Cell-mediated Cytotoxic Activity Research Articles

Functional and structural investigation of a broadly neutralizing SARS-CoV-2 antibody.

Since its emergence, SARS-CoV-2 has been continuously evolving, hampering the effectiveness of current vaccines against COVID-19. mAbs can be used to treat patients at risk of severe COVID-19. Thus, the development of broadly protective mAbs and an understanding of the underlying protective mechanisms are of great importance. Here, we isolated mAbs from donors with breakthrough infection with Omicron subvariants using a single-B cell screening platform. We identified a mAb, O5C2, which possesses broad-spectrum neutralization and antibody-dependent cell-mediated cytotoxic activities against SARS-CoV-2 variants, including EG.5.1. Single-particle analysis by cryo-electron microscopy revealed that O5C2 targeted an unusually large epitope within the receptor-binding domain of spike protein that overlapped with the angiotensin-converting enzyme 2 binding interface. Furthermore, O5C2 effectively protected against BA.5 Omicron infection in vivo by mediating changes in transcriptomes enriched in genes involved in apoptosis and interferon responses. Our findings provide insights into the development of pan-protective mAbs against SARS-CoV-2.

A bispecific antibody targeting HER2 and CLDN18.2 eliminates gastric cancer cells expressing dual antigens by enhancing the immune effector function.

Gastric cancer (GC) is widely regarded as one of the toughest cancers to treat. Trastuzumab, which targets the human epidermal growth factor receptor 2 (HER2) for GC treatment, has demonstrated clinical success. However, these patients have a high likelihood of developing resistance. Additionally, Claudin18.2 (CLDN18.2) is a promising emerging target for GC treatment. Therefore, therapies that simultaneously target both HER2 and CLDN18.2 targets are of great significance. Here, we constructed a bispecific antibody targeting both HER2 and CLDN18.2 (HC-2G4S; BsAb), which displayed satisfactory purity, thermostability and enhancing antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In a tumor spheroids model of GC, BsAb demonstrated greater therapeutic efficacy than monoclonal antibodies (mAb) or combination treatment strategies. We propose that the enhanced anti-tumor potency of BsAbs in vivo is due to the monovalent binding of single-chain antibodies to more targets due to weaker affinity, resulting in a more potent immune effect function. Therefore, HC-2G4S could be a productive agent for treating GC that is HER2-positive, CLDN18.2-positive, or both, with the potential to overcome trastuzumab resistance and provide significant clinical benefits and expanded indications.

ZGGS15, a Novel IgG4 Bispecific Monoclonal Antibody Targeting Both LAG-3 and Tigit, Demonstrates Outstanding Anti-Tumor Activity and Can be Used As Monotherapy or in Combination with Anti-PD-1 Antibodies for Cancer Immunotherapy

