Dual Antibody Research Articles

Case Report: Dual monoclonal antibody therapy in chronic rhinosinusitis with nasal polyps and severe eosinophilic asthma—a proteome analysis

Case Report: Dual monoclonal antibody therapy in chronic rhinosinusitis with nasal polyps and severe eosinophilic asthma—a proteome analysis

Enhancing Extracellular Vesicle Detection via Cotargeting Tetraspanin Biomarkers.

Extracellular vesicles (EVs) are emerging as key diagnostic biomarkers due to their widespread presence in body fluids and the proteins on their surfaces, which reflect the identity and condition of their parent cells. Research has focused on detecting EVs with biosensors that target individual transmembrane proteins (TMPs) like tetraspanins. However, due to TMP heterogeneity and the formation of tetraspanin-enriched microdomains (TEMs), cotargeting multiple TMPs is a promising strategy for enhancing EV detection. In this work, we introduce a dual-antibody surface functionalization approach using surface plasmon resonance (SPR) biosensors to cotarget tetraspanins on EVs derived from mouse macrophages. The expression of EV tetraspanin markers followed the trend of CD9 > CD63 > CD81, which was consistent with the EV detection targeting their nontetraspanin partners, exhibiting LFA-1 > ICAM-1 > VCAM-1, and suggesting a differential role of tetraspanins with their associated TMPs. Cotargeting EV tetraspanins via CD81/CD63, CD81/CD9, and CD63/CD9 dual monoclonal antibody surfaces resulted in higher EV detection compared to predictions based on binding with two monoclonal antibodies against tetraspanins without cotargeting. Furthermore, the optimization of dual monoclonal antibody surface ratios to improve cotargeting effect yielded a statistically significant enhancement in the sensitivity of EV detection. These findings underscore the importance of TEMs in designing EV-based biosensing platforms to achieve optimized sensitivity in EV detection.

ANCA and Anti-glomerular Basement Membrane (GBM) Dual Antibody Positivity: A Case Series and Proposal for a Clinical Classification

ANCA and Anti-glomerular Basement Membrane (GBM) Dual Antibody Positivity: A Case Series and Proposal for a Clinical Classification

Comparison of Dual Monoclonal Antibody Therapies for COVID-19 Evolution: A Multicentric Retrospective Study.

Neutralizing antibodies targeting the SARS-CoV-2 Spike protein reduce COVID-19-related risk of hospitalization, particularly in high-risk individuals. The COCOPREV-R study aimed to evaluate and compare clinical outcomes in high-risk SARS-CoV-2 patients treated with dual monoclonal antibody therapies and to identify associated virological factors. The COCOPREV-R study retrospectively collected real-world data from high-risk patients receiving Bamlanivimab/Etesevimab or Casirivimab/Imdevimab dual monoclonal antibody therapies (22 February 2021 to 15 June 2021). The study included 1004 patients with COVID-19, of whom 691 received Bamlanivimab/Etesevimab and 313 received Casirivimab/Imdevimab. The alpha variant represented 90.1% of those for whom data were available. The risk of hospitalization within 30 days was lower with Bamlanivimab/Etesevimab (12.7%, CI 95% [9.9-16.3%]) compared to Casirivimab/Imdevimab (28.4%, CI 95% [22.7-35.1%) (p < 0.001). The 30-day mortality rates were comparable between both groups (p = 0.982). Analysis of SARS-CoV-2 PCR negativity showed no difference between the two treatment groups (95.2% [93.0-96.9%] and 93.5% [89.1-96.6%] until day 30, p = 0.851 for Bamlanivimab/Etesevimab and Casirivimab/Imdevimab, respectively). Among persistently positive samples with available sequencing results (n = 43), Spike protein changes occurred only in Bamlanivimab/Etesevimab (42.9%) vs. Casirivimab/Imdevimab (0.0%) groups. Q493R (25.0%) and E484K (12.5%) were the most common mutations selected by Bamlanivimab/Etesevimab in follow-up samples. Other factors (immunodepression, comorbidities, and age) did not appear to be associated with the occurrence of Spike protein mutations. A higher rate of hospitalization was seen with Casirivimab/Imdevimab (RONAPREVE®) in comparison with Bamlanivimab/Etesevimab treatment, but with the emergence of Spike mutations only in the Bamlanivimab/Etesevimab group.

