Cellular Cytotoxicity Research Articles

Combining CD38 antibody with CD47 blockade is a promising strategy for treating hematologic malignancies expressing CD38.

CD38 and CD47 are expressed in many hematologic malignancies, including multiple myeloma (MM), B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia (ALL), and B-cell chronic lymphocytic leukemia (CLL). Here, we evaluated the antitumor activities of CD38/CD47 bispecific antibodies (BsAbs). Five suitable anti-CD38 antibodies for co-targeting CD47 and CD38 BsAb were developed using a 2 + 2 "mAb-trap" platform. The activity characteristics of the CD38/CD47 BsAbs were evaluated using in vitro and in vivo systems. Using hybridoma screening technology, we obtained nine suitable anti-CD38 antibodies. All anti-CD38 antibodies bind to CD38+ tumor cells and kill tumor cells via antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Five anti-CD38 antibodies (4A8, 12C10, 26B4, 35G5, and 65A7) were selected for designing CD38/CD47 BsAbs (IMM5605) using a "mAb-trap" platform. BsAbs had higher affinity and binding activity to the CD38 target than those to the CD47 target, decreasing the potential on-target potential and off-tumor effects. The CD38/CD47 BsAbs did not bind to RBCs and did not induce RBC agglutination; thus, BsAbs had much lower blood toxicity. The CD38/CD47 BsAbs had a greater ability to block the CD47/SIRPα signal in CD38+/CD47+ tumor cells than IMM01 (SIRPα Fc fusion protein). Through Fc domain engineering, CD38/CD47 BsAbs were shown to kill tumors more effectively by inducing ADCC and ADCP. IMM5605-26B4 had the strongest inhibitory effect on cellular CD38 enzymatic activity. IMM5605-12C10 had the strongest ability to directly induce the apoptosis of tumor cells. The anti-CD38 antibody 26B4 combined with the SIRPα-Fc fusion proteins showed strong antitumor effects, which were better than any of the mono-therapeutic agents used alone in the NCI-H929 cell xenograft model. The CD38/CD47 BsAbs exhibited strong antitumor effects; specifically, IMM5605-12C10 efficiently eradicated all established tumors in all mice. A panel of BsAbs targeting CD38 and CD47 developed based on the "mAb-tarp" platform showed potent tumor-killing ability in vitro and in vivo. As BsAbs had lower affinity for binding to CD47, higher affinity for binding to CD38, no affinity for binding to RBCs, and did not induce RBC agglutination, we concluded that CD38/CD47 BsAbs are safe and have a satisfactory tolerability profile.

Folate-Modified Curcumin-Loaded Nanoparticles for Overcoming Delivery Challenges in Cancer Treatment: A Narrative Review.

Curcumin, as an anti-tumor agent, is not widely used in cancer treatment due to the lack of effective levels of its metabolites in cancerous tissue. Addressing the barriers to the carrier and delivery of drugs to the specific sites of therapeutic action while reducing side effects is a priority. Folate receptor expression is high in malignant and low in normal cells. Folate as a targeted ligand could selectively target cancer cells. Thus, this narrative review aimed to provide an overview of the studies that have investigated the different types of folate-modified curcumin as a carrier and deliverer and their structural properties that enhance therapeutic drug efficacy. A literature search was performed using PubMed, Scopus, Web of Science, and Google Scholar databases. Thirty-eight preclinical studies addressing this topic were identified. The findings of the current review have shown that folate-modified nanoparticles containing curcumin as a promising therapeutic approach can be effective in improving different types of cancers. In vitro studies have shown a higher cellular uptake and cytotoxicity effect, higher cell inhibition, and anti-proliferation with a lower dosage of curcumin. In vivo studies have shown more tumor suppression and smaller tumor volume without toxicity after the administration of folate-modified nanoparticles containing curcumin. Future clinical trials are needed to confirm the beneficial effect of folate-modified curcumin as a new drug delivery platform for cancer treatment.

