Target Cell Population Research Articles

Insights into the binding recognition and computational design of IL-2 muteins with enhanced predicted binding affinity to the IL-2 receptor α

Interleukin-2 (IL-2) is an immune system regulator that has received approval for cancer treatment. However, high-dose IL-2 therapy has seen restricted use due to its low efficacy and on-target toxicity. To enhance the effectiveness of IL-2 therapy, it is essential to engineer IL-2 molecules to enhance their specificity toward target cell populations. In this study, molecular dynamics (MD) simulations and Rosetta software were utilized to design novel high-affinity IL-2Rα-binding IL-2 muteins. MD simulations were used to identify the target residues of IL-2 for design, and Rosetta software were then employed to predict potential IL-2 muteins with higher binding affinity toward IL-2Rα. Rosetta generated two potential designed IL-2 muteins. The results of the MD validation and MM/GBSA analysis indicated that both designed IL-2 muteins exhibited greater predicted binding affinities toward IL-2Rα than that of the native proteins. RMSF analysis demonstrated that the structural fluctuations of free IL-2 and designed muteins were similar, indicating that the mutations did not alter the intramolecular force responsible for IL-2's stability and folding. These designed IL-2 muteins may have potential benefits for cancer immunotherapy.

Mind the GAPS: Glia associated with psychological stress.

Glial cells are an integral component of the nervous system, performing crucial functions that extend beyond structural support, including modulation of the immune system, tissue repair, and maintaining tissue homeostasis. Recent studies have highlighted the importance of glial cells as key mediators of stress responses across different organs. This review focuses on the roles of glial cells in peripheral tissues in health and their involvement in diseases linked to psychological stress. Populations of glia associated with psychological stress ("GAPS") emerge as a promising target cell population in our basic understanding of stress-associated pathologies, highlighting their role as mediators of the deleterious effects of psychological stress on various health conditions. Ultimately, new insights into the impact of stress on glial cell populations in the periphery may support clinical efforts aimed at improving the psychological state of patients for improved health outcomes.

Partial chemogenetic inhibition of the locus coeruleus due to heterogeneous transduction of noradrenergic neurons preserved auditory salience processing in wild-type rats.

The acoustic startle reflex (ASR) and prepulse inhibition of the ASR (PPI) assess the efficiency of salience processing, a fundamental brain function that is impaired in many psychiatric conditions. Both ASR and PPI depend on noradrenergic transmission, yet the modulatory role of the locus coeruleus (LC) remains controversial. Clonidine (0.05 mg/kg, i.p.), an alpha2-adrenoreceptor agonist, strongly reduced the ASR amplitude. In contrast, chemogenetic LC inhibition only mildly suppressed the ASR and did affect the PPI in virus-transduced rats. The canine adenovirus type 2 (CAV2)-based vector carrying a gene cassette for the expression of inhibitory receptors (hM4Di) and noradrenergic cell-specific promoter (PRSx8) had high cell-type specificity (94.4±3.1%) but resulted in heterogeneous virus transduction of DbH-positive LC neurons (range: 9.2-94.4%). Clozapine-N-oxide (CNO; 1mg/kg, i.p.), a hM4Di actuator, caused the firing cessation of hM4Di-expressing LC neurons, yet complete inhibition of the entire population of LC neurons was not achieved. Case-based immunohistochemistry revealed that virus injections distal (> 150μm) to the LC core resulted in partial LC transduction, while proximal (< 50 μm) injections caused neuronal loss due to virus neurotoxicity. Neither the ASR nor PPI differed between the intact and virus-transduced rats. Our results suggest that a residual activity of virus-non-transduced LC neurons might have been sufficient for mediating an unaltered ASR and PPI. Our study highlights the importance of a case-based assessment of the virus efficiency, specificity, and neurotoxicity for targeted cell populations and of considering these factors when interpreting behavioral effects in experiments employing chemogenetic modulation.

Review on Monoclonal Antibodies (mAbs) as a Therapeutic Approach for Type 1 Diabetes.