Comparison of a dual antibody and antigen HCV immunoassay to standard of care algorithmic testing.

The Centers for Disease Control and Prevention (CDC) guidelines for hepatitis C virus (HCV) testing, although effective, may miss crucial diagnostic opportunities. The goal of this study was to assess the utility of an antibody (Ab) and antigen (Ag) combination immunoassay as an alternative to traditional HCV screening. Remnant specimens from 1,341 patients with concurrent third-generation serologic (Roche anti-HCV-II) and nucleic acid amplification testing (NAAT) were assessed using the HCV Duo Ab/Ag immunoassay (Roche). Patient demographics, risk factors, and standard of care (SOC) laboratory results from the medical records were recorded. Overall, 99.0% (197/199) of the HCV Duo Ab+/Ag+specimens accurately identified active infections as confirmed by NAAT, and 99.9% (670/671) Ab-/Ag- samples corresponded to those without HCV infections. Individually, the HCV Duo Ab component demonstrated a 95.6% positive percent agreement (PPA) (95% CI = 93.8-96.9) and 99.1% negative percent agreement (NPA) (98.8-99.6) compared with SOC anti-HCV II Ab assay. The HCV Duo Ag had a 73.5% PPA (67.9-78.4) and 99.8% NPA (99.3-100) with NAAT. Among RNA+ specimens, 73.4% (197/267) were HCV Duo Ag+, and 265/267 (99.3%) were successfully detected on the HCV Duo Ab component. Notably, 5/7 (71.4%) Ab-/RNA +specimens were detected by HCV Duo, which would have been missed by traditional algorithmic testing. Fourth generation HCV Duo Ab/Ag assay demonstrated comparable performance to SOC testing and shortens the diagnostic window but does not eliminate the need for NAAT in all patients. Ab/Ag testing identified several Ab-/RNA+ cases, a subgroup often undiagnosed by current algorithmic testing, demonstrating promise for improved diagnostic efficiency and accuracy in HCV detection.IMPORTANCEThis study highlights the potential of a combined hepatitis C virus (HCV) Duo antibody (Ab) and antigen (Ag) immunoassay to improve early detection of HCV infections. Traditional Ab-only screening methods recommended by the Centers for Disease Control and Prevention may miss early-stage infections. The HCV Duo assay showed high accuracy, detecting nearly all active infections confirmed by nucleic acid amplification testing. Dual detection of HCV Ab and Ag shortens the diagnostic window, enabling intervention and treatment in a single visit, which is crucial for improving patient outcomes and reducing HCV transmission, especially in areas with limited access to confirmatory molecular testing.

Protocols for circulating neutrophil depletion in neonatal C57Bl/6 mice.

Murine neonatal neutrophil depletion strategies have problems with achieving deep circulating neutrophil clearance and accurate residual neutrophil fraction detection. Boivin et al. demonstrated an isotype switch method of achieving profound neutrophil clearance using a combination of anti-Ly6G and anti-rat κ Ig light chain antibodies in adult C57Bl/6 mice, proven by extra- and intracellular Ly6G detection by flow cytometry. We adapted this technique to neonatal mice testing four neutrophil depletion strategies. Four protocols were tested: P3 Ly6G and P1-3 Ly6G (anti-Ly6G on postnatal days (P) 3 and 1-3 respectively), and P3 Dual and P1-3 Dual (anti-Ly6G and anti-rat κ Ig light chain on P3 and P1-3 respectively). Intracellular and extracellular Ly6G presence was detected using flow cytometry. Isotype control antibodies were used as controls. P1-3 Dual achieved significantly better neutrophil depletion than the P1-3 Ly6G or P3 Ly6G protocols (97% vs 74% and 97% vs 50%, respectively). The P3 Dual protocol alone was enough to achieve significantly better neutrophil clearance (93%) than any of the Ly6G alone protocols. The Ly6G alone protocols led to near-total elimination of extracellular Ly6G, but there was a significant presence of intracellular Ly6G in the CD45+ cell population, which evaded detection by extracellular Ly6G antibody-based detection methods. Anti-Ly6G antibody alone is ineffective in producing effective circulating neutrophil clearance in neonatal mice. Dual antibody-based neutrophil depletion strategies achieve>90% clearance in P4 mice. A single day of dual antibody treatment achieves 93% neutrophil depletion in neonatal mice and is an alternative to daily injections. Dual antibody-based neutrophil depletion effectively induces circulating neutrophil clearance in neonatal mice.

