Tumor Cell Surface Research Articles

Antibody-Drug Conjugates in the Treatment of Genitourinary Cancers: An Updated Review of Data.

The treatment landscape of genitourinary cancers has significantly evolved over the past few years. Renal cell carcinoma, bladder cancer, and prostate cancer are the most common genitourinary malignancies. Recent advancements have produced new targeted therapies, particularly antibody-drug conjugates (ADCs), due to a better understanding of the underlying oncogenic factors and molecular mechanisms involved. ADCs function as a 'drug delivery into the tumor' system. They are composed of an antigen-directed antibody linked to a cytotoxic drug that releases cytotoxic components after binding to the tumor cell's surface antigen. ADCs have been proven to be extremely promising in the treatment of several cancer types. For GU cancers, this novel treatment has only benefited patients with metastatic urothelial cancer (mUC). The rest of the GU cancer paradigm does not have any FDA-approved ADC treatment options available yet. In this study, we have thoroughly completed a narrative review of the current literature and summarized preclinical studies and clinical trials that evaluated the utility, activity, and toxicity of ADCs in GU cancers, the prospects of ADC development, and the ongoing clinical trials. Prospective clinical trials, retrospective studies, case reports, and scoping reviews were included.

Intratumor injection of BCG Ag85A high-affinity peptides enhanced anti-tumor efficacy in PPD-positive melanoma

As one of the scheduled immunization vaccines worldwide, virtually all individuals have been vaccinated with BCG vaccine. In order to verify the hypothesis that delivering BCG high-affinity peptides to tumor areas could activate the existing BCG memory T cells to attack tumor, we firstly predicted the HLA-A*0201 high-affinity peptides of BCG Ag85A protein (KLIANNTRV, GLPVEYLQV), and then, A375 melanoma cells and HLA-A*0201 PBMCs (from PPD-positive adults) were added to co-incubated with the predicted peptides in vitro. We found that the predicted BCG high-affinity peptides could be directly loaded onto the surface of tumor cells, enhancing the tumor-killing efficacy of PBMCs from PPD-positive volunteer. Then, we constructed PPD-positive mice model bearing B16F10 subcutaneous tumors and found that intratumor injection of BCG Ag85A high-affinity peptides (SGGANSPAL, YHPQQFVYAGAMSGLLD) enhanced the anti-tumor efficacy in PPD-positive melanoma mice. Along with the better anti-tumor efficacy, the expression of PDL1 on tumor cell surface was also increased, and stronger antitumor effects occurred when further combined with anti-PD1 antibody. For microenvironment analysis, the proportion of effector memory T cells was increased and the better treatment efficacy may be attributed to the elevated effector memory CD4 + T cells within the tumor. In conclusion, using the existing immune response of BCG vaccine by delivering high-affinity peptides of BCG to tumor area is a safe and promising therapy for cancer.

Co-encapsulation of granzyme B and perforin in nanocapsules for tumour therapy: biomimicking immune cells

Granzyme B (GrB)-based immunotherapy is of interest for cancer treatment. However, insufficient cellular uptake and a lack of targeting remain challenges to make use of GrB for solid tumour therapy. As GrB induced cell death requires the help of perforin (PFN), we designed a system (nGPM) for the co-delivery of GrB and PFN. Therefore, GrB and PFN were loaded in a porous polymeric nanocapsule rich in acetylcholine analogues and matrix metalloproteinase-2 (MMP-2) responsive peptides. The neutrally charged nGPM nanocapsules showed as long circulating time and accumulated at the tumour sites. Once in the tumour the outside shell of nanocapsules became degraded by overexpressed MMP-2 proteases, resulting in the release of GrB and PFN. We found that the PFN complex formed small pores on the surface of tumour cells which allow GrB to enter the cytoplasm of tumour cells inducing cell apoptosis and tumour suppression significantly.