Monoclonal antibodies have been successfully utilized in a variety of animal models to treat auto-immune illnesses for a long time. Immune system responses will either be less active or more active depending on how the immune system is operating abnormally. Immune system hypoactivity reduces the body's capacity to fight off various invading pathogens, whereas immune system hyperactivity causes the body to attack and kill its own tissues and cells. For maximal patient compliance, we will concentrate on a variety of antibody therapies in this study to treat Type 1 diabetes (an autoimmune condition). T-cells are responsible for the auto-immune condition known as T1D, which causes irregularities in the function of β-cells in the pancreas. As a result, for the treatment and prevention of T1D, immunotherapies that selectively restore continuous beta cellspecific self-tolerance are needed. Utilizing monoclonal antibodies is one way to specifically target immune cell populations responsible for the auto-immune-driven disease (mAb). Numerous mAbs have demonstrated clinical safety and varied degrees of success in modulating autoimmunity, including T1D. A targeted cell population is exhausted by mAb treatments, regardless of antigenic specificity. One drawback of this treatment is the loss of obtained protective immunity. Immune effector cell function is regulated by nondepleting monoclonal antibodies (mAb). The antigenfocused new drug delivery system is made possible by the adaptability of mAbs. For the treatment of T1D and T-cell-mediated autoimmunity, different existing and potential mAb therapy methods are described in this article.

The Projection-Specific Noradrenergic Modulation of Perseverative Spatial Behavior in Adult Male Rats.

Adaptive behavior relies on efficient cognitive control. The anterior cingulate cortex (ACC) is a key node within the executive prefrontal network. The reciprocal connectivity between the locus ceruleus (LC) and ACC is thought to support behavioral reorganization triggered by the detection of an unexpected change. We transduced LC neurons with either excitatory or inhibitory chemogenetic receptors in adult male rats and trained rats on a spatial task. Subsequently, we altered LC activity and confronted rats with an unexpected change of reward locations. In a new spatial context, rats with decreased noradrenaline (NA) in the ACC entered unbaited maze arms more persistently which was indicative of perseveration. In contrast, the suppression of the global NA transmission reduced perseveration. Neither chemogenetic manipulation nor inactivation of the ACC by muscimol affected the rate of learning, possibly due to partial virus transduction of the LC neurons and/or the compensatory engagement of other prefrontal regions. Importantly, we observed behavioral deficits in rats with LC damage caused by virus injection. The latter finding highlights the importance of careful histological assessment of virus-transduced brain tissue as inadvertent damage of the targeted cell population due to virus neurotoxicity or other factors might cause unwanted side effects. Although the specific role of ACC in the flexibility of spatial behavior has not been convincingly demonstrated, our results support the beneficial role of noradrenergic transmission for an optimal function of the ACC. Overall, our findings suggest the LC exerts the projection-specific modulation of neural circuits mediating the flexibility of spatial behavior.

Low intensity mechanical signals promote proliferation in a cell-specific manner: Tailoring a non-drug strategy to enhance biomanufacturing yields

Biomanufacturing relies on living cells to produce biotechnology-based therapeutics, tissue engineering constructs, vaccines, and a vast range of agricultural and industrial products. With the escalating demand for these bio-based products, any process that could improve yields and shorten outcome timelines by accelerating cell proliferation would have a significant impact across the discipline. While these goals are primarily achieved using biological or chemical strategies, harnessing cell mechanosensitivity represents a promising – albeit less studied – physical pathway to promote bioprocessing endpoints, yet identifying which mechanical parameters influence cell activities has remained elusive. We tested the hypothesis that mechanical signals, delivered non-invasively using low-intensity vibration (LIV; <1 ​g, 10–500 ​Hz), will enhance cell expansion, and determined that any unique signal configuration was not equally influential across a range of cell types. Varying frequency, intensity, duration, refractory period, and daily doses of LIV increased proliferation in Chinese Hamster Ovary (CHO)-adherent cells (+79% in 96 ​hr) using a particular set of LIV parameters (0.2 ​g, 500 ​Hz, 3 ​× ​30 ​min/d, 2 ​hr refractory period), yet this same mechanical input suppressed proliferation in CHO-suspension cells (−13%). Another set of LIV parameters (30 ​Hz, 0.7 ​g, 2 ​× ​60 ​min/d, 2 ​hr refractory period) however, were able to increase the proliferation of CHO-suspension cells by 210% and T-cells by 20.3%. Importantly, we also reported that T-cell response to LIV was in-part dependent upon AKT phosphorylation, as inhibiting AKT phosphorylation reduced the proliferative effect of LIV by over 60%, suggesting that suspension cells utilize mechanism(s) similar to adherent cells to sense specific LIV signals. Particle image velocimetry combined with finite element modeling showed high transmissibility of these signals across fluids (>90%), and LIV effectively scaled up to T75 flasks. Ultimately, when LIV is tailored to the target cell population, it's highly efficient transmission across media represents a means to non-invasively augment biomanufacturing endpoints for both adherent and suspended cells, and holds immediate applications, ranging from small-scale, patient-specific personalized medicine to large-scale commercial bio-centric production challenges.

