Targeting Moiety Research Articles

Interleukin-4 Receptor Targeting Peptide Decorated Extracellular Vesicles as a Platform for In Vivo Drug Delivery to Thyroid Cancer.

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have been demonstrated to deliver therapeutic drugs in preclinical studies. However, their use is limited, as they lack the ability to specifically deliver drugs to tumor tissues in vivo. In the present study, we propose the use of a targeting peptide, IL-4R-binding peptide (IL4RPep-1), to specifically deliver intravenously (i.v.) infused EVs to thyroid tumors. In vivo, a xenograft tumor model was treated with either the control peptide (NSSSVDK) or IL4RPep-1-Flamma; mice were fluorescently imaged (FLI) using an in vivo imaging system at 0–3 h post-treatment. EVs (labeled with DiD dye) were conjugated with IL4RPep-1 through a DOPE-NHS linker and administered to mice intravenously. FLI was performed 0–24 h post-injection, and the animals were sacrificed for further experiments. The morphology and size of EVs, the presence of EV markers such as CD63 and ALIX, and the absence of the markers GM130 and Cyto-C were confirmed. In vivo, FLI indicated an accumulation of i.v. injected IL4RPep-1-Flamma at the tumor site 90 min post-injection. No accumulation of NSSSVDK-Flamma was detected. In vivo, IL4RPep-1-EVs targeted the Cal-62 tumor 2 h post-injection. NSSSVDK-EVs were not even detected in the tumor 24 h post-injection. The quantification of FLI showed a significant accumulation of MSC-EVs in the tumor 2 h, 3 h, and 24 h post-injection. Furthermore, ex vivo imaging and an IF analysis confirmed the in vivo findings. Our results demonstrate the use of the IL4RPep-1 peptide as a targeting moiety of EVs for IL-4R-expressing anaplastic thyroid tumors.

SYST-12 D2C7 CAR: A NOVEL CAR T CELL THAT SIMULTANEOUSLY TARGETS WILDTYPE EGFR AND ITS MUTANT ISOFORM EGFRVIII FOR TREATMENT OF GLIOMA AND BRAIN METASTASES

INTRODUCTIONChimeric antigen receptor (CAR) T-cells represent a revolutionary class of immunotherapy, achieving considerable success in hematological cancers but generally failing to control solid tumors, including gliomas, partly due to the lack of a ubiquitously-expressed target antigen. In this study, we engineered a novel CAR T-cell consisting of the D2C7scfv targeting moiety that binds a shared epitope between EGFR and EGFRvIII. EGFR is the most homogeneous antigen on glial brain tumors, and the mutant EGFR variant, EGFRvIII, is present on a considerable subset of high grade gliomas. CAR T-cells targeting EGFRvIII alone fail to treat tumors possessing as few as 5-10% EGFRvIII-negative cell. Thus, D2C7 CAR is expected to be superior to the EGFRvIII CAR. METHODSWe retrovirally transduced T-cells with a vector encoding the D2C7scFv in tandem with intracellular signaling domains of CD28, 4-1BB, and CD3ζ to generate D2C7 CAR. We co-cultured D2C7 CAR or control CAR with fluorescently-tagged tumor cells expressing either EGFRwt or EGFRvIII to validate efficacy and specificity by flow cytometry. To determine in vivo efficacy, EGFRwt or EGFRvIII-expressing tumors were implanted intracranially in immunodeficient NSG mice. 48 hours later, D2C7 CAR, VIII CAR, or Mock CAR were administered intracranially and mice were monitored for survival. RESULTSD2C7 CAR specifically killed tumor cells that expressed either EGFRwt or EGFRvIII, but not cells that lacked EGFR. Intracranial D2C7 CAR administration resulted in significantly prolonged survival of mice bearing EGFRwt or EGFRvIII tumors compared to Mock CAR controls. Importantly, D2C7 CAR significantly benefitted mice bearing a heterogeneous mix of EGFRwt and EGFRvIII tumor cells, a model of tumor heterogeneity. CONCLUSIONSD2C7 CAR is efficacious against EGFRwt/EGFRvIII heterogeneous tumors. Intracranial administration of D2C7 CAR is predicted to safely and effectively treat a large cohort of patients due to the relatively high prevalence of EGFR and/or EGFRvIII-expressing brain tumors.

Protein Cages Engineered for Interaction-Driven Selective Encapsulation of Biomolecules.