Targeting PCSK9 to upregulate MHC-II on the surface of tumor cells in tumor immunotherapy.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), the last member of the proprotein convertase family, functions as a classic regulator of low-density lipoprotein (LDL) by interacting with low-density lipoprotein receptor (LDLR). Recent studies have shown that PCSK9 can affect the occurrence and development of tumors and can be used as a novel therapeutic target. However, a comprehensive pan-cancer analysis of PCSK9 has yet to be conducted. The potential oncogenic effects of PCSK9 in 33 types of tumors were explored based on the datasets of The Cancer Genome Atlas (TCGA) dataset. In addition, the immune regulatory role of PCSK9 inhibition was evaluated via in vitro cell coculture and the tumor-bearing mouse model. Finally, the antitumor efficacy of targeted PCSK9 combined with OVA-II vaccines was verified. Our results indicated that PCSK9 was highly expressed in most tumor types and was significantly correlated with late disease stage and poor prognosis. Additionally, PCSK9 may regulate the tumor immune matrix score, immune cell infiltration, immune checkpoint expression, and major histocompatibility complex expression. Notably, we first found that dendritic cell (DC) infiltration and major histocompatibility complex-II (MHC-II) expression could be upregulated by PCSK9 inhibition and improve CD8+ T cell activation in the tumor immune microenvironment, thereby achieving potent tumor control. Combining PCSK9 inhibitors could enhance the efficacies of OVA-II tumor vaccine monotherapy. Conclusively, our pan-cancer analysis provided a more comprehensive understanding of the oncogenic and immunoregulatory roles of PCSK9 and demonstrated that targeting PCSK9 could increase the efficacy of long peptide vaccines by upregulating DC infiltration and MHC-II expression on the surface of tumor cells. This study reveals the critical oncogenic and immunoregulatory roles of PCSK9 in various tumors and shows the promise of PCSK9 as a potent immunotherapy target.

Ultra‐Galactocation to Sialic Acid on Tumor Cells with A Penta‐Functional Dendritic Probe for Enhanced Immune‐Killing

AbstractGlycans on tumor cell surface have significant impacts in the immune‐killing process. Here an ultra‐galactocation to sialic acid (Sia) strategy is designed to hugely introduce galactose (Gal) to Sia and on tumor cells in vivo by using a penta‐functional dendritic probe (Den@5F), which efficiently enhances the immune‐killing of tumor cells. The Den@5F contains five different kinds of functional groups, including Gal, Cy5, amino, phenylboronic acid (PBA) and 4‐(4‐(hydroxymethyl)‐2‐methoxy‐5‐nitrophenoxy) butanoate (mNB), which can be conveniently prepared through a two‐step reaction. After injecting into the tumor‐bearing mouse, Den@5F can efficiently block Sia through the specific recognition between PBA and Sia on tumor cells and hugely introduce Gal through the subsequent photo‐crosslinking between mNB and amino groups to multiply conjugate excessive Den@5Fs. The comprehensively blocked Sia can prevent the immune escape, and the hugely introduced Gal can promote the immune stimulation of the immune cells, which lead to an efficient enhancement of the immune‐killing. The proposed strategy provides a significant and promising tool to promote the clinical immunotherapy of tumor.

Ultra-Galactocation to Sialic Acid on Tumor Cells with A Penta-Functional Dendritic Probe for Enhanced Immune-Killing.

Glycans on tumor cell surface have significant impacts in the immune-killing process. Here an ultra-galactocation to sialic acid (Sia) strategy is designed to hugely introduce galactose (Gal) to Sia and on tumor cells in vivo by using a penta-functional dendritic probe (Den@5F), which efficiently enhances the immune-killing of tumor cells. The Den@5F contains five different kinds of functional groups, including Gal, Cy5, amino, phenylboronic acid (PBA) and 4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butanoate (mNB), which can be conveniently prepared through a two-step reaction. After injecting into the tumor-bearing mouse, Den@5F can efficiently block Sia through the specific recognition between PBA and Sia on tumor cells and hugely introduce Gal through the subsequent photo-crosslinking between mNB and amino groups to multiply conjugate excessive Den@5Fs. The comprehensively blocked Sia can prevent the immune escape, and the hugely introduced Gal can promote the immune stimulation of the immune cells, which lead to an efficient enhancement of the immune-killing. The proposed strategy provides a significant and promising tool to promote the clinical immunotherapy of tumor.

Lymph Node Targeting Mediated by Albumin Hitchhiking of Synthetic Tn Glycolipid Leads to Robust In Vivo Antibody Production.