Background This study aims to evaluate the anti-tumor efficacy of ZGGS15, a novel IgG4 bispecific monoclonal antibody (BsAb) targeting TIGIT and LAG-3, for cancer immunotherapy. Methods: ZGGS15 was generated in-house with the standard recombinant BsAb technology. ZGGS15 proteins were purified with protein A affinity column chromatography and evaluated for purity by size-exclusion high-performance liquid chromatography. The ZGGS15 affinity was assessed using a biolayer interferometry (BLI) assay. The EC50s of ZGGS15 binding to TIGIT and LAG-3 were determined using the ELISA technique. The EC50s of ZGGS15 binding to REH, Hela, MCF-7, and HCC1954 cells were measured by flow cytometry. Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities were also evaluated by flow cytometry. BALB/c-hPD-1/hLAG-3 and BALB/c-hPD-1/hTIGIT subcutaneously transplanted with CT26.WT tumor mouse models, and peripheral blood mononuclear cells (PBMC) -humanized NOG bearing A375 tumor mouse model were used for in vivo anti-tumor efficacy evaluation. Results ZGGS15 binds specifically to activated CD4 + and CD8 + T cells and human LAG-3 and TIGIT proteins expressed by CHO-K1 cells identically with a dose-dependent nanomolar affinity of 0.69 nM and 1.87 nM for human LAG-3 and TIGIT, respectively. In vitro kinetic analysis BLI results revealed ZGGS15 has high affinity for human LAG-3 and human TIGIT proteins, with KDs of 3.05 nM and 2.65 nM, respectively. ZGGS15 binds to human LAG-3 and human TIGIT simultaneously with similar affinity. Furthermore, ZGGS15 competitively inhibited the binding of LAG-3 to MHC-II (IC50 = 0.77 nM) and the binding of TIGIT to CD155 (IC50=0.24 nM). ZGGS15 induced better T cell activation than single anti-LAG3 or anti-TIGIT mAb agent or combined with different stimuli with or without cancer cells. When combined with nivolumab, ZGGS15 can further enhance the T cell activation in a dose-dependent manner. ADCC/CDC assay results showed that ZGGS15 binding to target cells does not induce ADCC or CDC. ZGGS15 interacts with the human neonatal Fc receptor in a pH-dependent manner, which can promote recirculation of ZGGS15 with a prolonged half-life in plasma. Besides, ZGGS15 interacts with human Fc-γ receptors and the human complement component, C1q, with low binding affinities. However, ZGGS15 does not induce obvious cytokine production from PBMCs, including IFN-γ, IL-2, IL-6, IL-10, and TNF-α. In vivo anti-tumor efficacy for ZGG15 in PBMC humanized NOG mice bearing A375 tumors showed that ZGGS15 had significant anti-tumor effects in the subcutaneous A375 xenograft model. ZGGS15 showed a better anti-tumor inhibition than single anti-LAG-3 or anti-TIGIT agent and a synergetic effect when combined with nivolumab. On day 18 post-treatment, the ZSAB015 plus nivolumab treatment demonstrated a significantly tumor growth inhibition (TGI) of 95.80% ( p =0.001), compared with the vehicle group. In vivo efficacy tests in BALB/c-hPD-1/hLAG-3 mice subcutaneously transplanted with CT26.WT tumor model showed that both high and low doses of ZGGS15 could inhibit the growth of CT26.WT colon cancer. The combination of high-dose ZGGS15 plus nivolumab has better efficacy than high-dose ZGGS15 and is slightly better than nivolumab alone. The tumor volume inhibition rate (TGITV) for ZGGS15 at 2 mg/kg was 69.70%, and for ZGGS15 at 5 mg/kg plus nivolumab at 1 mg/kg, it was 94.03% ( p < 0.001). During the experiment, the tumor-bearing mice tolerated it well, and no adverse reactions were observed. In vivo efficacy tests of ZGG15 in BALB/c-hPD-1/hTIGIT mice subcutaneously transplanted with CT26.WT tumor model showed that ZGGS15 at a low dose (1.5 mg/kg) demonstrated a significant tumor inhibitory effect, with the TGITV of 51.42% ( p < 0.01). The combination of ZGGS15 high-dose with nivolumab (5 mg/kg + 1 mg/kg) showed a better tumor inhibitory effect compared with ZGGS15 high-dose (5 mg/kg), with the TGITV of 81.15% and 41.54%, respectively ( p < 0.05). Conclusions ZGGS15 has excellent anti-tumor activity by inhibiting LAG-3 and TIGIT as a new IgG4 BsAb. ZGGS15 also exhibits potent anti-tumor efficacy in transgenic mouse models combined with nivolumab. Additionally, ZGGS15 does not elicit ADCC or CDC, therefore avoiding the cross-reaction of non-target binding. Finally, ZGGS15 does not induce cytokine production, mitigating any potential adverse effects of CRS.

A novel Fc-enhanced humanized monoclonal antibody targeting B7-H3 suppresses the growth of ESCC

ABSTRACT Esophageal squamous cell carcinoma (ESCC) is a prevalent malignant tumor of the digestive tract with a low 5-year survival rate due to the lack of effective treatment methods. Although therapeutic monoclonal antibodies (mAbs) now play an important role in cancer therapy, effective targeted mAbs are still lacking for ESCC. B7-H3 is highly expressed in a variety of tumors and has emerged as a promising therapeutic target. Several mAbs against B7-H3 have advanced to clinical trials, but their development has not yet been pursued for ESCC. Here, we developed a humanized and Fc-engineered anti-B7H3 mAb 24F-Hu-mut2 and systematically evaluated its anti-tumor activity in vitro and in vivo. The 24F-Hu-mut2 was humanized and modified in Fc fragment to obtain stronger antibody-dependent cell-mediated cytotoxicity(ADCC) activity and nanomolar affinity. Furthermore, both of ESCC cell-derived xenograft (CDX) and patient-derived xenograft (PDX) mice models indicated that 24F-Hu-mut2 displayed potent in vivo anti-tumor activity. In addition, a computational docking model showed that the mAb bound to IgC1 and IgC2 domain of B7-H3, which is closer to the cell membrane. Consistently, our ELISA results verified the binding of 24F-Hu-WT and IgC1 and IgC2. Our results indicate that 24F-Hu-mut2 has significant anti-ESCC activity both in vitro and in vivo, and this monoclonal antibody may be a promising antibody against ESCC and other B7-H3 overexpressing tumors.

