Competitive Assay Research Articles

Preanalytical, Analytical and Postanalytical Analyses on Corynebacterium spp. and Actinomycetaceae in Urine Samples of Patients with Suspected Urinary Tract Infection-A Hypothesis-Forming Observational Study.

A hypothesis-forming exploratory cross-sectional assessment was conducted to assess the occurrence and relevance of Gram-positive rod-shaped bacteria like Corynebacterium spp. and Actinomycetaceae in human urine samples. In total, 1170 urine samples from 1031 inpatients with suspected urinary tract infection were assessed for culture-based growth of Gram-positive rod-shaped bacteria applying API Coryne assays, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and in-house 16S rRNA gene sequencing. Overall, 502 different bacterial colonies from 346 urine samples taken from 324 inpatients were observed. The three quantitatively most abundant genera or genus clusters were Corynebacterium (254 isolates, 62%), Actinomyces/Winkia (79 isolates, 19%), and Actinotignum/Actinobaculum (29 isolates, 7%). Compared to sequencing, the diagnostic accuracy of all assessed competitor assays from the diagnostic routine was <80% for differentiation on the genus level and <30% for differentiation on the species level. Prolongated incubation for 4 days compared to 2 days resulted in additional detection of 15% of the totally recorded Gram-positive rod-shaped bacteria. An approximately 5-fold increased detection rate in mid-stream urine compared to urine acquired applying alternative sampling strategies was observed. In conclusion, in the rare event of the suspected clinical relevance of such findings, confirmatory testing with invasively sampled urine should be considered due to the high contamination rate observed in mid-stream urine. Confirmatory testing by DNA-sequencing methods should be considered if an exact identification of genus or species is regarded as relevant for the individual choice of the therapeutic strategy.

An automated fluorescent immunoassay for on-site screening of AFM1 in raw milk at the ppt level

In the dairy industry, the quality of raw milk as it is collected from dairy farmers must be strictly ensured. Therefore, when on-site screening of typical carcinogens in raw milk, this technology must simultaneously be convenient to operate and highly sensitive. Here, an automated and fluorescent immunoassay system for screening trace amounts of aflatoxin M1 (AFM1) in raw milk was developed. The whole immunoassay procedure can be processed in a true “sample-to-results” paradigm, avoiding the tedious manual operation of the traditional indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method. In addition, we designed an integrated fluorescent spectrometer that can quantitatively measure fluorescent signals with high sensitivity. The automated and fluorescent immunoassay system can screen AFM1 in raw milk samples with an ultra low limit of detection (4.7 pg/mL in raw milk). The half maximal inhibitory concentration (IC50) was 8.3 pg/mL, which is approximately 4-fold lower than that of traditional ic-ELISA. When the system was applied to screen actual raw milk samples, the recovery rates ranged from 79.4% to 104.6%, with a coefficient of variation ranging from 8.9% to 15.2%. Overall, we believe that the automated and fluorescent immunoassay system can provide a one-stop solution that is user-friendly and highly sensitive for screening trace AFM1 contaminants in raw milk.

Diagnostic performance of a rapid immunochromatographic test for the simultaneous detection of antibodies to Theileria equi and Babesia caballi in horses and donkeys

BackgroundEquine piroplasmosis is caused by two tick-borne protozoan parasites, Theileria equi and Babesia caballi,, which are clinically relevant in susceptible horses, donkeys, and mules. Moreover, equine piroplasmosis significantly constrains international trading and equestrian events. Rapidly diagnosing both parasites in carrier animals is essential for implementing effective control measures. Here, a rapid immunochromatographic test for the simultaneous detection of antibodies to T. equi and B. caballi was evaluated using samples from horses and donkeys collected in Greece, Israel, and Italy. The results were compared with an improved competitive enzyme-linked immunosorbent assay (cELISA) for detecting antibodies to both parasites using the same panel of samples.MethodsBlood samples were collected from 255 horses and donkeys. The panel consisted of 129 horses sampled at four locations in northern Greece, 105 donkeys sampled at four locations in Sicily, and 21 horses sampled at two locations in Israel. The rapid test and the cELISA were performed according to the manufacturer’s instructions, and the results were subjected to a statistical analysis to determine the sensitivity and specificity of both tests and their association.ResultsThe immunochromatographic test provided a result within 15 min and can be performed in the field, detecting both pathogens simultaneously. The overall coincidence rate between the rapid test and the cELISA for detecting antibodies against T. equi was 93% and 92.9% for B. caballi. The rapid test’s sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for T. equi were above 91.5%. Sixteen samples were positive for both parasites in the rapid test and eight in the cELISA. Either test had no significant association between T. equi and B. caballi detection. The detection rates of both parasites were significantly higher in Italy than in Greece or Israel and in donkeys than in horses. The agreement for T. equi between the results of both tests was high in Greece (93.8%) and Italy (95.2%) and moderate in Israel (76.2%). For B. caballi, the specificity and NPV of the rapid test were high (94.2% and 98.3%, respectively), although the sensitivity and PPV were moderate (69.2% and 39.1%, respectively) due to the small sample size. However, for B. caballi, the sensitivity was higher with the rapid test.ConclusionsThe rapid test detected T. equi and B. caballi simultaneously in the field, potentially replacing laborious cELISA testing and is recommended for import/export purposes. The test can also be helpful for the differential diagnosis of clinical cases, since seropositivity may rule out equine piroplasmosis since it does not indicate current or active infection.Graphical

Gigantol ameliorates DSS-induced colitis via suppressing β2 integrin mediated adhesion and chemotaxis of macrophage