Tn antigen is a tumor-associated carbohydrate antigen, which is present prominently on the tumor cell surfaces and attracts an interest in vaccine development. This work demonstrates that a synthetic Tn antigen carrying glycoconjugate forms a complex with circulating albumin, delivers the antigen to lymph nodes (LNs), and leads to the efficient production of antibodies against the antigen. Synthetic Tn antigen glycoconjugate, possessing DSPE-PEG2000 linker and lipophilic moieties, undergoes micellization in PBS buffer. In the presence of bovine serum albumin (BSA), demicellization of the glycolipid occurs, with a rate constant of 0.18 min-1. In vitro studies show that the glycoconjugate binds preferentially to BSA in the presence of cells. Immunological assessments in mice models reveal the albumin-enabled delivery of the Tn glycoconjugate to antigen-presenting cells in the LNs, specifically leading to a robust humoral immune response. ELISA titers show superior binding, with a saturation dilution of 1:51 200 for Tn glycoconjugate, in comparison to that mediated by the Tn-BSA covalent conjugate with a saturation dilution of 1:6400. Immunohistochemical staining shows delivery of Tn glycoconjugate at the LNs, specifically at the subcapsular sinus and interfollicular areas. The work highlights the potential of albumin-mediated target delivery strategy for cancer immunotherapies.

Bacterial Biohybrids for Invasion of Tumor Cells Promote Antigen Cross-Presentation Through Gap Junction.

Due to inherent differences in cellular composition and metabolic behavior with host cells, tumor-harbored bacteria can discriminatorily affect tumor immune landscape. However, the mechanisms by which intracellular bacteria affect antigen presentation process between tumor cells and antigen-presenting cells (APCs) are largely unknown. The invasion behavior of attenuated Salmonella VNP20009 (VNP) into tumor cells is investigated and an attempt is made to modulate this behavior by modifying positively charged polymers on the surface of VNP. It is found that non-toxic chitosan oligosaccharide (COS) modified VNP (VNP@COS) bolsters the formation of gap junction between tumor cells and APCs by enhancing the ability of VNP to infect tumor cells. On this basis, a bacterial biohybrid is designed to promote in situ antigen cross-presentation through intracellular bacteria induced gap junction. This bacterial biohybrid also enhances the expression of major histocompatibility complex class I molecules on the surface of tumor cells through the incorporation of Mdivi-1 coupled with VNP@COS. This strategic integration serves to heighten the immunogenic exposure of tumor antigens; while, preserving the cytotoxic potency of T cells. A strategy is proposed to precisely controlling the function and local effects of microorganisms within tumors.

Abstract LB207: C5ar1 is a novel biomarker and potential therapeutic target in advanced cutaneous squamous cell carcinoma

Abstract Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer and the metastatic disease is associated with poor prognosis. At present, there are no established molecular markers to predict the metastasis risk and prognosis of patients with primary cSCC. Recently, the role of specific complement components in the progression of cSCC has been shown. Here, we have examined the role of complement C5a receptor C5aR1 in the progression and metastasis of cSCC. The results show that the expression of C5aR1 is increased in cSCC cells in 3D spheroid coculture model in the presence of fibroblasts and that treatment with recombinant C5a increases invasion of cSCC cells in this model. Staining for C5aR1 was detected on the surface of tumor cells in the invasive edge of human cSCC xenograft tumors in vivo. Staining of metastatic and non-metastatic primary human cSCCs, premalignant epidermal lesions and normal skin for C5aR1 with multiplexed fluorescence and chromogenic immunohistochemistry revealed stronger expression of C5aR1 on surface of cSCC tumor cells compared to epidermal cells in cSCC in situ, actinic keratosis and normal skin. Increased expression of C5aR1 on tumor cell surface was associated with the metastasis risk of primary cSCCs and independently predicted poor disease-specific survival of patients with primary cSCC. These results reveal the role of C5aR1 in cSCC progression and identify C5aR1 as a novel biomarker for metastasis risk and poor prognosis of patients with cSCC. The results also suggest C5aR1 as a novel therapeutic target for treatment of locally advanced and metastatic cSCC. Citation Format: Lauri Heiskanen, Liisa Nissinen, Elina Siljamäki, Jaakko Knuutila, Teijo Pellinen, Markku Kallajoki, Jyrki Heino, Pilvi Riihilä, Veli-Matti Kähäri. C5ar1 is a novel biomarker and potential therapeutic target in advanced cutaneous squamous cell carcinoma [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 LB207.