Regulation and distribution of European sea bass perforins point to their role in the adaptive cytotoxic response against NNV

Cell-mediated cytotoxicity is a complex immune mechanism that involves the release of several killing molecules, being perforin (PRF) one of the most important effector players. Perforin is synthesized by T lymphocytes and natural killer cells in mammals and responsible for the formation of pores on the target cell membrane during the killing process. Although perforin has been extensively studied in higher vertebrates, this knowledge is very limited in fish. Therefore, in this study we have identified four prf genes in European sea bass (Dicentrarchus labrax) and evaluated their mRNA levels. All sea bass prf genes showed the typical and conserved domains of its human orthologue and were closely clustered by the phylogenetic analysis. In addition, all genes showed constitutive and ubiquitous tissular expression, being prf1.9 gene the most highly expressed in immune tissues. Subsequently, in vitro stimulation of head-kidney (HK) cells with phytohemagglutinin, a T-cell activator, showed an increase of all prf gene levels, except for prf1.3 gene. European sea bass HK cells increased the transcription of prf1.2 and prf1.9 during the innate cell-mediated cytotoxic activity against xenogeneic target cells. In addition, sea bass infected with nodavirus (NNV) showed a similar expression pattern of all prf in HK and brain at 15 days post-infection, except for prf1.3 gene and in the gonad. Finally, the use of a polyclonal antibody against PRF1.9 showed an increase of positive cells in HK, brain and gonad from NNV-infected fish. Taken together, the data seem to indicate that all prf genes, except prf1.3, appear to be involved in the European sea bass immunity, and probably in the cell-mediated cytotoxic response, with PRF1.9 playing the most important role against nodavirus. The involvement of the PRFs and the CMC activity in the vertical transmission success of the virus is also discussed.

Development of a concise and reliable method for quantifying the antibody loaded onto lipid nanoparticles modified with Herceptin

Antibodies are essential components of the immune system with a wide range of molecular targets. They have been recognized as modalities for treating several diseases and more than 130 approved antibody-based therapeutics are available for clinical use. However, limitations remain associated with its efficacy, tissue permeability, and safety, especially in cancer treatment. Nanoparticles, particularly those responsive to external stimuli, have shown promise in improving the efficacy of antibody-based therapeutics and tissue-selective delivery. In this study, we developed a reliable and accurate method for quantifying the amount of antibody loaded onto lipid nanoparticles modified with Herceptin® (Trastuzumab), an antibody-based therapeutic used to treat HER2-positive cancers, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. This method proved to be a suitable alternative to commonly used protein quantification techniques, which are limited by lipid interference present in the samples. Furthermore, the amount of Herceptin modified on the liposomes, measured by this method, was confirmed by Herceptin's antibody-dependent cell-mediated cytotoxicity activity. Our results demonstrate the potential of this method as a critical tool for developing tissue-selective antibody delivery systems, leading to improved efficacy and reduced side effects of antibody-based therapeutics.

S-531011, a Novel Anti-Human CCR8 Antibody, Induces Potent Antitumor Responses through Depletion of Tumor-Infiltrating CCR8-Expressing Regulatory T Cells.

Although regulatory T cells (Treg) are inhibitory immune cells that are essential for maintaining immune homeostasis, Tregs that infiltrate tumor tissue promote tumor growth by suppressing antitumor immunity. Selective reduction of tumor-infiltrating Tregs is, therefore, expected to activate antitumor immunity without affecting immune homeostasis. We previously reported that selective Treg depletion targeted by a C-C motif chemokine receptor 8 (CCR8) resulted in induction of strong antitumor immunity without any obvious autoimmunity in mouse models. Thus, herein, we developed a novel humanized anti-CCR8 monoclonal antibody, S-531011, aimed as a cancer immunotherapy strategy for patients with cancer. S-531011 exclusively recognized human CCR8 among all chemokine receptors and showed potent antibody-dependent cell-mediated cytotoxicity activity toward CCR8+ cells and neutralization activity against CCR8-mediated signaling. We observed that S-531011 reduced tumor-infiltrating CCR8+ Tregs and induced potent antitumor activity in a tumor-bearing human-CCR8 knock-in mouse model. Moreover, combination therapy with S-531011 and anti-mouse programmed cell death 1 (PD-1) antibody strongly suppressed tumor growth compared with anti-PD-1 antibody alone with no observable adverse effects. S-531011 also depleted human tumor-infiltrating Tregs, but not Tregs derived from human peripheral blood mononuclear cells. These results suggest that S-531011 is a promising drug for inducing antitumor immunity without severe side effects in the clinical setting.