In situ borneol-modified polyester with antibacterial adhesion and long-term fungal-repellent properties

Polyester is widely used in biomedical, textile, and food packaging fields. Therefore, enhancing it with antimicrobial properties would be a significant advancement. In this paper, a series of borneol-triazine polyesters (BTPs) with different structures are synthesized through room temperature polycondensation. The structure and composition of BTPs are systematically characterized by 1H NMR, FTIR and GPC. Antimicrobial results reveal that the ability of BTPs to resist bacterial or fungal adhesion is directly related to the polymer structure. When the polymer chain of BTPs adopts a rigid structure, they exhibit excellent anti-adhesive and inhibitory performances against both Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus). Meanwhile, the as-synthesized BTPs poses a fungal-repelling effect on common fungal strains (Aspergillus niger) for up to 30 d. Further studies have shown that a stereochemical structure brought by borneol is key for imparting effective antimicrobial properties to BTPs. In addition, BTPs are non-leaching materials with low cellular cytotoxicity. Taking into consideration, BTP provides a potential strategy for preparing a new class of antimicrobial polyester materials.

N6-methyladenosine demethylase fat mass and obesity-associated protein suppresses hyperglycemia-induced endothelial cell injury by inhibiting reactive oxygen species formation via autophagy promotion

IntroductionHyperglycemia-induced endothelial cell injury is one of the main causes of diabetic vasculopathy. Fat mass and obesity-associated protein (FTO) was the first RNA N6-methyladenosine (m6A) demethylase identified; it participates in the pathogenesis of diabetes. However, the role of FTO in hyperglycemia-induced vascular endothelial cell injury remains unclear. Materials and methodsThe effects of FTO on cellular m6A, autophagy, oxidative stress, proliferation, and cytotoxicity were explored in human umbilical vein endothelial cells (HUVECs) treated with high glucose (33.3 mmol/mL) after overexpression or pharmacological inhibition of FTO. MeRIP-qPCR and RNA stability assays were used to explore the molecular mechanisms by which FTO regulates autophagy. ResultsHigh glucose treatment increased m6A levels and reduced FTO protein expression in HUVECs. Wild-type overexpression of FTO markedly inhibited reactive oxygen species generation by promoting autophagy, increasing endothelial cell proliferation, and decreasing the cytotoxicity of high glucose concentrations. The pharmacological inhibition of FTO showed the opposite results. Mechanistically, we identified Unc-51-like kinase 1 (ULK1), a gene responsible for autophagosome formation, as a downstream target of FTO-mediated m6A modification. FTO overexpression demethylated ULK1 mRNA and inhibited its degradation in an m6A-YTHDF2-dependent manner, leading to autophagy activation. ConclusionsOur study demonstrates the functional importance of FTO-mediated m6A modification in alleviating endothelial cell injury under high glucose conditions and indicates that FTO may be a novel therapeutic target for diabetic vascular complications.

Abstract PO-006: ABBV-319: A CD19–targeting glucocorticoid receptor modulator antibody-drug conjugate therapy for B-cell malignancies

Abstract Glucocorticoids such as prednisone (P) and dexamethasone (D) are key components in standard-of-care immuno-chemotherapy regimens (e.g., R-CHOP, EPOCH-R, Hyper-CVAD) for the treatment of B-cell malignancy. However, prolonged systemic glucocorticoid treatment is associated with several adverse events that often result in dose reduction and reduced therapeutic efficacy. ABBV-319 is a CD19-targeting antibody-drug conjugate (ADC) with a glucocorticoid receptor modulator (GRM) payload engineered to reduce glucocorticoid-associated toxicities while possessing three distinct mechanisms of action (MOA) to increase therapeutic efficacy: (1) CD19 receptor-mediated delivery of potent glucocorticoid receptor modulator (GRM) payload to induce apoptosis, (2) inhibition of B-cell receptor (BCR) stimulated CD19 signaling, and (3) enhanced Fc-mediated effector function via afucosylation of the antibody backbone. ABBV-319 elicited potent GRM-driven anti-tumor activity against multiple malignant B-cell lines in vitro as well as in cell line-derived xenografts (CDXs) and patient-derived xenografts (PDXs) in vivo. Remarkably, a single-dose of ABBV-319 induced sustained tumor regression and enhanced anti-tumor activity compared to repeat dosing of systemic prednisolone at the maximum tolerated dose (MTD) in mice. The unconjugated CD19 monoclonal antibody (mAb) also displayed anti-proliferative activity on a subset of B-cell lymphoma cell lines through the inhibition of PI3K signaling. Moreover, afucosylation of the CD19 mAb enhanced Fc-mediated antibody-dependent cellular cytotoxicity (ADCC), and this activity was maintained after conjugation with GRM payload. Notably, ABBV-319 displayed superior efficacy compared to afucosylated CD19 mAb in human CD34+ PBMC-engrafted NSG-tg(Hu-IL15) transgenic mice, demonstrating enhanced anti-tumor activity in vivo when multiple MOAs are enabled concurrently. ABBV-319 also showed potent anti-tumor activity across multiple B-cell lymphoma models, including non-germinal center B-cell (GCB) DLBCL and relapsed lymphoma treated with R-CHOP treatment. Collectively, these data support the ongoing evaluation of ABBV-319 for the treatment of B-cell malignancy in Phase I clinical trial (NCT05512390). Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: Chewei (Anderson) Chang. ABBV-319: A CD19–targeting glucocorticoid receptor modulator antibody-drug conjugate therapy for B-cell malignancies [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-006.

