonal antibody coupled to DNA damaging pyrrolobenzodiazepine (PBD) dimers (SG3199; drug-to-antibody ratio~1.8) via a cleavable linker in ACC pre-clinical models. ADCT-701 exhibited potent, nanomolar cytotoxicity in DLK1-expressing ACC cell lines and ACC patient-derived short-term organoid cultures. ADCT-701 cytotoxicity was dependent on DLK1 expression as DLK1 knockout and DLK1 overexpression abrogated and facilitated cytotoxicity, respectively. Mechanistically, ADCT-701 induced cytotoxicity through DLK1 dependent receptor-mediated internalization and DNA damage (γH2AX) as well as apoptosis (annexin V/PI positive cells, cleaved PARP, and cleaved caspase-3). In vivo studies showed that ADCT-701 was highly effective against DLK1-positive ACC xenografts and PDX models with durable anti-tumor activity. Our pre-clinical data demonstrate DLK1 as an important therapeutic target in ACC and support an upcoming phase I clinical trial of ADCT-701 in patients with neuroendocrine tumors including ACC (NCT06041516). Citation Format: Nai-Yun Sun, Suresh Kumar, Amber Weiner, Yoo Sun Kim, Arnulfo Mendoza, Rosa Nguyen, Reona Okada, Yves Pommier, Dan Martinez, Jennifer Pogoriler, Sharon Diskin, John Maris, Jaydira Del Rivero, Nitin Roper. Targeting DLK1, a Notch ligand, with an antibody-drug conjugate in adrenocortical carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6573.

Acidity-Triggered "Sticky Spotlight": CCK2R-Targeted TME-Sensitive NIR Fluorescent Probes for Tumor Imaging In Vivo.

Cancer which causes high mortality globally threatens public health seriously. There is an urgent need to develop tumor-specific near-infrared (NIR) imaging agents to achieve precise diagnosis and guide effective treatment. In recent years, imaging probes that respond to acidic environments such as endosomes, lysosomes, or acidic tumor microenvironments (TMEs) are being developed. However, because of their nonspecific internalization by both normal and tumor cells, resulting in a poor signal-to-noise ratio in diagnosis, these pH-sensitive probes fail to be applied to in vivo tumor imaging. To address this issue, a cholecystokinin-2 receptor (CCK2R)-targeted TME-sensitive NIR fluorescent probe R2SM was synthesized by coupling pH-sensitive heptamethine cyanine with a CCK2R ligand, minigastrin analogue 11 (MG11) for in vivo imaging, in which MG11 would target overexpressed CCK2Rs in gastrointestinal stromal tumors (GISTs). Cell uptake assay demonstrated that R2SM exhibited a high affinity for CCK2R, leading to receptor-mediated internalization and making probes finally accumulated in the lysosomes of tumor cells, which suggested in the tumor tissues, the probes were distributed in the extracellular acidic TME and intracellular lysosomes. With a pKa of 6.83, R2SM can be activated at the acidic TME (pH = 6.5-6.8) and lysosomes (pH = 4.5-5.0), exhibiting an apparent pH-dependent behavior and generating more intense fluorescence in these acidic environments. In vivo imaging showed that coupling of MG11 with a pH-sensitive NIR probe facilitated the accumulation of probe and enhanced the fluorescence in CCK2R-overexpressed HT-29 tumor cells. A high signal was observed in the tumor region within 0.5 h postinjection, indicating its potential application in intraoperative imaging. Fluorescence imaging of R2SM exhibited higher tumor-to-liver and tumor-to-kidney ratios (2.1:1 and 2.3:1, respectively), compared separately with the probes that are lipophilic, pH-insensitive, or MG11-free. In vitro and in vivo studies demonstrated that the synergistic effect of tumor targeting with pH sensitivity plays a vital role in the high signal-to-noise ratio of the NIR imaging probe. Moreover, different kinds of tumor-targeting vectors could be conjugated simultaneously with the NIR dye, which would further improve the receptor affinity and targeting efficiency.