Hollow protein cages have become attractive drug delivery vehicles with high biocompatibility and precise functional/structural manipulability. However, difficulties in effective cargo loading inside the cages have been limiting further development of protein cage-based drug carriers. Here, we developed a specific interaction-driven encapsulation and cellular delivery strategy for various biomolecules by engineering a porous protein cage. The computationally designed hyperstable mi3 protein cage was circularly permuted to fuse the cancer targeting RGD tripeptide to the cage surface and SpyTag (ST), which forms a covalent bond with SpyCatcher (SC), to the cage inner cavity. SC-fused proteins with different sizes and charges could be stably and actively encapsulated in the engineered nanocage via the ST/SC reaction. Cargo protein encapsulation inside the cage was directly confirmed by cryo-electron microscopy (EM) structure determination. In addition, SC-fused monomeric avidin was added to the nanocage to encapsulate various biotinylated (nonprotein) cargos such as oligonucleotides and the anticancer drug doxorubicin. All cargo molecules loaded onto the engineered mi3 were effectively delivered to cells. This work introduces a highly versatile cargo loading/delivery strategy, where loading/delivery interactions, cargo molecules, and cell targeting moieties can be further varied for optimal cellular drug delivery.

Anti-Fn14-Conjugated Prussian Blue Nanoparticles as a Targeted Photothermal Therapy Agent for Glioblastoma.

Prussian blue nanoparticles (PBNPs) are effective photothermal therapy (PTT) agents: they absorb near-infrared radiation and reemit it as heat via phonon-phonon relaxations that, in the presence of tumors, can induce thermal and immunogenic cell death. However, in the context of central nervous system (CNS) tumors, the off-target effects of PTT have the potential to result in injury to healthy CNS tissue. Motivated by this need for targeted PTT agents for CNS tumors, we present a PBNP formulation that targets fibroblast growth factor-inducible 14 (Fn14)-expressing glioblastoma cell lines. We conjugated an antibody targeting Fn14, a receptor abundantly expressed on many glioblastomas but near absent on healthy CNS tissue, to PBNPs (aFn14-PBNPs). We measured the attachment efficiency of aFn14 onto PBNPs, the size and stability of aFn14-PBNPs, and the ability of aFn14-PBNPs to induce thermal and immunogenic cell death and target and treat glioblastoma tumor cells in vitro. aFn14 remained stably conjugated to the PBNPs for at least 21 days. Further, PTT with aFn14-PBNPs induced thermal and immunogenic cell death in glioblastoma tumor cells. However, in a targeted treatment assay, PTT was only effective in killing glioblastoma tumor cells when using aFn14-PBNPs, not when using PBNPs alone. Our methodology is novel in its targeting moiety, tumor application, and combination with PTT. To the best of our knowledge, PBNPs have not been investigated as a targeted PTT agent in glioblastoma via conjugation to aFn14. Our results demonstrate a novel and effective method for delivering targeted PTT to aFn14-expressing tumor cells via aFn14 conjugation to PBNPs.

Application of Nano-based Drug Loading Systems in the Treatment of Neurological Infections: An Updated Review

Infection of the central nervous system (CNS) is a global healthcare concern with high rates of death and disease. CNS infections mainly include meningitis, encephalitis, and brain abscesses. Bacteria, viruses, fungi, protozoa, and parasites are the most common causes of neuroinfections. There are many types of medications used in the treatment of CNS infections, but drug delivery through the blood-brain barrier (BBB) is a major challenge to overcome. The BBB is a specialized multicellular barrier separating the neural tissue from the peripheral blood circulation. Unique characteristics of the BBB allow it to tightly control the movement of ions and molecules. Thus, there is a critical need to deal with these conditions with the aim of improving novel antimicrobial agents. Researchers are still struggling to find effective drugs to treat CNS infections. Nanoparticle (NP)-mediated drug delivery has been considered a profound substitute to solve this problem because NPs can be tailored to facilitate drug transport across the BBB. NPs are colloidal systems with a size range of 1-1000 nm, which can be used to encapsulate therapeutics, improve drug transport across the BBB, and target specific brain areas in CNS infections. A wide variety of NPs has been displayed for the CNS delivery of therapeutics, especially when their surfaces are coated with targeting moieties. This study aimed to review the available literature on the application of NPs in CNS infections.