An armed anti-immunoglobulin light chain nanobody protects mice against influenza A and B infections.

The immune system eliminates pathogen intruders such as viruses and bacteria. To recruit immune effectors to virus-infected cells, we conjugated a small molecule, the influenza neuraminidase inhibitor zanamivir, to a nanobody that recognizes the kappa light chains of mouse immunoglobulins. This adduct was designed to achieve half-life extension of zanamivir through complex formation with the much-larger immunoglobulins in the circulation. The zanamivir moiety targets the adduct to virus-infected cells, whereas the anti-kappa component simultaneously delivers polyclonal immunoglobulins of indeterminate specificity and all isotypes. Activation of antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity promoted elimination of influenza neuraminidase-positive cells. A single dose of the conjugate protected mice against influenza A or B viruses and was effective even when given several days after infection with a lethal dose of virus. In the absence of circulating immunoglobulins, we observed no in vivo protection from the adduct. The type of conjugates described here may thus find application for both anti-influenza prophylaxis and therapy.

Abstract 2938: Preclinical development of a novel bispecific mAb-Trap fusion protein, IMM2902, targeting both HER2 and CD47 as cancer immunotherapy

Abstract The overexpression of human epidermal growth factor receptor 2 (HER2) has been identified in a variety of solid tumors including breast cancer (BC), gastric cancer and is associated with tumor recurrence, brain metastasis, and an overall poor prognosis. As such, HER2 has become a major target in the research and development of anticancer drugs. CD47, an integrin-associated protein, is expressed in many human cancer cells, especially upregulated preferentially in HER2-expressing cells. Co-expression of HER2 and CD47 is more frequently detected in recurrent BC patients with poor prognosis when comparing with the counterpart primary tumors. IMM2902 is a first-in-class, clinical-stage, novel recombinant bispecific mAb-Trap fusion protein targeting both CD47 and HER2 simultaneously. The binding activities of IMM2902, measured as EC50, to total CD47+ and HER2+ in BT474, NCI-N87 and SKOV-3 cancer cell lines are 16, 11 and 5 nM respectively. The affinity of IMM2902 to HER2 is about 15 times higher than that of CD47. IMM2902 not only prevents the engagement of CD47 with SIRPα and hence blocks “don’t eat me” signal of CD47/SIRPα, but also induces strong antibody-dependent cell-mediated cytotoxicity (ADCC, EC50 = 0.0045 - 0.0544 nM) and antibody-dependent cellular phagocytosis (ADCP, EC50 = 0.05 - 0.13 nM) activity with specifically engineered IgG1 Fc fragment. Most importantly, IMM2902 induces accelerated HER2 degradation in tumor tissues. Despite its high affinity to CD47, IMM2902 does not binds to CD47 on erythrocyte membrane, therefore, unlike conventional CD47 mAb, IMM2902 demonstrated a minimal impact on red blood cells (RBC). In preclinical research, IMM2902 has demonstrated a much stronger antitumor activity with excellent tolerability in a mouse model of human breast cancer (dose range 3.5 - 10 mg/kg) and a mouse model of human gastric cancer (dose range 1 -18 mg/kg) than that of trastuzumab (targeting HER2 only) alone, IMM01 (a SIRPα-Fc fusion protein) alone, and the combination of trastuzumab and IMM01. In HER2-low SUN-1 xenograft models, IMM2902 has showed a strong tumor growth inhibition at doses between 2 to 18 mg/kg. Intriguingly, IMM2902 also has exhibited potent tumor killing activity against breast cancer resistant to trastuzumab (HCC-1954 and patient-derived xenografts herceptin-resistant model) at doses between 3.5 to 10 mg/kg. Given the strong preclinical antitumor activity as well as the favorable safety profile, IMM2902 may serve as a potent immunotherapy for HER2-expressing cancers via dual blockade of CD47 and HER2. A Phase 1 clinical trial exploring safety, tolerability, and preliminary efficacy of IMM2902 in patients with HER2-expressing advanced solid tumors is currently ongoing in both China (CXSL2101035) and USA (NCT05076591). Citation Format: Binglei Zhang, Song Li, Dianze Chen, Dandan Liu, Huiqin Guo, Chunmei Yang, Li Zhang, Wei Zhang, Xiaoping Tu, Liang Peng, Gui Zhao, Ruliang Zhang, Frank X. Gan, Wenzhi Tian, Fan Zhang, Yongping Song. Preclinical development of a novel bispecific mAb-Trap fusion protein, IMM2902, targeting both HER2 and CD47 as cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2938.

Application of Pseudotyped Viruses.

