mAb Targeting Research Articles

Characterization of a plant-derived monoclonal antibody targeting extracellular enveloped virions of Monkeypox virus.

In 2022, the global outbreak of monkeypox virus (MPXV) with increased human-to-human transmission triggered urgent public health interventions. Plant-derived monoclonal antibodies (mAbs) are being explored as potential therapeutic strategies due to their diverse mechanisms of antiviral activity. MPXV produces two key infectious particles: the mature virion (MV) and the extracellular enveloped virion (EV), both essential for infection and spread. Effective therapies must target both to halt replication and transmission. Our prior research demonstrated the development of a potent neutralizing mAb against MPXV MV. This study focuses on developing a plant-derived mAb targeting MPXV EV, which is critical for viral dissemination within the host and generally resistant to antibody neutralization. Our findings reveal that the mAb (H2) can be robustly produced in Nicotiana benthamiana plants via transient expression. The plant-made H2 mAb effectively targets MPXV EV by binding specifically to the A35 MPXV antigen. Importantly, H2 mAb shows notable neutralizing activity against the infectious MPXV EV particle. This investigation is the first to report the development of a plant-derived anti-EV mAb for MPXV prevention and treatment, as well as the first demonstration of anti-MPXV EV activity by an mAb across any production platform. It highlights the potential of plant-produced mAbs as therapeutics for emerging infectious diseases, including the MPXV outbreak.

Anti-Hcp1 Monoclonal Antibody Is Protective against Burkholderia pseudomallei Infection via Recognizing Amino Acids at Asp95-Leu114.

Melioidosis, a severe tropical illness caused by Burkholderia pseudomallei, poses significant treatment challenges due to limited therapeutic options and the absence of effective vaccines. The pathogen's intrinsic resistance to numerous antibiotics and propensity to induce sepsis during acute infections further complicate management strategies. Thus, exploring alternative methods for prevention and treatment is crucial. Monoclonal antibodies (mAbs) have emerged as a promising strategy for the prevention and treatment of infectious diseases. This study focused on generating three mAbs (13F1, 14G11, and 15D9) targeting hemolysin-coregulated protein 1 (Hcp1), a protein involved in the type VI secretion system cluster 1 (T6SS1) of B. pseudomallei. Notably, pretreatment with 13F1 mAb significantly reduced the intracellular survival of B. pseudomallei and inhibited the formation of macrophage-derived multinucleated giant cells (MNGCs). This protective effect was also observed in vivo. We identified a sequence of amino acids (Asp95-Leu114) within Hcp1 as the likely binding site for 13F1 mAb. In summary, our findings reveal that 13F1 mAb counteracts infection by targeting Hcp1, offering potential new targets and insights for melioidosis prevention.

Automated Bioanalytical Workflow for Ligand Binding-Based Pharmacokinetic Assay Development.

The growth of therapeutic monoclonal antibodies (mAbs) continues to accelerate due to their success as treatments for many diseases. As new therapeutics are developed, it is increasingly important to have robust bioanalytical methods to measure the pharmacokinetics (PK) of circulating therapeutic mAbs in serum. Ligand-binding assays such as enzyme-linked immunosorbent assays (ELISAs) with anti-idiotypic antibodies (anti-IDs) targeting the variable regions of the therapeutic antibody are sensitive and specific bioanalytical methods to measure levels of therapeutic antibodies in a biological matrix. However, soluble circulating drug mAb targets can interfere with the anti-IDs binding to the therapeutic mAb, thereby resulting in an underestimation of total drug concentration. Therefore, in addition to a high binding affinity for the mAb, the selection of anti-IDs and the assay format that are not impacted by soluble antigens and have low matrix interference is essential for developing a robust PK assay. Standardized automated approaches to screen and select optimal reagents and assay formats are critical to increase efficiency, quality, and PK assay robustness. However, there does not exist an integrated screening and analysis platform to develop robust PK assays across multiple formats. We have developed an automated workflow and scoring platform with multiple bioanalytical assay parameters that allow for ranking of candidate anti-IDs. A primary automated indirect electrochemiluminescence (ECL) was utilized to shortlist the anti-IDs that were selected for labeling and screening in pairs. A secondary screen using an ECL sandwich assay with labeled-anti-ID pairings was used to test multiple PK assay formats to identify the best anti-ID pairing/PK assay format. We developed an automated assay using fixed plate maps combined with a human-guided graphical user interface-based scoring system and compared it to a data-dependent scoring system using Gaussian mixture models for automated scoring and selection. Our approach allowed for screening of anti-IDs and identification of the most robust PK assay format with significantly reduced time and resources compared with traditional approaches. We believe that such standardized, automated, and integrated platforms that accelerate the development of PK assays will become increasingly important for supporting future human clinical trials.

Safety and Pharmacokinetic Assessment of the FIC CLDN18.2/4-1BB Bispecific Antibody in Rhesus Monkeys.