Retro-inversion follicle-stimulating hormone peptide-modified nanoparticles for delivery of PDK2 shRNA against chemoresistant ovarian cancer by switching glycolysis to oxidative phosphorylation

BackgroundMost ovarian cancers are diagnosed at advanced stages characterized by abdominal dissemination and frequently exhibit chemoresistance. Pyruvate dehydrogenase kinase 2 (PDK2) regulates the switch between glycolysis and oxidative phosphorylation and contributes to tumor progression and chemoresistance. Here, we investigated the effects of PDK2 blockade on metabolic reprogramming and cisplatin sensitivity and evaluated the in vivo antitumor effects of PDK2 shRNA in chemoresistant ovarian cancer using retro-inverso follicle-stimulating hormone peptide-modified nanoparticle as carriers.MethodsThe expression of PDK2 was detected by immunohistochemistry, Western blot and real-time PCR. Cell proliferation and apoptosis were detected using CCK-8 and flow cytometry. Cell migration was detected by Transwell assay. Seahorse Analyzer was used to evaluate metabolic changes. The cisplatin-resistant ovarian cancer cells A2780cp were used to establish the mouse model of peritoneal metastatic ovarian cancer.ResultsA higher expression level of PDK2 was observed in chemoresistant ovarian cancer tissues and cell lines and was associated with shorter progression-free survival. PDK2 knockdown inhibited proliferation and migration and promoted apoptosis of both cisplatin-sensitive and cisplatin-resistant ovarian cancer cells. Cisplatin sensitivity was increased even in cisplatin-resistant ovarian cancer cells. Mechanistically, PDK2 knockdown resulted in an increased oxygen consumption rate and decreased extracellular acidification rate, along with reduced lactate production, increased PDHC activity and increased levels of electron transport chain complexes III and V. The metabolism switched from glycolysis to oxidative phosphorylation. Finally, to specifically and effectively deliver PDK2 shRNA in vivo, we formulated a targeted delivery system containing retro-inverso follicle-stimulating hormone peptide as a targeting moiety and polyethylene glycol–polyethylenimine copolymers as carriers. The nanoparticle complex significantly suppressed tumor growth and peritoneal metastasis of cisplatin-resistant ovarian cancer without obvious toxicities.ConclusionsOur findings showed the link between metabolic reprogramming and chemoresistance in ovarian cancer and provided an effective targeting strategy for switching metabolic pathways in cancer therapy.

Lung Cancer Targeted Chemoradiotherapy via Dual-Stimuli Responsive Biodegradable Core-Shell Nanoparticles.

Lung cancer is one of the major causes of cancer-related deaths worldwide, primarily because of the limitations of conventional clinical therapies such as chemotherapy and radiation therapy. Side effects associated with these treatments have made it essential for new modalities, such as tumor targeting nanoparticles that can provide cancer specific therapies. In this research, we have developed novel dual-stimuli nanoparticles (E-DSNPs), comprised of two parts; (1) Core: responsive to glutathione as stimuli and encapsulating Cisplatin (a chemo-drug), and (2) Shell: responsive to irradiation as stimuli and containing NU7441 (a radiation sensitizer). The targeting moieties on these nanoparticles are Ephrin transmembrane receptors A2 (EphA2) that are highly expressed on the surfaces of lung cancer cells. These nanoparticles were then evaluated for their enhanced targeting and therapeutic efficiency against lung cancer cell lines. E-DSNPs displayed very high uptake by lung cancer cells compared to healthy lung epithelial cells. These nanoparticles also demonstrated a triggered release of both drugs against respective stimuli and a subsequent reduction in in vitro cancer cell survival fraction compared to free drugs of equivalent concentration (survival fraction of about 0.019 and 0.19, respectively). Thus, these nanoparticles could potentially pave the path to targeted cancer therapy, while overcoming the side effects of conventional clinical therapies.

Programmable Attenuation of Antigenic Sensitivity for a Nanobody-Based EGFR Chimeric Antigen Receptor Through Hinge Domain Truncation.

Epidermal growth factor family receptor (EGFR) is commonly overexpressed in many solid tumors and an attractive target for chimeric antigen receptor (CAR)-T therapy, but as EGFR is also expressed at lower levels in healthy tissues a therapeutic strategy must balance antigenic responsiveness against the risk of on-target off-tumor toxicity. Herein, we identify several camelid single-domain antibodies (also known as nanobodies) that are effective EGFR targeting moieties for CARs (EGFR-sdCARs) with very strong reactivity to EGFR-high and EGFR-low target cells. As a strategy to attenuate their potent antigenic sensitivity, we performed progressive truncation of the human CD8 hinge commonly used as a spacer domain in many CAR constructs. Single amino acid hinge-domain truncation progressively decreased both EGFR-sdCAR-Jurkat cell binding to EGFR-expressing targets and expression of the CD69 activation marker. Attenuated signaling in hinge-truncated EGFR-sdCAR constructs increased selectivity for antigen-dense EGFR-overexpressing cells over an EGFR-low tumor cell line or healthy donor derived EGFR-positive fibroblasts. We also provide evidence that epitope location is critical for determining hinge-domain requirement for CARs, as hinge truncation similarly decreased antigenic sensitivity of a membrane-proximal epitope targeting HER2-CAR but not a membrane-distal EGFRvIII-specific CAR. Hinge-modified EGFR-sdCAR cells showed clear functional attenuation in Jurkat-CAR-T cells and primary human CAR-T cells from multiple donors in vitro and in vivo. Overall, these results indicate that hinge length tuning provides a programmable strategy for throttling antigenic sensitivity in CARs targeting membrane-proximal epitopes, and could be employed for CAR-optimization and improved tumor selectivity.