Enhanced Prostate-specific Membrane Antigen Targeting by Precision Control of DNA Scaffolded Nanoparticle Ligand Presentation.

Targeted nanoparticles have been extensively explored for their ability to deliver their payload to a selective cell population while reducing off-target side effects. The design of actively targeted nanoparticles requires the grafting of a ligand that specifically binds to a highly expressed receptor on the surface of the targeted cell population. Optimizing the interactions between the targeting ligand and the receptor can maximize the cellular uptake of the nanoparticles and subsequently improve their activity. Here, we evaluated how the density and presentation of the targeting ligands dictate the cellular uptake of nanoparticles. To do so, we used a DNA-scaffolded PLGA nanoparticle system to achieve efficient and tunable ligand conjugation. A prostate-specific membrane antigen (PSMA) expressing a prostate cancer cell line was used as a model. The density and presentation of PSMA targeting ligand ACUPA were precisely tuned on the DNA-scaffolded nanoparticle surface, and their impact on cellular uptake was evaluated. It was found that matching the ligand density with the cell receptor density achieved the maximum cellular uptake and specificity. Furthermore, DNA hybridization-mediated targeting chain rigidity of the DNA-scaffolded nanoparticle offered ∼3 times higher cellular uptake compared to the ACUPA-terminated PLGA nanoparticle. Our findings also indicated a ∼ 3.7-fold reduction in the cellular uptake for the DNA hybridization of the non-targeting chain. We showed that nanoparticle uptake is energy-dependent and follows a clathrin-mediated pathway. Finally, we validated the preferential tumor targeting of the nanoparticles in a bilateral tumor xenograft model. Our results provide a rational guideline for designing actively targeted nanoparticles and highlight the application of DNA-scaffolded nanoparticles as an efficient active targeting platform.

Chimeric antigen receptor T cell therapy for autoimmune disease.

Infusion of T cells engineered to express chimeric antigen receptors (CARs) that target B cells has proven to be a successful treatment for Bcell malignancies. This success inspired the development of CAR T cells to selectively deplete or modulate the aberrant immune responses that underlie autoimmune disease. Promising results are emerging from clinical trials of CAR T cells targeting the B cell protein CD19 in patients with B cell-driven autoimmune diseases. Further approaches are being designed to extend the application and improve safety of CAR T cell therapy in the setting of autoimmunity, including the use of chimeric autoantibody receptors to selectively deplete autoantigen-specific B cells and the use of regulatory T cells engineered to express antigen-specific CARs for targeted immune modulation. Here, we highlight important considerations, such as optimal target cell populations, CAR construct design, acceptable toxicities and potential for lasting immune reset, that will inform the eventual safe adoption of CAR T cell therapy for the treatment of autoimmune diseases.

Bulk lysis procedures alter target cell population counts.

To achieve high-sensitivity cell measurements (<1 in 105 cells) by flow cytometry (FCM), the minimum number of acquired cells must be considered and conventional immunophenotyping protocols fall short of these numbers. The bulk lysis (BL) assay is a standardized erythrocyte lysing approach that allows the analysis of the millions of cells required for high-sensitivity measurable residual disease (MRD) detection. However, this approach has been associated with significant cell loss, along with potential over or underestimates of rare cells when using this method. The aim of this study was to evaluate bulk lysis protocols and compare them with minimal sample perturbation (MSP) protocols, which are reported to better preserve the native cellular state and avoid significant cell loss due to washing steps. To achieve this purpose, we first generated an MRD model by spiking fresh peripheral blood with K562 cells, stably expressing EGFP, at known percentages of EGFP positive cells to leukocytes. Samples were then prepared with BL and MSP protocols and analyzed using FCM. For all percentages of K562 cells established and evaluated, a significant decrease of this population was detected in BL samples compared with MSP samples, even at low K562 cell percentages. Significant decreases for non-necrotic cells were also observed in BL samples relative to MSP samples. In conclusion, the evaluation of the potential effects of BL protocols in obtaining the final count is of great interest, especially for over- or under-estimation of target cells, as in the case of measurable residual disease. Since conventional flow cytometry or minimal sample perturbation assays fall short in obtaining the minimum numbers required to reach high sensitivity measurements, significant efforts may be needed to improve bulk lysis solution reagents.

Inhibition of Microbicidal Activity of Canine Macrophages DH82 Cell Line by Capsular Polysaccharides from Cryptococcus neoformans.