Gastric cancer is one of the most common cancers worldwide, particularly in China, with over half a million new cases and over 400 thousand deaths in 2022. Zolbetuximab, a first-in-class investigational monoclonal antibody (mAb) targeting tumor-associated antigen CLDN18.2 which is highly expressed on gastric cancer cells, was recently reported to meet the primary endpoint in Phase III trial as first-line treatment in CLDN18.2 positive and HER2-negative gastric cancers. In the present study, we developed a humanized bispecific antibody (bsAb) CLDN18.2/4-1BB named PM1032. PM1032 activates immune cells via CLDN18.2 mediated crosslinking of 4-1BB, a potent stimulator of T/NK cells. It induced strong immunological memory in multiple tumor-bearing animal models, indicating significant potential as an effective treatment for CLDN18.2 positive cancers such as gastric cancer. Since liver and gastrointestinal (GI) related toxicities were reported in 4-1BB and CLDN18.2 targeting programs during the clinical development, respectively, extensive pharmacokinetics (PK) and safety profile characterization of PM1032 was performed in rhesus monkeys. PM1032 had a half-life comparable to a conventional IgG1 mAb, and serum drug concentration increased in a dose-dependent pattern. Furthermore, PM1032 was generally well tolerated, with no significant abnormalities observed in toxicity studies, including the liver and stomach. In summary, PM1032 demonstrated good PK and an exceptional safety profile in rhesus monkeys supporting further investigation in clinical studies.

Abstract A150: Effects of radioimmunotherapy on human and canine osteosarcoma microenvironment

Abstract Introduction: Osteosarcoma (OS) is a type of primary bone cancer that predominantly affects children and adolescents with an unacceptably high mortality rate of 30% within 5 years of diagnosis. Though several treatment options including radiotherapy, chemotherapy, and amputation surgery exist, these are either ineffective or very invasive. Also, no significant advancements in treatment options have occurred in the last 30 years. The incidence of OS among companion dogs is much higher than in humans and phenotypically OS is very similar between humans and canines. We are using a comparative oncology approach to OS treatment by developing radioimmunotherapy (RIT) which targets the insulin-like growth factor receptor type 2 (IGF2R) on OS cells. IGF2R is abundant in human and canine OS and has shown promise a novel target for RIT of OS using a comparative oncology approach. It is therefore important to investigate the mechanistic underpinnings of RIT efficacy by investigating its interaction with OS microenvironment to develop improved RIT regimens for OS. Materials and Methods: Severe combined immunodeficiency (SCID) female mice bearing human and canine patient-derived OS xenografts were treated with RIT utilizing IF3, a novel full-length human mAb targeting IGF2R obtained by phage-display technology. The IF3 antibody was radiolabeled with an alpha emitter 225Actinium (225Ac) or a beta emitter 177Lutetim (177Lu) radionuclides. The tumors were removed at 1, 3 and 7 days post-RIT and the effects of RIT on IGF2R-positive tumor cells, OS stem cells, and immune components of the tumor microenvironment such as natural killer (NK) cells and tumor-associated macrophages (TAMs) were assessed using quantitative immunohistochemistry (IHC). Results: In the untreated tumors IHC detected the presence of IGF2R-positive tumor cells, OS stem cells, and immune components of the tumor microenvironment such as NK cells and TAMs, in both human and canine OS tumors obtained from mouse models. Quantitative analysis of IHC results in the tumors post-RIT demonstrated a time-dependent decrease in IGF2R-positive OS tumor cells and OS stem cells and an increase in TAMs and NK cells. Conclusions: Deciphering the mechanisms of RIT interaction with tumor microenvironment can assist in the design of the clinical trials in companion dogs with OS, which will be followed by the trials in children with this cancer. Also, it supports combining RIT with immunotherapy targeting OS stem cells and other immune components of tumor microenvironments such as NK cells and TAMS, which can result in improved patient outcomes. Citation Format: Sabeena Giri, Kevin J.H. Allen, Maruti Uppalapati, Ekaterina Dadachova. Effects of radioimmunotherapy on human and canine osteosarcoma microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A150.

A novel anti-galectin-9 immunotherapy limits the early progression of pancreatic neoplastic lesions in transgenic mice.

Pancreatic adenocarcinoma (PDAC) is a devastating disease with an urgent need for therapeutic innovation. Immune checkpoint inhibition has shown promise in a variety of solid tumors, but most clinical trials have failed to demonstrate clinical efficacy in PDAC. This low efficacy is partly explained by a highly immunosuppressive microenvironment, which dampens anti-tumor immunity through the recruitment or induction of immunosuppressive cells, particularly regulatory T cells (Tregs). In this context, our laboratory has developed a novel immunotherapeutic strategy aimed at inhibiting the suppressive activity of Tregs, based on a patented (EP3152234B1) monoclonal antibody (mAb) targeting galectin-9 (LGALS9). CD4+ conventional T cells (TCD4 or Tconv), Treg ratio, and LGALS9 expression were analyzed by immunohistochemistry (IHC) and cytometry in blood and pancreas of K-rasLSL.G12D/+;Pdx-1-Cre (KC) and K-rasWildType (WT);Pdx1-Cre (WT) mice aged 4-13 months. Pancreatic intraepithelial neoplasm (PanIN) progression and grade were quantified using FIJI software and validated by pathologists. The anti-galectin-9 mAb was validated for its use in mice on isolated murine C57BL/6 Treg by immunofluorescence staining and cytometry. Its specificity and functionality were validated in proliferation assays on rLGALS9-immunosuppressed murine Tconv and in suppression assays between murine Treg and Tconv. Finally, 2-month-old KC mice were treated with anti-LGALS9 and compared to WT mice for peripheral and infiltrating TCD4, Treg, and PanIN progression. IHC and cytometry revealed a significant increase in LGALS9 expression and Treg levels in the blood and pancreas of KC mice proportional to the stages of precancerous lesions. Although present in WT mice, LGALS9 is expressed at a basal level with low and restricted expression that increases slightly over time, while Treg cells are few in number in their circulation and even absent from the pancreas over time. Using our anti-LGALS9 mAb in mice, it is shown that (i) murine Treg express LGALS9, (ii) the mAb could target and inhibit recombinant murine LGALS9, and (iii) neutralize murine Treg suppressive activity. Finally, the anti-LGALS9 mAb in KC mice reduced (i) LGALS9 expression in pancreatic cancer cells, (ii) the Treg ratio, and (iii) the total surface area and grade of PanIN. We demonstrate for the first time that an anti-LGALS9 antibody, by specifically targeting endogenous LGALS9 tumor and exogenous LGALS9 produced by Treg, was able to limit the progression of pancreatic neoplastic lesions in mice, opening up new prospects for its use as an immunotherapeutic tool in PDAC.