The Association of Chronic Pulmonary Aspergillosis and Chronic Pulmonary Histoplasmosis with MDR-TB Patients in Indonesia.

In Indonesia, 2.4% of all new tuberculosis patients had multi-drug resistant disease (MDR-TB); an estimated 24,000 incidences. Historical case series of MDR-TB described a high frequency of cavitation and poor prognosis. The diagnosis of chronic pulmonary aspergillosis (CPA) relies on raised levels of Aspergillus IgG antibodies, and detectable Histoplasma IgG antibodies are suspicious for chronic pulmonary histoplasmosis (CPH). We investigated whether MDR-TB patients might have concurrent CPH or CPA. This was a cross-sectional study with 50 MDR-TB patients. ELISA was used to detect Histoplasma IgG antibodies and lateral flow assay was used to detect Aspergillus IgG/IgM antibodies. Several other possible disease determinants were assessed by multivariate analysis. Of the 50 MDR-TB patients, 14 (28%) and 16 (32%) had positive Histoplasma or Aspergillus serology; six patients (12%) had dual antibody reactivity. Radiological abnormalities in positive patients included diffuse or local infiltrates, nodules, consolidation, and apical cavities, consistent with CPH and CPA. Patients with detectable fungal antibodies tended to have worse disease, and 4 of 26 (15.3%) died in the first 5 months of dual infection (p = 0.11 compared with no deaths in those with only MDR-TB). The criteria for the diagnosis of CPH and CPA were fulfilled in those with moderately and far advanced disease (13 of 14 or 93%) and 12 of 16 (75%), respectively. Damp housing was the only determinant associated with Histoplasma antibodies (PR 2.01; 95%CI 0.56-7.19), while pets were associated with the Aspergillus antibody (PR 18.024; 95%CI 1.594-203.744). CPA or CPH are probably frequent in MDR-TB patients in Indonesia and may carry a worse prognosis.

First-in-human study of ZGGS15, a dual specific antibody targeting LAG-3 and TIGIT, as monotherapy in patients with advanced solid tumors.

e14678 Background: ZGGS15 is a recombinant humanized bispecific antibody composed of a monoclonal antibody against LAG-3 and TIGIT. It can specifically bind LAG-3 and TIGIT. It reverses LAG-3 and TIGIT immune suppression to T and NK cells, relieves suppression of Treg to T and NK cells, promotes the activation and proliferation of T and NK cells, and cytokines release. ZGGS15 restores T and NK cell functions to kill tumor cells. In non-clinical studies, we found that ZGGS15 had significant anti-tumor effects and synergistic anti-tumor effects with Anti-PD-1 antibody. Therefore, we conducted a Phase 1 clinical study to assess the tolerability, safety, and preliminary efficacy of this drug as monotherapy in patients with advanced solid tumors. Methods: During the dose escalation stage, an accelerated titration design (ATD) in the first dose (1 subject if ≤Grade 2 treatment-related adverse event occurred) and the standard "3+3" design in the rest doses were used. Subjects were patients with advanced solid tumors who failed to the available standard treatments. The dose groups were set from 0.3 to 30 (0.3, 1, 3, 10, 20, 30) mg/kg, intravenous infusion, once every 3 weeks. The first 21-day period was defined as the dose-limiting toxicity (DLT) observation period. Subjects could continue to receive ZGGS15 until treatment discontinuation criteria were met. Tumor response was assessed by RECIST1.1 and iRECIST. Pharmacokinetics (PK) and receptor occupancy (RO) profiles were also assessed in this study. Results: A total of 12 patients (4 males and 8 females) completed the DLT observation in the first four dose groups (0.3 to 10 mg), with a median age of 57 years. Eight (66.7%) of them received at least 3 lines of antineoplastic therapies, and five (41.7%) have been exposed to PD-1 or PD-L1 inhibitors. There was no DLT event observed. Thirty-eight treatment-related adverse events (TRAEs) occurred. Up to now, TRAEs were all grade 1 ~ 2. The most frequent TRAEs were proteinuria (33.3%, 4/12), hypokalemia (25%, 3/12), hypoalbuminemia (25%, 3/12), and hypertriglyceridemia (25%, 3/12). The Cmax and AUC increased from 0.3-1 mg/kg approximately in dose proportion. Nearly complete and sustained RO of T and NK cells in PBMCs was observed at 1 mg/kg dose of ZGGS15 for at least 3 weeks. Conclusions: The first four dose levels of ZGGS15 were very well tolerated. A good PK profile, and a long-lasting RO were observed in this escalation study. It is anticipated that this product could provide an additional benefit only when use in combination with an anti-PD-1 antibody. Clinical trial information: NCT05864573 .

