Cancer Cell Lysates Research Articles

Human Cripto-1 and Cripto-3 Protein Expression in Normal and Malignant Settings That Conflicts with Established Conventions.

Background/Objectives: Cripto-1 (CR1) is a plurifunctional embryonic protein required for implantation and re-expressed in the adult during wound repair, inflammation, and tumorigenesis. CR1 and its predicted CR1 pseudogene product Cripto-3/CR3 are highly homologous proteins, and given this physical attribute, commercially available antibodies cannot discriminate between CR1 and CR3. Methods: A series of mouse monoclonal antibodies [MoAbs] were developed with a high-affinity binding that can differentiate human CR1/CR3 proteins and showed no measurable cross-reactivity. Results: Using these reagents, we confirm that CR3 is a bona fide translated protein found in human tumor tissue, cancer cell lysates, and in normal/cancer patient donor sera. We also reveal that CR1 and CR3 compete for binding to signal transduction protein Nodal, glucose-regulated protein 78Da (GRP78), and activin receptor-like kinase 4 (Alk4). Our discriminatory MoAbs provide new reagents to help clarify current CR1/CR3 protein expression vagaries in the Cripto field of study, challenging established CR1 conventions. In addition, our data validate CR3 involvement in human carcinogenesis and cell signaling pathways, with potential clinical relevance in determining cancer patient prognosis and disease severity.

Tumor microenvironment-activated polypeptide nanoparticles for oncolytic immunotherapy

Cationic oncolytic polypeptides have gained increasing attention owing to their ability to directly lyse cancer cells and activate potent antitumor immunity. However, the low tumor cell selectivity and inherent toxicity induced by positive charges of oncolytic polypeptides hinder their systemic application. Herein, a tumor microenvironment-responsive nanoparticle (DNP) is developed by the self-assembly of a cationic oncolytic polypeptide (PLP) with a pH-sensitive anionic polypeptide via electrostatic interactions. After the formation of DNP, the positive charges of PLP are shielded. DNPs can keep stable in physiological conditions (pH 7.4) but respond to acidic tumor microenvironment (pH 6.8) to release oncolytic PLP. As a result, DNPs evoke potent immunogenic cell death by disrupting cell membranes, damaging mitochondria and increasing intracellular levels of reactive oxygen species. In vivo results indicate that DNPs significantly improve the biocompatibility of PLP, and inhibit tumor growth, recurrence and metastasis by direct oncolysis and activation of antitumor immune responses. In summary, these results indicate that pH-sensitive DNPs represent a prospective strategy to improve the tumor selectivity and biosafety of cationic polymers for oncolytic immunotherapy.

Oncolytic Vaccinia Virus Encoding Aphrocallistes vastus Lectin Suppresses the Proliferation of Gastric Cancer Cells.

Our previous research has demonstrated that the oncolytic vaccinia virus encoding Aphrocallistes vastus lectin (oncoVV-AVL), an oncolytic vaccinia virus engineered to carry the AVL, exhibits potent cytotoxic effects on colorectal and hepatocellular cancer cells. Based on this foundation, we undertook a series of experiments to explore its efficacy on gastric cancer (GC) cells. Our findings revealed that oncoVV-AVL significantly increased reactive oxygen species levels and suppressed the expression of nuclear factor erythroid 2-related factor 2, thereby enhancing viral replication and disrupting the cellular redox balance, ultimately leading to the demise of cancer cells. Additionally, our investigations uncovered that oncoVV-AVL reprogrammed the metabolic microenvironment to favor viral replication, culminating in the lysis of cancer cells. Furthermore, we observed that oncoVV-AVL not only regressed tumor growth but also induced tumor tissue necrosis. These promising results suggest potential new avenues for the therapeutic management of GC.

Ultrasensitive Electrochemical Biosensor with Powerful Triple Cascade Signal Amplification for Detection of MicroRNA.