Patterns of CS1 Expression on Natural Killer Cells and Correlated Factors in Plasma Cell Dyscrasias

Abstract BackgroundCS1 is overexpressed on myeloma cells, making it a promising target for immunotherapy in plasma cell dyscrasias (PCDs). However, monoclonal antibody (mAb) targeting CS1 showed limited efficacy when used alone. In addition, few clinical data of anti-CS1 chimeric antigen receptor T cell (CAR-T) have been published to date, suggesting that there may be some barriers to CS1-targeted immunotherapy. From previous studies, CS1 is expressed on some normal lymphocytes and plays an immunomodulatory role, and NK cells in particular play an important role in CS1-mediated immunity against MM. However, there are few studies on CS1 expression by NK cells in patients with plasma cell disease. Therefore, we investigated the expression of CS1 on NK cells, normal lymphocytes, and plasma cells in plasma cell dyscrasias and examined factors related to the CS1 expression level of NK cells and its relationship to survival. Methods Between January 2018 and September 2019, a retrospective analysis was performed in 236 patients with multiple myeloma (MM) and 30 patients with other PCDs, including systemic light chain amyloidosis (AL), monoclonal gammopathy of undetermined significance (MGUS), POEMS syndrome, and monoclonal gammopathy of renal significance (MGRS). CS1 expression levels in NK cells, lymphocytes, and monoclonal plasma cells were assessed in patients with plasma cell dyscrasias. Membrane-bound CS1 of these three cell types was measured by multiparameter flow cytometry. Associations of CS1 expression with clinical and laboratory features were determined by univariate and multivariate analyses. Correlations between CS1 expression and survival were computed using the Cox proportional hazards regression and by plotting Kaplan-Meier survival plots. Results When subgroups of MM and other PCDs were compared, there were no significant differences in CS1 expression in lymphocytes and NK cells alone. MM Patients had significantly higher CS1 expression levels in monoclonal plasma cells than other PCDs patients. In both MM and other PCDs patients, CS1 expression was significantly higher on monoclonal plasma cells than on NK cells and lymphocytes. There was a significant correlation between CS1 expression of monoclonal plasma cells (r = 0.60; P < 0.001) and that of NK cells (r = 0.79; P < 0.001) on univariate and multivariate analysis(Figure 1). Factors such as cytogenetic abnormalities, disease progression, or survival were not associated with CS1 expression level on NK cells. Neither CS1 expression level of monoclonal plasma cells nor that of NK cells was an independent predictor of patient survival. Conclusion We demonstrated that CS1 expression level in NK cells is consistent in plasma cell dyscrasias. CS1 expression in NK cells positively correlates with that in clonal plasma cells. This positive correlation may have implications for the development of immunotherapy targeting CS1.

Anti-Hemojuvelin Monoclonal Antibody DISC-0974 Elicits a Durable and Consistent Response with Repeated Dosing in Cynomolgus Monkeys

Introduction DISC-0974 is a monoclonal antibody (mAB) targeting hemojuvelin and is currently in clinical trials evaluating effects on anemia in patients with myelofibrosis or chronic kidney disease. Inhibition of hemojuvelin (HJV) results in decreased hepcidin and reduction in ferroportin degradation, enabling more iron to be exported out of cells and into the bloodstream for transport to other cells and tissues. Methods A study was conducted in healthy, naïve cynomolgus monkeys to evaluate the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of DISC-0974 following once-monthly dosing for 6 months. Male and female cynomolgus monkeys (n=6/sex/dose group) were intravenously administered DISC-0974 at doses of 0, 0.3, 1, or 6 mg/kg on Days 1, 29, 57, 85, 113, and 141. An additional IV dose was administered on Day 165 for males and Day 166 for females. Dose levels were selected where the high dose would maximally mobilize iron for sustained periods over the treatment regimen, with lower doses selected to provide a dose-response. Male and female monkeys (n=4/sex/group) were euthanized on Day 168 and 169, respectively, and subgroups were monitored for an additional 6 months (n=2/sex/group) to evaluate reversibility of any effects. PK was evaluated after the 1 st, 3 rd, and 6 th dose in all monkeys, as well as during the 6-month period after the final dose in the remaining monkeys. PD effects (i.e., serum iron and transferrin saturation [TSAT]) were monitored at the same timepoints as those collected for PK. Results DISC-0974 was well tolerated, and no DISC-0974-related adverse effects were observed. Systemic exposure, based on area under the curve (AUC) and maximum plasma concentrations (C max), increased with dose. Systemic exposures were achieved that reached and exceeded those observed in clinical trials. The high dose of 6 mg/kg achieved systemic exposures, based on AUC, that were >15-fold those observed in humans. Half-lives were consistent with an mAb, and repeated exposure did not result in accumulation with monthly dosing. Increases in serum iron (up to approximately 3-fold baseline) (Figure 1) and TSAT (>90%), representative of PD activity, correlated with systemic exposures of DISC-0974, with similar activity maintained after each of the 7 monthly doses. Conclusion Overall, these data demonstrate that DISC-0974 is well tolerated after repeated monthly exposure in cynomolgus monkeys, and an appropriate and durable PK/PD profile was observed, supporting clinical development of chronic once-monthly dosing.