Phase 1/2 study of NGM707, an ILT2/ILT4 dual antagonist antibody, in advanced solid tumors: Interim results from dose-escalation.

Phase 1/2 study of NGM707, an ILT2/ILT4 dual antagonist antibody, in advanced solid tumors: Interim results from dose-escalation.

Hypersensitive detection of CYFRA21-1 by SERS dual antibody sandwich method

PurposeAs an important reference index for the early diagnosis of non-small cell lung cancer (NSCLC), CYFRA21-1 still lacks the detection of low equipment cost, wide linear range and high sensitivity. Surface-enhanced Raman scattering (SERS) is a vibration spectrum technology based on the surface plasma of precious metal nanoparticles, which has been effectively applied to the detection of tumor markers. The combination of SERS technology and sensors has great potential in the ultrasensitive detection of tumor markers. The purpose of this study was to develop a sensitive method using SERS to quantitatively detect CYFRA21-1 for early diagnosis of NSCLC. MethodsA double-antibody sandwich immunoassay based on SERS was designed and tested to implement the ultrasensitive detection of CYFRA21-1 in the serum of NSCLC patients. ResultsGold @Ag nanorods (Au @Ag NRs) with higher Raman signals were prepared and used as probes, while magnetic graphene oxide was used as a magnetic substrate. The immunized probe, immune substrate and CYFRA21-1 standard substance in the buffer system formed a double-antibody sandwich structure. The standard curve displayed a liner range from 1pg mL−1 to 10 ng mL−1, and the detection limit (LOD) is 0.8943pg mL−1. The Raman intensity exhibited a wide linear relationship with the logarithm of CYFRA21-1 concentration. ConclusionOur study successfully established a double-antibody sandwich immunoassay based on SERS. This method demonstrated high specificity and sensitivity for detecting CYFRA21-1 protein content in serum. It has the potential to be applied for early detection of lung cancer biomarkers.

E027 A rare cause of shoulder pain: calcific tendonitis at the pectoralis major humeral insertion

Abstract Background/Aims Shoulder pain in the elderly is very common and often caused by calcific tendonitis of the rotator cuff muscles. We report the case of a lady presenting with unilateral shoulder pain who had an X-Ray which showed calcification over the proximal humerus. This calcification turned out to be due to calcific tendonitis of the humeral insertion of the pectoralis major, which is very rare. Unfamiliarity with this unusual condition could lead to inappropriate investigation, such as invasive biopsy, and possible mismanagement. Methods A 61-year-old lady of Pakistani origin with dual antibody positive rheumatoid arthritis, well controlled on methotrexate, reported new and severe right shoulder pain during her outpatient appointment. Her past history included coeliac disease, osteoarthritis, hypertension, iron and vitamin D deficiency, and she was not currently taking any calcium supplements. The pain had rapidly progressed over weeks to months without any trauma history and was significantly limiting all shoulder movements, particularly shoulder abduction and rotation. Her rheumatoid arthritis was in remission. Concerningly, she reported nocturnal pain, which alongside the rapidly progressive nature of the pain raises suspicions of a more sinister cause like malignancy. Results Blood tests showed a normal CRP, calcium profile and ALP. An X-Ray of the right shoulder showed no calcification or osteoarthritis affecting the glenohumeral joint, but demonstrated the unusual finding of a prominent focus of calcification close to the lateral aspect of the proximal humerus. An MSK radiologist advised further imaging to confirm nature of lesion. Subsequent USS confirms a focus of calcification (14mm x 18mm) in the pectoralis major humeral insertion, adjacent to the long head of the biceps tendon. Features that are suggestive of benign calcification rather than neoplasm include the characteristic location of the calcification and absence of an overlying soft tissue mass. The patient was reassured regarding the benign nature of the calcification and was managed conservatively with simple analgesia and physiotherapy. Conclusion Calcific tendinopathy is a common disease, but calcification of the pectoralis major at its humeral insertion point is very rare. This case highlights the importance of familiarity with this condition to enable the clinician to make an accurate diagnosis, and reduce the likelihood of unnecessary investigations and stress for the patient. Conservative management, such as simple analgesia and physiotherapy, is very effective for the majority of patients and should be used as first-line treatment. Steroid injection can be considered with orthopaedic intervention advised for those who have not responded to conservative management. Disclosure E.M. Quinlan: None. M. Kazmi: None.