Ethnopharmacological relevanceDendrobium, recognized as “Shihu” in traditional Chinese medicine, holds a rich history of medicinal utilization documented in the Chinese Pharmacopoeia. Ancient texts like “Shen Nong Ben Cao Jing” extol Dendrobium's virtues as a superior herbal medicine fortifying “Yin” and invigorating the five viscera. Dendrobium is extensively employed for the treatment of gastrointestinal inflammatory disorders, showcasing significant therapeutic efficacy, particularly against ulcerative colitis (UC), within the realm of Chinese ethnopharmacology. Dendrobium plays crucial pharmacological roles due to its rich content of polysaccharides, alkaloids, phenanthrenes, and bibenzyls. Gigantol, a prominent bibenzyl compound, stands out as one of the most vital active constituents within Dendrobium, the gigantol content of Dendrobium leaves can reach approximately 4.79 μg/g. Its significance lies in being recognized as a noteworthy anti-inflammatory compound derived from Dendrobium. Aim of the studyGiven the pivotal role of gigantol as a primary active substance in Dendrobium, the therapeutic potential of gigantol for gastrointestinal diseases remains enigmatic. Our present investigation aimed to evaluate the therapeutic effects of gigantol on dextran sulfate sodium (DSS)-induced colitis and reveal its potential mechanism in countering UC activity. Materials and methodsThe protective efficacy of gigantol against colitis was assessed by examining the histopathological changes and conducting biochemical analyses of colon from DSS-challenged mice. Assessments focused on gigantol's impact on improving the intestinal epithelial barrier and its anti-inflammatory effects in colonic tissues of colitis mice. Investigative techniques included the exploration of the macrophage inflammatory signaling pathway via qPCR and Western blot analyses. In vitro studies scrutinized macrophage adhesion, migration, and chemotaxis utilizing transwell and Zigmond chambers. Furthermore, F-actin and Rac1 activation assays detailed cellular cytoskeletal remodeling. The potential therapeutic target of gigantol was identified and validated through protein binding analysis, competitive enzyme-linked immunosorbent assay (ELISA), cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assay. The binding sites between gigantol and its target were predicted via molecular docking. ResultsGigantol ameliorated symptoms of DSS-induced colitis, rectified damage to the intestinal barrier, and suppressed the production of pro-inflammatory cytokines in colonic tissues. Intriguingly, gigantol significantly curtailed NF-κB signaling activation in the colons of DSS-induced colitis mice. Notably, gigantol impaired the β2 integrin-dependent adhesion and migratory capacity of RAW264.7 cells. Moreover, gigantol notably influenced the cytoskeleton remodeling of RAW264.7 cells by suppressing Vav1 phosphorylation and Rac1 activation. Mechanistically, gigantol interacted with β2 integrin, subsequently diminishing binding affinity with intercellular adhesion molecule-1 (ICAM-1). ConclusionsIn conclusion, these findings elucidate that gigantol ameliorates DSS-induced colitis by antagonizing β2 integrin-mediated macrophage adhesion, migration, and chemotaxis, thus it may impede macrophage recruitment and infiltration into colonic tissues. This study suggests that gigantol shows promise as a viable candidate for clinical colitis therapy.

ADVANCING PARKINSON’S DISEASE DETECTION WITH FLISA: A NOVEL APPROACH TARGETING MISFOLDED ALPHA-SYNUCLEIN

A Fluorescence-Linked Immunosorbent Assay (FLISA) targeting β-cross-sheet α-synuclein protein, a crucial marker in Parkinson’s Disease (PD), was assessed using in vitro assay with a rotenone-induced PD cellular model. The study aimed to evaluate FLISA’s applicability with the inhouse developed labelled polyclonal antibody (pAb). Results are promising the competitive assay successfully distinguished misfolded α-synuclein in SH-SY5Y cells. Quantitative analysis revealed a direct correlation between competitive antigen concentration and decreased fluorescence, showcasing FLISA’s sensitivity. Controls exhibited maximal fluorescence, confirming the absence of misfolded proteins, while rotenone exposed cells displayed reduced fluorescence, suggesting their presence. This approach enhances PD understanding and supports potential interventions.

Evaluation of a Commercial Serum Competitive Enzyme-Linked Immunosorbent Assay for Detection of Neospora caninum-Specific Antibodies in Raw Milk of Ruminants

Bovine neosporosis is an infection caused by the protozoan parasite Neospora caninum and has substantial veterinary hazards. Neosporosis cannot be controlled by vaccination or chemotherapy. Thus, accurate diagnosis followed by isolation and culling of infected animals is regarded as the most efficient method of control. In vivo diagnosis often relies on serologic testing of the animals, and milk represents a non-invasive and easy-to-collect sample matrix. However, indirect enzyme-linked immunosorbent assay (ELISA) specifically designed for antibody detection in milk are sometimes not easily available and it is tempting to use ELISA kits that are originally designed for use in serum in milk samples instead. Herein, we evaluated a widely used commercial ELISA (ID Screen® Neospora caninum competition Multispecies ELISA (ID. Vet, Grabels, France)), developed for detection of N. caninum antibodies in serum samples, for its performance on milk samples. Milk samples from dairy ruminants (cows, buffaloes, sheep, and goats; n = 149) were tested in parallel with the serum ELISA and a commercial milk ELISA as a standard test (Neospora caninum Milk Competitive ELISA, ID. Vet, Grabels, France). The detected prevalence values were 28.2% (42/149), 17.4% (26/149), and 17.4% (26/149) using milk ELISA, serum ELISA, and both ELISAs, respectively. Sensitivity, specificity, positive predictive value, and negative predictive value for the serum ELISA used with milk samples were 61.9%, 100%, 100%, and 87%, respectively. The agreement and kappa value between the two ELISAs were 89.3% and 0.70, respectively, suggesting substantial agreement. High values of Pearson correlation coefficient (0.904, p ≥ 0.0001) and area under the receiver operating characteristic (ROC) curve (0.789, p ≥ 0.0001) demonstrated the high diagnostic performance of the serum ELISA in milk samples. Also, a Bland–Altman Plot and histogram describing the frequency of distribution of ELISA optical densities confirmed the high agreement of both serum and milk ELISAs. The current results revealed the high specificity but moderate sensitivity of the serum ELISA used for milk samples compared with the milk ELISA. However, the excellent positive predictive value of the serum ELISA makes it an alternative option in case of the unavailability of milk ELISAs. With this study, we provided additional evidence that a widely used serum ELISA test kit may also be used for the detection of N. caninum antibodies in milk samples.