Broadening anticancer spectrum by preprocessing and treatment of T- lymphocytes expressed FcγRI and monoclonal antibodies for refractory cancers.

Chimeric antigen receptor T (CAR-T) cell therapies have achieved remarkable success in the treatment of hematological tumors. However, given the distinct features of solid tumors, particularly heterogeneity, metabolic aggressiveness, and fewer immune cells in tumor microenvironment (TME), the practical utility of CAR-T cells for solid tumors remains as a challenging issue. Meanwhile, although anti-PD-1 monoclonal antibody (mAb) has shown clinical efficacy, most mAbs also show limited clinical benefits for solid tumors due mainly to the issues associated with the lack of immune cells in TME. Thus, the infiltration of targeted immunological active cells into TME could generate synergistic efficacy for mAbs. We present a combinational strategy for solid tumor treatment, which combines armored-T cells to express Fc-gamma receptor I (FcγRI) fragment on the surfaces for targeting various tumors with therapeutically useful mAbs. Choosing CD20 and HER-2 as the targets, we characterized the in vitro and in vivo efficacy and latent mechanism of the combination drug by using flow cytometry, ELISA and other methods. The combination and preprocessing of armored T-cells with corresponding antibody of Rituximab and Pertuzumab exerted profound anti-tumor effects, which is demonstrated to be mediated by synergistically produced antibody-dependent cellular cytotoxicity (ADCC) effects. Meanwhile, mAb was able to carry armored-T cell by preprocessing for the infiltration to TME in cell derived xenograft (CDX) model. This combination strategy showed a significant increase of safety profiles from the reduction of antibody doses. More importantly, the present strategy could be a versatile tool for a broad spectrum of cancer treatment, with a simple pairing of engineered T cells and a conventional antibody.

Selective depletion of tumor-infiltrating regulatory T cells with BAY 3375968, a novel Fc-optimized anti-CCR8 antibody

Regulatory T cells (Tregs) are known to facilitate tumor progression by suppressing CD8+ T cells within the tumor microenvironment (TME), thereby also hampering the effectiveness of immune checkpoint inhibitors (ICIs). While systemic depletion of Tregs can enhance antitumor immunity, it also triggers undesirable autoimmune responses. Therefore, there is a need for therapeutic agents that selectively target Tregs within the TME without affecting systemic Tregs. In this study, as shown also by others, the chemokine (C–C motif) receptor 8 (CCR8) was found to be predominantly expressed on Tregs within the TME of both humans and mice, representing a unique target for selective depletion of tumor-residing Tregs. Based on this, we developed BAY 3375968, a novel anti-human CCR8 antibody, along with respective surrogate anti-mouse CCR8 antibodies, and demonstrated their in vitro mode-of-action through induction of potent antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) activities. In vivo, anti-mouse CCR8 antibodies effectively depleted Tregs within the TME primarily via ADCP, leading to increased CD8+ T cell infiltration and subsequent tumor growth inhibition across various cancer models. This monotherapeutic efficacy was significantly enhanced in combination with ICIs. Collectively, these findings suggest that CCR8 targeting represents a promising strategy for Treg depletion in cancer therapies. BAY 3375968 is currently under investigation in a Phase I clinical trial (NCT05537740).

The combination of three CD4-induced antibodies targeting highly conserved Env regions with a small CD4-mimetic achieves potent ADCC activity.