Cryptococcus neoformans is a lethal fungus that primarily affects the respiratory system and the central nervous system. One of the main virulence factors is the capsule, constituted by the polysaccharides glucuronoxylomannan (GXM) and glucuronoxylomanogalactan (GXMGal). Polysaccharides are immunomodulators. One of the target cell populations for modulation are macrophages, which are part of the first line of defense and important for innate and adaptive immunity. It has been reported that macrophages can be modulated to act as a "Trojan horse," taking phagocytosed yeasts to strategic sites or having their machinery activation compromised. The scarcity of information on canine cryptococcosis led us to assess whether the purified capsular polysaccharides from C. neoformans would be able to modulate the microbicidal action of macrophages. In the present study, we observed that the capsular polysaccharides, GXM, GXMGal, or capsule total did not induce apoptosis in the DH82 macrophage cell line. However, it was possible to demonstrate that the phagocytic activity was decreased after treatment with polysaccharides. In addition, recovered yeasts from macrophages treated with polysaccharides after phagocytosis could be cultured, showing that their viability was not altered. The polysaccharides led to a reduction in ROS production and the mRNA expression of IL-12 and IL-6. We observed that GXMGal inhibits MHC class II expression and GXM reduces ERK phosphorylation. In contrast, GXMGal and GXM were able to increase the PPAR-γ expression. Furthermore, our data suggest that capsular polysaccharides can reduce the microbicidal activity of canine macrophages DH82.

Gli1+ Progenitors Mediate Glucocorticoid-Induced Osteoporosis In Vivo.

For a wide range of chronic autoimmune and inflammatory diseases in both adults and children, synthetic glucocorticoids (GCs) are one of the most effective treatments. However, besides other adverse effects, GCs inhibit bone mass at multiple levels, and at different ages, especially in puberty. Although extensive studies have investigated the mechanism of GC-induced osteoporosis, their target cell populations still be obscure. Here, our data show that the osteoblast subpopulation among Gli1+ metaphyseal mesenchymal progenitors (MMPs) is responsive to GCs as indicated by lineage tracing and single-cell RNA sequencing experiments. Furthermore, the proliferation and differentiation of Gli1+ MMPs are both decreased, which may be because GCs impair the oxidative phosphorylation(OXPHOS) and aerobic glycolysis of Gli1+ MMPs. Teriparatide, as one of the potential treatments for GCs in bone mass, is sought to increase bone volume by increasing the proliferation and differentiation of Gli1+ MMPs in vivo. Notably, our data demonstrate teriparatide ameliorates GC-caused bone defects by targeting Gli1+ MMPs. Thus, Gli1+ MMPs will be the potential mesenchymal progenitors in response to diverse pharmaceutical administrations in regulating bone formation.

Stem Cells Collection and Mobilization in Adult Autologous/Allogeneic Transplantation: Critical Points and Future Challenges.

Achieving successful hematopoietic stem cell transplantation (HSCT) relies on two fundamental pillars: effective mobilization and efficient collection through apheresis to attain the optimal graft dose. These cornerstones pave the way for enhanced patient outcomes. The primary challenges encountered by the clinical unit and collection facility within a transplant program encompass augmenting mobilization efficiency to optimize the harvest of target cell populations, implementing robust monitoring and predictive strategies for mobilization, streamlining the apheresis procedure to minimize collection duration while ensuring adequate yield, prioritizing patient comfort by reducing the overall collection time, guaranteeing the quality and purity of stem cell products to optimize graft function and transplant success, and facilitating seamless coordination between diverse entities involved in the HSCT process. In this review, we aim to address key questions and provide insights into the critical aspects of mobilizing and collecting hematopoietic stem cells for transplantation purposes.