Tissue Factor Pathway Inhibitor (TFPI) Beta and Gamma Isoforms Contribute to Bleeding in Hemophilia Mice

Key Points: Expression of any single TFPI isoform (α, β, or γ) is sufficient for mouse survival.Two-Kunitz domain TFPI isoforms, anchored β and circulating γ, each contribute to hemophilia bleeding.In vivo, TFPIβ is a more potent anticoagulant than TFPIα. Upon injury, tissue factor (TF) triggers coagulation by complexing with factor (F)VIIa to activate FIX and FX, leading to thrombin generation and a fibrin clot. Tissue factor pathway inhibitor limits TF-triggered coagulation. Humans and mice express transcripts encoding for 3-Kunitz domain TFPIα and membrane-anchored 2-Kunitz TFPIβ. Mice also express transcripts encoding untethered, 2-Kunitz TFPIγ. In humans, proteolysis of TFPIα and/or β produces “γ-like” 2-Kunitz TFPI. Hemophilia A and B, caused by deficiency in factors VIII and IX, respectively, are congenital X-linked recessive bleeding disorders that affect an estimated 1.1 million males worldwide. Hemophilia can cause excessive bleeding spontaneously or in response to trauma that can be life-threatening or significantly decrease the quality of life. Approved treatments for hemophilia include factor replacement, bypassing agents, the bispecific antibody Emicizumab, and recently approved gene therapies. Also in development are agents that reduce endogenous anticoagulant activity to counter the procoagulant deficiency of hemophiliacs, including monoclonal antibodies (Mab) to TFPI. These Mab target all TFPI isoforms to improve coagulation and correct bleeding in hemophilia patients, but with some demonstrated thrombosis risk. We investigated if TFPI-isoform-specific inhibition could provide equivalent efficacy which might reduce thrombotic risk. We generated TFPI-isoform-specific exon deletions in mice bred into hemophilia (FVIII-null) background. We find mice expressing any single TFPI isoform (α, β, or γ) appear healthy and reproduce. Our in vivo tail-vein re-bleeding assay is based on serial clot disruptions over 15 minutes, which we have shown is sensitive to anticoagulant and antiplatelet treatment, as well as factor replacement therapy. In this assay, we find C57Bl/6 WT mice clot 25.6 + 0.8 times (mean + SEM), whereas FVIII-null hemophilia mice clot 5.3 +0.4 times. In hemophilia mice, TFPIα-specific deletion is without impact, forming clots 5.1 + 0.8 times, whereas TFPIβ-specific or TFPIγ-specific deletion improves clot formation to 16.8 + 2.0 and 15.2 + 1.5 times, respectively (p<0001). Even hemophilia mice with heterozygotic deletion of the β exon form clots 11.8 + 0.8 times (p<0001). Thus, in vivo 2-Kunitz TFPI isoforms, anchored β and untethered γ each provides greater anticoagulant activity and impact on bleeding than 3-Kunitz TFPIα. Ex vivo plasma-based thrombin generation assays show that TFPIγ provides more anticoagulant activity than TFPIα. As shown in the figure, the deletion of TFPIγ increases thrombin generation to a greater extent than the deletion of TFPIα. The addition of an anti-TFPI antibody that effectively blocks TFPI activity shows a significant increase in endogenous thrombin potential in the plasma of TFPIαβ-deleted (γ-only) mice (370 nM.min), versus TFPIβγ-deleted (α-only) plasma (70 nM.min) and TFPIαγ-deleted (β-only) mice plasma (22 nM.min), (p<0.001). As expected, membrane-anchored TFPIβ is not detected in plasma. It is well established that 3-Kunitz TFPIα is a potent anticoagulant in plasma-based clotting time assays, while truncated 2-Kunitz TFPI, like mouse TFPIγ, appears inactive. Based on such assays, it was unexpected that TFPIγ-only expressing mice thrive, and in vivo, TFPIα is not the dominant endogenous anticoagulant. If mouse models are to be useful in providing insights into human hemostasis and TF-associated diseases, then it is critical to understand the similarities and differences between mice and humans. As humans do not express TFPIγ, the observed impact of depleting circulating 2-Kunitz TFPI on bleeding and thrombin generation in hemophilic mice is unlikely to translate to man. By contrast, our results demonstrating that, in vivo, TFPIβ is a more potent anticoagulant than TFPIα in mice has potential therapeutic implications in humans.