Structural and kinetic analysis of the monofunctional Staphylococcus aureus PBP1

Staphylococcus aureus, an ESKAPE pathogen, is a major clinical concern due to its pathogenicity and manifold antimicrobial resistance mechanisms. The commonly used β-lactam antibiotics target bacterial penicillin-binding proteins (PBPs) and inhibit crosslinking of peptidoglycan strands that comprise the bacterial cell wall mesh, initiating a cascade of effects leading to bacterial cell death. S. aureus PBP1 is involved in synthesis of the bacterial cell wall during division and its presence is essential for survival of both antibiotic susceptible and resistant S. aureus strains. Here, we present X-ray crystallographic data for S. aureus PBP1 in its apo form as well as acyl-enzyme structures with distinct classes of β-lactam antibiotics representing the penicillins, carbapenems, and cephalosporins, respectively: oxacillin, ertapenem and cephalexin. Our structural data suggest that the PBP1 active site is readily accessible for substrate, with little conformational change in key structural elements required for its covalent acylation of β-lactam inhibitors. Stopped-flow kinetic analysis and gel-based competition assays support the structural observations, with even the weakest performing β-lactams still having comparatively high acylation rates and affinities for PBP1. Our structural and kinetic analysis sheds insight into the ligand–PBP interactions that drive antibiotic efficacy against these historically useful antimicrobial targets and expands on current knowledge for future drug design and treatment of S. aureus infections.

Brucellosis in camel, small ruminants, and Somali pastoralists in Eastern Ethiopia: a One Health approach.

Brucellosis is a neglected bacterial zoonotic disease with economic and public health importance in pastoral communities of sub-Saharan Africa. A cross-sectional study was conducted from December 2021 to April 2022, to estimate the prevalence and identify the associated risk factors causing brucellosis in animals and associated with occupational diseases in humans from three selected districts of "the Somali Pastoral region," Eastern Ethiopia. In this study, 1,000 serum samples were screened for anti-Brucella spp. antibodies using Rose Bengal Plate Test (RBPT) and further confirmed using a competitive enzyme-linked immunosorbent assay (cELISA). A structured questionnaire was used to collect the biodata of tested animals and animal attendants to test the association between explanatory and outcome variables. The overall animal level prevalence was 5% (95% CI, 6.1-7.2.0) in small ruminants, 2.9% (95% CI, 1.5-4.9) in camels, and 2.0% (95% CI, 0.2-3.7) in occupationally linked humans. Herd size and herd history of retained fetal membranes were risk factors associated with Brucella spp. seropositivity in animals (p < 0.05). Disposing of retained fetal membranes was significantly associated (p < 0.05) with Brucella spp. seropositivity in humans. Evidence of brucellosis in various livestock species and associated seropositivity in humans indicates the need for a coordinated One Health approach, considering sociocultural dynamics of pastoral communities in controlling brucellosis to safe guard public health and increase livestock productivity.

PEDF-derived peptide protects against Amyloid-β toxicity in vitro and prevents retinal dysfunction in rats

Amyloid-beta (Aβ), a family of aggregation-prone and neurotoxic peptides, has been implicated in the pathophysiology of age-related macular degeneration (AMD). We have previously shown that oligomeric and fibrillar species of Aβ42 exerted retinal toxicity in rats, but while the consequences of exposure to amyloid were related to intracellular effects, the mechanism of Aβ42 internalization in the retina is not well characterized. In the brain, the 67 kDa laminin receptor (67LR) participates in Aβ-related neuronal cell death. A short peptide derived from pigment epithelium-derived factor (PEDF), formerly designated PEDF-335, was found to mitigate experimental models of ischemic retinopathy via targeting of 67LR. In the present study, we hypothesized that 67LR mediates the uptake of pathogenic Aβ42 assemblies in the retina, and that targeting of this receptor by PEDF-335 may limit the internalization of Aβ, thereby ameliorating its retinotoxicity. To test this assumption ARPE-19 cells in culture were incubated with PEDF-335 before treatment with fibrillar or oligomeric structures of Aβ42. Immunostaining confirmed that PEDF-335 treatment substantially prevented amyloid internalization into ARPE-19 cells and maintained their viability in the presence of toxic oligomeric and fibrillar Aβ42 entities in vitro. FRET competition assay was performed and confirmed the binding of PEDF-335 to 67LR in RPE-like cells. Wild-type rats were treated with intravitreal PEDF-335 in the experimental eye 2 days prior to administration of retinotoxic Aβ42 oligomers or fibrils to both eyes. Retinal function was assessed by electroretinography through 6 weeks post injection. The ERG responses in rats treated with oligomeric or fibrillar Aβ42 assemblies were near-normal in eyes previously treated with intravitreal PEDF-335, whereas those measured in the control eyes treated with injection of the Aβ42 assemblies alone showed pathologic attenuation of the retinal function through 6 weeks. The retinal presence of 67LR was determined ex vivo by immunostaining and western blotting. Retinal staining demonstrated the constitutional expression of 67LR mainly in the retinal nuclear layers. In the presence of Aβ42, the levels of 67LR were increased, although its retinal distribution remained largely unaltered. In contrast, no apparent differences in the retinal expression level of 67LR were noted following exposure to PEDF-335 alone, and its pattern of localization in the retina remained similarly concentrated primarily in the inner and outer nuclear layers. In summary, we found that PEDF-335 confers protection against Aβ42-mediated retinal toxicity, with significant effects noted in cells as well as in vivo in rats. The effects of PEDF-335 in the retina are potentially mediated via binding to 67LR and by at least partial inhibition of Aβ42 internalization. These results suggest that PEDF-335 may merit further consideration in the development of targeted inhibition of amyloid-related toxicity in the retina. More broadly, our observations provide evidence on the importance of extracellular versus intracellular Aβ42 in the retina and suggest concepts on the molecular mechanism of Aβ retinal pathogenicity.

Abstract 4659: A genome-wide CRISPR screen identifies ARID1A as a potential resistance marker of IDH1 inhibitor in IDH1-mutant cholangiocarcinoma cell