In this work, by ingeniously integrating catalytic hairpin assembly (CHA), double-end Mg2+-dependent DNAzyme, and hybridization chain reaction (HCR) as a triple cascade signal amplifier, an efficient concatenated CHA-DNAzyme-HCR (CDH) system was constructed to develop an ultrasensitive electrochemical biosensor with a low-background signal for the detection of microRNA-221 (miRNA-221). In the presence of the target miRNA-221, the CHA cycle was initiated by reacting with hairpins H1 and H2 to form DNAzyme structure H1-H2, which catalyzed the cleavage of the substrate hairpin H0 to release two output DNAs (output 1 and output 2). Subsequently, the double-loop hairpin H fixed on the electrode plate was opened by the output DNAs, to trigger the HCR with the assistance of hairpins Ha and Hb. Finally, methylene blue was intercalated into the long dsDNA polymer of the HCR product, resulting in a significant electrochemical signal. Surprisingly, the double-loop structure of the hairpin H could prominently reduce the background signal for enhancing the signal-to-noise ratio (S/N). As a proof of concept, an ultrasensitive electrochemical biosensor was developed using the CDH system with a detection limit as low as 9.25 aM, achieving favorable application for the detection of miRNA-221 in various cancer cell lysates. Benefiting from its enzyme-free, label-free, low-background, and highly sensitive characteristics, the CDH system showed widespread application potential for analyzing trace amounts of biomarkers in various clinical research studies.

Ingenious dual-circle DNA walker-mediated electrochemical biosensor for rapid and ultrasensitive detection of microRNA

In this work, an ingenious dual-circle DNA walker (DCDW) with pretty fast walking speed and high amplification efficiency was developed for rapid and ultrasensitive electrochemical detection of microRNA-221 (miRNA-221) related to liver cancer, combined with the toehold-mediated strand-displacement reactions (TSDRs). Impressively, compared with the traditional DNA walker, the DCDW with unique double-stranded interlocked DNA nanostructure not only possesses higher stability, flexibility, and anti-entanglement ability, but also enables more functional domain in a smaller area, thereby enhancing the local concentration, which can greatly improve the working efficiency. As a validation, the electrochemical biosensor realized rapid and ultrasensitive detection of miRNA-221 with a reaction time of 15 min and detection limit down to 1.9 aM, and had been applied in MHCC97L and HeLa cancer cell lysates, thus providing an innovative insight to design intelligent functional interlocked DNA walkers for ultimate application in the construction of biosensing platform and miRNA detection in biological sample.

Investigating the effect of reduced temperatures on the efficacy of rhabdovirus-based viral vector platforms.

Rhabdoviral vectors can induce lysis of cancer cells. While studied almost exclusively at 37 °C, viruses are subject to a range of temperatures in vivo, including temperatures ≤31 °C. Despite potential implications, the effect of temperatures <37 °C on the performance of rhabdoviral vectors is unknown. We investigated the effect of low anatomical temperatures on two rhabdoviruses, vesicular stomatitis virus (VSV) and Maraba virus (MG1). Using a metabolic resazurin assay, VSV- and MG1-mediated oncolysis was characterized in a panel of cell lines at 28, 31, 34 and 37 °C. The oncolytic ability of both viruses was hindered at 31 and 28 °C. Cold adaptation of both viruses was attempted as a mitigation strategy. Viruses were serially passaged at decreasing temperatures in an attempt to induce mutations. Unfortunately, the cold-adaptation strategies failed to potentiate the oncolytic activity of the viruses at temperatures <37 °C. Interestingly, we discovered that viral replication was unaffected at low temperatures despite the abrogation of oncolytic activity. In contrast, the proliferation of cancer cells was reduced at low temperatures. Equivalent oncolytic effects could be achieved if cells at low temperatures were treated with viruses for longer times. This suggests that rhabdovirus-mediated oncolysis could be compromised at low temperatures in vivo where therapeutic windows are limited.

Oncolytic Viral Therapy in Osteosarcoma.

Primary bone malignancies, including osteosarcoma (OS), are rare but aggressive. Current OS treatment, involving surgical resection and chemotherapy, has improved survival for non-metastatic cases but remains ineffective for recurrent or metastatic OS. Oncolytic viral therapy (OVT) is a promising alternative, using naturally occurring or genetically modified viruses to selectively target and lyse cancer cells and induce a robust immune response against remaining OS cells. Various oncolytic viruses (OVs), such as adenovirus, herpes simplex virus, and measles virus, have demonstrated efficacy in preclinical OS models. Combining OVT with other therapeutics, such as chemotherapy or immunotherapy, may further improve outcomes. Despite these advances, challenges in reliability of preclinical models, safety, delivery, and immune response must be addressed to optimize OVT for clinical use. Future research should focus on refining delivery methods, exploring combination treatments, and clinical trials to ensure OVT's efficacy and safety for OS. Overall, OVT represents a novel approach with the potential to drastically improve survival outcomes for patients with OS.