Abstract 2832: Mouse models advancing cancer research - The Jackson Laboratory

Abstract Genetically defined mice are a cornerstone of cancer research. The Jackson Laboratory (JAX) offers more than 13,500 unique mouse lines to the scientific community, many useful in advancing cancer research. Available are mouse models for specific cancers, xenograft recipients, immunodeficient platforms for PDX studies and multi-purpose tool strains having CRISPR/Cas9 utility, conditional/inducible expression (Cre-lox, FLP-frt, Tet-On/-Off), optogenetic function and calcium-sensing technologies. This poster will highlight some recently added models, more than 100 of which have cancer research applications. The portfolio of strains useful for transplantation/engraftment and “humanized” mouse research continues to expand and complement the NSG (NOD scid IL2rγc-/-) model as ideal tools for engraftment with a wide range of malignant or non-malignant human or mouse tissues. Fluorescent protein-expressing strains include Traffic Light Reporters for tracking of DNA repair pathways, Gene Editing Reporters for detection of homology directed repair (HDR) and non-homologous end joining (NHEJ) repair events, as well as several CRISPR Editing, Bxb1 Integrase Editing, Base Editor and Prime Editor tools. For example, the Rosa26PE2 mouse has Cre-inducible expression of a prime editor enzyme and a fluorescent reporter, allowing viral delivery of prime editing guide RNAs to introduce cancer mutations into the genome. Recently added CRISPR Array Repair Lineage Tracing (CARLIN and DARLIN) component strains allow researchers to achieve high barcode diversity in targeted cell populations. In addition to safeguarding each strain by cryopreservation, JAX has a comprehensive genetic quality control program that confirms mutation identity and genetic background, and screens for unwanted alleles (such as stray GFP, Cre, neo, etc.). Search the comprehensive collection of innovative mouse models using our improved JAX Mouse Search site (mice.jax.org). This resource includes models created by many generous donating institutions. Researchers are encouraged to donate their mouse lines via a very short Strain Submission form (jax.org/donate-a-mouse). Please also visit the JAX Oncology webpage for cancer research related strains and resources (jax.org/jax-mice-and-services/solutions-by-therapeutic-area/oncology). JAX is supported by the NIH, The Howard Hughes Medical Institute and other private charitable foundations. Citation Format: Jason Beckwith, Jennifer Merriam, Alicia Valenzuela, Darcy Pomerleau, Jennifer Kelmenson. Mouse models advancing cancer research - The Jackson Laboratory [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 2832.

Abstract 2477: Selective IL-21 and IFN-alpha immunocytokines engineered using the AlphaSeq platform

Abstract Although cytokine therapies have demonstrated curative effects in some cancer patients, clinical use remains limited due to undesired toxicity profiles accompanying systemic administration. Next generation cytokine approaches include conditional signaling focused on sites of interest, such as the tumor microenvironment or specific immune cell populations. Here, we share a novel approach for generating detuned cytokine therapeutic candidates using the AlphaSeq platform, which involves the re-engineering of yeast agglutination and mating to quantitatively measure protein-protein interactions at a library-on-library scale. Engineering interferon-alpha (IFNα) and interleukin-21 (IL-21), we show how AlphaSeq measures cytokine-receptor interactions and identifies cytokine variants with a broad range of affinities. A saturated mutational library was created for IFNα and IL-21and subsequently screened against a second library consisting of human IFNAR2 or human IL-21R, species orthologs and off-target receptors. AlphaSeq enabled identification of hundreds of detuned IFNα and IL-21 variants against both human and mouse receptors in parallel assays. Cytokine variants with lower affinity than parental IFNα or IL-21 were recombinantly expressed as Fc fusion proteins to orthogonally measure affinity with biolayer interferometry and characterize potency with an in vitro human PBMC phosflow assay, which showed strong correlation with AlphaSeq affinity measurements. Finally, detuned cytokine candidates were fused to anti-CD8 and other localizing antibodies to demonstrate cell population-specific signaling. Candidate molecules showed 1000-fold or greater potency in the targeted cell population than non-targeted populations. Our results show the AlphaSeq platform can accurately quantitate thousands of cytokine variant affinities simultaneously against multiple relevant receptors, enabling the selection of candidate immunocytokine antibody fusion proteins with targeted cell biased signaling. AlphaSeq’s rapid, comprehensive affinity determination is being used to develop clinically relevant IFNα and IL-21 therapeutic immunocytokines with accompanying preclinical data. Citation Format: Charles Lin, Colleen Shikany, Leah Homad, Jessica Fint, Ruchi Bansal, Davis Goodnight, Jeff Adamo, Danielle Van Citters, Ryan Swanson, Randolph Lopez. Selective IL-21 and IFN-alpha immunocytokines engineered using the AlphaSeq platform [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 2477.

Abstract 2046: Targeting cellular models of glioblastoma versus non-target and off-target cell populations using U87-MG membrane lipid-modified nanoliposomes