Abstract Background: Isocitrate dehydrogenase 1 (IDH1) mutations have been identified in approximately 20% of intrahepatic cholangiocarcinoma cases. These predominant gain-of-function mutations at codon 132 lead to abnormal production of the oncometabolite D-2-hydroxyglutarate. Targeted pharmacological disruption of the IDH1 mutation demonstrates tumor growth inhibition in IDH1-mutated cholangiocarcinoma. However, the efficacy of an IDH1 inhibitor (AG-120) as monotherapy for IDH1-mutated cholangiocarcinoma has been proven clinically insufficient, suggesting a need for investigating predictive biomarkers. In this study, we employed a genome-wide CRISPR screen to pursue resistance mechanisms to pharmacological IDH1 inhibition. Methods: The screening utilized SNU1079 cells harboring the IDH1 R132C mutation and the Brunello genome-wide CRISPR knockout (KO) pooled library (Addgene #73178, Broad Institute GPP, 4 guides/gene). The read couns of sgRNAs were normalized to reads per million, and then log2 was transformed. The log fold change (LFC) of each sgRNA of the AG-120-treated group was determined relative to that of the DMSO-treated group. Hypergeometric distribution was used to determine statistical significance. P values per gene were calculated as the average -log10(P value) of individual sgRNAs. We validated the CRISPR screening outcomes in vitro using both IDH1R132C mutant (SNU1079) and IDH1R132S mutant (RBE) cells by WST-1 assays, clonogenic assay and competition assay. (Tsujino T et al. Nat Commun. 2023) In competition assay, GFP was constitutively expressed in ARID1A-knockout cells and cultured together with ARID1A intact cells, under treatment with 1 µM AG120 or DMSO for 21 days. Relative cell numbers of GFP-positive (ARID1A-KO) cells in comparison with GFP-negative (ARID1A intact) cells were quantified using a BD LSRFortessa Analyzer (BD Biosciences). Results: Based on a volcano plot analysis, we identified three genes (SMCHD1, ARID1A, and PGP) with LFC &amp;gt; 1.5. Following a recent report of frequent ARID1A alteration (20%; 84/412) in a clinical study (Boerner, et al. Hepatology. 2021), we focused our research on ARID1A (LFC = 1.531; −log10 P = 3.75). WST-1 assays and clonogenic assays as short-term (3-7 days) assays failed to validate the results of CRISPR screening; however, in competition assays under treatment with AG120, cells with ARID1A-KO exhibited significantly greater proliferation in comparison with ARID1A intact cells in two different cell lines (SNU1079, RBE). Conclusions: The results demonstrated that ARID1A disruption reduced the efficacy of the IDH1 inhibitor against IDH1-mutatedcholangiocarcinoma cells. Based on these findings, we are planning comprehensive analyses employing in vivo models and clinical data to substantiate these observations. Citation Format: Tomohiro Kondo, Osamu Kikuchi, Yafeng Wang, Yukie Nakai, Tomomi Ida, Yang Cao, Trang H. Vu, Yuki Kondo, Tomohiko Sunami, Yoshihiro Yamamoto, Tomoki Saito, Shigeki Kataoka, Atsushi Yamada, Masashi Kanai, Shinya Ohashi, Manabu Muto. A genome-wide CRISPR screen identifies ARID1A as a potential resistance marker of IDH1 inhibitor in IDH1-mutant cholangiocarcinoma cell [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 4659.

Abstract 7141: The novel microtubule-destabilizing compound AUS_001 maintains unique binding to the colchicine site of tubulin and elicits reversible cellular effects relative to other anti-tubulin agents

Abstract Our previous work showed that the prenylated hydroxy-stilbene AUS_001 exerts a strong safety profile and potent anti-proliferative responses, as assessed in 30 types of cancer cell lines, stemming from its microtubule destabilization activity. The aim of the current study was to delineate the molecular mechanism of microtubule destabilization by AUS_001 and to provide insight into its high potency and low toxicity. Surface plasmon resonance (SPR) experiments revealed the direct interaction of AUS_001 with tubulin with an apparent equilibrium dissociation constant, Kd, of 2.13 × 10−5M. Size exclusion competition assays and an X-ray crystal structure of the tubulin-AUS_001 complex at 2.1 Å resolution establish ligand binding within the colchicine site on tubulin. Since colchicine-site ligands are well known to inhibit the curved-to-straight conformational transition of tubulin, which is an essential process for microtubule formation, these results readily explain the microtubule-destabilizing activity of the compound. Notably, while both combretastatin A4 (CA-4) and AUS_001 are both colchicine-site stilbene ligands, CA-4 binds in the cis configuration, while AUS_001 maintains a trans configuration leading to a substantially different pocket engagement. Comparison of the tubulin-AUS_001 complex structure with the ones of tubulin-colchicine and tubulin-plinabulin revealed that AUS_001 elicits different rearrangements in the T7 loop of β-tubulin versus colchicine and occupies a different zone of the pocket relative to plinabulin, further elaborating its unique binding properties. Interestingly, AUS_001 exhibits a higher relative affinity than colchicine itself, as assessed by i) an ultrafiltration-based tubulin-ligand release assay and ii) a radioligand colchicine competition assay. However, fitting steady state SPR data for AUS_001 binding to tubulin revealed fast association and dissociation rates and was indicative of reversible binding. The latter finding is corroborated by AUS_001-triggered reverse biological effects including morphological alterations, cell cycle G2/M arrest, and by a decrease of cell viability upon discontinuation of the drug treatment in glioma and pancreatic cancer cells. Conversely, colchicine, CA-4 or paclitaxel administered at the same doses as those applied for AUS_001 failed to reverse the drug-induced phenotypes upon removal resulting in sustained cytotoxicity. In conclusion, we fully characterized the unique binding mode of AUS_001 to the colchicine site of tubulin and elucidated the reversible nature of the target engagement. AUS_001 features a distinctive and reversible molecular interaction with tubulin which provides a plausible explanation for its favorable safety profile compared to other microtubule targeting agents. Citation Format: Herman Lelie, Yao-Chieh Chou, Alastair J. King, Zlata Boiarska, Andrea E. Prota, Michel O. Steinmetz, Marina Koutsioumpa. The novel microtubule-destabilizing compound AUS_001 maintains unique binding to the colchicine site of tubulin and elicits reversible cellular effects relative to other anti-tubulin agents [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 7141.

Abstract 6577: Chemoproteomic evaluation of PROTACs targeting estrogen receptor alpha for ER+ breast cancer

Abstract In our investigation of estrogen receptor (ER) targeted protein degraders for advanced ER+ breast cancer, we assessed three proteolysis targeting chimeras (PROTACs), ARV471, ERD308, and ERD3111. Global proteomic analysis unveiled off-target protein degradation of progesterone receptor (PR) and phosphodiesterase 6D (PDE6D) by ER targeted PROTACs, but not selective ER degraders (SERDs). PR and PDE6D have previously been implicated as targets in cancer treatment. Protein degradation is rescued in CRBN- or VHL-knockout cells. Co-treating ER+ breast cancer cells with ligands for PR or PDE6D outcompeted PROTAC-mediated degradation and stabilized the respective protein, demonstrating the PROTACs are targeting the PR and PDE6D ligand-binding domains. Competition assays in ER-negative cell lines confirmed PR and PDE6D protein degradation is ER-independent. These findings underscore the importance of understanding off-target effects for developing more selective and effective therapeutics in ER+ breast cancer. Citation Format: Zachary Sandusky, Myles Brown. Chemoproteomic evaluation of PROTACs targeting estrogen receptor alpha for ER+ breast cancer [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 6577.