The majority of naturally-elicited antibodies against the HIV-1 envelope glycoproteins (Env) are non-neutralizing (nnAbs), because they are unable to recognize the Env timer in its native "closed" conformation. Nevertheless, it has been shown that nnAbs have the potential to eliminate HIV-1-infected cells by Antibody-Dependent Cellular Cytotoxicity (ADCC) provided that Env is present on the cell surface in its "open" conformation. This is because most nnAbs recognize epitopes that become accessible only after Env interaction with CD4 and the exposure of epitopes that are normally occluded in the closed trimer. HIV-1 limits this vulnerability by downregulating CD4 from the surface of infected cells, thus preventing a premature encounter of Env with CD4. Small CD4-mimetics (CD4mc) sensitize HIV-1-infected cells to ADCC by opening the Env glycoprotein and exposing CD4-induced (CD4i) epitopes. There are two families of CD4i nnAbs, termed anti-cluster A and anti-CoRBS Abs, which are known to mediate ADCC in the presence of CD4mc. Here, we performed Fab competition experiments and found that anti-gp41 cluster I antibodies comprise a major fraction of the plasma ADCC activity in people living with HIV (PLWH). Moreover, addition of gp41 cluster I antibodies to cluster A and CoRBS antibodies greatly enhanced ADCC mediated cell killing in the presence of a potent indoline CD4mc, CJF-III-288. This cocktail outperformed broadly-neutralizing antibodies and even showed activity against HIV-1 infected monocyte-derived macrophages. Thus, combining CD4i antibodies with different specificities achieves maximal ADCC activity, which may be of utility in HIV cure strategies.

Antibody Recognition of Human Epidermal Growth Factor Receptor-2 (HER2) Juxtamembrane Domain Enhances Anti-Tumor Response of Chimeric Antigen Receptor (CAR)-T Cells.

Chimeric antigen receptor (CAR) T cell therapy shows promise in treating malignant tumors. However, the use of human epidermal growth factor receptor-2 (HER2) CAR-T cells carries the risk of severe toxicity, including cytokine release syndrome, due to their "on-target off-tumor" recognition of HER2. Enhancing the quality and functionality of HER2 CARs could greatly improve the therapeutic potential of CAR-T cells. In this study, we developed a novel anti-HER2 monoclonal antibody, Ab8, which targets domain III of HER2, distinct from the domain IV recognition of trastuzumab. Although two anti-HER2 mAbs induced similar levels of antibody-dependent cellular cytotoxicity, trastuzumab-based CAR-T cells exhibited potent antitumor activity against HER2-positive cancer cells. In conclusion, our findings provide scientific evidence that antibody recognition of the membrane-proximal domain promotes the anti-tumor response of HER2-specific CAR-T cells.

Chiral silver nanoparticles with surface-anchored L(D)-Cys exhibit dissimilar biological characteristics in vitro but not in vivo

This work investigated the influence of surface chirality on cellular internalization, cytotoxicity, and tissue distribution of silver nanoparticles (AgNPs). D-cysteine and L-cysteine are chiral forms of the amino acid cysteine. These enantiomers exhibit distinct spatial arrangements, with D-cysteine having a different configuration from L-cysteine. This structural dissimilarity can lead to variations in how these forms interact with biological systems, potentially impacting their cytotoxic responses. Four distinct types of AgNPs were synthesized, each possessing a unique surface coating: pristine AgNPs (pAgNPs), L-cysteine coated AgNPs (AgNPs@L-Cys), D-cysteine coated AgNPs (AgNPs@D-Cys), and racemic AgNPs coated with both L-Cys and D-Cys (AgNPs@L/D-Cys). We found chiral-dependent cytotoxicity of AgNPs on J774A.1 cells. Specifically, AgNPs@L-Cys exhibited the highest toxicity, and AgNPs@D-Cys exhibited the lowest toxicity. Meanwhile, the cellular uptake of the AgNPs correlated nicely with their cytotoxicity, with AgNPs@L-Cys being internalized to the greatest extent while AgNPs@D-Cys displays the least internalization. Scavenger receptors and clathrin predominantly mediate the cellular internalization of these AgNPs. Strikingly, the dissimilar cellular internalization and cytotoxicity of AgNPs with different chirality were eliminated upon protein corona coverage. Notably, following intravenous injection in mice, these four types of AgNPs showed similar patterns among various organs due to the inevitable protein adsorption in the bloodstream. These findings underscored the pivotal role of surface chirality in governing the biological interactions and toxicity of AgNPs.