Abstract LB429: Discovery and identification of a novel cancer associated fibroblast target using Oxford Biotherapeutics (OBTs) OGAP discovery platform

Abstract Cancer Associated Fibroblasts (CAFs) are a major component of the tumor stromal microenvironment and have been indicated in promoting tumor growth as well as inhibiting tumor suppression mechanisms. One of the challenges in this field has been a lack of molecules that target CAFs specifically and can interfere with the CAF function. Here we describe a novel target discovered using OBTs proprietary OGAP platform, that appears to be specific for CAFs in a variety of tumor tissues. Interestingly this target appears to be minimally expressed in normal tissue fibroblasts by in situ hybridization RNA analysis but is highly upregulated in solid tissue stroma fibroblasts of colon, breast and pancreatic cancer tissues. The OGAP Discovery Platform utilizes a mass spectrometry approach to identify membrane targets on the surface of tumor cells as well as tumor microenvironment associated cells. This suggests that this novel target is expressed on the membrane of the CAFs making it a unique target for drug intervention. The identification of such a novel target is both exciting and a wonderful validation of OBTs OGAP Discovery Platform. Citation Format: Jeremy Sarnecky, Joseph Lewis, Jason Allen, Daniel Davies, Matthew Metzger, Ben Thomas, Christian Rohlff, Belinda Cairns. Discovery and identification of a novel cancer associated fibroblast target using Oxford Biotherapeutics (OBTs) OGAP discovery platform [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 LB429.

Abstract LB187: Investigating the roles of ROR1/2 in non-small cell lung carcinoma NSCLC and potential therapeutic applications

Abstract Lung cancer is the leading cause of cancer mortality worldwide, representing an unmet medical need. Receptor tyrosine kinase-like orphan receptors (RORs) are oncofetal antigens that act as receptors/co-receptors of WNT5A and other related noncanonical WNT proteins, in transducing β-catenin independent WNT signaling. Dysregulated ROR/WNT have been linked to cancer progression and therapy resistance. Recently, ROR receptors have drawn increasing attention as anti-cancer targets of antibody-drug conjugates (ADCs) due to their selective expression on the tumor cell surface. The ROR family contains ROR1 and ROR2, which share 58% sequence homology. However, their functional interactions between ROR/WNT5A and whether RORs have distinct or redundant roles in tumor phenotypes remain unclear. In this study, we sought to address these biological questions in NSCLC, and investigate the potential application of anti-ROR therapies. To this end, we used CRISPR/Cas9 to generate clones of ROR1 or ROR2 single knockouts (KOs) in the NSCLC LCLC-103H cell line and implemented an array of assays to characterize the effect of single ROR KOs. ROR KOs were confirmed by both western blot and flow cytometry. We found reduced ROR2 mean fluorescence intensity (MFI) on ROR1 KOs and increased ROR1 MFI on ROR2 KOs in flow cytometrical analysis. Phenotypically, ROR1 KOs did not affect tumor doubling time, but ROR2 KOs promoted cell proliferation in 2D cultures. Unlike 2D cultures, tumor 3D spheroid proliferation was significantly inhibited in both ROR1 and ROR2 KOs, implying that each ROR is indispensable in maintaining tumor growth featured by cell-cell/extracellular matrix interactions. Mechanistically, ROR1 KOs tended to decrease ROR2 and WNT5A/B protein expression, while ROR2 KOs caused compensatory upregulation of ROR1 and WNT5A/B. Both ROR1 and ROR2 KOs decreased SNAIL expression, which is a key driver of epithelial-to-mesenchymal plasticity (EMP). Furthermore, alterations in ROR1/2 signaling pathways were observed. We have identified pathways downstream of ROR1/2 including WNT, EMP, and survival, which may shed light on the biological roles of RORs in cancer and potential therapeutic intervention. Finally, the therapeutic application of ROR1/2 was evaluated by monomethyl auristatin F (MMAF) containing anti-ROR ADCs. ROR1 KOs demonstrated the on-target cytotoxicity of anti-ROR1 ADC, while ROR2 KOs showed increased sensitivity to anti-ROR1 ADC due to the increased ROR1 expression in ROR2 KOs. In vivo analysis is currently underway. Collectively, our results demonstrate that RORs have both distinct and compensatory functions in regulating tumor proliferation. We believe a greater understanding in ROR1/2 biology may lead to better targeted therapeutic applications. Citation Format: Wen-An Pan, Xin Guo, Christina Galang, Mira Ko, Jeff Kucharski, Nicole Miller, Haim Moskowitz, Jeff Watkins, Brian Lannutti, Katti Jessen, Hao Liu. Investigating the roles of ROR1/2 in non-small cell lung carcinoma NSCLC and potential therapeutic applications [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 LB187.