Highly pathogenic emerging and reemerging viruses have serious public health and socioeconomic implications. Although conventional live virus research methods can more reliably investigate disease pathogenicity and evaluate antiviral products, they usually depend on high-level biosafety laboratories and skilled researchers; these requirements hinder in vitro assessments of efficacy, as well as efforts to test vaccines and antibody drugs. In contrast, pseudotyped viruses (i.e., single-round infectious viruses that mimic the membrane structures of various live viruses) are widely used in studies of highly pathogenic viruses because they can be handled in biosafety level 2 facilities. This chapter provides a concise overview of various aspects of pseudotyped virus technologies, including (1) exploration of the mechanisms of viral infection; (2) evaluation of the efficacies of vaccines and monoclonal antibodies based on pseudovirion-based neutralization assay; (3) assessment of antiviral agents (i.e., antibody-based drugs and inhibitors); (4) establishment of animal models of pseudotyped virus infection in vivo; (5) investigation of the evolution, infectivity, and antigenicity of viral variants and viral glycosylation; and (6) prediction of antibody-dependent cell-mediated cytotoxic activity.

HTLV-2 Enhances CD8+ T Cell-Mediated HIV-1 Inhibition and Reduces HIV-1 Integrated Proviral Load in People Living with HIV-1.

People living with HIV-1 and HTLV-2 concomitantly show slower CD4+ T cell depletion and AIDS progression, more frequency of the natural control of HIV-1, and lower mortality rates. A similar beneficial effect of this infection has been reported on HCV coinfection reducing transaminases, increasing the spontaneous clearance of HCV infection and delaying the development of hepatic fibrosis. Given the critical role of CD8+ T cells in controlling HIV-1 infection, we analysed the role of CD8+ T cell-mediated cytotoxic activity in coinfected individuals living with HIV-1. One hundred and twenty-eight individuals living with HIV-1 in four groups were studied: two groups with HTLV-2 infection, including individuals with HCV infection (N = 41) and with a sustained virological response (SVR) after HCV treatment (N = 25); and two groups without HTLV-2 infection, including individuals with HCV infection (N = 25) and with a sustained virological response after treatment (N = 37). We found that CD8+ T cell-mediated HIV-1 inhibition in vitro was higher in individuals with HTLV-2. This inhibition activity was associated with a higher frequency of effector memory CD8+ T cells, higher levels of granzyme A and granzyme B cytolytic enzymes, and perforin. Hence, cellular and soluble cytolytic factors may contribute to the lower HIV-1 pre-ART viral load and the HIV-1 proviral load during ART therapy associated with HTLV-2 infection. Herein, we confirmed and expanded previous findings on the role of HTLV-2 in the beneficial effect on the pathogenesis of HIV-1 in coinfected individuals.

An Fc variant with two mutations confers prolonged serum half-life and enhanced effector functions on IgG antibodies

The pH-selective interaction between the immunoglobulin G (IgG) fragment crystallizable region (Fc region) and the neonatal Fc receptor (FcRn) is critical for prolonging the circulating half-lives of IgG molecules through intracellular trafficking and recycling. By using directed evolution, we successfully identified Fc mutations that improve the pH-dependent binding of human FcRn and prolong the serum persistence of a model IgG antibody and an Fc-fusion protein. Strikingly, trastuzumab-PFc29 and aflibercept-PFc29, a model therapeutic IgG antibody and an Fc-fusion protein, respectively, when combined with our engineered Fc (Q311R/M428L), both exhibited significantly higher serum half-lives in human FcRn transgenic mice than their counterparts with wild-type Fc. Moreover, in a cynomolgus monkey model, trastuzumab-PFc29 displayed a superior pharmacokinetic profile to that of both trastuzumab-YTE and trastuzumab-LS, which contain the well-validated serum half-life extension Fcs YTE (M252Y/S254T/T256E) and LS (M428L/N434S), respectively. Furthermore, the introduction of two identified mutations of PFc29 (Q311R/M428L) into the model antibodies enhanced both complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity activity, which are triggered by the association between IgG Fc and Fc binding ligands and are critical for clearing cancer cells. In addition, the effector functions could be turned off by combining the two mutations of PFc29 with effector function-silencing mutations, but the antibodies maintained their excellent pH-dependent human FcRn binding profile. We expect our Fc variants to be an excellent tool for enhancing the pharmacokinetic profiles and potencies of various therapeutic antibodies and Fc-fusion proteins.

Specific location of galactosylation in an afucosylated antiviral monoclonal antibody affects its FcγRIIIA binding affinity.