Abstract Background: Glioblastoma Multiform (GBM) is an aggressive and malignant form of brain cancer known for its invasiveness and significant heterogenicity. The majority of GBM cases are linked to genetic mutations (ie. TP53, EGFR, and PTEN), which disrupt the normal regulation of cell growth and division, resulting in uncontrolled cell proliferation. The utilization of nanoparticles presents a promising approach for addressing the complexities associated with selective drug delivery to GMB and treatment. The purpose of this study was to investigate the effect of including U87-MG membrane lipid extracts in nanoliposomes using target, non-target, and normal healthy cellular populations in vitro. Methods: The nanoliposomes employed for the experiments included various ratios of U87-MG lipid extracts (lipids derived from U87-MG cells), DOPC, Cholesterol, and DPPE-Rhodamine for fluorescence studies. The particle size and zeta potential values were measured and monitored throughout for consistency. Fluorescence intensity values (arbitrary units) were used to assess extent of cell binding, and uptake at the various time points. In vitro studies were employed to evaluate the cellular uptake activity of U87-MG-modified nanoliposome preparations using mixed ratios and compositions of lipid materials. Cell lines employed were, LN-18 (human glioma), T98G (human glioma), U87-MG (human glioma), 4TI (murine breast), and HBEC-5i (normal healthy) cells. Results: The average size for the nanoliposomes (with and without lipid extract) fell within the range of 110 to 220 nm, while the zeta potential for the CLENs was negatively-charged. In vitro studies revealed that the inclusion of U87MG lipid extract in nanoliposomes increased cellular uptake in glioma cells when compared to control. Current investigations focus on different microenvironments, including non-target and off target cell populations. The findings from the cellular uptake analyses to date suggest that the in vitro cellular uptake of the CLENs varied in accordance with the total lipid extract content for all cell lines. Citation Format: Kaylee Janton, Duha Baamir, Aesha Patel, Sheren Boco, Robert Campbell. Targeting cellular models of glioblastoma versus non-target and off-target cell populations using U87-MG membrane lipid-modified nanoliposomes [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 2046.

Abstract 4708: Potency and selectivity of ERBB2-targeting antibody drug conjugates in vitro

Abstract Previously we have reported on the relevance of the joint ProLiFiler and Cancer DataMiner platforms when studying the antitumor efficacy of novel antibody-drug conjugates (ADCs), such as Sacituzumab govitecan. In this study, we use our platforms to investigate and compare the selectivity of two FDA-approved Trastuzumab-based ADCs targeting ERBB2: Kadcyla, the first-in-class ADC employing a microtubule inhibitor bound via a non-cleavable linker, and Enhertu, which combines a topoisomerase inhibitor with a protease-cleavable linker. We performed a cell proliferation and survival assay using the ProLiFilerTM (Reaction Biology) to characterize the in vitro antitumor effects of Enhertu, Kadcyla, and their individual cytotoxins, deruxtecan &amp; mertansine on 160 human cancer cell lines (CLs). The resulting data were uploaded to 4HF´s Cancer DataMiner platform for data analysis. First, we confirmed high potency and limited selectivity of mertansine, as it displayed an IC50 &amp;lt; 250 nM (mean: 27.3 nM) in 95% of cell lines (CLs). A COMPARE analysis confirmed that mertansine data correlated best with those of other microtubule inhibitors in our drug databases (e.g., MMAE, Spearman rho = 0.79, p=9.6E-27). Conversely, a paired analysis showed that CLs were less sensitive to Kadcyla than to mertansine except for CLs overexpressing ERBB2. The 160-CL panel included a total of nine CLs (six mammary, two gastric, one ovarian) with strong ERBB2 overexpression (Affymetrix data &amp;gt;10), mostly associated with ERBB2 gene amplification; seven of them were the most sensitive CLs to Kadcyla (IC50 &amp;lt; 6 nM). Focusing solely on breast cancer cell lines (breast cancer is the approved indication for Kadcyla), we observed a strong correlation between sensitivity to Kadcyla and ERBB2 expression (Spearman r = -0.78, p = 0.003). Interestingly, in line with published data, the ERBB2-overexpressing cell lines SNU-216 (stomach cancer) and JIM-T1 (breast cancer) were less sensitive to Kadcyla. In non-ERBB2-amplified CLs, Kadcyla sensitivity did not correlate with ERBB2 expression levels. Focusing on ERBB2-negative CLs such as hematological CLs, we unexpectedly observed consistent cell inhibition (mean IC50 =33 nM) with Kadcyla, suggesting that the ADC was cleaved, and the payload released into the medium. Overall, this study has demonstrated the relevance of our platforms to study and compare newly developed ADCs. Cytotoxic activities recorded for conjugates and free payloads, along with molecular data for the test cell lines, provides insight in the mode of action of conjugates and their target cell populations and hence can support the development of ADCs. Investigations are underway with Enhertu and its payload deruxtecan, and results will be compared with those obtained for Kadcyla and mertansine. Citation Format: Anne-Lise Peille, Kaja Holtorf, Pablo Anton Garcia, Nadine Obier, Daniel Feger, Thomas Metz, Sebastian Dempe, Heinz-Herbert Fiebig, Jan Ehlert, Vincent Vuaroqueaux. Potency and selectivity of ERBB2-targeting antibody drug conjugates in vitro [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 4708.