Abstract LB340: Harnessing the gp70 endogenous retrovirus envelope protein as a universal tumor antigen for T cell therapy in mice

Abstract FDA approval for chimeric antigen receptor (CAR) T cell therapies targeting hematological cancers has unleashed the potential of utilizing live T cells as therapeutics. However, CAR-mediated targeting of solid tumors is currently limited by a paucity of suitable tumor antigens that are both specific and highly expressed on the tumor cell surface. In this report we explore the possibility of harnessing tumor-specific expression of endogenous retroviruses (ERVs) as a universal target for CAR T cell therapy. We found that gp70, the envelope protein from a murine ERV, is highly expressed on the surface of tumor cell lines derived from a diverse array of murine tumor types. We utilized the B16 melanoma model as a representative poorly inflamed tumor that does not respond to existing immunotherapies to examine efficacy of gp70 CAR T cells. T cells engineered to express a gp70 CAR suppress tumor growth in vivo and recognize B16 tumor cells with improved sensitivity compared to T cells expressing two previously described dominant tumor antigens (gp100 or P15e) due to low expression of MHC-I on B16 tumors. Co-incubation of B16 tumor cells with gp70 CAR T cells results in upregulation of MHC-I on the tumor cell surface suggesting that in addition to killing tumor cells directly, gp70 CAR T cells could facilitate recognition of the tumor by endogenous T cells through local release T cell derived IFNγ. We postulate that utilization of CAR T cells specific for cell surface ERV envelope proteins offers a promising strategy that could be harnessed to achieve local release of T cell derived payloads specifically within the tumor microenvironment. Citation Format: Sophie M. Lehar, Srirupa Roy, Lisa Liao, Haiyin Chen, Yongmei Chen, Mayra Cruz Tleugabulova, Eric Janezic, Azadeh Madjidi, Tamaki Jones, Vincent Javinal, James Ziai, Ira Mellman, Jill Schartner, Andrey Shaw. Harnessing the gp70 endogenous retrovirus envelope protein as a universal tumor antigen for T cell therapy in mice [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 LB340.

Abstract LB125: Tri-specific T cell engager targeting of selective surface cancer-selective ligands recruits both T cells and APC targeting of cancer neoantigens of tumor-initiating stem-like cells

Abstract Chemotherapy does not sufficiently eliminate tumor-initiating cells (TICs) of hepatocellular carcinoma (HCC). To remove TICs and achieve personalized cancer therapy, dendritic cells (DC) were vaccinated against tumor germline mutations, but metastatic cancers recurred in many cases. Emerging data suggest that nanoparticle-based siRNA delivered targeting of stemness transcription factor NANOG (pluripotency transcription factor) potentiates anti-tumor immunity. We hypothesized that pre-treatment of selective TIC inhibitors targeting stemness genes sensitized the efficacy of immunotherapy targeting of liver TICs. Novel TIC-targeting immunoglobulin, variable chain fragments were identified by phage-display library screening. The top single chain camelid antibody (scFv) candidates were identified and individually cloned into mammalian expression vectors to test binding to TICs. A Tri-specific T cell engager (TriTE) was constructed to connect TIC-targeting scFv with anti-dendritic cell (DC) scFv (anti-CD14) and CD3. Such scFv vectors were tested for target cell lysis. Candidate ligands of anti-TIC ScFvs present on the tumor cell surface were identified through a series of bioinformatic analysis., The latter provided the preconditions for one of our aims to identify the target, tumor surface ligand(s) of anti-TIC ScFv to better understand the mechanism of TriTE action upon a tumor cell. IP-Mass spectrometry analyses demonstrated that two target surface antigens, TBCE and CEP170, along with their respective mRNAs were highly expressed in CD133+ TIC Huh7 cells when compared to CD133- non-TIC Huh7 cells. This accounted for specific TriTE binding to TIC cells, but not to CD133(-) Huh7 cells or primary hepatocytes. These data demonstrated that these unique tumor antigens, primarily arising during neoplastic transformation, elicited T and/or monocyte immunity capable of protecting the host from cancer progression. These conjunctive with PD1 antibody therapeutic modality leads to the personalized immune/chemotherapy of HCC patients. Human monocyte-derived DCs, TIC and T cells were incubated with TriTE to measure IFNγ production during CTL killing effects against TICs. TriTE treatment significantly reduced tumor growth of PDX tissues in NSG-SG3 mice with humanized immunity. Other novel surface antigens on TIC were identified by mass spectroscopy analyses. These data demonstrated that the presence of unique tumor antigens, primarily arising during neoplastic transformation, which elicited T and/or myeloid cell immunity capable of protecting the host from cancer progression. Self-adapting antigen-targeting machinery with Trite therapy is adaptable and fine-tuning next-generation immunotherapy. Citation Format: Wei Ma, Nuan Wang, Hye Yeon Choi, Lin Ziying, Nicolette Fajardo, Royce Ilaga, Risa Machida, Zhou Lin, Ambika Ramrakhiani, Yvonne Y. Chen, Linda Sher, Stanley M. Tahara, Keigo Machida. Tri-specific T cell engager targeting of selective surface cancer-selective ligands recruits both T cells and APC targeting of cancer neoantigens of tumor-initiating stem-like cells [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 LB125.