Abstract 5899: Evaluation of Up284 as a selective proteasome inhibitor for prostate cancer therapy

Abstract Recent research into prostate cancer treatment has unveiled a promising contender known as Up284. In laboratory experiments, this compound has exhibited a remarkable preference for targeting prostate cancer cells as opposed to normal cells. Specifically, in vitro cell viability assays using MTT have shown that prostate cancer cells display high sensitivity to Up284 at low nanomolar IC50 values, while primary human prostate epithelial cells and mouse keratinocytes exhibit minimal sensitivity. This establishes a notable therapeutic window between cancerous and non-cancerous cells, with Up284 significantly inhibiting colony formation in PC3 cells. Additional functional assays have confirmed Up284's potential, including the 4UbFL Reporter Gene assay, which has revealed robust, dose-dependent proteasome inhibition. This effect is supported by the accumulation of polyubiquitinated proteins in various prostate cancer cell lines following Up284 treatment, suggesting a distinct mechanism of toxicity compared to bortezomib. Furthermore, Up284 has been found to induce apoptosis in PC3 cells, surpassing the efficacy of bortezomib. Competitive binding assays have shown that Up284 exhibits a stronger binding affinity to the proteasomal receptor RPN13 than the prototype inhibitor RA190B. This is further supported by a Cellular Thermo Stabilization Assay (CETSA), which indicates that Up284 stabilizes RPN13 in cells. A preliminary in vivo study utilizing the bone metastatic prostate cancer genetically engineered mouse model(BMPC GEMM) model for metastatic prostate adenocarcinoma has demonstrated that Up284 leads to significant tumor regression and increased levels of poly-ubiquitinated proteins in residual tumor tissues. Moreover, when Up284 is conjugated with an E3 ligase ligand, it successfully degrades RPN13, resulting in the death of PC3 cells. Up284 has also shown favorable pharmacokinetics/pharmacodynamics (PK/PD) profiles and safety in animal models. It is well-tolerated in mice and holds promise for oral administration, demonstrating a proteasome-dependent reporter protein stabilization effect superior to ixazomib. Importantly, Up284 has induced tumor regression and improved survival rates in various cancer models, including ovarian and breast cancers. In conclusion, Up284 emerges as a lead candidate for targeted prostate cancer therapy due to its high specificity, substantial therapeutic index, and potent anti-cancer activity both in vitro and in vivo. Further development and clinical investigation of Up284 are warranted to fully explore its therapeutic potential. Citation Format: Balasubramanyam Karanam, Ravi Anchoori, Yung Chang. Evaluation of Up284 as a selective proteasome inhibitor for prostate cancer 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 5899.

Abstract 5318: SM2275, A tetraspecific, dual EGFR and PD-L1-targeting, conditionally activating co-stimulatory CD28 and simultaneously blocking PD-1/PD-L1 signaling to promoting fully activated T cell killing effects on tumor cells

Abstract Background: T cells require both signal 1 TCR engagement and signal 2 co-stimulatory signal to achieve complete activation and tumor recognition. CD28 is a signal 2 co-stimulatory receptor and activating CD28 increases T cell sensitivity, cytokine production and T cell survival. PD-1/PD-L1 signaling attenuates the T cells functional activity by inhibiting CD28 co-stimulation in the presence of TCR stimulation. Therefore, we developed SM2275, a tetraspecific VHH antibody against EGFR, PD-L1, CD28 and HSA, for the treatment of EGFR+/PD-L1+ solid tumors. Preclinical study demonstrated promising efficacy and favorable safety profile via a unique mechanism of action. Methods: SM2275 was developed using StarMab’s proprietary Quadbody multifunctional VHH antibody platform. It features two monovalent, low-affinity binding domains for EGFR and PD-L1, along with nanomolar low affinity CD28 binding domains arranged in a tandem format, followed by a half-life extending HSA binding domain. Differential binding affinities of SM2275 to EGFR/PD-L1 single-expressing versus double-expressing cell lines were analyzed by flow cytometry. Avidity-enhanced blockade of PD-1 on EGFR+PD-L1+ double-positive compared to PD-L1+ single-positive cells was analyzed via competitive binding assay and Jurkat PD1 NFAT Luciferase reporter assay. EGFR/PD-L1 dual-target-dependent CD28 activation was confirmed using a primary human T cell IL2 &amp; IFN-γ release assay in the presence of TCR stimulation. In vivo efficacy against EGFR+/PD-L1+ double-positive human tumors were evaluated using a syngeneic tumor transplantation model of MC38-hEGFR/hPDL1 cell line into hPD-L1/hCD28 humanized mice. Results: SM2275 displays dramatically increased affinity for cancer cells co-expressing EGFR/PD-L1 compared to single-expressing cells. This leads to an avidity-enhanced PD-L1 blockade specifically on the surface of EGFR+PD-L1+ double-positive cancer cells, enabling localized reactivation of tumor-infiltrating T cells without systemic immune toxicity. Simultaneously blocking the PD-L1 signaling unleashed the inhibition of CD28 co-stimulation for the full T cell activation. In the presence of SM2275, EGFR/PD-L1 double-positive cancer cells induce significantly enhanced CD28 activation and cytokine secretion compared to single-antigen-expressing cells. In a syngeneic tumor transplantation model, SM2275 effectively inhibits the growth of MC38-hEGFR/hPDL1 tumors in hPD-L1-hCD28 humanized mice, with no apparent cytokine release and immune toxicity. Conclusions: SM2275 is a novel dual-EGFR &amp; PD-L1-targeting, conditional CD28 agonistic antibody. It demonstrated promising efficacy and favorable safety profile in preclinical studies via a unique mechanism of action. Citation Format: Shihao Lu, Xiaodan Liu, Sheila Zhou, Wenjing Song, Huiqin Geng, Simin Yang, Hong Zhou, Siyao Xie, Yi Wei, Xing Zhang, Yanbin Liang. SM2275, A tetraspecific, dual EGFR and PD-L1-targeting, conditionally activating co-stimulatory CD28 and simultaneously blocking PD-1/PD-L1 signaling to promoting fully activated T cell killing effects on tumor 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 5318.