Monoclonal antibodies (mAbs) comprise an essential type of biologic therapeutics and are used to treat diseases because of their anti-cancer and anti-inflammatory properties, and their ability to protect against respiratory infections. Its production involves post-translational glycosylation, a biosynthetic process that conjugates glycans to proteins, which plays crucial roles in mAb bioactivities including effector functions and pharmacokinetics. These glycans are heterogeneous and have diverse chemical structures whose composition is sensitive to manufacturing conditions, rendering the understanding of how specific glycan structures affect mAb bioactivity challenging. There is a need to delineate the effects of specific glycans on mAb bioactivity to determine whether changes in certain glycosylation profiles (that can occur during manufacturing) will significantly affect product quality. Using enzymatic transglycosylation with chemically-defined N-glycans, we show that galactosylation at a specific location of N-glycans in an afucosylated anti-viral mAb is responsible for FcγRIIIA binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. We report a facile method to obtain purified asymmetric mono-galactosylated biantennary complex N-glycans, and their influence on bioactivity upon incorporation into an afucosylated mAb. Using ELISA, surface plasmon resonance and flow cytometry, we show that galactosylation of the α6 antenna, but not the α3 antenna, consistently increases FcγRIIIA binding affinity. We confirm its relevance in an anti-viral model of respiratory syncytial virus (RSV) using an adapted ADCC reporter assay. We further correlate this structure-function relationship to the interaction of the galactose residue of the α6 antenna with the protein backbone using 2D-1H-15N-NMR, which showed that galactosylation of at this location exhibited chemical shift perturbations compared to glycoforms lacking this galactose residue. Our results highlight the importance of identifying and quantifying specific glycan isomers to ensure adequate quality control in batch-to-batch and biosimilar comparisons.

Abstract 2871: NM28-2746, a reduced affinity bivalent mesothelin-binding MATCH4 T cell engager, with half-life extension, increases selectivity for killing of mesothelin-overexpressing cells

Abstract CD3-based T cell engagers are highly potent therapeutic molecules which enable T cell-mediated cytotoxic activity toward cells expressing selected tumor-associated antigens. Alongside a highly potent anti-tumor activity, is the risk of on-target off-tumor side effects due to low levels of expression of the target antigen in normal tissue. We have sought to overcome this issue with the design and generation of a target-density-dependent activation mechanism. We have generated reduced-affinity antibody fragments to the tumor associated antigen mesothelin and constructed a multi-domain MATCH4 molecule encompassing bivalent mesothelin binding domains, a CD3 binding domain, and a human serum albumin (HSA)-binding domain for half-life extension. Here we report the design of the MATCH4 molecule and the preclinical activity of the molecule in vitro and in vivo. We also report the biochemical characteristics of the therapeutic candidate molecule showing its highly favorable properties for clinical development. We demonstrate that the bivalent mesothelin T cell engager has increased in vitro potency in T cell activation and tumor cell killing in the presence of high mesothelin expressing cells, when compared to a higher-affinity monovalent counterpart. We also demonstrate that the activity on low mesothelin expressing cells, such as healthy mesothelial cells, is reduced for the bivalent molecule compared to the higher affinity monovalent molecule. Due to the shedding of mesothelin from the surface of cancer cells and the high circulating levels of soluble mesothelin in patient sera, we also demonstrate that the bivalent molecule is still highly potent in cytotoxic activity in the presence of concentrations of soluble mesothelin up to 500 ng/ml. We also demonstrate dose dependent anti-tumor activity in in vivo efficacy studies in PBMC-reconstituted mice, and combination therapeutic activity with an anti-PD-L1, anti-4-1BB bispecific molecule (NM21-1480). Collectively, these data demonstrate an increased selectivity to mesothelin-overexpressing cells by this novel MATCH4 reduced affinity bivalent T cell engager. These data indicate the potential of this molecule to increase the therapeutic window by reducing safety concerns on normal tissue where mesothelin expression is low, and yet promote cytotoxicity on mesothelin over-expressing cancer cells. Citation Format: Bithi Chatterjee, Christian Hess, Daniel Snell, Tea Gunde, Stefan Warmuth, Alexandre Simonin, Matthias Brock, Fabio Spiga, Maria Johansson, Christopher Weinert, Julia Tietz, Niels Kirk, Nicole Bassler, Dana Mahler, Dania Diem, Alessandra Carrella, Noreen Giezendanner, Alessandra Alberti, Giorgio Gambino, Belinda Wickihalder, Bettina Bommer, Simone Muntwiler, Yasemin Yaman, Naomi Flueckiger, Robin Heiz, Sandro Wagen, David Urech. NM28-2746, a reduced affinity bivalent mesothelin-binding MATCH4 T cell engager, with half-life extension, increases selectivity for killing of mesothelin-overexpressing cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2871.