Enabling multi-modal imaging and photoimmunotherapy of oral cancer spheroids through an EGFR-targeted antibody conjugate

SignificanceDelineating tumor margins during surgery and eliminating residual disease is a major challenge in the treatment of oral cancers. The dual function antibody conjugate (DFAC) developed in this study can enable 3D-imaging and destroy residual microscopic disease by photoimmunotherapy in a single intra-operative setting. ApproachDFAC was synthesized by routine carbodiimide crosslinker chemistry. Multi-modal imaging (Photoacoustic and fluorescence imaging) and photoimmunotherapy was performed on oral cancer spheroids. ResultsPhotoacoustic imaging could detect tumor spheroids, even of the smallest size (0.01 mm3), while fluorescence imaging could detect only larger spheroids with volumes greater than 0.049 mm3 and treated with a higher DFAC concentration. Photoimmunotherapy showed a spheroid size dependent response, with smaller spheroids of volumes 0.018 mm3 and lower showing no recurrence. ConclusionsThe complementary imaging and treatment capabilities of DFAC could help in minimizing healthy tissue resection and enhance recurrence free survival in oral cancer patients.

Abstract 2670: Development of novel anti-SIRPα/β dual antibody with single-agent phagocytosis activity

Abstract Signal-regulatory protein alpha (SIRPα) is an inhibitory receptor expressed on macrophages that interacts with CD47, resulting a "don't eat me" signal on cancer cells. This interaction suppresses macrophage dependent phagocytosis and contributes to the evasion of host immune system. Consequently, the SIRPα-CD47 axis has become a well sought after and established therapeutic angle to treat cancer. While CD47-targeting agents have shown promise in clinical trials, the ubiquitous expression of CD47 raises the concerns about hematologic toxicity, including anaemia and thrombocytopenia, hindering the development of CD47-targeting therapies. This has prompted exploration of another end of the axis i.e., targeting SIRPα. Moreover, targeting SIRPβ protein, expressed on myleoid cells, via agonistic approach would generate ITAM-mediated activating signal through DAP12 protein contributing to cancer cell death. Considering the limited expression of SIRPα and β, a dual antibody capable of blocking the CD47-SIRPα pathway and activating SIRPβ downstream signaling may result in a desirable efficacy and safety profile. In this study, we present novel anti-SIRP α/β dual antibody clones discovered from a proprietary human antibody discovery platform consisting of a large human antibody library, along with in vitro and in silico screening technologies. The platform was explored against native SIRPα/β antigens to identify novel human antibody clones based on affinity-based epitope coverage. Subsequently, antibody clones were produced and evaluated for preclinical functional relevance. Novel full-length SIRPα/β dual antibody clones demonstrated high affinity binding to soluble human SIRPα and SIRPβ proteins as well as these receptors expressed on CHO-K1 cells and macrophages. The antibody clones effectively disrupted CD47-SIRPα interaction in biochemical assays and significantly enhanced activation of macrophages as observed by increased cytokine release. Most importantly, the dual antibody clones demonstrated potent in vitro single-agent phagocytosis activity against tumor cells. In summary, the novel SIRPα/β dual antibodies activating macrophage mediated phagocytosis of tumor cells without the need for tumor opsonizing antibodies or agents capable of inducing “eat-me-signals” highlight their potential as a single-agent therapy. These findings prompt us to continue the development of the anti-SIRP α/β dual antibody for use in cancer therapy. Citation Format: Subhra Prakash Chakrabarty, Sandeep Sadashiv Patil, Sunaina Singh Rajput, Amrita Mukerji, Sandhya Aurumugam, Pooja Balu Gade, Sidhesha S, Anup Kumar Patra, Shivaranjini C, Vaishnavi Shivaji Khamkar, Lavanya P, Lakshmeesha K N, Ujjwal Kapoor, Jagadeesh R, Jwala Nagaraj, Vijaya Shankar Nataraj, Kavitha Nellore, Thomas Antony, Susanta Samajdar, Murali Ramachandra. Development of novel anti-SIRPα/β dual antibody with single-agent phagocytosis activity [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 2670.