A Critical Role of Culture Medium Selection in Maximizing the Purity and Expansion of Natural Killer Cells.

Natural killer (NK) cells hold promise in cancer treatment due to their ability to spontaneously lyse cancer cells. For clinical use, high quantities of pure, functional NK cells are necessary. Combining adherence-based isolation with specialized media showed the unreliability of the isolation method, but demonstrated the superiority of the NK MACS® medium, particularly in suboptimal conditions. Neither human pooled serum, fetal calf serum (FCS), human platelet lysate, nor chemically defined serum replacement could substitute human AB serum. Interleukin (IL-)2, IL-15, IL-21, and combined CD2/NKp46 stimulation were assessed. IL-21 and CD2/NKp46 stimulation increased cytotoxicity, but reduced NK cell proliferation. IL-15 stimulation alone achieved the highest proliferation, but the more affordable IL-2 performed similarly. The RosetteSep™ human NK cell enrichment kit was effective for isolation, but the presence of peripheral blood mononuclear cells (PBMCs) in the culture enhanced NK cell proliferation, despite similar expression levels of CD16, NKp46, NKG2D, and ICAM-1. In line with this, purified NK cells cultured in NK MACS® medium with human AB serum and IL-2 demonstrated high cytotoxicity against primary glioblastoma stem cells.

Design, Synthesis, and Bioactivity Evaluation of a TF-Based Cancer Vaccine Candidate Using Lipid A Mimetics As a Built-In Adjuvant.

This study describes the design and synthesis of five TF-based cancer vaccine candidates using a lipid A mimetic as the carrier and a built-in adjuvant. All synthesized conjugates elicited robust and consistent TF-specific immune responses in mice without external adjuvants. Immunological studies subsequently conducted in wild-type and TLR4 knockout C57BL/6 mice demonstrated that the activation of TLR4 was the main reason that the synthesized lipid A mimetics increased the TF-specific immune responses. All antisera induced by these conjugates can specifically recognize, bind to, and induce the lysis of TF-positive cancer cells. Moreover, representative conjugates 2 and 3 could effectively reduce the growth of tumors and prolong the survival time of mice in vivo, and the efficacies were better than glycoprotein TF-CRM197 with alum adjuvant. Lipid A mimetics could therefore be a promising platform for the development of new carbohydrate-based vaccine carriers with self-adjuvanting properties for the treatment of cancer.

Single-Atom Iron Doped Carbon Dots with Highly Efficient Electrochemiluminescence for Ultrasensitive Detection of MicroRNAs.

Herein, single-atom iron doped carbon dots (SA Fe-CDs) were successfully prepared as novel electrochemiluminescence (ECL) emitters with high ECL efficiency, and a biosensor was constructed to ultrasensitively detect microRNA-222 (miRNA-222). Importantly, compared with the conventional without single-atom doped CDs with low ECL efficiency, SA Fe-CDs exhibited strong ECL efficiency, in which single-atom iron as an advanced coreactant accelerator could significantly enhance the generation of reactive oxygen species (ROS) from the coreactant S2O82- for improving the ECL efficiency. Moreover, a neoteric amplification strategy combining the improved strand displacement amplification with Nt.BbvCI enzyme-induced target amplification (ISDA-EITA) could produce 4 output DNAs in every cycle, which greatly improved the amplification efficiency. Thus, a useful ECL biosensor was built with a detection limit of 16.60 aM in the range of 100 aM to 1 nM for detecting traces of miRNA-222. In addition, miRNA-222 in cancer cell lysate (MHCC-97L) was successfully detected by using the ECL biosensor. Therefore, this strategy provides highly efficient single-atom doped ECL emitters for the construction of sensitive ECL biosensing platforms in the biological field and clinical diagnosis.