Active targeted photodynamic therapeutic effect of silver-based nanohybrids on melanoma cancer cells

Malignant melanoma is aggressive cancer that metastasizes along with the heterogeneity at the molecular and cellular levels, thereby reducing overall therapeutic efficacy. In the present research, the concept of photodynamic therapy (PDT) was investigated on silver-based nanohybrids for the treatment of A375 melanoma cancer cells. Hence, two different nanoparticles (NPs) namely Ag-PEG NPs and core/shell Ag@mSiO2 NPs were fabricated and conjugated to zinc phthalocyanine tetrasulfonate (ZnPcS4) photosensitizer (PS). Folic acid (FA) as a targeting moiety was also decorated onto the surface of nanohybrids to selectively target the folate receptors that are overexpressed on the A375 cells. Ultimately, the PDT efficacy of both ZnPcS4/Ag-PEG-FA and ZnPcS4/Ag@mSiO2-FA nanohybrids were compared via ATP viability, flow cytometry, and reactive oxygen species (ROS) assays. The obtained near-spherical shaped nanohybrids had zeta potential of -4.03 ± 0.3 mV for ZnPcS4/Ag-PEG-FA, and -14.4 ± 0.6 mV for the ZnPcS4/Ag@mSiO2-FA. A significant PDT effect was observed for the cells exposed to 674 nm laser irradiation after incubation with ZnPcS4/Ag@mSiO2-FA with ∼92%* ± 1.1 cell death compared to ∼70%* ± 2.9 cell death for ZnPcS4/Ag-PEG-FA nanohybrids owing to the higher generation of ROS for the former nanohybrids. The majority of the cell death was induced via apoptosis rather than necrosis as the nanohybrids successfully localized in mitochondria. The overall finding of this study concluded that an active targeting strategy significantly enhanced the cellular uptake of the nanohybrids compared to passive targeting. Moreover, strong surface plasmon-PS resonance coupling in ZnPcS4/Ag@mSiO2-FA nanohybrids enhanced singlet oxygen generation in comparison to the PS alone or ZnPcS4/Ag-PEG-FA nanohybrids.

Emerging role of exosomes in hematological malignancies.

Hematological malignancies are a heterogeneous group of neoplasms in the blood characterized by dysregulated hematopoiesis and classified as leukemia, lymphoma, and myeloma. The occurrence and progression of hematological malignancies depend on transformed hematopoietic stem cells, which refract to chemotherapy and often cause relapse. In recent years, monoclonal antibody therapies are preferred for hematopoietic cancers, owing to their inherent mechanisms of action and improved outcomes. However, efficient drug delivery methods and the establishment of novel biomarkers are currently being investigated and warranted to improve the outcome of patients with hematological malignancies. For instance, non-viral-mediated, natural carriers have been suggested for latent intracellular drug delivery. In this purview, repurposing small vesicles (e.g., exosomes) is considered a latent approach for myeloma therapy. Exosomes (nano-vesicles) have many advantages in that they are secreted by various animals and plants and become sought after for therapeutic and diagnostic purposes. The size of the cellular membrane of exosomes (30-150nm) facilitates ligand binding and targeted delivery of the loaded molecules. Furthermore, exosomes can be modified to express specific target moiety on their cell membrane and can also be featured with desired biological activity, thereby potentially employed for various convoluted diseases, including hematological malignancies. To advance the current knowledge, this review is focused on the source, composition, function and surface engineering of exosomes pertaining to hematological malignancies.