Abstract 3641: Pharmacodynamic and predictive biomarker results from the phase I dose escalation study of NGM707, an ILT2/ILT4 dual antagonist antibody, in patients with advanced solid tumors

Abstract Background: NGM707 is a dual antagonist antibody that binds a shared epitope of the immune inhibitory receptors ILT2 (LILRB1) and ILT4 (LILRB2). In preclinical studies, NGM707 reprogrammed suppressive myeloid cells via blockade of ILT2/ILT4 and enhanced NK and CD8+ T cell activity via blockade of ILT2; furthermore, NGM707 and pembrolizumab acted additively in vitro to enhance CD4+ T cell activation in mixed lymphocyte reactions. NGM707-IO-101 (NCT04913337) is a first-in-human Phase 1/2 clinical trial of NGM707 monotherapy and combination with pembrolizumab. Here we report pharmacodynamic and predictive biomarker data from NGM707-IO-101 monotherapy and combination dose escalation cohorts. Methods: Eligible patients with advanced or metastatic solid tumors were enrolled into escalating dose cohorts from 6 to 1800 mg NGM707, alone or in combination with 200 mg pembrolizumab, administered Q3W iv. Biomarker assessments of tumors comprised RNA-sequencing and immunohistochemistry (including ILT2, ILT4, CD163) of archival/baseline (N= 41 IHC, 47 RNA) and paired on-treatment tumor biopsies (N=13 IHC, 21 RNA). Peripheral blood biomarker samples were collected on days 1, 4, 8, and 15 of cycle 1 and prior to dosing at each subsequent cycle. Blood biomarker assessments included circulating cytokines/chemokines, PBMC RNA-sequencing, and flow cytometry immunophenotyping. Baseline biomarkers were associated with anti-tumor activity per RECIST v1.1. Results: Pre-treatment biopsies from patients with disease control (DC) showed higher expression of genes in the 18-gene tumor inflammation signature (TIS) than patients with progressive disease (PD). A composite gene signature incorporating gene sets related to TIS, NK cell activity, and tumor cell proliferation had a statistically stronger association with DC than TIS genes alone. Baseline ILT2/ILT4 target expression (≥1% of tumor area) was observed in 31 (75.6%) patients, with similar levels observed in patients with DC and PD. Pharmacodynamic changes were observed in tumor samples following treatment, including increased expression of gene signatures that reflect myeloid cell activation and a proinflammatory tumor microenvironment. Decreased protein expression of the immunosuppressive myeloid marker CD163 was observed following treatment in both blood and tumor for a subset of patients. Proinflammatory chemokines including CXCL9 increased in serum following treatment. Conclusion: Baseline gene expression signatures have shown an encouraging association with clinical benefit from NGM707 and merit further investigation as promising predictive biomarkers. Protein and gene expression changes reflecting the expected modes of action of NGM707 and pembrolizumab were observed in both peripheral blood and tumor biopsies following treatment. Citation Format: Lisa K. Blum, Hung-I H. Chen, Anushka De Costa, Anjushree Iyer, Geoffrey W. Stone, Nicole D. Galicia, Jerry Chan, Brenda Dampier, Katherine Wu, Ursula Jeffry, Kefei Zhou, Joanne Sloan-Lancaster, Dhiraj Abhayankar, Vladimir Hanes, Hsiao D. Lieu, Daniel D. Kaplan, Julie M. Roda. Pharmacodynamic and predictive biomarker results from the phase I dose escalation study of NGM707, an ILT2/ILT4 dual antagonist antibody, in patients with advanced 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 3641.