Abstract LB282: Therapeutic efficacy and dosimetry for a 177-lutetium radiolabeled minibody targeting DLL3 in a preclinical model of small cell lung cancer

Abstract Small cell lung cancer (SCLC) is the most aggressive subtype of lung carcinoma with a 5-year survival rate below 7%. While the addition of anti-PD-1 antibody treatment has improved SCLC patient outcomes, nearly all patients relapse, indicating an unmet need for more efficacious therapeutics. Delta-like ligand 3 (DLL3) is an inhibitory Notch ligand that is over-expressed on the surface of SCLC tumor cells. Due to this, DLL3 has become a target of interest for SCLC treatment and several agents are in preclinical and clinical evaluation using different strategies such as antibody-drug conjugates, bispecific T cell engagers, and DLL3-targeting CAR-T. Minibodies are antibody fragments that provide optimal pharmacokinetics for the delivery of radioactive payloads. Their molecular weight of 80 kDa allows for rapid tumor uptake and faster circulation clearance than full-length antibodies. These scaffolds also present deeper tumor penetration relative to full-length antibodies, and higher target specificity and retention at the tumor site, with respect to small molecules. We hypothesized that a minibody targeting DLL3 (IAB57) would be a valid approach for radiopharmaceutical therapy (RPT) of SCLC and tested its efficacy in a preclinical model of SCLC. We conjugated the IAB57 minibody with DOTAGA and radiolabeled the protein with the β-emitter 177-Lu. Nu/J female mice were inoculated subcutaneously with the non-encapsulated lung carcinoma cell line SHP77, and the tumor size was measured 3 times per week until it reached 100-150 mm3. 24-hour biodistribution results revealed a tumor uptake of 18.8% injected dose per gram (%ID/gr). Mice were treated with 13.3 MBq and 13.6 MBq doses of 177-Lu-DOTAGA-IAB57 7 days apart to determine if there was a therapeutic effect and improved animal survival. A subgroup of mice was not treated and was considered controls. A time course biodistribution was performed to assess organs dosimetry. The dosimetry results were extrapolated to human organ exposure showing kidneys and liver uptakes of 2.2 and 4.6 Gy which is 10 times and 7 times below published exposure thresholds (23 Gy and 32 Gy respectively). Exposure at bone marrow (b.m., 0.1 Gy), spleen (1.5 Gy) and lungs (0.4 Gy) were 20 times, 27 times and 68 times below published toxicity thresholds (b.m.: 2 Gy; spleen: 40 Gy; lungs: 27 Gy). The RPT data showed a significant decrease in tumor size beginning on day 11 in the treated group compared to the control group. The decreased tumor size also coincided with a doubling in the survival rate. Overall, these results indicate that the IAB57 minibody targeting DLL3 is a promising and effective agent for RPT treatment of SCLC via β-emitting radionuclides. Citation Format: Kelley C. Atkinson, Leticia M. De Souza Cordeiro, Fang Jia, Argin Aivazian, Gareth E. Smith, Ian Wilson, Alessandro Mascioni. Therapeutic efficacy and dosimetry for a 177-lutetium radiolabeled minibody targeting DLL3 in a preclinical model of small cell lung cancer [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 LB282.

A multi-mode Rhein-based nano-platform synergizing ferrotherapy/chemotherapy-induced immunotherapy for enhanced tumor therapy