Monoclonal Antibodies Targeting LIGHT Block Ltbr Signalling and Eliminate Acute Myeloid Leukemia Stem Cells

Introduction Acute myeloid leukemia (AML) is a molecular heterogeneous disease that originates from the malignant transformation of a hematogenic stem- or progenitor cells resulting in a leukemia stem cell (LSC). LSCs are a main cause of resistance to therapy and relapse. Despite recent advances in targeted therapy and immunotherapy, prognosis for elderly patients not fit do undergo intensive therapy remains poor. We recently documented that targeting CD70, the ligand of the tumor necrosis factor receptor (TNFR) CD27, by the antibody-dependent cell-mediated cytotoxicity (ADCC)-enhanced monoclonal antibody (mAb) cusatuzumab eliminates LSC. We now studied the role of the related TNFR lymphotoxin b receptor (LTbR) with its ligand LIGHT in the pathogenesis of AML in mice and humans and developed a LIGHT targeting mAb to specifically eliminate LSCs. Experimental design We investigated the impact of LIGHT/LTbR signalling in AML development by using the retrovirally-induced MLL-AF9 syngeneic AML mouse model. Light-, Ltbr -deficient and control MLL-AF9 transduced LSCs were injected intravenously into non-irradiated BL/6 recipient mice. AML development was evaluated by analysing MLL-AF9-GFP-positive leukemic cells in blood, spleen, and bone marrow (BM). BM LSC were phenotypically characterized by FACS and functionally by performing colony forming unit (CFU) assays in vitro and by secondary transplantations of LSCs including limited dilution assays (LDA) in vivo. Correlative survival analysis for LIGHT and LTbR expression was performed on a publicly available gene dataset (GSE6891). LIGHT and LTBR protein expression was analysed on primary AML CD34 + leukemic stem and progenitor cells (LSPCs) and control human CD34 + hematopoietic stem cell and progenitor cells (HSCPs). LIGHT/LTbR signalling was blocked by either siRNA treatment or by treatment with novel LIGHT-targeting mAb. The effect of blocking LIGHT/LTbR-signalling was analysed in vitro by CFU assays, RNA-sequencing and xenotransplant experiments in vivo. LIGHT-targeting mAbs were generated by immunization of llamas and phage display selections followed by cloning of FABs into a human backbone (hIgG1). Clones were selected based on their blocking potency of receptor/ligand interaction using competition ELISA and FACS-based cell binding affinity assays. ADCC-enhanced anti-LIGHT mAbs were generated via POTELLIGENT® technology. Anti-LIGHT mAbs that specifically block the interaction of LIGHT/LTBR with deficiency in effector functions (Fc-death) were produced by amino acid substitutions in the CH2 domain of the parental clone. Results LTbR was expressed by HSCs and AML LSCs. In contrast, LIGHT was only expressed on murine and human LSCs but not normal HSCs. The absence of LIGHT or its receptor LTbR on LSCs resulted in significantly prolonged survival and decreased numbers of LSC in BM of AML mice. Light -/- and Ltbr -/- LSCs had a reduced CFU capacity compared to control LSCs. Similarly, inhibition of LIGHT-signalling on LSCs via LTbR-Ig significantly reduced colony formation. Moreover, secondary transplantation of Light -/-LSCs resulted in prolonged survival compared to Light-proficient LSCs. An LDA analysis revealed a 11-fold reduction of the LSC frequency in BM in the absence of LIGHT or LTBR. Similarly, inhibiting LIGHT signalling by siLIGHT RNA in human AML LSPCs resulted in reduced CFU capacity compared to control siRNA treatment. In addition, a GEO analysis revealed that low LIGHT or LTbR mRNA levels correlated with improved overall survival in AML patients. After immunizations of llamas and phage-display selection three humanized anti-LIGHT mAbs with high blocking and high binding efficiency were selected (mAb10F11 and 12C2 specific for human LIGHT and the cross-reactive murine/human 9F6 mAB). Incubation of LSCs with the blocking mAbs significantly reduced CFU capacity. HSPCs from healthy donors remained unaffected by the treatment. A gene expression analysis revealed that blocking LIGHT/LTbR-signalling in LSCs reduced stemness and promoted differentiation. In addition, ADCC-enhanced mAbs activated NK cells to eliminate LIGHT-expressing LSCs. Our findings reveal that LIGHT/LTbR-signalling is crucial for the pathogenesis of AML and especially for the maintenance and expansion of LSCs. We developed and validated novel LIGHT targeting mAbs in vitro and in vivo for further development in clinical trials.

Development and characterisation of highly specific monoclonal antibody-based immunoassays for the detection and quantification of genistein-7-O-[α-rhamnopyranosyl-(1→6)]-β-glucopyranoside in Derris scandens (Roxb.) Benth.

The stem of the plant species Derris scandens (Roxb.) Benth. (DS) contains genistein-7-O-[α-rhamnopyranosyl-(1→6)]-β-glucopyranoside (GTG), which is a unique marker. Previous analyses of GTG using antibody-based immunoassays were compromised because of their high cross-reactivity with structurally related compounds of DS, thereby limiting their applicability in DS quality control. Conjugation of GTG with carrier proteins was achieved using the Mannich reaction to produce a highly specific monoclonal antibody (mAb) targeting GTG (anti-GTG mAb). The anti-GTG mAb was generated using hybridoma technology and characterised using an indirect competitive enzyme-linked immunosorbent assay (icELISA). Both lateral-flow immunoassay (LFIA) and icELISA were developed to detect and quantify GTG in DS raw materials and associated products. icELISA using the anti-GTG mAb showed 100% specificity for GTG, with only 1.77% cross-reactivity with genistin and less than 0.01% cross-reactivity with other compounds. icELISA demonstrated a linear range for GTG determination between 62.5 and 2000 ng/mL. The limits of detection (LOD) and quantification were 49.68 and 62.50 ng/mL for GTG, respectively. The precision of the analysis ranged from 1.28% to 4.20% for repeatability and from 1.03% to 7.05% for reproducibility. The accuracy of the analysis ranged from 101.97% to 104.01% for GTG recovery. GTG levels determined via icELISA were consistent with those confirmed via high-performance liquid chromatography (HPLC) (R2 = 0.9903). Moreover, the LOD of LFIA for GTG was 500 ng/mL. Immunoassays utilising specific anti-GTG mAbs were successfully developed, including LFIA for rapid GTG detection and icELISA for GTG quantification.