Abstract 3196: Overcoming lung cancer drug resistance: Enhanced cellular delivery using lipid-extracted nanoliposomes

Abstract Background: Lung cancer causes substantial cancer deaths in the U.S. Drug resistance remains a major issue. Nanoliposomes demonstrate enhanced drug targeting and resistance-overcoming potential. Incorporating lipid extracts derived from target cells shows improved delivery over conventional nanoliposomes. This study examined the effects of lung cancer cell lipid extract nanoliposomes against target and non-target cell populations from different environments. Method: Cellular Membrane Lipid-Extracted Nanoliposomes (CLENs) were fabricated with DOPC, DOTAP, ChaGo-K-1 lipid extract, cholesterol, PEG5000, and rhodamine fluorescent label was incorporated for uptake studies. The liposome size was reduced by probe sonication. Particle size and polydispersity index (PDI) were measured by dynamic light scattering. Zeta potential was quantified by laser Doppler micro-electrophoresis. Four cell lines were utilized: ChaGo-K-1 NSCLC target, A549 pulmonary microenvironment control, 4T1 extra-pulmonary control, and normal lung fibroblasts off-target control. All cell lines were cultured in 48-well plates at a seeding density of 10,000 cells/mL and incubated for 24 hours. Freshly prepared CLENs were added to all cell lines and incubated for an additional 24 hours. Cells were washed with PBS, and fluorescence intensity was then quantified and qualified by a microplate reader to measure the cellular uptake of the CLENs. Results: The mean particle size and PDI of the ChaGo-K-1 CLENs were 117 ± 0.7 nm and 0.216 ±0.009, compared to 126 ± 0.5 nm and 0.195 ± 0.008 for the control, respectively. Both formulations were within the optimal size range for drug delivery and tumor cell penetration. The zeta potential of the ChaGo-K-1 CLENs was -1.75 ± 1.81 mV compared to -1.74 ± 6 mV for the control. Quantification and qualification of cellular uptake using fluorescence intensity measurements revealed a 3.2-fold increase in ChaGo-K-1 CLENs compared to conventional liposomes without ChaGo-K-1 lipid extracts. This demonstrates that the inclusion of the lipid extracts in the CLEN significantly enhanced selectivity and delivery to target cancer cell populations. Conclusion: Higher uptake occurred with target cells using CLENs with lipid extracts versus standard liposomes. NSCLC-derived CLENs show potential to improve selective delivery and overcome drug resistance. Further, in vivo studies evaluating selective NSCLC tumor accumulation and efficacy enhancement are warranted. Citation Format: Mohammed Alqaryan, Rashad Allahyani, Dayeoun Lee, Robert B. Campbell. Overcoming lung cancer drug resistance: Enhanced cellular delivery using lipid-extracted nanoliposomes [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 3196.

Abstract 4010: Assessing impacts of tumor burden and CAR-T cell dosage on the toxicity and efficacy profile of CD19 CAR-T therapy in a PBMC-humanized mouse model