Au-doped Fe3O4 nanoparticle as an effective nanocarrier for delivery of 5-fluorouracil anti-liver cancer drug

5-Fluorouracil (5-FU) is an important chemotherapy that is classified as an antimetabolite and is widely used in cancer treatment, particularly for liver cancer. Yet, administering high doses of 5-FU for liver cancer can result in a range of adverse effects. Therefore, employing a suitable delivery system for this important drug can greatly enhance its safety and effectiveness, leading to improved treatment outcomes and better quality of life for patients. Au-doped Fe3O4 nanoparticles have attracted significant interest in scientific research due to their unique physical and chemical properties. This study presents a straightforward and cost-effective method for synthesizing Au-doped Fe3O4 nanoparticles by using a natural reducing agent. The study involved a comprehensive analysis of the Au-doped Fe3O4 nanoparticles using various techniques such as FT-IR, XRD, XPS, FE-SEM, TEM, UV–Vis, EDS, VSM, and DLS. The delivery experiments indicated a loading capacity of approximately 78 %. The release profile showed slow release profile, dependent on pH. Therefore, 41 % release was detected under physiological pH 7.4, while a much faster release was observed at pH 5.7 (84 %) within 48 h. Moreover, the high crystallinity degree of approximately 84 % was attained for Au-doped Fe3O4, confirming this nanoparticle as an efficient drug delivery system. In another attempt, PDI and hydrated particle size distribution analyses on Au-doped Fe3O4 showed no significant change in size after 72 h. Therefore, it can be concluded that drug loaded Au-doped Fe3O4 is stable enough and maintained its efficacy after a long time of 3 days. Finally, an in vitro assessment of cellular cytotoxicity was performed on a liver cancer cell line (HepG2) to assess the safety and efficacy of the Au-doped Fe3O4 nanoparticles. The results from the MTT assay confirmed the potential of these nanoparticles as a promising vehicle for delivering 5-Fluorouracil.

Near-infrared fluorescent probe to track Cys in plant roots under heavy metal hazards and its application in cells and zebrafish

Heavy metals, including Hg2+, Cr6+ and Cd2+, have always been a major issue in environmental pollution, leading to abnormal changes in the levels of biologically active molecules including Cys in plants, seriously affecting all aspects of the growth and development of plants. This makes it essential to develop a simple and practical method to study the potential impact of heavy metals on plants. In this paper, our research group has developed near-infrared fluorescent probe WRM-S, which has the advantages of fast response, sensitivity to Cys, and successfully applying it to cells and zebrafish. Moreover, it combined the close relationship between heavy metal stress on plants and Cys, using Cys as the detection target, monitoring the internal environment changes of two plants under Hg2+, Cr6+, and Cd2+ stress in the environment, and then conducting 3D imaging. The results indicated that the probe has strong penetration ability in plant tissues, and revealed abnormal changes in plant Cys levels caused by heavy metal stress-induced cellular oxidative stress or cytotoxicity. Thus, the in-situ imaging detection of this probe provides a direction for the physiological dynamics research of plant environmental stress.

Three families of CD4-induced antibodies are associated with the capacity of plasma from people living with HIV to mediate ADCC in presence of CD4-mimetics.

CD4-mimetics (CD4mcs) are small molecule compounds that mimic the interaction of the CD4 receptor with HIV-1 envelope glycoproteins (Env). Env from primary viruses normally samples a "closed" conformation which occludes epitopes recognized by CD4-induced (CD4i) non-neutralizing antibodies (nnAbs). CD4mcs induce conformational changes on Env resulting in the exposure of these otherwise inaccessible epitopes. Here we evaluated the capacity of plasma from a cohort of 50 people living with HIV to recognize HIV-1-infected cells and eliminate them by antibody-dependent cellular cytotoxicity (ADCC) in the presence of a potent indoline CD4mc. We observed a marked heterogeneity among plasma samples. By measuring the levels of different families of CD4i Abs, we found that the levels of anti-cluster A, anti-coreceptor binding site and anti-gp41 cluster I antibodies are responsible for plasma-mediated ADCC in presence of CD4mc.

Adeno-associated viral delivery of Env-specific antibodies prevents SIV rebound after discontinuing antiretroviral therapy.

An alternative to lifelong antiretroviral therapy (ART) is needed to achieve durable control of HIV-1. Here we show that adeno-associated virus (AAV)-delivery of two rhesus macaque antibodies to the SIV envelope glycoprotein (Env) with potent neutralization and antibody-dependent cellular cytotoxicity can prevent viral rebound in macaques infected with barcoded SIV mac 239M after discontinuing suppressive ART. Following AAV administration, sustained antibody expression with minimal anti-drug antibody responses was achieved in all but one animal. After ART withdrawal, SIV replication rebounded within two weeks in all of the control animals but remained below the threshold of detection in plasma (<15 copies/mL) for more than a year in four of the eight animals that received AAV vectors encoding Env-specific antibodies. Viral sequences from animals with delayed rebound exhibited restricted barcode diversity and antibody escape. Thus, sustained expression of antibodies with potent antiviral activity can afford durable, ART-free containment of pathogenic SIV infection.