BCKDK modification enhances the anticancer efficacy of CAR-T cells by reprogramming branched chain amino acid metabolism

Altered Branched Chain Amino Acids (BCAA), including leucine, isoleucine and valine, are frequently observed in patients with advanced cancer. We evaluated the efficacy of Chimeric Antigen Receptor (CAR) T cell-mediated cancer cell lysis potential in the immune microenvironment of BCAA supplementation and deletion. BCAA supplementation increased cancer cell killing percentage, while accelerating BCAA catabolism and deceasing BCAA transporter decreased cancer cell lysis efficacy. We thus designed BCKDK engineering CAR T cells for the reprogramming of BCAA metabolism in the tumor microenvironment based on the genotype and phenotype modification. BCKDK overexpression (OE) in CAR-T cells significantly improved cancer cell lysis, while BCKDK knockout (KO) resulted in inferior lysis potential. In an in vivo experiment, BCKDK-OE CAR-T cells treatment significantly prolonged the survival of mice bearing NALM6-GL cancer cells, with the differentiation of central memory cells and the increasing proportion of CAR-T cells in peripheral circulation. BCKDK-KO CAR-T cells treatment resulted in shorter survival and decreasing percentage of CAR-T cells in peripheral circulation. In conclusion, BCKDK engineered CAR-T cells exert distinct phenotype for the superior anticancer efficiency.

Detection of Uracil-Excising DNA Glycosylases in Cancer Cell Samples Using a Three-Dimensional DNAzyme Walker.

DNA glycosylase dysregulation is implicated in carcinogenesis and therapeutic resistance of cancers. Thus, various DNA-based detection platforms have been developed by leveraging the base excision activity of DNA glycosylases. However, the efficacy of DNA-based methods is hampered due to nonspecific degradation by nucleases commonly present in cancer cells and during preparations of cell lysates. In this report, we describe a fluorescence-based assay using a specific and nuclease-resistant three-dimensional DNAzyme walker to investigate the activity of DNA glycosylases from cancer cell lysates. We focus on DNA glycosylases that excise uracil from deoxyuridine (dU) lesions, namely, uracil DNA glycosylase (UDG) and single-stranded monofunctional uracil DNA glycosylase (SMUG1). The limits of detection for detecting UDG and SMUG1 in the buffer were 3.2 and 3.0 pM, respectively. The DNAzyme walker detected uracil excision activity in diluted cancer cell lysate from as few as 48 A549 cells. The results of the UDG inhibitor experiments demonstrate that UDG is the predominant uracil-excising glycosylase in A549 cells. Approximately 500 nM of UDG is present in each A549 cell on average. No fluorescence was generated in the samples lacking DNAzyme activation, indicating that there was no nonspecific nuclease interference. The ability of the DNAzyme walker to respond to glycosylase activity illustrates the potential use of DNAzyme walker technology to monitor and study biochemical processes involving glycosylases.

Reducing Cathodic Drift during Isoelectric Focusing Using Microscale Immobilized pH Gradient Gels.

Microfluidic analytical tools play an important role in miniaturizing targeted proteomic assays for improved detection sensitivity, throughput, and automation. Microfluidic isoelectric focusing (IEF) can resolve proteoforms in lysate from low-to-single cell numbers. However, IEF assays often use carrier ampholytes (CAs) to establish a pH gradient for protein separation, presenting limitations like pH instability in the form of cathodic drift (migration of focused proteins toward the cathode). Immobilized pH gradient (IPG) gels reduce cathodic drift by covalently immobilizing the pH buffering components to a matrix. To our knowledge, efforts to implement IPG gels at the microscale have been limited to glass microdevices. To adapt IEF using IPGs to widely used microfluidic device materials, we introduce a polydimethylsiloxane (PDMS)-based microfluidic device and compare the microscale pH gradient stability of IEF established with IPGs, CAs, and a hybrid formulation of IPG gels and CAs (mixed-bed IEF). The PDMS-based IPG microfluidic device (μIPG) resolved analytes differing by 0.1 isoelectric point within a 3.5 mm separation lane over a 20 min focusing duration. During the 20 min duration, we observed markedly different cathodic drift velocities among the three formulations: 60.1 μm/min in CA-IEF, 2.5 μm/min in IPG-IEF (∼24-fold reduction versus CA-IEF), and 1.4 μm/min in mixed-bed IEF (∼43-fold reduction versus CA-IEF). Lastly, mixed-bed IEF in a PDMS device resolved green fluorescent protein (GFP) proteoforms from GFP-expressing human breast cancer cell lysate, thus establishing stability in lysate from complex biospecimens. μIPG is a promising and stable technique for studying proteoforms from small volumes.