AK117 (anti-CD47 monoclonal antibody) in Combination with Azacitidine for Newly Diagnosed Higher Risk Myelodysplastic Syndrome (HR-MDS): AK117-103 Phase 1b Results

Background: CD47, an immune checkpoint overexpressed by tumor cells, plays a crucial role in evading the antitumor immune response. AK117, an IgG4 monoclonal antibody (mAb) targeting CD47, has emerged as a potential best-in-class therapy with high affinity binding to CD47 and without inducing hemagglutination effects. By blocking CD47, AK117 effectively triggers macrophage-mediated phagocytosis of hematological malignancy cells, and in combination with azacitidine (AZA), it synergistically enhances the activation of “eat me” signals. In this study, we present the Phase 1b data from the AK117-103 study conducted in patients with newly diagnosed higher risk myelodysplastic syndrome (HR-MDS). Methods: Eligible patients with newly diagnosed intermediate/high/very high-risk MDS, determined by the Revised International Prognostic Scoring System (IPSS-R) prognostic risk score >3, were enrolled to receive combination therapy with AK117 and AZA. AK117 was administered intravenously (IV) following various dosing regimens (20-30 mg/kg once weekly [QW], or 20-45 mg/kg every 2 weeks [Q2W], or 30 mg/kg every 4 weeks [Q4W]), whereas AZA was administered subcutaneously (SC) at the standard dosage (75 mg/m 2, Day 1-7, Q4W). The primary endpoints of this study were safety/tolerability and the complete remission (CR) rate based on the International Working Group (IWG) 2006 response criteria. Results: As of June 9, 2023, 72 patients were enrolled (median age: 66 years, 69.4% males). Most patients exhibited abnormal hematologic conditions at baseline, including decreased hemoglobin, neutrophil count, and platelet count. Specifically, 69.4% of patients had Grade ≥3 anemia, 61.1% had Grade ≥3 neutrophil count decrease, and 47.2% had Grade ≥3 platelet count decrease. For the recommended phase 2 dose (RP2D) group, treated with a combination of AK117 30 mg/kg Q2W and AZA, 30 patients were enrolled with a median age of 67 years, 80.0% of whom were males. According to IPSS-R risk assessment, 33.3%, 23.3%, and 43.3% of patients had intermediate, high, and very high risk, respectively. 30.0% of patients had poor-risk cytogenetics, with 23.0% of those being complex. After a median follow-up of 6.6 months (ranging from 0.4-17.2 months), anemia (a primary adverse event associated with CD47 blocking antibodies) occurred in 30.6% (22/72) of the patients, of which Grade ≥ 3 anemia accounted for 22.2% (16/72). Other commonly observed treatment-emergent adverse events (TEAEs) (≥30%) included decreased neutrophil count (77.8%), decreased white blood cell count (72.2%), decreased platelet count (69.4%), pyrexia (58.3%), decreased lymphocyte count (41.7%), constipation (41.7%), and vomiting (31.9%). Three patients (4.2%) discontinued treatment due to TEAEs. The efficacy of AK117 was compared at different dosages, and ultimately, 30mg/kg Q2W was chosen as the RP2D. As of June 21, 2023, among 27 evaluable patients, the CR rate was 48.1%. 8 patients achieved marrow CR (5 also with hematologic improvement [HI]), and 2 patients achieved HI alone. Among 24 evaluable patients with ≥ 3 months follow-up, the CR rate further escalated to 54.2%. Of 13 evaluable patients who initially required red blood cells (RBC) transfusions, 8(61.5%) became independent of RBC transfusion. Conclusions: AK117 in combination with AZA was well tolerated with low incidence of anemia and demonstrated promising efficacy in patients with newly diagnosed HR-MDS.

Development of a Novel CLDN18.2-directed Monoclonal Antibody and Antibody-Drug Conjugate for Treatment of CLDN18.2-Positive Cancers.

Gastric and pancreatic cancers are malignancies of high unmet clinical need. Expression of CLDN18.2 in these cancers, coupled with it's absence from most normal tissues, provides a potential therapeutic window against this target. We present preclinical development and characterization of a novel therapeutic mAb and antibody-drug conjugate (ADC) targeting CLDN18.2. A humanized CLDN18.2 specific mAb, CLDN18.2-307-mAb, was generated through immunization in mice followed by full humanization of the mouse mAb sequences. Antibody clones were screened by flow cytometry for selective binding to membrane bound CLDN18.2. A CLDN18.2-directed ADC (CLDN18.2-307-ADC) was also generated by conjugating MMAE to CLDN18.2 mAb using a cleavable linker. Tissue expression of CLDN18.2 was determined by IHC assay using a CLDN18.2-specific mAb. CLDN18.2-307-mAb binds with high affinity to CLDN18.2-positive (CLDN18.2+) cells and induces antibody-dependent cell-mediated cytotoxicity (ADCC). Treatment with this CLDN18.2-mAb blocked the growth of CLDN18.2+ gastric and pancreas cancer cell line xenograft (CDX) models. Upon binding to the extracellular domain of this target, the CLDN18.2-ADC/CLDN18.2 protein was internalized and subsequently localized to the lysosomal compartment inducing complete and sustained tumor regressions in CLDN18.2+ CDXs and patient-derived pancreatic cancer xenografts (PDX). A screen of human cancer tissues, by IHC, found 58% of gastric, 60% of gastroesophageal junction, and 20% of pancreatic adenocarcinomas to be positive for membrane expression of CLDN18.2. These data support clinical development of the CLDN18.2-307-mAb and CLDN18.2-307-ADC for treatment of CLDN18.2+ cancers. Both are now being investigated in phase I clinical studies.

Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326).

Syphilis, a sexually transmitted infection caused by the spirochete Treponema pallidum (Tp), is resurging globally. Tp's repertoire of outer membrane proteins (OMPs) includes BamA (β-barrel assembly machinery subunit A/TP0326), a bipartite protein consisting of a 16-stranded β-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptide transport-associated) domains. BamA ECL4 antisera promotes internalization of Tp by rabbit peritoneal macrophages. Three overlapping BamA ECL4 peptides and a two-stage, phage display strategy, termed "Epivolve" (for epitope evolution) were employed to generate single-chain variable fragments (scFvs). Additionally, antisera generated by immunizing mice and rabbits with BamA ECL4 displayed by a Pyrococcus furiosus thioredoxin scaffold (PfTrxBamA/ECL4). MAbs and antisera reactivities were evaluated by immunoblotting and ELISA. A comparison of murine and rabbit opsonophagocytosis assays was conducted to evaluate the functional ability of the Abs (e.g., opsonization) and validate the mouse assay. Sera from Tp-infected mice (MSS) and rabbits (IRS) were evaluated for ECL4-specific Abs using PfTrxBamA/ECL4 and overlapping ECL4 peptides in immunoblotting and ELISA assays. Each of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of PfTrxBamA/ECL4. One mAb, containing a unique amino acid sequence in both the light and heavy chains, showed activity in the murine opsonophagocytosis assay. Mice and rabbits hyperimmunized with PfTrxBamA/ECL4 produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides, including S2. In contrast, Abs generated during Tp infection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope. Epivolve produced mAbs target subdominant opsonic epitopes in BamA ECL4, a top syphilis vaccine candidate. The murine opsonophagocytosis assay can serve as an alternative model to investigate the opsonic potential of vaccinogens. Detailed characterization of BamA ECL4-specific Abs provided a means to dissect Ab responses elicited by Tp infection.

SYST-07 PLASMA MEMBRANE PROFILING REVEALS A TARGETABLE CELL-SURFACE PHENOTYPE OF WNT-MEDULLOBLASTOMA

Abstract WNT-medulloblastoma has an excellent prognosis, with an overall survival rate of 90% among children receiving standard-of-care surgical resection, radiotherapy, and chemotherapy. While curative, this treatment is associated with major, long-term, debilitating side effects. Therefore, it is crucial we develop effective, less toxic therapies for these children. The Gilbertson lab has previously shown that WNT-medulloblastomas are devoid of a Blood-Brain-Barrier (BBB). This disruption allows large molecules such as monoclonal antibodies (mAbs) to penetrate WNT-medulloblastomas. To discover WNT-medulloblastoma specific antigens for mAb targeting, we have first generated bulk and single-cell RNA sequencing profiles of the wild-type and WNT-medulloblastoma-prone mossy-fibre progenitor lineages in the lower rhombic lip, as well as human medulloblastoma patient derived orthotopic xenograft (PDOX) tumours. We next generated plasma membrane proteome profiles of hindbrain germinal zones and WNT-medulloblastomas. Plasma Membrane Profiling (PMP) deploys an isobaric chemical labelling approach -Tandem Mass Tags (TMT) - that allows multiplexing of up to sixteen variables per mass spectrometry experiment and ex vivo TMT-labelling of primary cells. Using this approach, we have mapped the cell surface proteome of our medulloblastoma models. Interrogation of these developmental and tumour data has unmasked WNT-medulloblastoma associated antigens that are specific for prenatal brain and WNT-medulloblastoma, but that are not expressed on critical postnatal tissues. Our workflow integrates multi-omic data sets to uncover a targetable landscape on the surface of WNT-medulloblastoma cells that we have cross-validated with a large cohort of patient RNA sequencing profiles and histological samples of both mouse and human tumours. To translate this dataset to real patient benefit, we are generating high affinity antibodies of these validated cell-surface proteins for assessment in our pre-clinical mouse models. Through our comparative analyses we have additionally identified non-WNT-specific targets and thus aim to expand this approach to identifying targets in other more agressive CNS tumours and brain metastases.

Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis

Morphine is a frequently used analgesic that activates the mu-opioid receptor (MOR), which has prominent side effects of tolerance. Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance, currently, there is no effective therapy to treat morphine tolerance. In the current study, we aimed to develop a monoclonal antibody (mAb) precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms. We successfully prepared a mAb targeting MOR, named 3A5C7, by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization, and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation. Treatment of two cell lines, HEK293T and SH-SY5Y, with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2 (GRK2)/β-arrestin2-dependent mechanism, as demonstrated by immunofluorescence staining, flow cytometry, Western blotting, coimmunoprecipitation, and small interfering ribonucleic acid (siRNA)-based knockdown. This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR. We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid. Western blot, enzyme-linked immunosorbent assays, and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase, the in vitro biomarker of morphine tolerance, via the GRK2/β-arrestin2 pathway. Furthermore, in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alleviated morphine tolerance in mice, and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence. Finally, intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/β-arrestin2 pathway. Collectively, our study provided a therapeutic mAb, 3A5C7, targeting MOR to treat morphine tolerance, mediated by enhancing morphine-induced MOR endocytosis. The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.