Abstract 1389: In vitro killing assays for evaluation of immuno-oncology drug candidates

Abstract The need to better understand the tumor microenvironment (TME) dictates the characterization of the cell types involved, the roles they play and how they respond to treatment. To develop novel cancer immunotherapies, in vitro primary immune cell bioassays offer an early assessment of their effects on the various players of the TME. Cytotoxic cells are key players in the anti-tumor immune response. CD8+ T cells, also known as cytotoxic T lymphocytes, recognize and kill cells presenting antigens bound to MHC class I molecules, such as neoantigens present on the tumor cell surface. Activation of CD8+ T cells requires at least two signals from antigen presenting cells: binding of their T cell receptor (TCR) to the appropriate peptide presented on MHC I and co-stimulation by B7 binding to CD28. However, the TME possesses several mechanisms to reduce immune cell activity, such as the expression of checkpoint inhibitors dampening the immune system. Natural killer (NK) cells represent another type of cytotoxic cells, with their innate immune activity dictating an important role in anti-tumor immunity. NK cells induce apoptosis via the release of perforin and granzymes from granules, eliminating cells lacking expression MHC I, including cancer cells that have lost expression of MHC I. However, also the activity of NK cells can be hampered in the TME, notably by the PD1/PDL1 interaction.Increasing the activity of CD8+ T and NK cells as a therapeutic anti-tumor strategy has become an important point of attention. Such strategies can have several shapes, with Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of IgG1 isotypes being one of the mechanisms that can be exploited by immunoglobulin-based therapeutics. Another way to increase the killing of tumor cells is to re-direct T cells to tumor cells by employing bi-specific antibodies targeting specific tumor associated antigens. Using this strategy, T cell activation can be concentrated in the TME, thereby also avoiding off-tumor activity. Real-time in vitro assessment of the potential enhanced killing capacity of CD8+ T and NK cells elicited by test molecules could further provide pivotal information on the functional dynamics of test molecules. Using high quality primary human immune cells, in vitro bioassays were developed with improved robustness and reproducibility to screen for the potential to enhance tumor cell killing by novel therapeutics. The ability to evaluate the compounds capacity to increase or induce a cytotoxic activity profile and thereby facilitate the anti-tumor immune response is essential in the early drug development process. Citation Format: Johan Arnold, Thibaut J. Janss, Simon Lefevre, Martijn Vlaming, Ellen Boelen, Sofie Pattijn. In vitro killing assays for evaluation of immuno-oncology drug candidates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1389.

Identification, Efficacy, and Stability Evaluation of Succinimide Modification With a High Abundance in the Framework Region of Golimumab.

Succinimide (Asu) is the intermediate for asparagine deamidation in therapeutic proteins, and it can be readily hydrolyzed to form aspartate and iso-aspartate residues. Moreover, Asu plays an important role in the protein degradation pathways, asparagine deamidation, and aspartic acid isomerization. Here, Asu modification with a high abundance in the framework region (FR) of golimumab was first reported, the effect of denaturing buffer pH on the Asu modification homeostasis was studied, and the results revealed that it was relatively stable over a pH range of 6.0–7.0 whereas a rapid decrease at pH 8.0. Then, the peptide-based multi-attribute method (MAM) analyses showed that the Asu formation was at Asn 43 in the FR of the heavy chain. Meanwhile, the efficacy [affinity, binding and bioactivity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cell-mediated cytotoxicity (ADCC) activity] and stability of the Asu modification of golimumab were evaluated, and the current results demonstrated comparable efficacy and stability between the Asu low- and high-abundance groups. Our findings provide valuable insights into Asu modification and its effect on efficacy and stability, and this study also demonstrates that there is a need to develop a broad-spectrum, rapid, and accurate platform to identify and characterize new peaks in the development of therapeutic proteins, particularly for antibody drugs.

Perspectives on the Efficacy of Benralizumab for Treatment of Eosinophilic Granulomatosis With Polyangiitis.