Current common prodrug delivery methods and biochemical property

Prodrugs are pivotal in overcoming drug delivery and absorption challenges. These bioreversible derivatives are strategically designed to address medicines poor bioavailability and administration constraints. Prodrugs offer versatile solutions for various delivery methods, including oral, dermal, nasal, and central nervous system (CNS) routes and tumor targeting. This literature review aims to summarize several prodrugs delivery methods. The report also focuses on the common concepts of prodrug design. The utilization of prodrugs also includes innovative strategies, such as receptor-mediated internalization and responsive drug release, demonstrating promising outcomes for cancer therapy. The application of prodrug methods is comprehensive. Prodrugs emerged as a universal approach to improve drug administration and absorption, with their applications spanning diverse medical contexts. By tailoring prodrugs to specific delivery routes and addressing unique challenges, researchers open avenues for more effective and targeted therapies. Further exploration and optimization of prodrug strategies hold the potential to revolutionize drug delivery in clinical settings. Although prodrugs have many advantages, there are still many possible future improvements. Many diseases or target organs still need help applying prodrugs even though the current drug design needs this technology for administration. Future research should focus on the lack of chemical stability, including considering the balance between aqueous solubility and lipophilicity. Also, the elimination of the formation of degradation by-products or the design of acceptable metabolism pathways is another aspect that should be considered.

Tumor integrin targeted theranostic iron oxide nanoparticles for delivery of caffeic acid phenethyl ester: preparation, characterization, and anti-myeloma activities.

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells preferentially in the bone marrow. Currently, emerging chemotherapy drugs with improved biosafety profiles, such as immunomodulatory agents and protease inhibitors, have been used in clinics to treat MM in both initial therapy or maintenance therapy post autologous hematopoietic stem cell transplantation (ASCT). We previously discovered that caffeic acid phenethyl ester (CAPE), a water-insoluble natural compound, inhibited the growth of MM cells by inducing oxidative stress. As part of our continuous effort to pursue a less toxic yet more effective therapeutic approach for MM, the objective of this study is to investigate the potential of CAPE for in vivo applications by using magnetic resonance imaging (MRI)-capable superparamagnetic iron oxide nanoparticles (IONP) as carriers. Cyclo (Arg-Gly-Asp-D-Phe-Cys) (RGD) is conjugated to IONP (RGD-IONP/CAPE) to target the overexpressed αvβ3 integrin on MMcells for receptor-mediated internalization and intracellular delivery of CAPE. A stable loading of CAPE on IONP can be achieved with a loading efficiency of 48.7% ± 3.3% (wt%). The drug-release studies indicate RGD-IONP/CAPE is stable at physiological (pH 7.4) and basic pH (pH 9.5) and subject to release of CAPE at acidic pH (pH 5.5) mimicking the tumor and lysosomal condition. RGD-IONP/CAPE causes cytotoxicity specific to human MM RPMI8226, U266, and NCI-H929 cells, but not to normal peripheral blood mononuclear cells (PBMCs), with IC50s of 7.97 ± 1.39, 16.75 ± 1.62, and 24.38 ± 1.71μM after 72-h treatment, respectively. Apoptosis assays indicate RGD-IONP/CAPE induces apoptosis of RPMI8226 cells through a caspase-9 mediated intrinsic pathway, the same as applying CAPE alone. The apoptogenic effect of RGD-IONP/CAPE was also confirmed on the RPMI8226 cells co-cultured with human bone marrow stromal cells HS-5 in a Transwell model to mimic the MM microenvironment in the bone marrow. In conclusion, we demonstrate that water-insoluble CAPE can be loaded to RGD-IONP to greatly improve the biocompatibility and significantly inhibit the growth of MMcells in vitro through the induction of apoptosis. This study paves the way for investigating the MRI-trackable delivery of CAPE for MM treatment in animal models in the future.

Dally is not essential for Dpp spreading or internalization but for Dpp stability by antagonizing Tkv-mediated Dpp internalization

Dpp/BMP acts as a morphogen to provide positional information in the Drosophila wing disc. Key cell-surface molecules to control Dpp morphogen gradient formation and signaling are heparan sulfate proteoglycans (HSPGs). In the wing disc, two HSPGs, the glypicans Division abnormally delayed (Dally) and Dally-like (Dlp) have been suggested to act redundantly to control these processes through direct interaction of their heparan sulfate (HS) chains with Dpp. Based on this assumption, a number of models on how glypicans control Dpp gradient formation and signaling have been proposed, including facilitating or hindering Dpp spreading, stabilizing Dpp on the cell surface, or recycling Dpp. However, how distinct HSPGs act remains largely unknown. Here, we generate genome-engineering platforms for the two glypicans and find that only Dally is critical for Dpp gradient formation and signaling through interaction of its core protein with Dpp. We also find that this interaction is not sufficient and that the HS chains of Dally are essential for these functions largely without interacting with Dpp. We provide evidence that the HS chains of Dally are not essential for spreading or recycling of Dpp but for stabilizing Dpp on the cell surface by antagonizing receptor-mediated Dpp internalization. These results provide new insights into how distinct HSPGs control morphogen gradient formation and signaling during development.