Abstract 2715: Preclinical characterization of SM2248: A dual-action bispecific antibody enhancing immunotherapy for solid tumors

Abstract Background: Innate immune cells, such as macrophages and myeloid-derived suppressor cells (MDSCs), dominate the immune landscape within "cold" solid tumors, which typically exhibit poor responses to immune checkpoint inhibitors (ICIs). The dearth of tumor-infiltrating T cells and the prevailing immunosuppressive microenvironment contribute to this ineffectiveness. To bolster immunotherapy against such tumors, we developed SM2248, a bispecific VHH antibody. SM2248 simultaneously obstructs the CD47-SIRPα phagocytosis checkpoint pathway and conditionally activates CD40 on tumor-infiltrating innate immune cells. CD47/SIRPa blockade enhances macrophage and dendritic cell (DC) phagocytosis and cross-presentation of antigens to cytotoxic T cells. CD40 activation fosters DC licensing, reprograms macrophages, and guides MDSC differentiation towards DCs and macrophages. The combined action of CD47 blockade and CD40 stimulation remodels the tumor microenvironment, fostering T cells infiltration and activation. Methods: SM2248 was developed by fusing a highly selective CD47-blocking VHH module - minimizing binding to human erythrocytes and platelets, with a conditional CD40 agonistic VHH module - selectively activating immune cells upon CD47 engagement, incorporated into an inactivated IgG1 Fc backbone. The specificity and affinity of SM2248 binding to human CD47 and CD40 were assessed via ELISA and flow cytometry assays. CD47 and SIRPα interaction blockade was confirmed through a competitive ligand binding assay. Target-dependent CD40 activation was evaluated via a CD40-NFκB-Luc reporter assay. In vivo anti-tumor efficacy was determined using a syngeneic transplantation model of MC38-hCD47 cancer cells in B-hCD40 humanized mice. Binding to erythrocytes and platelets, compared with magrolimab, was assessed using flow cytometry. Hemolysis toxicity was evaluated through an in vitro erythrocyte lysis assay. Results: SM2248 effectively inhibits cell surface CD47-SIRPα interaction at nanomolar concentrations and activates CD40-NF-κB-luciferase reporters at picomolar concentrations in a target-dependent manner. In contrast to the CD47 monoclonal antibody magrolimab, SM2248 does not bind to human erythrocytes or platelets and does not induce hemolysis. In murine syngeneic tumor models, SM2248 completely halts the growth of MC38-hCD47 tumors in humanized B-hCD40 mice and does not induce dose-dependent hepatotoxicity at doses up to 10 mg/kg. Conclusions: SM2248, a humanized bispecific tetravalent VHH antibody targeting CD47 and CD40, exhibits promising in vivo efficacy and a favorable safety profile in preclinical studies, offering the potential for enhancing immunotherapy in solid tumors. Citation Format: Xiaodan Liu, Hong Zhou, Shihao Lu, Huiqin Geng, Wenjing Song, Siyao Xie, Yi Wei, Xing Zhang, Simin Yang, Sheila Zhou, Yanbin Liang. Preclinical characterization of SM2248: A dual-action bispecific antibody enhancing immunotherapy for 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 2715.

Abstract 40: Persistence but not antitumor efficacy of CAR-engineered lymphocytes is governed by a FAS/FAS ligand auto-regulatory circuit

Abstract Adoptive cell transfer of chimeric antigen receptor (CAR) engineered T and NK cells has revolutionized the treatment of many B-lymphoid malignancies; however, limited persistence of infused lymphocytes can restrain the full potential of this approach. Apoptosis induction through the FAS ligand/FAS pathway is a key negative regulator of lymphocyte survival. To define which cellular subsets express FASLG, the gene encoding FAS ligand, we generated a single-cell transcriptomic atlas from 37 patients with diverse cancer types. We discovered that FASLG expression is highly constrained and limited primarily to T and NK cells while stromal and tumor cells express minimal to no FASLG. Multiplex RNA in situ hybridization of clinical samples from patients treated with anti-CD19 CAR-T cells confirmed these results and revealed significantly higher FASLG content in CAR-expressing compared with non-CAR expressing T cells. Based on these findings, we hypothesized that FAS ligand expression by CAR+ T and NK cells will engage FAS+CAR+ cells to induce apoptosis and limit cellular persistence. To test this hypothesis, we performed competitive fitness assays using two phenotypically discernable populations of human CAR-T and CAK-NK cells that are either responsive or unresponsive to FAS-signaling through expression of a FAS dominant negative receptor (ΔFAS). ΔFAS+ and ΔFAS− CAR-expressing cells were co-infused 1:1 into tumor bearing hosts and persistence of each population was quantified. For both CAR-T and CAR-NK, we measured significant enrichment in ΔFAS-expressing cells across multiple tissues, demonstrating that FAS-signaling regulates CAR+ cell survival in a cell-intrinsic manner. Knock out (KO) of FASLG using CRISPR/Cas9 prevented population skewing while KO of the AAVS1 control locus had no effect, indicating that CAR-derived FASLG is sufficient to restrain the persistence of transferred cells. By contrast, we discovered that FASLG was dispensable for CAR-T and CAR-NK mediated tumor killing both in vitro and in vivo. Finally, using two different tumor models, we found that adoptive transfer of CAR-T or CAR-NK cells that co-express ΔFAS resulted in superior antitumor efficacy compared with control cells that express the CAR alone. Taken together, these findings reveal that CAR-engineered lymphocyte persistence is governed by a FAS/FAS ligand auto-regulatory circuit. Further, they indicate that the therapeutic benefit of a FAS-dominant negative receptor is broadly applicable and agnostic of whether FASLG is expressed by cells in the tumor microenvironment. Citation Format: Fei Yi, Tal Cohen, Paul Zumbo, Razan Eltilib, Hannah Arkin, Michael Gormally, Christopher S. Hackett, Korbinian Kropp, Smita Chandran, Jae H. Park, Doron Betel, Christopher A. Klebanoff. Persistence but not antitumor efficacy of CAR-engineered lymphocytes is governed by a FAS/FAS ligand auto-regulatory circuit [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 40.