Abstract 7092: Cancer biomarker discovery with scalable biofluid proteomics

Abstract Biofluid proteomics opens opportunities for the discovery of early detection, diagnostic, and prognostic cancer biomarkers through non-invasive or minimally invasive screening. Plasma and serum are accessible sample types, however, the high dynamic range of proteins creates analytical challenges for the sensitive detection of potential biomarkers. In this work, we compare two proteomic approaches for large-scale cancer biomarker discovery: the Olink Proximity Extension Assay and the Seer Proteograph Product Suite. Olink Explore 3072 is an immunoassay that gains specificity by using dual antibody recognition to measure approx. 3,000 proteins in each sample. Antibody pairs are tagged with oligonucleotides, which hybridize and are amplified before measurement by next generation sequencing. Seer Proteograph is an enrichment strategy that uses a proprietary mix of functionalized magnetic nanoparticles with affinity for certain amino acids and functional groups in native state proteins. The resulting peptides are measured using untargeted mass spectrometry to identify proteins at the peptide level, paving the way for the identification of proteoforms and peptide variants. Aliquots of human plasma from 78 samples representing a range of cancer indications were obtained through the Discovery Life Sciences biobank and used for analysis by both the Olink and Proteograph workflows. Olink analysis achieved Pearson correlation scores of greater than or equal to 0.8 when correlating each of seven process controls to each other for all approx. 3000 proteins. On average, greater than or equal to 75% of the proteins were detected across all panels in all sample groups except for Normal and Benign groups (64% and 74% respectively). After stringent FDR filtering to ensure only high-quality data was analyzed, the Seer Proteograph workflow quantified 4,323 proteins across all 78 samples. Pearson correlation scores of greater than or equal to 0.96 were achieved when comparing digestion controls, process controls, and sample cleanup controls. Both platforms achieved detection of proteins across the dynamic range of plasma, including low abundance proteins. The Olink Explore 3072 and Seer Proteograph platforms were found to be complementary, with approx. 15% overlap in measured proteins. Cancer biomarker discovery at the proteomic level is bolstered by recent advances in analytical capabilities, as revealed by the Olink Explore and Seer Proteograph platforms. Importantly, complementary implementation of these two workflows will enable comprehensive biomarker analysis. Citation Format: Danielle B. Gutierrez, Jessica L. Moore, Jaison Arivalagan, Tiffany Louie, Leslie Wilkinson, Ray Clemens. Cancer biomarker discovery with scalable biofluid proteomics [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 7092.

An rolling circle amplification-assisted CRISPR/Cas12a-based biosensor for protein detection

Glycated hemoglobin (HbA1c) has been extensively studied as a pivotal marker for monitoring and diagnosing diabetes. In this study, we introduce a highly sensitive and rapid enzyme-catalyzed rolling circle amplification (RCA)-assisted nicked-PAM CRISPR/Cas12a assay (termed “RANPC”). The RANPC system initially recognizes and captures the target using immunomagnetic beads and antibodies, forming a sandwich structure. The antibodies, upon ligating the primers, trigger the RCA reaction, resulting in the generation of single-stranded DNA with numerous repetitive sequences. The product strand of RCA then initiates the trans-cutting activity of nicked-PAM CRISPR/Cas12a, ultimately reporting the target concentration through fluorescent signaling. This study combines dual antibody capture, signal amplification through RCA, and signal output by Cas12a, resulting in high sensitivity, high specificity, and high signal-to-noise ratio. Moreover, the strategic incorporation of an incomplete PAM design in the CRISPR/Cas12a system substantially reduces background interference and enhances assay specificity relative to previous conventional Cas12a detection methods. RANPC possesses advantages such as ultra-sensitivity (with the lowest detection limit up to 0.129 pg/mL and a linear range of 12.8 pg/mL to 1 µg/mL), high specificity, rapid reaction time (approximately 50 min), and straightforward operation. Under optimized experimental conditions, we successfully detected HbA1c in eight real-world blood samples, and the results were in good agreement with the theoretical values. This suggests that this method holds promise as a new approach for the analysis of HbA1c levels in the clinical setting.

Efficacy and safety of combining ginkgolide diterpene glucosamine injection with dual antibodies in elderly patients with acute ischemic stroke