Highly sensitive and selective demethylase FTO detection using a DNAzyme-mediated CRISPR/Cas12a signal cascade amplification electrochemiluminescence biosensor with C–CN/PCNV heterojunction as emitter

Fat mass and obesity-associated protein (FTO) has gained attention as the first RNA N6-methyladenosine (m6A) modification eraser due to its overexpression being associated with various cancers. In this study, an electrochemiluminescence (ECL) biosensor for the detection of demethylase FTO was developed based on DNAzyme-mediated CRISPR/Cas12a signal cascade amplification system and carboxylated carbon nitride nanosheets/phosphorus-doped nitrogen-vacancy modified carbon nitride nanosheets (C–CN/PCNV) heterojunction as the emitter. The biosensor was constructed by modifying the C–CN/PCNV heterojunction and a ferrocene-tagged probe (ssDNA-Fc) on a glassy carbon electrode. The presence of FTO removes the m6A modification on the catalytic core of DNAzyme, restoring its cleavage activity and generating activator DNA. This activator DNA further activates the trans-cleavage ability of Cas12a, leading to the cleavage of the ssDNA-Fc and the recovery of the ECL signal. The C–CN/PCNV heterojunction prevents electrode passivation and improves the electron-hole recombination, resulting in significantly enhanced ECL signal. The biosensor demonstrates high sensitivity with a low detection limit of 0.63 pM in the range from 1.0 pM to 100 nM. Furthermore, the biosensor was successfully applied to detect FTO in cancer cell lysate and screen FTO inhibitors, showing great potential in early clinical diagnosis and drug discovery.

The carbon dots with near-infrared electrochemiluminescence emission and low excitation potential for ultrasensitive analysis of microRNA-222

In this work, nitrogen and sulfur codoped near-infrared emission carbon dots (N,S-NIR-CDs) were used as neoteric electrochemiluminescence (ECL) emitters with high efficiency to construct a biosensor for ultrasensitive analysis of microRNA-222 (miRNA-222), which has been regarded as prospective biomarkers for early cancer diagnosis. Impressively, compared to conventional CDs with short ECL wavelength and large excitation voltage, N,S-NIR-CDs possessed wide sp2 domains and narrow band gap, leading to the near-infrared ECL emission at 680 nm and low excitation potential from −1.0 to 0 V, which not only decreased the photochemical damage for biomolecules but also reduced the side reaction. Moreover, an improved T7 Exonuclease induced cascade amplification (EICA) strategy was designed to transform trace miRNA-222 into plentiful output DNA in dual-cycle for greatly improving the target conversion efficiency. Therefore, by combining the N,S-NIR-CDs as neoteric ECL emitters and EICA strategy as signal amplifier, the constructed ECL biosensor could realize the ultrasensitive analysis of miRNA-222 with a identification limit of 5.65 aM and successfully employed in miRNA-222 analysis from liver cancer cell (HCCLM3 and MHCC-97L) lysate. This strategy developed a kind of neoteric CDs with NIR ECL emission and low excitation potential to establish a highly efficient biosensor for biomarker analysis, which prepared a road for early diseases diagnosis and lied a foundation for biological ECL imaging.

Abstract 1239: SAIL66, a next generation T cell engager targeting CLDN6, potentiates efficacy by binding to CD3/CD137