Dally is not essential for Dpp spreading or internalization but for Dpp stability by antagonizing Tkv-mediated Dpp internalization.

Dpp/BMP acts as a morphogen to provide positional information in the Drosophila wing disc. Key cell-surface molecules to control Dpp morphogen gradient formation and signaling are heparan sulfate proteoglycans (HSPGs). In the wing disc, two HSPGs, the glypicans Division abnormally delayed (Dally) and Dally-like (Dlp) have been suggested to act redundantly to control these processes through direct interaction of their heparan sulfate (HS) chains with Dpp. Based on this assumption, a number of models on how glypicans control Dpp gradient formation and signaling have been proposed, including facilitating or hindering Dpp spreading, stabilizing Dpp on the cell surface, or recycling Dpp. However, how distinct HSPGs act remains largely unknown. Here, we generate genome-engineering platforms for the two glypicans and find that only Dally is critical for Dpp gradient formation and signaling through interaction of its core protein with Dpp. We also find that this interaction is not sufficient and that the HS chains of Dally are essential for these functions largely without interacting with Dpp. We provide evidence that the HS chains of Dally are not essential for spreading or recycling of Dpp but for stabilizing Dpp on the cell surface by antagonizing receptor-mediated Dpp internalization. These results provide new insights into how distinct HSPGs control morphogen gradient formation and signaling during development.

Purinergic Receptor P2Y12-Mediated Tau Internalization in Microglia.

Microglia are the resident brain macrophage cells that are involved in constant surveillance of brain microenvironment. In Alzheimer's disease, microglia get over activated upon the accumulation of Tau and amyloid-β species in the extracellular space, ultimately leading to neurodegeneration. Microglia phagocytose the extracellular Tau species by several mechanisms among which P2Y12 receptor-mediated internalization of extracellular Tau is recently studied. Extracellular Tau activates microglia and directly interacts with the P2Y12 receptor. Tau-receptor complex is then internalized followed by perinuclear accumulation and lysosomal degradation. Upon microglial activation by extracellular Tau, P2Y12 receptor is also involved in membrane-associated actin remodeling which has its key role in active migration and phagocytosis.

Interaction of Tau with G-Protein-Coupled Purinergic P2Y12 Receptor by Molecular Docking and Molecular Dynamic Simulation.

Alzheimer's disease, a progressive neurological disorder, is characterized by the accumulation of neurofibrillary tangles and senile plaques by Tau and amyloid-β, respectively, in the brain microenvironment. The misfolded protein aggregates interact with several components of neuronal and glial cells such as membrane lipids, receptors, transporters, enzymes, cytoskeletal proteins, etc. Under pathological conditions, Tau interacts with several G-protein-coupled receptors (GPCRs), which undergoes either receptor signaling or desensitization followed by internalization of the protein complex. The purinergic GPCR, P2Y12 which is expressed in microglial cells, plays a key role in its activation and migration. Microglial cells sense and migrate to the site of injury aided by P2Y12 receptor that interacts with ADP released from damaged cells. P2Y12 receptor also interacts with misfolded Tau accumulated at the extracellular space and promotes receptor-mediated internalization. Immunocolocalization and co-immunoprecipitation studies demonstrated the interaction of Tau species with the P2Y12 receptor. Later, in-silico analyses were carried out with the repeat domain of Tau (TauRD), which has been identified as the interacting partner of P2Y12 receptor by in-vitro studies. Molecular docking and molecular dynamics simulation studies show the stability and the type of interaction in TauRD-receptor complex. Tau interaction with P2Y12 receptor plays a significant role in maintaining the active state of microglia which could lead to neuroinflammation and neuronal damage in AD brain. Hence, blocking P2Y12-Tau interaction and P2Y12-mediated Tau internalization in microglial cells could be possible therapeutic strategies in downregulating the severity of neuroinflammation in AD.