Micelles as potential drug delivery systems for colorectal cancer treatment

Despite the significant progress in cancer therapy, colorectal cancer (CRC) remains one of the most fatal malignancies worldwide. Chemotherapy is currently the mainstay therapeutic modality adopted for CRC treatment. However, the long-term effectiveness of chemotherapeutic drugs has been hampered by their low bioavailability, non-selective tumor targeting mechanisms, non-specific biodistribution associated with low drug concentrations at the tumor site and undesirable side effects. Over the last decade, there has been increasing interest in using nanotechnology-based drug delivery systems to circumvent these limitations. Various nanoparticles have been developed for delivering chemotherapeutic drugs among which polymeric micelles are attractive candidates. Polymeric micelles are biocompatible nanocarriers that can bypass the biological barriers and preferentially accumulate in tumors via the enhanced permeability and retention effect. They can be easily engineered with stimuli-responsive and tumor targeting moieties to further ensure their selective uptake by cancer cells and controlled drug release at the desirable tumor site. They have been shown to effectively improve the pharmacokinetic properties of chemotherapeutic drugs and enhance their safety profile and anticancer efficacy in different types of cancer. Given that combination therapy is the new strategy implemented in cancer therapy, polymeric micelles are suitable for multidrug delivery and allow drugs to act concurrently at the action site to achieve synergistic therapeutic outcomes. They also allow the delivery of anticancer genetic material along with chemotherapy drugs offering a novel approach for CRC therapy. Here, we highlight the properties of polymeric micelles that make them promising drug delivery systems for CRC treatment. We also review their application in CRC chemotherapy and gene therapy as well as in combination cancer chemotherapy.

Effect of High-Frequency Ultrasound on Targeted Liposomes

Delivering highly toxic drugs inside a safe carrier to tumors while achieving controlled and effective drug release at the targeted sites represents an attractive approach to enhance drug efficiency while reducing its undesirable side effects. Functionalization of highly biocompatible nanocarriers such as liposomes conjugated with targeting moieties enhances their ability to target specific cancer cells overexpressing the targeted receptors. Furthermore, upon their accumulation at the target site, high-frequency ultrasound (HFUS) can be used to stimulate the controlled release of the loaded drugs. Here, the US-mediated drug release from calcein-loaded non-pegylated, pegylated as well as targeted-pegylated liposomes modified with human serum albumin (HSA) and transferrin (Tf) was investigated. HFUS at two different frequencies (1 MHz and 3 MHz) was found to trigger calcein release, with higher release rates recorded at the lower frequency (i.e., 1 MHz) compared to the higher frequency (i.e., 3 MHz) despite a higher power density. Pegylation was found to enhance liposomal sensitivity to HFUS. In addition, targeted pegylated liposomes were more susceptible to HFUS than non-targeted pegylated (control) liposomes. These findings show that pegylation and targeting moieties directly influence liposomal sensitivity to HFUS. Therefore, combining targeted-pegylated liposomes with HFUS represents a promising controlled and effective drug delivery system.

Next-Generation Diprovocims with Potent Human and Murine TLR1/TLR2 Agonist Activity That Activate the Innate and Adaptive Immune Response.

The diprovocims, a new class of toll-like receptor (TLR) agonists, bear no similarity to prior TLR agonists, act through a well-defined mechanism (TLR1/TLR2 agonist), exhibit exquisite structure-activity relationships, and display in vivo adjuvant activity. They possess potent and efficacious agonist activity toward human TLR1/TLR2 but modest agonism toward the murine receptor. A manner by which diprovocims can be functionalized without impacting hTLR1/TLR2 activity is detailed, permitting future linkage to antigenic, targeting, or delivery moieties. Improvements in both potency and its low efficacy in the murine system were also achieved, permitting more effective use in animal models while maintaining the hTLR1/TLR2 activity. The prototypical member diprovocim-X exhibits the excellent potency/efficacy of diprovocim-1 in human cells, displays substantially improved potency/efficacy in mouse macrophages, and serves as an adjuvant in mice when coadministered with a nonimmunogenic antigen, indicating stimulation of the adaptive as well as innate immune response.

Theranostic Small-Molecule Prodrug Conjugates for Targeted Delivery and Controlled Release of Toll-like Receptor 7 Agonists.

We previously reported the design and synthesis of a small-molecule drug conjugate (SMDC) platform that demonstrated several advantages over antibody–drug conjugates (ADCs) in terms of in vivo pharmacokinetics, solid tumor penetration, definitive chemical structure, and adaptability for modular synthesis. Constructed on a tri-modal SMDC platform derived from 1,3,5-triazine (TZ) that consists of a targeting moiety (Lys-Urea-Glu) for prostate-specific membrane antigen (PSMA), here we report a novel class of chemically identical theranostic small-molecule prodrug conjugates (T-SMPDCs), [18/19F]F-TZ(PSMA)-LEGU-TLR7, for PSMA-targeted delivery and controlled release of toll-like receptor 7 (TLR7) agonists to elicit de novo immune response for cancer immunotherapy. In vitro competitive binding assay of [19F]F-TZ(PSMA)-LEGU-TLR7 showed that the chemical modification of Lys-Urea-Glu did not compromise its binding affinity to PSMA. Receptor-mediated cell internalization upon the PSMA binding of [18F]F-TZ(PSMA)-LEGU-TLR7 showed a time-dependent increase, indicative of targeted intracellular delivery of the theranostic prodrug conjugate. The designed controlled release of gardiquimod, a TLR7 agonist, was realized by a legumain cleavable linker. We further performed an in vivo PET/CT imaging study that showed significantly higher uptake of [18F]F-TZ(PSMA)-LEGU-TLR7 in PSMA+ PC3-PIP tumors (1.9 ± 0.4% ID/g) than in PSMA− PC3-Flu tumors (0.8 ± 0.3% ID/g) at 1 h post-injection. In addition, the conjugate showed a one-compartment kinetic profile and in vivo stability. Taken together, our proof-of-concept biological evaluation demonstrated the potential of our T-SMPDCs for cancer immunomodulatory therapies.