Abstract Background: Claudin-6 (CLDN6), one of over 20 known human CLDN family members, is upregulated in many solid tumors but shows minimal or no expression in healthy adult tissues. This makes it an ideal target for cancer therapy. Although several therapeutic modalities targeting CLDN6 are currently under investigation—including T cell engagers (TCE), CAR-T, and antibody drug conjugates—a more potent therapeutic option is still desirable. TCEs are a promising avenue within cancer immunotherapy, but their current efficacy and safety must be improved, particularly against solid tumors. Methods and Results: To enhance immune activation and achieve successful clinical outcomes against solid tumors expressing CLDN6, we generated SAIL66, a CLDN6-targeting next generation TCE, using a new technological format called Dual-Ig®. SAIL66 is a tri-specific monoclonal antibody designed to bind to CLDN6 on cancer cells with one Fab arm and to both CD3 and CD137 with the other Fab arm. The preclinical characterization of SAIL66 was performed in a series of in vitro and in vivo studies which included comparisons to a conventional TCE targeting CLDN6 and CD3. SAIL66 demonstrated high specificity for CLDN6 despite its similarity to CLDN3, CLDN4, and CLDN9, suggesting a lack of off-target toxicity in patients. Using a Jurkat cell system harboring NFAT or NF-κB reporter genes, we confirmed that SAIL66 exerts CD3 and CD137 signal induction activity depending on CLDN6 expression. SAIL66 induced activation of T cells, release of cytokines, and lysis of CLDN6-positive cancer cells. In vivo studies with a humanized mouse (huNOG) model showed that SAIL66 had better anti-tumor efficacy in eliminating CLDN6-positive cancer cells than a conventional TCE. Flow cytometry analysis in the huNOG model revealed that SAIL66 also more significantly increased CD3+ T cell infiltration of tumor tissue and resulted in a smaller percentage of exhausted T cells in tumor. Moreover, transcriptome analysis in the huNOG model showed that both SAIL66 and the conventional TCE increase expression of genes associated with immune activation, with a durable increase observed especially with SAIL66. Further, we clarified that T cells repeatedly stimulated with SAIL66 had a greater ability to inhibit the growth of CLDN6-positive cancer cells than T cells stimulated with the conventional TCE in vitro. Conclusion and Discussion: Our study shows that SAIL66 has better anti-tumor efficacy against CLDN6-positive cancer cells than a conventional TCE. This is due to effective immune activation by SAIL66 which triggers CD3 signaling (signal 1) combined with CD137 co-stimulatory signaling (signal 2) in T cells. Thus, SAIL66 represents a promising therapeutic option for solid tumors expressing CLDN6. A clinical trial for CLDN6-positive cancer patient is now ongoing (NCT05735366). Citation Format: Moe Yoshimoto, Naoki Kimura, Takayuki Kamikawa, Shinya Ishii, Masaru Muraoka, Kenji Taniguchi, Ryo Uchikawa, Momoko Okuda-Miura, Sho Akai, Tatsushi Kodama, Hirofumi Sakumoto, Shigeto Kawai, Mei Shimada, Mika Kamata-Sakurai, Hiroyuki Aburatani, Takehisa Kitazawa, Tomoyuki Igawa. SAIL66, a next generation T cell engager targeting CLDN6, potentiates efficacy by binding to CD3/CD137 [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 1239.

Abstract 482: Advancing treatment for metastatic triple negative breast cancer: Liposomal oncolytic adenovirus as a novel neoadjuvant therapy

Abstract Introduction: Adenovirus type-5 (Ad) is considered a prominent viral vector in cancer therapy due to its modifiability for the selective lysis of cancer cells. However, challenges are posed in Ad delivery to cancers lacking coxsackievirus and adenovirus receptors (CARs). In response to this limitation, Ad-encapsulated DOTAP-folate liposomes were developed, which improved Ad delivery and heightened therapeutic efficacy through membrane fusion and folate receptor-mediated endocytosis. Methods: The 4T1 mammary carcinoma model, a CAR-negative triple-negative breast cancer (TNBC) cell line (ER-, PR-, HER2-), resistant to adenoviral infection, was utilized for studying the comparative in vitro transduction of green fluorescent protein (GFP)-expressing Ad (Ad-GFP). The study assessed Ad-GFP with and without DOTAP-folate liposomal encapsulation at a multiplicity of infection (MOI) of 50. Additionally, an oncolytic adenovirus, Ad-hTERT, with a human telomerase promoter, underwent liposomal encapsulation. CAR-negative 4T1 GFP-expressing cells and CAR-positive MDA-MB-231-GFP human breast adenocarcinoma TNBC cells were transduced in vitro at MOI 100 with Ad-hTERT, both with and without DOTAP-folate liposomal encapsulation. Results: CAR-negative 4T1 cells exhibited superior Ad-GFP transduction when encapsulated in DOTAP-folate liposomes, compared to the unencapsulated form (p &amp;lt; 0.001). For oncolytic adenovirus Ad-hTERT, encapsulation resulted in 100% cell lysis within 24 hours in 4T1-GFP cells, contrasting with 13% with unencapsulated Ad-hTERT (P &amp;lt; 0.0001). In CAR-positive MDA-MB-231-GFP TNBC cells, encapsulated Ad-hTERT showed enhanced efficacy, resulting in 100% cell lysis within 24 hours compared to 75% with unencapsulated Ad-hTERT (P &amp;lt; 0.001). Discussion: DOTAP-folate liposomal encapsulation enhances the efficacy of oncolytic Ad-hTERT in both CAR-negative and CAR-positive TNBC cells, establishing it as an optimal therapy for tumors with CAR heterogeneity. Ongoing in vivo studies with Balb/C immune-competent mice and the 4T1 tumor model will assess the effectiveness of encapsulation through intratumoral administration. This approach may extend the recurrence-free period post-surgical tumor removal. Citation Format: Jaimin R. Shah, Tao Dong, Abraham Phung, Sarah L. Blair, Omonigho Aisagbonhi, William C. Trogler, Andrew C. Kummel. Advancing treatment for metastatic triple negative breast cancer: Liposomal oncolytic adenovirus as a novel neoadjuvant therapy [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 482.