A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy.

Interleukin (IL)-7 is broadly active on T-cell populations, and modified versions have been clinically evaluated for a variety of therapeutic applications, including cancer, lymphopenia, and infectious diseases; and found to be relatively well-tolerated and biologically active. Here we describe novel IL-7R agonists that are unrelated in structure to IL-7, bind to the receptor subunits differently from IL-7, but closely emulate IL-7 biology. The small size, low structural complexity, and the natural amino acid composition of the pharmacologically active peptide MDK1472 allows facile incorporation into protein structures, such as the IgG2-Fc fusion MDK-703. This molecule possesses properties potentially better suited to therapeutic applications than native IL-7 or its derivatives. We compared these compounds with IL-7 for immune cell selectivity, induction of IL-7R signaling, receptor-mediated internalization, proliferation, and generation of immune cell phenotypes in human and non-human primate (NHP) peripheral blood cells in vitro; and found them to be similar in biological activity to IL-7. In cynomolgus macaques, MDK-703 exhibits a circulating half-life of 46 hr and produces sustained T-cell expansion characteristic of IL-7 treatment. In the huCD34+-engrafted NSG mouse model of the human immune system, MDK-703 induces an immune cell profile very similar to that generated by IL-7-derived compounds; including the pronounced expansion of memory T-cells, particularly the population of stem-like memory T-cells (Tscm) which may be important for anti-tumor activities reported with IL-7 treatment. Clinical administration of IL-7 and modified variants has been reported to induce anti-drug antibodies (ADAs), including IL-7 neutralizing antibodies. The novel peptide agonist reported here scores very low in predicted immunogenicity, and because the peptide lacks sequence similarity with IL-7, the problematic immunogenic neutralization of endogenous cytokine should not occur. The properties we report here implicate MDK-703 as a candidate for clinical evaluation in oncology, anti-viral and other infectious disease, vaccine enhancement, and treatment of lymphopenia.

Dual-targeted delivery system using hollow silica nanoparticles with H+-triggered bubble generating characteristic coated with hyaluronic acid and AS1411 for cancer therapy

Objective Herein, a dual-targeting delivery system using mesoporous silica nanoparticles with hollow structures (HMSNs) was developed for the specific delivery of epirubicin (EPI) to cancer cells and introducing a H+-triggered bubble generating nanosystem (BGNS). HMSNs containing EPI are covered by hyaluronic acid (HA) shell and AS1411 aptamer to create the BGNS-EPI-HA-Apt complex, which is highly selective against CD44 marker and nucleolin overexpressed on the surface of tumor cells. Methods MTT assay compared the cytotoxicity of different treatments in CHO (Chinese hamster ovary) cells as well as 4T1 (murine mammary carcinoma) and MCF-7 (human breast adenocarcinoma) cells. The internalization of Epi was assessed by flow cytometry along with fluorescence imaging. In vivo studies were conducted on BALB/c mice bearing a tumor from 4T1 cell line where monitoring included measuring tumor volume, mouse weight changes over time alongside mortality rate; accumulation levels for Epi within organs were also measured during this process. Results The collected data illustrated that BGNS-EPI-HA-Apt complex controlled the release of EPI in a sustained method. Afterward, receptor-mediated internalization via nucleolin and CD44 was verified in 4T1 and MCF-7 cells using fluorescence microscopy assay and flow cytometry analysis. The results of tumor inhibitory effect study exhibited that BGNS-EPI-HA-Apt complex decreased off-target effect and improved on-target effects because of its targeting ability. Conclusion The data acquired substantiates that HA-surface modified HMSNs functionalized with aptamers possess significant potential as a focused platform for efficient transportation of anticancer agents to neoplastic tissues.

Longitudinal evaluation of the biodistribution and cellular internalization of the bispecific CD3xTRP1 antibody in syngeneic mouse tumor models

BackgroundCD3 bispecific antibodies (CD3-bsAbs) require binding of both a tumor-associated surface antigen and CD3 for their immunotherapeutic effect. Their efficacy is, therefore, influenced by the tumor uptake and the extracellular...