Sex Differences during Influenza A Virus Infection and Vaccination and Comparison of Cytokine and Antibody Responses between Plasma and Serum Samples.

In this study, we evaluated sex differences during infection with mouse-adapted H1N1 and H3N2 influenza A viruses (IAVs) in the C57BL/6J mouse model and compared the cytokine and antibody responses between plasma and serum samples during IAV infection and vaccination. Lethal doses for both H1N1 and H3N2 IAVs were lower for adult females and they suffered with greater morbidity than adult males when infected with sublethal doses. In influenza virus-infected mice, cytokine responses differed between plasma and serum samples. After inactivated influenza virus vaccination and drift variant challenge, adult female mice had greater antibody responses and were better protected. In influenza-vaccinated and challenged mice, binding antibodies were unaffected between paired plasma or serum samples. However, functional antibody assays, including hemagglutination inhibition, microneutralization, and antibody-dependent cellular cytotoxicity assays, were affected by the use of plasma and serum sample types. Our results indicate that careful consideration is required while selecting plasma versus serum samples to measure cytokine and antibody responses during IAV infection and vaccination.

Fc–Fc interactions and immune inhibitory effects of IgG4: implications for anti-PD-1 immunotherapies

BackgroundThe majority of anti-programmed cell-death 1 (PD-1) monoclonal antibodies (mAbs) use S228P mutation IgG4 as the structural basis to avoid the activation of immune cells or complement. However, little attention...

Safety and efficacy results from the phase I/IIa study of FG-M108 plus CAPOX as first-line (1L) treatment for patients with CLDN18.2+/HER2- locally advanced unresectable or metastatic gastric or gastroesophageal junction (G/GEJ) adenocarcinoma.

4049 Background: There is an unmet medical need for 1L treatment of patients with HER2- G/GEJ adenocarcinoma. CLDN18.2 is expressed in normal gastric mucosa cells and retained in G/GEJ tumor cells. FG-M108 is a monoclonal antibody specifically targeting CLDN18.2 with optimal affinity and enhanced antibody-dependent cellular cytotoxicity. FG-M108 has demonstrated potent anti-tumor activity and excellent safety profile in preclinical studies. Methods: This cohort of a phase I/IIa study evaluated the safety and preliminary efficacy of FG-M108 300 mg/m2 every 3 weeks plus CAPOX in patients with locally advanced unresectable or metastatic G/GEJ adenocarcinoma. Patients had measurable lesions and were HER2- and CLDN18.2+ (immunohistochemistry [IHC] 1+/2+/3+≥10%). At baseline, 22 patients (43.1%) had liver metastases and 15 (29.4%) had peritoneal metastases. Treatment-emergent adverse events (TEAEs) were categorized by CTCAE v5.0. Efficacy was evaluated every 6 weeks per RECIST1.1. Results: As of January 12, 2024, 52 patients were treated. Most TEAEs were grade 1-2; grade ≥3 TEAEs occurred in 55.8% of patients and serious TEAEs in 25.0%. The most common TEAEs were neutropenia (63.5%), thrombocytopenia (44.2%), and hypoalbuminemia (42.3%). FG-M108–related grade ≥3 TEAEs occurred in 36.5% of patients and FG-M108–related serious TEAEs in 11.5%. No FG-M108–related grade 4-5 TEAEs were reported. No patients withdrew due to severe nausea, vomiting or hypoalbuminemia during the treatment period. Confirmed objective response rate (ORR) and disease control rate (DCR) were 46.7% (7/15) and 100.0% (15/15), respectively, in 15 patients with low CLDN18.2 expressions (IHC 1+/2+/3+ ≥10% &amp; 2+/3+ &lt;40%). Median progression-free survival (mPFS) in patients with low CLDN18.2 expressions was 5.0 months, similar to treatment effect of CAPOX. For 36 patients with medium or high expressions of CLDN18.2 (IHC 2+/3+ ≥40%), confirmed ORR was 77.8% (28/36) and DCR was 97.2% (35/36). The median follow-up was 218 days, and mPFS and median duration of response (mDOR) have not been reached yet; However, FG-M108 plus CAPOX have shown a clear trend to prolong the PFS and DOR in this group as compared to patients with low CLDN18.2 expressions (estimated HR=0.33). Median overall survival has not been reached. Conclusions: FG-M108 plus CAPOX as 1L treatment for CLDN18.2+/HER2- advanced G/GEJ cancer showed good tolerability and improved efficacy compared to historical CAPOX control, with an apparent trend of better efficacy with higher CLDN18.2 expression. Based on these results, pivotal phase 3 study of FG-M108 plus CAPOX versus CAPOX as 1L treatment of locally advanced unresectable or metastatic G/GEJ adenocarcinoma expressing medium/high CLDN18.2 has been initiated and is ongoing. Clinical trial information: NCT06177041 .