Abstract 3968: Class I HLA-independent lysis of cancer cells by tumor-infiltrating CD8 T cells

Abstract T cell-based therapies have transformed cancer treatment, although intrinsic or acquired resistance develops in nearly half of the patients. Tumor-infiltrating CD8+ T lymphocytes (TILs) are key determinants of anti-tumor immunity by secretion of cytotoxic granules, such as granzymes and perforin, and effector cytokines, such as TNFa and IFNg. A deep understanding of how CD8+ TILs eliminate cancer cells will help identify resistance mechanisms and develop new therapeutic strategies. Here, we demonstrate that TILs eliminated matched melanoma cells in a time and dose-dependent manner but in a class I HLA-independent fashion. Specifically, blocking class I HLA-TCR interaction either by the deletion of B2M (resulting in loss of class I HLA surface expression) or TCR genes did not alter the activity of these TILs. We further confirmed that TIL-mediated killing is contact-dependent. After excluding the potential roles of CD1d and MR1-restrictive T cells, gamma delta T cells, and NKG2D in TIL-mediated killing, a whole-genome CRISPR/Cas9 screen was performed in cancer cells. We identified that genes involved in JAK-STAT and cell-extrinsic apoptosis pathways are necessary for TIL-mediated killing. Together, these findings demonstrate that expanded CD8 TILs from melanoma patients are capable of eliminating melanoma cells via a novel, class I MHC-independent mechanism by coactivation of both JAK-STAT and cell-extrinsic apoptosis pathways. Citation Format: Hongyan Xie, Aiping Jiang, Jonathan Perera, Aonkon Dey, Neal Smith, Moshe Sade-Feldman, Robert T. Manguso, Nir Hacohen, Russell W. Jenkins. Class I HLA-independent lysis of cancer cells by tumor-infiltrating CD8 T cells [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 3968.

Abstract 2821: Development of therapeutic monoclonal antibodies against DKK1 peptide-HLA-A2 complex to treat human cancers

Abstract Immunotherapy is a promising option for cancer treatment. Targeted immunotherapy with monoclonal antibodies (mAbs) is an effective and safe method for the treatment of malignancies. In recent years, efforts have been made to identify potential therapeutic mAbs by defining alternative or novel target antigens. We reported that Dickkopf-1 (DKK1) is a good target for immunotherapy of human cancers based on its wide expression in different cancers but not in normal tissues. As DKK1 is a secreted protein, mAbs directly binding to DKK1 may have limited effects on cancer cells in vivo. Our previous studies demonstrated that DKK1 peptide (such as P20 and P66v, which bind with HLA-A*0201 (HLA-A2) molecule) specific cytotoxic T cells specifically kill myeloma and other cancer cells that express DKK1 and HLA-A2, but not HLA-A2+ normal cells, indicating that DKK1+ tumor cells naturally express these peptides, in the context of HLA-A2 molecules, on their surface. To develop cancer therapeutic antibodies, DKK1 peptide P20-HLA-A2 monomer was synthesized and used to immunize mice. Hybridomas secreting mAbs recognizing cell surface-expressed DKKl P20-HLA-A2 complex (DKK1-A2) were obtained and analyzed. In this study, we generated novel (DKK1-A2) mAbs recognizing DKK1 (P20) peptide in the context of human HLA-A*0201 (HLA-A2) molecules (DKK1-A2 complexes) that are naturally expressed by HLA-A2+DKK1+ cancer cells. These mAbs bound to HLA-A2+DKK1+ hematologic (myeloma, lymphoma, and leukemia) and solid (breast, pancreatic, and prostate) cancer cells and directly induced apoptosis in the cancer cells by activating the caspase-9 cascade. DKK1-A2 mAbs also effectively lysed cancer cells in vitro by mediating CDC and ADCC activities. Furthermore, DKK1-A2 mAbs were therapeutic against established hematologic and solid cancers in their xenograft mouse models. To enhance the therapeutic efficacy of DKK1-A2 mAbs, we investigated the effect of combination treatment of DKK1-A2 mAbs with lenalidomide. Our results showed that there is an additive therapeutic effect between DKK1-A2 mAbs and lenalidomide in vivo, underscoring a potential clinical development strategy for combining DKK1-A2 mAb with lenalidomide to treat cancer patients. As DKK1 is not detected in most human tissues, DKK1-A2 mAbs did not bind to or kill HLA-A2+ blood cells in vitro nor caused tissue damage in vivo in tumor-free or tumor-bearing HLA-A2-transgenic mice. Thus, our study suggests that DKK1-A2 mAbs may be a promising therapeutic agent to treat human cancers and warrants further preclinic studies to determine their translatability to clinic. Citation Format: Jianfei Qian, Wang Qiang, Liuling Xiao, Wei Xiong, Miao Xian, Chuanchao Zhang, Yabo Li, Qing Yi. Development of therapeutic monoclonal antibodies against DKK1 peptide-HLA-A2 complex to treat human cancers [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 2821.