Targeted peptide-modified oxidized mesoporous carbon nanospheres for chemo-thermo combined therapy of ovarian cancer in vitro

Ovarian cancer remains one of serious hazards to human health due to many drawbacks of existing available treatment options. In this study, a multifunctional chemo-thermo combined therapy nanoplatform (OMCNPID) was successfully prepared, which is composed of I6P8 peptide as a targeting moiety to interleukin-6 receptors (IL-6Rs), oxidized mesoporous carbon nanospheres (OMCN) as a near infrared (NIR)-triggered drug carrier and doxorubicin (DOX) as a chemotherapeutic drug and fluorescent agent. The synthesized multifunctional nanoplatform displayed high storage capacity for drugs and excellent photothermal properties. Besides, DOX was rapidly released from OMCNPID at the condition of low pH and NIR laser irradiation due to the dissociation of DOX from graphitic cores of OMCN. In vitro experimental results verified that OMCNPID could be markedly taken up by SKOV-3 monolayer cells and tumor spheroids, and revealed a remarkable synergistic chemo-photothermal effect against ovarian cancer. All the results demonstrated that OMCNPID is a pH/NIR dual-stimulus responsive nanoplatform and can achieve efficient chemo-thermo combined therapy.

New Triazacycloalkane Derivatives as Cytotoxic Agents for CLL Treatment: From Proof of Concept to the Targeting Biomolecule.

The 1,4,7-tris-(2-pyridinylmethyl)-1,4,7-triazacyclononane ligand (no3py) and its bifunctional analogue no3pyCOOK were synthesized to investigate their action toward zinc(II) depletion related to the apoptosis phenomenon in chronic lymphocytic leukemia (CLL) cells. no3py was used as the "free" ligand, while its "graftable" derivative was conjugated on a newly synthesized bifunctional sialoglycan, 6'-SL-NH2, selected to specifically bind CD22 biomarker expressed on the B-CLL cell surface. Both compounds were produced with good yields thanks to a Sonogashira coupling reaction and an orthoester function, respectively, for the chelator and the targeting moiety. The newly reported bioconjugate 6'-SL-no3py was then obtained through a peptidic coupling reaction. Biological in vitro studies of no3py and 6'-SL-no3py consisting of real-time detection of cell health (cytotoxicity and proliferation) and caspases 3/7 activation (crucial enzymes whose activation triggers cell death signaling pathways) have been investigated. First, Ramos, Daudi, and Raji B-cell lines, which present different sensitivity to zinc(II) content variation, were incubated with no3py and 6'-SL-no3py. Then, a videomicroscope allowed the real-time monitoring of the morphological changes leading to cell death from the detection of the cytotoxicity, the antiproliferative effect, and the caspasic activity. In terms of mechanism, the Zn2+ chelator cytotoxic effect of no3py has been evidenced by a culture medium ion supplementation study and by the decrease of intracellular fluorescence of Zn-specific fluorophore zinquin in the presence of no3py and 6'-SL-no3py chelators. Finally, flow cytometry analysis with classical Annexin V staining was conducted to detect no3py- and 6'-SL-no3py-induced apoptotic cell death in B-CLL cells. Time-course analysis, using the Incucyte Live-Cell Analysis System, demonstrated that no3py induced cell death in a time- and dose-dependent manner with variability across cell lines. 6'-SL-no3py exhibited the same dose-dependent trend as no3py, showing the efficiency of the targeting moiety. In both cases, the chelators depicted proliferation curves that were inversely correlated with kinetic death. Morphological changes specific to apoptosis and caspase 3/7 activation were observed for the three cell lines treated with no3py and 6'-SL-no3py, highlighting their role as apoptotic agents. A higher concentration of 6'-SL-no3py is needed to reach 50% of the B-CLL mortality, confirming a targeting of the chelator to the cell membrane. Overall, our results proved that the biological properties of the triazamacrocyclic chelator still remain even after addition of the targeting moiety. The free chelator as well as the bioconjugate constitute promising cytotoxic agents for CLL therapy through apoptosis induction.