Abstract Chimeric antigen receptor T-cell (CAR-T) therapy is a significant advancement in treating hematological malignancies, yet it faces challenges due to its variable therapeutic responses and the risk of severe toxicities. This study delves into the effects of tumor burden and CAR-T cell doses on the toxicity profile of the therapy, employing a PBMC-humanized mouse model engrafted with luciferase-labeled Raji B cell lymphoma (Raji-luc). By exploring high vs. low tumor burden and high vs. low CAR-T dose scenarios, we aim to gain insights into the dynamic relationship between these factors and CAR-T-induced toxicity and efficacy. In the first experiment, we established PBMC humanized mouse models with a high tumor burden and a low tumor burden. 10 days (high burden) or 7 days (low burden) after the Raji-luc inoculation, mice were engrafted with 10M human PBMCs and dosed with CD19 autologous CAR-Ts. In mice with low tumor burden, CAR-T treatment demonstrated significant efficacy, as evidenced by IVIS imaging, while the high tumor burden model exhibited reduced efficacy. CAR-T treatment in Raji-bearing mice did not induce weight loss in either high or low burden models. CD19 CAR-T cells effectively reduced the target cell population in peripheral blood, with greater expansion observed in the higher tumor burden model. We also evaluated human cytokine levels post-CAR-T treatment, revealing higher cytokine induction in the high tumor burden model, peaking at 2 days post-treatment. Secondly, in a CAR-T dose-response study, we treated PBMC-humanized Raji-bearing mice with 10M, 15M, and 20M CAR-T cells. Higher CAR-T cell doses (15M and 20M) resulted in observable toxicity measured by body weight loss, while the 10M dose did not. All CAR-T doses effectively slowed tumor growth and induced significant CAR-T cell expansion. Selected human cytokines, such as IL-5 and RANTES, demonstrated a dose-response correlation with CAR-T treatment. Additionally, IL-6 and IL-10 were significantly correlated with tumor burden rather than CAR-T doses. The differential cytokine responses observed in our study provide valuable insights into the utility of the PBMC-humanized mouse model for investigating the biological responses associated with CD19 CAR-T therapy. Our findings underscore the utility of the PBMC-humanized mouse model in assessing variability in toxicity and cytokine responses to CAR-T therapy. This model offers valuable insights into the factors influencing CAR-T treatment outcomes and provides a platform for planning more precise treatment and enhancing the safety and efficacy of CAR-T therapy. Citation Format: Won Lee, Destanie Rose, James G. Keck, Jiwon Yang. Assessing impacts of tumor burden and CAR-T cell dosage on the toxicity and efficacy profile of CD19 CAR-T therapy in a PBMC-humanized mouse model [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 4010.

Abstract 1171: Estimating sensitivity of exploratory cellular biomarkers in oncology clinical trials

Abstract Background: In the prolonged and complex process of assessing immunotherapeutic oncology drug candidates, flow cytometry plays an essential role. In oncology studies, biomarker measurement requires robust and reliable flow cytometric assays. A crucial element of these assays is to understand the lower limits of measurable surface biomarkers. Methods: We incorporated into the development and validation of flow cytometry assays the measurement of the lower limit of quantitation (LLOQ). The LLOQ is the lowest number of a reportable parameter in a blood or bone marrow study sample that can be quantitatively determined with acceptable precision and accuracy. Two steps are necessary to determine the LLOQ. First in these assessments a measurable range of lower limits of a particular cell type or receptor is estimated. That is, in validating an assay LLOQ, there are three options to select cells expressing target surface biomarkers of interest: using cultured or bioengineered cells, using samples of known high expression of a biomarker, or using pre-titered positive samples. Then decreasing concentrations of the target cell or receptor may be used to precisely determine the LLOQ of a specific assay reportable parameter. Results: By acquiring at least ten negative control sample results, the limit of the blank (LOB) and limit of detection (LOD) of a biomarker is determined. LOB is calculated as mean +1.645 SD, and LOD as mean +3 SD. The negative control samples either lack the fluorescent monoclonal antibodies against the markers of interest or lack the target cell population. Different reportable parameters, detected by one or more biomarkers have varying LLOQ. The LLOQ for a reportable parameter is a precise measurement of a parameter above or equal to the LOD. We found that typical LLOQ values are from 4 to 145 positive cells, with a frequency of 0.01 to 0.1% among a parent population. For example, in the case of tetramer assays, flow cytometry can reliably detect as few as 25 CD8+ antigen-specific cells among three million lymphocytes. Conclusions: LLOQ determinations for exploratory flow cytometry biomarkers are required for clinical research in cancer immunotherapy and cancer vaccine development. This valuable information allows for interpretation of data at the low end of a measurable range. Citation Format: Chengsen Xue, Christina D. Swenson, Thomas W. Mc Closkey. Estimating sensitivity of exploratory cellular biomarkers in oncology clinical trials [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 1171.

The role of tissue-resident memory T cells as mediators for response and toxicity in immunotherapy-treated melanoma-two sides of the same coin?

Tissue-resident memory T cells (TRM cells) have become an interesting subject of study for antitumor immunity in melanoma and other solid tumors. In the initial phases of antitumor immunity, they maintain an immune equilibrium and protect against challenges with tumor cells and the formation of primary melanomas. In metastatic settings, they are a prime target cell population for immune checkpoint inhibition (ICI) because they highly express inhibitory checkpoint molecules such as PD-1, CTLA-4, or LAG-3. Once melanoma patients are treated with ICI, TRM cells residing in the tumor are reactivated and expand. Tumor killing is achieved by secreting effector molecules such as IFN-γ. However, off-target effects are also observed. Immune-related adverse events, such as those affecting barrier organs like the skin, can be mediated by ICI-induced TRM cells. Therefore, a detailed understanding of this memory T-cell type is obligatory to better guide and improve immunotherapy regimens.