Abstract 6720: HINT1 steric masked, protease-activatable bispecific protein nanorings direct human T and NK cells to eradicate EGFR+ solid tumors

Abstract Introduction/Purpose: The Wagner lab has developed a protein nanoring based platform to direct human immune cells to combat solid tumors. These nanorings are bispecific, comprised of tumor-targeting and immune cell-targeting dihydrofolate reductase (DHFR2) fusion proteins. These protein nanorings direct immune cells to target and destroy cancer cells. For treating solid tumors, we aimed to target epidermal growth factor receptor (EGFR). However, targeting EGFR could lead to potential “on-target, off-tumor” effects due to expression of EGFR in healthy tissue. Matrix metalloproteinases are overexpressed in a variety of solid tumors. Therefore, to improve tumor specificity, we hypothesized that masking the immune cell targeting DHFR2 protein through a matrix metalloproteinase-2 (MMP-2) sensitive linker will prevent engagement and activation of immune cells outside the tumor microenvironment (TME). Once these protease-activatable nanorings enter the TME and bind to EGFR, cleavage by MMP-2 will lead to unmasking of the immune cell binder, thereby crosslinking and activating immune cells, causing targeted cancer cell lysis. Methods: We designed human histidine triad nucleotide binding protein-1 (HINT1) masked anti-immune cell targeting nanobody-DHFR2 fusion proteins. HINT1 was hypothesized to sterically block the engagement of immune cells outside the tumor. HINT1-anti-TCR-DHFR2 (TCR = T cell receptor) and HINT1-anti-CD16-DHFR2 were cloned, expressed, purified, and characterized. Their apparent binding affinities were determined using flow cytometry. The anti-tumor efficacy of these nanorings was evaluated against EGFR overexpressing A431 (human epidermoid carcinoma) cells. Results: HINT1-anti-TCR-DHFR2 and HINT1-anti-CD16-DHFR2 rings showed reduced binding to T and NK-92-CD16 cells, respectively. In vitro MMP-2 cleavage assay showed complete removal of the HINT1 mask within 2 hours. Western blot analysis showed that both proteins were stable in human serum up to 3 days. 2D cytotoxicity assays with protein nanorings assembled using anti-EGFR-DHFR2 and anti-TCR-DHFR2 or anti-CD16-DHFR2 with T and NK-92-CD16 cells respectively, showed a potent lysis of A431 cells. The EGFR-targeted lysis was diminished in the presence of HINT1 mask, thereby providing proof-of-concept for the steric masking of the anti-immune cell targeting nanobodies. Conclusion: Protease-activatable nanorings can selectively lyse EGFR+ tumor cells in vitro. Their antitumor efficacy and safety are currently being evaluated using a previously established in vivo mouse model. Therefore, HINT1-steric masking can serve as a simple and effective strategy to address off-tumor toxicities associated with bispecific immunotherapies. Citation Format: Abhishek Kulkarni, Debasmita Paul, Declan Dahlberg, Kostana Ligori, Freddys Rodriguez, Lakmal Rozumalski, Bruce Walcheck, Carston R. Wagner. HINT1 steric masked, protease-activatable bispecific protein nanorings direct human T and NK cells to eradicate EGFR+ solid tumors [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 6720.