MDB-06. PLASMA MEMBRANE PROFILING REVEALS A TARGETABLE CELL-SURFACE PHENOTYPE OF WNT-MEDULLOBLASTOMA

Abstract WNT-medulloblastoma has an excellent prognosis, with an overall survival rate of 90% among children receiving standard-of-care surgical resection, radiotherapy, and chemotherapy. While curative, this treatment is associated with major, long-term, debilitating side effects. Therefore, it is crucial we develop effective, less toxic therapies for these children. The Gilbertson lab has previously shown that WNT-medulloblastomas are devoid of a Blood-Brain-Barrier (BBB). This disruption allows large molecules such as monoclonal antibodies (mAbs) to penetrate WNT-medulloblastomas. To discover WNT-medulloblastoma specific antigens for mAb targeting, we have first generated bulk and single-cell RNA sequencing profiles of the wild-type and WNT-medulloblastoma-prone mossy-fibre progenitor lineages in the lower rhombic lip, as well as human medulloblastoma patient derived orthotopic xenograft (PDOX) tumours. We next generated plasma membrane proteome profiles of hindbrain germinal zones and WNT-medulloblastomas. Plasma Membrane Profiling (PMP) deploys an isobaric chemical labelling approach -Tandem Mass Tags (TMT) - that allows multiplexing of up to sixteen variables per mass spectrometry experiment and ex vivo TMT-labelling of primary cells. Using this approach, we have mapped the cell surface proteome of our medulloblastoma models. Interrogation of these developmental and tumour data has unmasked WNT-medulloblastoma associated antigens that are specific for prenatal brain and WNT-medulloblastoma, but that are not expressed on critical postnatal tissues. Our workflow integrates multi-omic data sets to uncover a targetable landscape on the surface of WNT-medulloblastoma cells that we have cross-validated with a large cohort of patient RNA sequencing profiles and histological samples of both mouse and human tumours. To translate this dataset to real patient benefit, we are generating high affinity antibodies of these validated cell-surface proteins for assessment in our pre-clinical mouse models. mAbs directed towards essential receptors may have a therapeutic effect alone, however we will additionally modify mAbs to specifically deliver cytotoxic therapeutics - curing children whilst sparing healthy brain tissue.

Selected Articles from This Issue

Highlights| May 04 2023 Selected Articles from This Issue Author & Article Information Online ISSN: 1538-8514 Print ISSN: 1535-7163 ©2023 American Association for Cancer Research2023American Association for Cancer Research Mol Cancer Ther (2023) 22 (5): 553. https://doi.org/10.1158/1535-7163.MCT-22-5-HI Related Content A commentary has been published: Discovery of a Small-Molecule Inhibitor Targeting the Androgen Receptor N-Terminal Domain for Castration-Resistant Prostate Cancer A commentary has been published: Novel Arginase Inhibitor, AZD0011, Demonstrates Immune Cell Stimulation and Antitumor Efficacy with Diverse Combination Partners A commentary has been published: Preclinical Evaluation of ON203, A Novel Bioengineered mAb Targeting Oxidized Macrophage Migration Inhibitory Factor as an Anticancer Therapeutic Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record May 4 2023 Citation Selected Articles from This Issue. Mol Cancer Ther 1 May 2023; 22 (5): 553. https://doi.org/10.1158/1535-7163.MCT-22-5-HI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search The oxidized macrophage migration inhibitory factor (oxMIF) is an emerging therapeutic target for many cancers due to oxMIF's selective occurrence in tumor lesions. Here, Rossmueller and colleagues describe ON203, a novel anti-oxMIF antibody with optimized physicochemical characteristics, in-vitro safety, and enhanced ADCC and ADCP effector functions. ON203 significantly reduced tumor growth and tumor cell proliferation as well as tumor cell intravasation and neo-angiogenesis in xenograft models. These findings highlight the potential of ON203 as a standalone immunotherapy or for enhancing efficacy in combination with angiogenesis and immune checkpoint-inhibitors in patients with solid tumors. Current mainstay treatment for prostate cancer is to suppress the androgen receptor (AR) signaling by castration and antiandrogens that target the ligand-binding domain (LBD). However, emergence of AR variants, such as AR-V7 lacking the LBD, have conferred severe drug resistance to all LBD-targeting AR inhibitors. AR-V7 has been established as a driver for the sustained AR signaling... You do not currently have access to this content.

B Cell Targeting mAb–siRNA Conjugates Reduced Autoantibody in Experimental Myasthenia

Abstract Conjugates of monoclonal antibodies (mAbs) and siRNA have been used for target cell-specific gene repression. B cells are integrally involved in the anti-Acetylcholine receptor autoantibody-mediated pathogenic development of autoimmune myasthenia gravis (MG). Here we show that simultaneous targeting of the critical receptors expressed on pathogenic B-cell subsets with the optimized dose of mAb-siRNA conjugates markedly depleted target B cells and substantially reduced pathogenic autoantibody in a mouse model of MG. The results show the potential of mAb-conjugates in the treatment of myasthenia gravis. Supported by grants from AFM-Telethon, France

Real-World Effectiveness of Fremanezumab in Patients with Migraine who Switched From Another mAb Targeting the CGRP Pathway (Subgroup Analysis From FINESSE) (P13-12.007)

Real-World Effectiveness of Fremanezumab in Patients with Migraine who Switched From Another mAb Targeting the CGRP Pathway (Subgroup Analysis From FINESSE) (P13-12.007)