Abstract 4274: Unraveling collagen signatures in cancer-associated fibroblasts: A biomarker-driven approach

Abstract Background: The tumor microenvironment (TME) plays a crucial role in driving tumor development. Among the constituents of the tumor stroma, cancer-associated fibroblasts (CAFs) are a significant component. CAFs are actively involved in tumor progression by modulating the architecture of the TME through increased deposition of various collagens resulting in tumor fibrosis. Several studies have shown that CAFs have heterogeneity within, and between, individual tissues. TGF-β is thought to be the main driver of tumor fibrosis, however, the field lacks a characterization of the specific collagen deposition of CAFs from different tissues. In this study, we investigated the fibrotic activity of CAFs from various tissues by measuring the production of three specific collagen peptides in vitro by use of non-invasive clinically validated biomarkers. Methods: Primary human CAFs from pancreas (pCAF), colon (cCAF), breast (bCAF) and lung (lCAF) were cultured over a 12-day period in ficoll-based media under both unstimulated conditions and TGF-β stimulation. Additionally, cells were subjected to treatment with the ALK5/TGF-β1 receptor kinase inhibitor (ALK5i). The assessment of collagen I (PRO-C1), collagen III (PRO-C3), and collagen VI (PRO-C6) formation was conducted in the cell supernatant at day 3, day 6, day 9, and day 12 using competitive enzyme-linked immunosorbent assay (ELISA). Results: For lCAFs, cCAFs, and pCAFs, the addition of TGF-β led to a notable elevation in the levels of PRO-C1, PRO-C3, and PRO-C6 within the supernatant. Conversely, in the case of bCAFs, stimulation with TGF-β did not elicit remarkable alterations in the levels of PRO-C1 and PRO-C3 compared to unstimulated cells. Notably, bCAFs exhibit elevated intrinsic activity (high PRO-C3) even in the absence of TGF-β stimulation whereas lCAFs and cCAFs exhibited relatively low level of PRO-C3. Additionally, across all different CAFs, the introduction of ALK5 inhibitor (ALK5i) to the culture medium resulted in a considerable reduction in PRO-C1, PRO-C3, and PRO-C6 levels, returning them to the baseline of the unstimulated control or even lower. Distinct collagen formation patterns were observed among the various CAFs. CAFs derived from breast cancer exhibited the highest release of PRO-C1 and PRO-C3, while CAFs isolated from colon cancer displayed the highest levels of PRO-C6. Conclusion: These findings underscore the heterogeneity in collagen production among different CAFs, providing valuable insights into the extracellular matrix dynamics within distinct tumor microenvironments. Moreover, non-invasive biomarkers demonstrate the capability to differentiate between different CAF subtypes. Lastly, our model proves to be a valuable tool for anti-fibrotic drug screening, as evidenced by the consistent suppression of TGF-β1 signaling and reduced levels of PRO-C1, PRO-C3, and PRO-C6 upon treatment with ALK5i across all cases. Citation Format: Annika Hettich, Neel I. Nissen, Morten A. Karsdal, Nicholas Willumsen. Unraveling collagen signatures in cancer-associated fibroblasts: A biomarker-driven approach [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 4274.

Abstract 3055: Characterizing the role of HNF4α isoforms in human PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly and recalcitrant malignancy with a 5-year survival rate of 12%. Transcriptomic studies have classified PDAC into a classical or basal-like subtype. The classical subtype has a better prognosis, response to chemotherapy, and express higher levels of endodermal lineage specifiers compared to the basal-like subtype. However, the field lacks a comprehensive understanding of the key subtype-specific regulators. Functional analysis of transcription factors associated with each subtype are needed to elucidate their roles in regulation of PDAC subtypes and to identify therapeutically targetable vulnerabilities. Previous work in our lab demonstrated that HNF4α is a key regulator of the classical subtype. Using genetically engineered mouse models, our lab showed that loss of HNF4α leads to an increase in tumor burden, a shift from glandular to poorly differentiated histology, and a decrease in the classical subtype score. However, while murine PDAC only express one set of HNF4α isoforms, human PDAC express both sets of HNF4α isoforms, suggesting murine PDAC may not fully recapitulate human disease. There are 2 subsets of HNF4α isoforms driven by expression from 2 distinct promoters, P1 and P2. P1 and P2 isoforms are differentially expressed within the gastrointestinal (GI) tract and play dichotomous roles in GI malignancies. In HNF4α-positive human PDAC, P2 isoforms are always expressed but there is variable expression of P1 isoforms. Based on the differential role of HNF4α isoforms in both normal tissue and cancer, we hypothesize that P1 isoforms will restrain tumor growth and promote greater classical/GI differentiation compared to P2 isoforms. We utilized CRISPRi to selectively inhibit transcription at the P1 or P2 promoter in HNF4α-positive, human cell lines expressing both isoforms. To test for sufficiency, we exogenously expressed P1 or P2 isoforms in HNF4α-negative, human cell lines. We then performed Incucyte, CRISPRi competition, and anchorage-independent growth assays. We performed RNA-seq and ChIP-seq on both exogenous and endogenous HNF4α isoforms. Functional assays revealed that HNF4α isoforms regulated growth in a context dependent manner. Exogenous P1 isoforms significantly inhibited growth while loss of endogenous P1 isoforms was dispensable for growth. Exogenous P2 isoforms inhibited growth to a lesser degree while loss of endogenous P2 isoforms led to a significant reduction in growth. Despite ChIP-seq analysis revealing very similar binding profiles, RNAseq analysis revealed that P1 isoforms are significantly more transcriptionally active than P2 isoforms and are stronger inducers of GI differentiation. Taken together, our data demonstrates that P2 isoforms are more compatible with growth in PDAC compared to P1 isoforms, which promote a more differentiated cell state. Mechanistically, this may be due to the increased transcriptional activity of P1 isoforms. Citation Format: Pengshu Fang, Acramul Kabir, Gabriela Fort, Eric L. Snyder. Characterizing the role of HNF4α isoforms in human PDAC [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 3055.