Phase I study of CN201, a novel CD3xCD19 bispecific antibody, in patients with relapsed or refractory B-cell non-Hodgkin lymphoma.

7040 Background: CN201 is a novel CD19-targeting T-cell-engager IgG4 bispecific antibody, designed to reduce cytokine release syndrome (CRS) while retaining potent T-cell mediated cytotoxicities. A silenced Fc fragment was used to prevent antibody-dependent cellular cytotoxicity while prolonging half-life of the molecule. Here, we report results of the phase I study of CN201 in patients with relapsed or refractory B-cell non-hodgkin lymphoma (R/R B-NHL) (ClinicalTrials.gov identifier: NCT06189391). Methods: The i3+3 dose-escalation design was used in adult patients with CD19+ R/R B-NHL to investigate safety, tolerability, maximum tolerated dose, and preliminary anti-tumor activity. Patients with R/R DLBCL who failed≧2 prior lines of therapy, or R/R indolent NHL(iNHL) who had at least 1 prior systemic therapy were eligible for enrollment. Patients received CAR-T therapy can be enrolled after 90 days washout periord provided that CD19 was still positive on tumor cells. Response were evaluated according to the Lugano 2014 criteria. Single-agent CN201 was administered intravenously once per week. Intial dose escalation was conducted in the patients at fixed ascending doses; later, a step-up dosing regimen was adopted, including a priming dose on cycle 1 Day 1 followed by an intermediate dose on Day 8 and the target dose administered on Day 15 and thereafter. Results: As of December 29, 2023, 58 adult patients received CN201, including 19 patients with fixed doses from 2.5μg to 600μg, 39 patients with step-up doses from 1.2mg to 40mg of the target doses. Maximum tolerated dose has not been reached. The most common treatment-related adverse effects (AEs) (≥20%) of any grade were white blood cell decreased (32%), neutropenia (30%), lymphopenia (29%), anemia (29%), pyrexia (25%) and platelet decreased (23%). The most common AEs (≥10%) of grade 3 or higher were lymphopenia (24%) and neutropenia (14%). CRS occurred in 4 (7%) patients, mainly occurred following the first dose. All of those CRS were low-grade, no ≥Grade 3. No immune effector cell-associated neurotoxicity syndrome was observed. In patients with fixed doses up to 600 μg and target dose up to 2.5mg, stable diseases (SD) were observed. The best overall responses dramatically improved in patients received full doses ≥ 5mg, ranging from 5 mg to 40 mg investigated so far. Among 22 evaluable patients with full doses ≥ 5mg, the objective response rate (ORR) was 77% and the complete remission (CR) rate was 22%. Among 11 patients with indolent B-NHL, the ORR was 91%, CR rate was 45.5%, including one patient had failed previous CAR-T therapy. The responses at higher doses are still under evaluation, and additional data will be reported. Conclusions: CN201 has a well tolerable safety profile and promising anti-tumor activity in patients with R/R B-NHL. Clinical trial information: NCT06189391 .

FG-M108 plus nab-paclitaxel and gemcitabine (AG) as first-line (1L) treatment for patients with Claudin-18.2 (CLDN18.2) positive locally advanced unresectable or metastatic pancreatic cancer (PC): Preliminary results from the phase 1b study.