Ferroptosis has emerged as a promising strategy for treating triple-negative breast cancer (TNBC) due to bypassing apoptosis and triggering immunogenic cell death (ICD) of tumor cells. However, the antitumor efficacy has been limited by the insufficient intracellular ferrous iron concentration required for ferroptosis and inadequate antitumor immune response. To address these limitations, we designed a multi-mode nano-platform (MP-FA@R-F NPs), which exhibited a synergistic effect of ferroptosis, apoptosis and induced immune response for enhanced antitumor therapy. MP-FA@R-F NPs target folate receptors, which are over-expressed on the tumor cell's surface to promote intracellular uptake. The cargoes, including Rhein and Fe3O4, would be released in intracellular acid, accelerating by NIR laser irradiation. The released Rhein induced apoptosis of tumor cells mediated by the caspase 3 signal pathway, while the released Fe3O4 triggered ferroptosis through the Fenton reaction and endowed the nanoplatform with magnetic resonance imaging (MRI) capabilities. In addition, ferroptosis-dying tumor cells could release damage-associated molecular patterns (DAMPs) to promote T cell activation and infiltration for immune response and induce immunogenic cell death (ICD) for tumor immunotherapy. Together, MP-FA@R-F NPs represent a potential synergistic ferro-/chemo-/immuno-therapy strategy with MRI guidance for enhanced antitumor therapy. Statement of significanceThe massive strategies of cancer therapy based on ferroptosis have been emerging in recent years, which provided new insights into designing materials for cancer therapy. However, the antitumor efficacy of ferroptosis is still unsatisfactory, mainly due to insufficient intracellular pro-ferroptotic stimuli. In the current study, we designed a multi-mode nano-platform (MP-FA@R-F NPs), which represented a potential synergistic ferro-/chemo-/immuno-therapy strategy with MRI guidance for enhanced antitumor therapy.

Assessment of Inhibition of the Growth of Breast and Colon Tumors when Blocking VEGFR-1 with Monoclonal Antibodies

Vascular endothelial growth factor receptor-1 (VEGFR-1) plays a critical role in tumor-associated angiogenesis. VEGFR-1 is found on the surface of tumor cells and cells in the tumor microenvironment. Blocking this receptor leads to the suppression of proliferation and increased apoptosis of tumor cells, reduction of tumor vascularization, inhibition of the production of immunosuppressive cytokines by tumor-associated macrophages, and the suppression of tumor invasion and metastasis. The creation of monoclonal antibody drugs that block VEGFR-1 is an urgent task in the development of potential antitumor therapeutic drugs. Target molecules created on the basis of antibodies that bind to VEGFR-1 are a promising basis for the creation of theranostic radiopharmaceuticals for the diagnosis and treatment of malignant neoplasms. To study the therapeutic potential of VEGFR-1 inhibition in breast and colon cancers using antibodies, monoclonal antibodies against recombinant human VEGFR-1 protein are developed. The resulting monoclonal antibodies bind to the VEGFR-1 receptor on the cell surface and effectively inhibit the proliferation of breast and colon cancer cells in vitro, reduce the growth rate of the tumor node in vivo, and prolong the survival of tumor-inoculated mice.

Folate-modified liposomes mediate the co-delivery of cisplatin with miR-219a-5p for the targeted treatment of cisplatin-resistant lung cancer

Cisplatin (DDP) resistance, often leading to first-line chemotherapy failure in non-small cell lung cancer (NSCLC), poses a significant challenge. MiR-219a-5p has been reported to enhance the sensitivity of human NSCLC to DDP. However, free miR-219a-5p is prone to degradation by nucleases in the bloodstream, rendering it unstable. In light of this, our study developed an efficient nanodrug delivery system that achieved targeted delivery of DDP and miR-219a-5p by modifying liposomes with folate (FA). Based on the results of material characterization, we successfully constructed a well-dispersed and uniformly sized (approximately 135.8 nm) Lipo@DDP@miR-219a-5p@FA nanodrug. Agarose gel electrophoresis experiments demonstrated that Lipo@DDP@miR-219a-5p@FA exhibited good stability in serum, effectively protecting miR-219a-5p from degradation. Immunofluorescence and flow cytometry experiments revealed that, due to FA modification, Lipo@DDP@miR-219a-5p@FA could specifically bind to FA receptors on the surface of tumor cells (A549), thus enhancing drug internalization efficiency. Safety evaluations conducted in vitro demonstrated that Lipo@DDP@miR-219a-5p@FA exhibited no significant toxicity to non-cancer cells (BEAS-2B) and displayed excellent blood compatibility. Cellular functional experiments, apoptosis assays, and western blot demonstrated that Lipo@DDP@miR-219a-5p@FA effectively reversed DDP resistance in A549 cells, inhibited cell proliferation and migration, and further promoted apoptosis. In summary, the Lipo@DDP@miR-219a-5p@FA nanodrug, through specific targeting of cancer cells and reducing their resistance to DDP, significantly enhanced the anti-NSCLC effects of DDP in vitro, providing a promising therapeutic option for the clinical treatment of NSCLC.

Cx1Mab-1: A Novel Anti-mouse CXCR1 Monoclonal Antibody for Flow Cytometry.