Strategies to mitigate the on- and off-target toxicities of recombinant immunotoxins: an antibody engineering perspective.

Targeted cancer therapies using immunotoxins have achieved remarkable efficacy in hematological malignancies. However, the clinical development of immunotoxins is also faced with many challenges like anti-drug antibodies and dose-limiting toxicity issues. Such a poor efficacy or safety ratio is also the major hurdle in the research and development of antibody-drug conjugates. From an antibody engineering perspective, various strategies were summarized or proposed to tackle the notorious on-target off-tumor toxicity issues, including passive strategy (XTENylation of immunotoxins) and active strategies (modulating the affinity and valency of the targeting moiety of immunotoxins, conditionally activating immunotoxins in the tumor microenvironments and reconstituting split toxin to reduce systemic toxicity, etc.). By modulating the functional characteristics of the targeting moiety and the toxic moiety of immunotoxins, selective tumor targeting can be augmented while sparing the healthy cells in normal tissues expressing the same target of interest. If successful, the improved therapeutic index will likely help to address the dose-limiting toxicities commonly observed in the clinical trials of various immunotoxins.

Abstract 3513: INBRX-121, a safe and efficacious molecular targeted cytokine that enhances NK cell-mediated tumor killing

Abstract IL-2 is a powerful cytokine that enhances the activity of and induces the expansion of tumoricidal effector cells including T and NK cells. Despite promising signs of activity, the clinical utility of IL-2 is limited due to severe treatment-associated toxicities including vascular leak syndrome and the concurrent expansion of immunosuppressive regulatory T cells, which blunts efficacy. We developed a molecular targeted cytokine (MTC) platform that combines an engineered IL-2 variant with reduced affinity for the IL-2 receptor and high affinity single-domain antibodies (sdAb) to restrict IL-2 receptor signaling to cells expressing the target antigen (termed cis-signaling). INBRX-121 is an MTC designed to deliver IL-2 to NK cells using an NKp46-specific sdAb as the targeting moiety. In vitro, INBRX-121 induces dose-dependent STAT5 phosphorylation and proliferation in NK cells with comparable potency to wild-type IL-2, while showing no NKp46-independent activity at concentrations of more than three magnitudes higher than that required for NK cell activity. INBRX-121 enhances NK cell activation and cytotoxic capacity, thereby reducing the NK-to-target cell ratio as well as the target receptor density needed for optimal tumor cell killing. Furthermore, INBRX-121 lowers the threshold for antibody-dependent cellular cytotoxicity (ADCC) and increases maximal target cell killing in combination with ADCC-competent antibodies to an extent that may lessen the need for Fc engineering to enhance binding to the low affinity CD16A variant. In vivo, INBRX-121 demonstrates potent anti-tumor activity with increased intra-tumoral NK cell accumulation and upregulation of NK cell activation markers. INBRX-121 monotherapy reduces primary tumor growth and the formation of metastasis, while combination with ADCC-competent antibodies further increases NK cell activity to convert tumor growth suppression into complete tumor elimination. INBRX-121 is well tolerated in rodents and non-human primates and no overt signs of toxicity have been observed in cynomolgus monkeys up to the highest administered dose of 30 mg/kg. Our MTC platform provides a novel path to overcoming the limitations of IL-2 therapy through the pinpointed delivery of IL-2 activity to target cells of interest. In the case of INBRX-121 this ensures potent enhancement of the naturally broad anti-tumor activity of NK cells, which have distinct advantages over T cell therapeutics. Unlike T cells, tumor recognition by NK cells is independent of MHC-presented antigens and can elicit immediate cytotoxic activity. Further, many of the toxicities associated with T cell therapeutics have not been observed with NK-centric treatments. INBRX-121 therefore provides a promising safe and efficacious treatment option for a broad array of cancer indications both as a monotherapy and in combination with ADCC-enabled antibodies. Citation Format: Heather Kinkead, Florian J. Sulzmaier, Anya Polovina, Nadja Kern, Angelica Sanabria, Jason Ho, Chelsie Macedo, Ryan Henderson, Abrahim Hussain, Rajay Pandit, William Crago, Emily Rowell, Brendan P. Eckelman. INBRX-121, a safe and efficacious molecular targeted cytokine that enhances NK cell-mediated tumor killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3513.