4142 Background: PC is one of the most lethal cancers with limited treatment options and poor outcomes. The CLDN18.2 antigen is frequently expressed in malignant gastrointestinal cancers including pancreatic cancer. FG-M108 is a human monoclonal antibody specifically targeting CLDN18.2 with optimal affinity and enhanced antibody-dependent cellular cytotoxicity. Methods: A multi-center phase 1b study of FG-M108 (NCT04894825) is ongoing, with one cohort evaluating the safety, tolerability, preliminary efficacy, pharmacokinetics, and immunogenicity of FG-M108 plus AG in patients with locally advanced unresectable or metastatic PC. Only patients with positive CLDN18.2 expression (IHC 1+/2+/3+≥10%) were eligible. This cohort of patients received FG-M108 (intravenous, 300 mg/m2 day 1) plus gemcitabine (intravenous, 1000 mg/m2, days 1 and 8) and nab-paclitaxel (intravenous, 125 mg/m2, days 1 and8) every 3 weeks until disease progression, unacceptable toxicity, or discontinuation. The primary safety endpoint was the incidence of adverse events (AEs) and the secondary efficacy endpoints included objective response rate (ORR), disease control rate (DCR), duration of response, progression-free survival (PFS), and overall survival (OS). Responses were assessed by RECIST 1.1 every 6 weeks. AEs were graded using CTCAE v5.0. Results: As of January 15, 2024, 31 patients received 300 mg/m2 FG-M108 plus chemotherapy. The most common FG-M108 related AEs were nausea (43.5%), emesis (30.4%), neutropenia (21.7%), and anemia (21.7%). The most common grade ≥3 AEs of any cause were neutropenia (26.1%). FG-M108–related serious AEs occurred in 29.0% of patients. No FG-M108–related grade 4 or 5 AEs and no treatment-related deaths were reported. Median PFS and median OS have not been reached yet. Of the 14 patients who received tumor evaluation at least once, ORR was 50.0% (7/14) and DCR was 100% (14/14). Conclusions: FG-M108 plus AG showed excellent safety profile and promising clinical efficacy as 1L treatment for patients with CLDN18.2+ locally advanced unresectable or metastatic PC. The safety and efficacy, especially PFS and OS, of FG-M108 plus AG treatment will be further evaluated. Clinical trial information: NCT04894825 .

CD19 expression in diffuse large B-cell lymphoma (DLBCL) patient biopsies after treatment with tafasitamab.

e19050 Background: Tafasitamab (tafa) is a CD19-targeting immunotherapy that induces enhanced antibody dependent cellular cytotoxicity and phagocytosis. Tafa received accelerated FDA approval in 2020 for treating relapsed or refractory DLBCL in combination with lenalidomide in patients ineligible for autologous stem cell transplant, based on results from L-MIND, a multicenter, open-label, single-arm, phase 2 trial. FirmMIND (NCT05429268) is an ongoing identical study, further evaluating efficacy and safety of tafa in this setting. Several other CD19 targeting agents are available; however, CD19 antigen loss following treatment with chimeric antigen receptor T-cell therapy targeting CD19 (CART19) has raised concerns over CD19-targeted therapy sequencing. Loss of CD19 has not been observed to date in tafa clinical trials or practice. Methods: To evaluate CD19 expression at screening and after disease progression in firmMIND, we developed an immunohistochemistry (IHC) assay using a mAb that binds to an intracellular epitope of CD19 clone LE-CD19. To validate that CD19 expression is detectable after tafa treatment, we treated cultured SU-DHL-4 or Jurkat cells with tafa at a saturating concentration for 24 hours. After washing out unbound tafa, cells were harvested at specific timepoints. Resulting cell line blocks were assessed for CD19 expression by IHC. We also monitored CD19 trafficking in response to fluorescently labeled tafa by confocal microscopy. Results: By IHC, CD19 expression was comparable between treatment conditions, suggesting the assay can effectively measure CD19 expression in lymphoma biopsy samples post-tafa. Confocal microscopy showed a fraction of tafa-bound CD19 internalized and colocalized with a CD19 intracellular pool labeled with anti-CD19 mAb clone EPR5906; this mAb also detected CD19 that remained localized on the cell surface. The data demonstrated that CD19 persists on tumor cells after tafa treatment at levels that could be bound by other CD19-targeting therapies. As of the January 25, 2024 data cutoff, we received 6 end-of-treatment (EOT) samples from patients in firmMIND. Biopsies were collected 14-26 days after the last tafa dose; 5 of 6 samples were biopsies from lymph nodes or liver and showed ≥80% of tumor cells staining CD19+ on membranes with 3+ intensity by IHC, except 1 that was collected only 6 days after last dose and showed 50% positivity. The sixth biopsy (extranodal subcutaneous lesion) showed no CD19 expression at baseline or EOT. Conclusions: Patient biopsy analysis and preclinical data suggest that following tafa treatment, CD19 antigen is preserved on the surface of tumor cells after short and transient internalization and could be targeted by other CD19 agents, such as CART therapy. This conclusion is supported by sequential treatment with tafa followed by CART in vivo (Sakemura, 2024). Analyses are ongoing to confirm these findings.