Purpose: To investigate the clinical effects and safety of ginkgo biloba diterpene lactone glucosamine injection, combined with dual antibiotic therapy for acute ischemic stroke in elderly individuals within a hyper thrombolytic time window. &#x0D; Methods: 100 elderly patients with acute ischemic stroke at Changsha First Hospital, Changsha, China from March 2022 to March 2023 were randomly and equally assigned to study and control groups (50 patients each). Study group received aspirin and clopidogrel, while control group received ginkgo biloba diterpene lactone glucosamine injection along with dual antibodies. Clinical efficacy, National Institutes of Health Stroke Scale (NIHSS) score, Instrumental Activities of Daily Living (IADL) score, cerebral hemodynamics, and safety were compared between the two groups. &#x0D; Results: There was no significant difference in effective rate between the two groups (p &gt; 0.05). However, following treatment, study group showed superior NIHSS and IADL scores compared to control group, with greater statistical significance as the disease progressed. Doppler ultrasound revealed that study group had smaller Pulsatility index (PI) values and larger mean cerebral artery blood flow velocity (Vm), and systolic value (Vs) compared to control group (p &lt; 0.05). In study group, three adverse reactions were reported (gastrointestinal discomfort, abnormal liver/kidney function, and skin itching), whereas the control group experienced five adverse reactions (gastrointestinal discomfort, abnormal liver/kidney function, dizziness, and skin itching). &#x0D; Conclusion: The combination of Ginkgo biloba diterpene lactone glucosamine injection and dual antibodies in elderly individuals with acute ischemic stroke within a super thrombolytic time window enhances intracerebral blood flow levels, effectively promotes neurological recovery, improves daily living abilities, and safety. Further research is needed to elucidate the exact pathways involved.

Steroid-responsive dual neuronal antibody-positive paraneoplastic encephalitis

Here, we report the case of an elderly African male with multifocal neuraxial involvement in the form of progressive parkinsonism and ataxia over a year. On evaluation, dual neuronal antibody positivity was detected. A diagnosis of paraneoplastic encephalitis was made without any detection of primary neoplasm. He was successfully managed with pulse steroid therapy followed by oral steroid and steroid-sparing oral immunosuppressive drug without any need for intravenous immunoglobulin or plasma exchange.

Better safe than sorry: dual targeting antibodies for cancer immunotherapy.

Antibody-based therapies are revolutionizing cancer treatment and experience a steady increase from preclinical and clinical pipelines to market share. While the clinical success of monoclonal antibodies is frequently limited by low response rates, treatment resistance and various other factors, multispecific antibodies open up new prospects by addressing tumor complexity as well as immune response actuation potently improving safety and efficacy. Novel antibody approaches involve simultaneous binding of two antigens on one cell implying increased specificity and reduced tumor escape for dual tumor-associated antigen targeting and enhanced and durable cytotoxic effects for dual immune cell-related antigen targeting. This article reviews antibody and cell-based therapeutics for oncology with intrinsic dual targeting of either tumor cells or immune cells. As revealed in various preclinical studies and clinical trials, dual targeting molecules are promising candidates constituting the next generation of antibody drugs for fighting cancer.

Establishment and application of a gold nanoparticle-based immunochromatographic test strip for the detection of avian leukosis virus P27 antigen in egg white samples.

Avian leukemia is an infectious tumorous disease of chickens caused by subgroup A of the avian leukemia virus (ALV-A), which mainly causes long-term viremia, slow growth, immune suppression, decreased production performance, multi-tissue tumors, and even death. The infection rate of this disease is very high in chicken herds in China, causing huge economic losses to the poultry industry every year. We successfully expressed the specific antigen protein of ALV (P27) through recombinant protein technology and screened a pair of highly sensitive monoclonal antibodies (mAbs) through mouse immunity, cell fusion, and antibody pairing. Based on this pair of antibodies, we established a dual antibody sandwich ELISA and gold nanoparticle immunochromatographic strip (AuNP-ICS) detection method. In addition, the parameters of the dual antibody sandwich ELISA and AuNP-ICS were optimized under different reaction conditions, which resulted in the minimum detection limits of 0.2 ng mL-1 and 1.53 ng ml-1, respectively. Commonly available ELISA and AuNP-ICS products on the market were compared, and we found that our established immune rapid chromatography had higher sensitivity. This established AuNP-ICS had no cross-reactivity with Influenza A (H1N1), Influenza A (H9N2), respiratory syncytial virus (RSV), varicella-zoster virus (VZV), Listeria monocytogenes listeriolysin (LLO), and Staphylococcal enterotoxin SED or SEC. Finally, the established AuNP-ICS was used to analyze 35 egg samples, and the results showed 5 positive samples and 30 negative samples. The AuNP-ICS rapid detection method established by our group had good specificity, high sensitivity, and convenience, and could be applied to the clinical sample detection of ALV-A.