Abstract 2579: A nectin-4 PET radiotracer for pharmacodynamic evaluation of enfortumab vedotin (EV) therapy in urothelial carcinoma

Abstract Introduction: Enfortumab vedotin (EV), an antibody-drug conjugate targeting nectin-4, offers hope for urothelial carcinoma patients (UC) but prolonged use leads to treatment-related toxicities often necessitating dose adjustments. How EV engages nectin-4 at the tumor and its relevance to efficacy is unknown. To understand the pharmacodynamics (target engagement) of EV at the tumor across FDA approved dosing levels, we developed a nectin-4 specific peptide-based radiotracer, [68Ga]AJ647, for positron emission tomography (PET) imaging. Methods: Six UC cell lines of luminal (HT-1376, UC-14, RT-112) and basal (UC-9, SCaBER, T24) morphological type were selected for in vitro and in vivo testing of [68Ga]AJ647 and nectin-4 target engagement by EV. Cell uptake and competitive binding assays were conducted to test [68Ga]AJ647 specificity for nectin-4 in the presence or absence of EV. Flow cytometry derived receptor density was used to correlate [68Ga]AJ647 uptake in cells with nectin-4 expression. Pharmacokinetics, biodistribution and specificity of [68Ga]AJ647 for nectin-4 expression was evaluated in six xenograft models by PET imaging and biodistribution studies. Target engagement by EV was quantified by measuring unoccupied nectin-4 levels by [68Ga]AJ647-PET in HT-1376 xenografts at different doses (15, 9, and 6 mg/kg) and time (1, 3, and 6 days). Results: [68Ga]AJ647 was generated in 20-30% radiochemical yield with &amp;gt;95% purity. In vitro assays showed distinctive uptake in cell lines (HT-1376 &amp;gt; UC-14 &amp;gt; RT-112; UC-9 &amp;gt; SCaBER &amp;gt; T24) that correlated with nectin-4 density (R2=0.85). PET and biodistribution studies showed tractable pharmacokinetics of [68Ga]AJ647 enabling high contrast images in 60 min and exhibiting nectin-4 dependent uptake in all the six xenografts. Uptake of [68Ga]AJ647 in UC cells reduced in a dose dependent manner in the presence of EV, indicating that [68Ga]AJ647 measures unoccupied nectin-4, and paving the way for EV target engagement evaluation. For target engagement evaluation, [68Ga]AJ647 accumulation in HT-1376 tumors in saline treated mice was assumed as 100% unoccupied nectin-4 levels. In mice treated with EV for 24 hrs (n=3-5), unoccupied nectin-4 levels were 21.1±11.6, 23.2±14.5, and 24.3±18.8 for 15, 9, and 6 mg/kg doses, respectively. Whereas on day 6, unoccupied nectin-4 levels showed significant differences at 29.5±15.8, 64.2±21.0, and 89.5±18.9 for 15, 9, and 6 mg/kg dose, respectively. These data indicate that FDA approved doses of EV show higher target engagement at 24 h but a dramatic reduction at lower doses over time. Conclusion: [68Ga]AJ647-PET quantifies variable nectin-4 expression in tumors. Furthermore, [68Ga]AJ647-PET quantifies EV therapy induced pharmacodynamics at the tumor. Such real-time ADC target engagement assessment could inform patient selection and dosing strategies to enhance efficacy and minimize toxicities. Citation Format: Akhilesh Mishra, Ajay K. Sharma, Kuldeep Gupta, Burles A. Johnson, David J. McConkey, Jeannie Hoffman-Censits, Sridhar Nimmagadda. A nectin-4 PET radiotracer for pharmacodynamic evaluation of enfortumab vedotin (EV) therapy in urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2579.

Abstract 1341: SM2235: A bispecific EGFR x CD16A innate immune cell engager for enhanced treatment of EGFR-expressing solid tumors, regardless of RAS mutation status

Abstract Background: Monoclonal antibodies (mAbs) targeting the epidermal growth factor receptor (EGFR) are frontline therapies for numerous EGFR-dependent epithelial tumors. However, their efficacy faces challenges due to on-target skin toxicity and intrinsic or acquired resistance. Within the tumor microenvironment, innate immune cells, including macrophages and natural killer (NK) cells, play a significant role but are hindered by the lack of specific cancer cell targeting and a suppressive microenvironment. To enhance innate immune cell cytotoxicity, we have developed SM2235, a humanized tetravalent bispecific VHH antibody. SM2235 partially blocks EGFR and EGF interaction while conditionally activating innate immune cells through CD16A upon engagement with cancer cells. Methods: SM2235 was engineered by combining a partially blocking EGFR single-domain antibody with a high-affinity CD16A-activating single-domain antibody on an inactivated human IgG1 Fc backbone. The binding affinity and specificity were confirmed using bio-layer interferometry and ELISA with recombinant proteins, followed by flow cytometry with EGFR+ human cancer cell lines and CD16A+ primary NK cells. SM2235's efficacy in blocking EGFR-EGF interaction and downstream signaling was assessed through competitive ELISA and proliferation assays in both EGFR-dependent and independent cancer cell lines. Its ability to induce target-dependent NK cell and macrophage activation was evaluated through activation markers as well as cytotoxicity and phagocytosis assays. In vivo efficacy of SM2235 against EGFR+ human tumors was measured using xenograft assays in NSG mice, covering cetuximab-sensitive and resistant cancer cell lines. Preliminary pharmacokinetics and safety studies were conducted in cynomolgus monkeys. Results: SM2235 exhibits sub-nanomolar affinity for EGFR+ human cancer cell lines and CD16A+ primary human NK cells. It partially inhibits EGFR-dependent cancer cell proliferation. Compared to cetuximab, SM2235 significantly enhances target-dependent CD16A-mediated NK cell and macrophage activation and cytotoxicity against EGFR+ cancer cells in ADCC and ADCP assays in vitro. SM2235 effectively inhibits EGFR+ xenograft tumors in vivo, irrespective of KRAS mutation status. In cynomolgus monkeys, SM2235 demonstrates excellent tolerance with a 20-hour serum half-life of approximately 20 hours and no dose-dependent skin toxicity. Conclusions: Preclinical characterization of SM2235 shows promising in vivo efficacy against EGFR+ human cancers, regardless of KRAS mutation, with a favorable safety profile marked by minimal skin toxicity. Citation Format: Huiqin Geng, Wenjing Song, Xiaodan Liu, Shihao Lu, Hong Zhou, Simin Yang, Siyao Xie, Yi Wei, Xing Zhang, Sheila Zhou, Yanbin Liang. SM2235: A bispecific EGFR x CD16A innate immune cell engager for enhanced treatment of EGFR-expressing solid tumors, regardless of RAS mutation status [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 1341.