The C-X-C motif chemokine receptor-1 (CXCR1) is a rhodopsin-like G-protein-coupled receptor, expressed on the cell surface of immune cells and tumors. CXCR1 interacts with some C-X-C chemokines, such as CXCL6, CXCL7, and CXCL8/interleukin-8, which are produced by various cells. Since CXCR1 is involved in several diseases including tumors and diabetes mellitus, drugs targeting CXCR1 have been developed. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CXCR1 has been desired for the diagnosis and treatment. This study established a novel anti-mouse CXCR1 (mCXCR1) mAb, Cx1Mab-1 (rat IgG1, kappa), using the Cell-Based Immunization and Screening method. Cx1Mab-1 reacted with mCXCR1-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR1) and mCXCR1-overexpressed LN229 glioblastoma (LN229/mCXCR1) in flow cytometry. Cx1Mab-1 demonstrated a high binding affinity for CHO/mCXCR1 and LN229/mCXCR1 with a dissociation constant of 2.6 × 10-9 M and 2.1 × 10-8 M, respectively. Furthermore, Cx1Mab-1 could detect mCXCR1 by Western blot analysis. These results indicated that Cx1Mab-1 is useful for detecting mCXCR1, and provides a possibility for targeting mCXCR1-expressing cells in vivo experiments.

Bioorthogonal In Situ Polymerization of Dendritic Agents for Hijacking Lysosomes and Enhancing Antigen Presentation in Cancer Cells.

A low-generation lysine dendrimer, SPr-G2, responds to intracellular glutathione to initiate bioorthogonal in situ polymerization, resulting in the formation of large assemblies in mouse breast cancer cells. The intracellular large assemblies of SPr-G2 can interact with lysosomes to induce lysosome expansion and enhance lysosomal membrane permeabilization, leading to major histocompatibility complex class I upregulation on tumor cell surfaces and ultimately tumor cell death. Moreover, the use of the SPr-G2 dendrimer to conjugate the chemotherapeutic drug, camptothecin (CPT), can boost the therapeutic potency of CPT. Excellent antitumor effects in vitro and in vivo are obtained from the combinational treatment of the SPr-G2 dendrimer and CPT. This combinational effect also enhances antitumor immunity through promoting activation of cytotoxic T cells in tumor tissues and maturation of dendritic cells. This study can shed new light on the development of peptide dendritic agents for cancer therapy.

Abstract 5157: Development and evaluation of a high-throughput method for rapid detection of surface antigen expression in fixed cells

Abstract Cell surface proteins are increasingly considered in the development of novel anticancer agents, as demonstrated by recent clinical successes in the field of immuno-oncology and antibody-drug conjugates (ADCs). These conjugates specifically target antigens present on the surface of tumor cells, enabling the delivery of cytotoxic agents to specific tumor sites. During the preclinical development of such compounds, for example, when they are tested on a wide panel of cell lines, it is essential to have a rapid and reliable method for monitoring target expression and distribution. Complementing our 160 Cell line panel ProLiFiler™, we report here the development of a novel method named oncoFLOW-Profiler™ which includes steps of cell fixation, micronic storage, staining, and measurement by flow cytometry. This method is applied in a 96-well format, enabling screening of all cell lines in parallel and analysis of the expression of different targets in just one day. In this proof-of-concept study, we investigated the level of ERBB2 expression across the ProLiFiler panel of 160-cell lines in order to validate this methodology. We first compared the impact of fixation methods and sample storage on knowingly positive and negative control cell lines over a period of several months. ERBB2 measurements from the oncoFLOW-Profiler™ will be analyzed and validated by comparison with various OMICs datasets from the Cancer DataMiner platform (4HF Biotec), including RNA expression, mutational status, and protein expression levels. To determine the expression cutoff of ERBB2 amplified models, results will be analyzed according to gene copy number variation. Finally, the flow cytometry data generated will be investigated for use as a predictor of response to clinically approved ADCs targeting ERBB2: Kadcyla and Enhertu. Here we present a method that we have demonstrated is feasible for investigating cell surface receptor/antigens during preclinical phase of drug development. Our method has enabled us to successfully assess ERBB2 in biological samples and to identify models sensitive to ADCs targeting ERBB2. We will also discuss the results of ongoing experiments analyzing the applicability of this screen to intracellular targets and carbohydrate-based surface antigens, which cannot be analyzed either by transcriptomics or proteomics. Citation Format: Kaja Holtorf, Anne-Lise Peille, Daniel Feger, Vincent Vuaroqueaux, Jan Erik Ehlert, Nadine Obier. Development and evaluation of a high-throughput method for rapid detection of surface antigen expression in fixed 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 5157.