Abstract 5579: First-in-human study of ICAM-1-specific affinity tuned CAR T cells against advanced thyroid cancer

Abstract Background: The use of chimeric antigen receptor (CAR) T cells for solid tumors has a number of challenges, such as lack of tumor-specific targets, CAR T cell exhaustion, and the immunosuppressive tumor microenvironment. To address these challenges, AffyImmune has developed technologies to affinity tune and track CAR T cells in patients. The targeting moiety is affinity tuned to preferentially bind to tumor cells overexpressing the target while leaving normal cells with low basal levels untouched, thereby increasing the therapeutic window and allowing for more physiological T cell killing. The CAR T cells are engineered to co-express somatostatin receptor 2 (SSTR2), which allows for the tracking of CAR T cells in vivo via PET/CT scan using FDA-approved DOTATATE. Methods: AIC100 was generated by affinity tuning the I-domain of LFA-1, the physiological ligand to ICAM-1. Various mutants with 106-fold difference in affinity were evaluated for structure activity relationships using targets with varying antigen densities. The AIC100 with micromolar affinity was clearly the most effective in non-clinical animal models. AIC100 is currently being evaluated to assess safety, CAR T expansion, tumor localization, and preliminary activity in patients with advanced thyroid cancer (ATC) in a phase I study (NCT04420754). Our study uses a modified toxicity probability interval design with three dosage groups of 10 × 106, 100 × 106, and 500 × 106 cells. Results: Preclinical studies demonstrated greater in vivo anti-tumor activity and safety with micromolar affinity CAR T cells. A single dose of AIC100 resulted in tumor elimination and significantly improved survival of animals bearing ATC xenografts. AIC100 activity was confirmed in other high ICAM-1 tumor models including breast cancer, gastric cancer, and multiple myeloma. In a Phase I patient given 10 × 106 CAR T cells, near synchronous imaging of FDG and DOTATATE revealed preliminary evidence of transient CAR T expansion and tumor reduction at multiple tumor lesions, with the peak of CAR T cell density coinciding with the spike in CAR T cell numbers in blood. Conclusion: We have developed affinity tuned CAR T cells designed to selectively target ICAM-1 overexpressing tumor cells and to spatiotemporally image CAR T cells. Near-synchronous FDG and DOTATATE scans will enhance patient safety by early detection of off-tumor CAR T activity and validation of tumor response. We anticipate that our “tune and track” technology will be widely applicable to developing potent yet safe CAR T cells against hard-to-treat solid cancers. Citation Format: Jingmei Hsu, Yen-Michael Hsu, Koen van Besien, Thomas J. Fahey, Jana Ivanidze, Janusz Puc, Karrie Du, Yanping Yang, Yogindra Vedvyas, Irene M. Min, Eric von Hofe, Moonsoo M. Jin. First-in-human study of ICAM-1-specific affinity tuned CAR T cells against advanced thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5579.

Abstract 3520: Designing a dendritic cell-targeted immunotherapy for the treatment of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death. Low response rate, acquired resistance, severe side effects, highly desmoplastic, and immunosuppressive microenvironment have limited the clinical outcomes of immunotherapy in PDAC. To overcome these hurdles, we set to design novel immunotherapy that would be safe, efficacious, and selective to PDAC. The failure of immunotherapies to yield clinical response in PDAC is partially attributed to the inefficient antigen presentation by dendritic cells (DC) which results in insufficient T cells stimulation despite their expression of tumor-associated antigens (TAA). DC have an important role in the initiation and regulation of the innate and adaptive immune system. By efficient presentation of antigens, DC elicit an antigen-specific immune response. Trp53R172H is among the most common TP53 mutations in human PDAC, and is known as one of PDAC TAA. Despite the poor clinical outcomes, longer survival time was observed in PDAC patients with tumors that express high number of TAA combined with an abundant presentation of CD8+ T cell. To that end, we designed a DC-targeted nanoparticle (NP) containing PDAC Trp53R172H TAA and immune potentiators, with the DC targeting moiety, mannose, that was covalently conjugated to the NP, to enhance TAA presentation on DC. Thus, promote specific activation of T cells against PDAC’s TAA. The therapeutic activity of the NP was evaluated in vitro by our newly established 3D PDAC spheroids model, which composed of freshly isolated murine stromal cells, therefore, better imitates the complex microenvironment found in PDAC in vitro. Along with an in vivo PDAC model, we evaluated the safety profile and therapeutic effect of our NP. The NP proposed herein successfully activated splenocytes and induced a specific cytotoxic reaction against PDAC cells ex vivo, which may result in an improved survival rate. Citation Format: Daniella Vaskovich-Koubi, Ron kleiner, Anat Eldar-Boock, Sabina Pozzi, Dikla Dikla Ben-Shushan, Helena F. Florindo, Ronit Satchi-Fainaro. Designing a dendritic cell-targeted immunotherapy for the treatment of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3520.