Two types of interleukin (IL)-5 antibody biologics, anti-IL-5 antibodies (mepolizumab) and anti-IL-5α receptor antibodies (benralizumab), are indicated for severe asthma. While high-dose mepolizumab is also indicated for EGPA, benralizumab is indicated only for severe asthma. Benralizumab is characterized by antibody-dependent cell-mediated cytotoxicity activity, giving them specific and rapid anti-IL-5α receptor binding abilities and the ability to target a high number of eosinophils in tissues as well as peripheral blood. Recently, reports on the efficacy of benralizumab as a treatment for EGPA have been published, along with reports on some cases that are difficult to treat with existing oral corticosteroids and mepolizumab. Therefore, we focus on the perspective of the efficacy and safety of benralizumab as a treatment for EGPA patients with steroid dependence in this review. A total of 41 patients with EGPA were treated with benralizumab. After the introduction of benralizumab, oral corticosteroids could be reduced to 10 mg/day or less in all cases and to less than 5 mg/day in 80% or more of the cases. Discontinuation of oral corticosteroids was achieved in more than 40% of patients with EGPA. Benralizumab was effective in patients with mepolizumab-refractory EGPA and intractable cardiac and neuropathy complications. Efficient elimination of eosinophils is expected to improve the remission rate of EGPA with benralizumab treatment. Although the total number of patients was small, benralizumab was safe and tolerable in a wide range of age groups, suggesting efficacy in severe cases with EGPA.

Abstract P2-13-04: Final survival analysis of a phase 3 study comparing SB3 (trastuzumab biosimilar) and reference trastuzumab in HER2-positive early or locally advanced breast cancer

Abstract Background: SB3 (trastuzumab-dttb) is a biosimilar approved globally based on its similarity with reference trastuzumab (TRZ) demonstrated by thorough comparability exercises in analytical, biological, and clinical studies. In a randomized, double-blind, multicenter Phase 3 study of 875 patients with HER2-positive early or locally advanced breast cancer in the neoadjuvant setting, equivalent efficacy, similar safety, pharmacokinetics, and immunogenicity between SB3 and TRZ were shown. However, when quality attributes of TRZ were examined, downward drifts in antibody-dependent cell-mediated cytotoxicity activities (ADCC) were observed in the TRZ lots with expiry dates ranging from Aug 2018 to Dec 2019. Some of these lots of the reference product were found to be used in the Phase 3 study. After completing the Phase 3 study, patients from select countries were included in a follow-up observational study to monitor cardiac safety and survival. Here, we report the final survival results, including post-hoc subgroup analysis based on ADCC status, at a median follow-up of 68 months. Methods: During the follow-up observational study, the protocol was amended to include additional patients who originally were enrolled in the Phase 3 study but had not been followed in the observational study, in order to collect a larger sample of survival data. For these additional patients, medical records from the last assessment in the Phase 3 study through the date of enrollment in the follow-up study were collected retrospectively. As post-hoc analysis, patients in the TRZ arm were stratified into two subgroups: patients who received during neoadjuvant treatment at least one vial of TRZ with downward drift in ADCC as “Drifted TRZ”, and the others as “Non-drifted TRZ”. Event-free survival (EFS) and overall survival (OS) were assessed. Results: Of 875 patients randomized in the Phase 3 study, 538 patients (SB3, N=267; TRZ, N=271) were enrolled in the follow-up observational study: 367 patients were initially enrolled in the follow-up study, and 171 patients were additionally enrolled following the protocol amendment. The median follow-up duration was 68 months from randomization in the Phase 3 study. 54 events (20.2%) in the SB3 arm, and 67 events (24.7%) in the TRZ arm were reported (HR 0.84 [0.58, 1.20], p=0.335). 22 deaths (8.2%) and 38 deaths (14%) were reported in SB3 and TRZ arms, respectively (HR 0.61 [0.36, 1.05], p=0.073). In post-hoc analysis, of 271 patients in TRZ arm, 107 patients were grouped as “Non-drifted TRZ”, and 164 patients as “Drifted TRZ”. 19 events (17.8%) in the Non-drifted TRZ group and 48 (29.3%) events in the Drifted TRZ group occurred (HR 2.57 [1.28, 5.14], p=0.008). 9 deaths (8.4%) in the Non-drifted TRZ group and 29 deaths (17.7%) in the Drifted TRZ group were reported (HR 3.87 [1.37, 10.93], p=0.011). No difference was observed between SB3 arm and Non-drifted TRZ group in terms of EFS (HR 1.28 [0.73, 2.22], p=0.391) and OS (HR 0.99 [0.42, 2.31], p=0.975). Conclusions: Comparable long-term efficacy results in EFS and OS were shown at 68 months of follow-up, further supporting biosimilarity of SB3 to the reference product. Currently, these follow-up results represent the longest monitoring data of patients treated with a trastuzumab biosimilar for HER2-positive early or locally advanced breast cancer. Citation Format: Xavier Pivot, Mark D Pegram, Javier Cortes, Diana Lüftner, Gary H Lyman, Giuseppe Curigliano, Igor M Bondarenko, Mikhail Dvorkin, Jin Hee Ahn, Seock-Ah Im, Maria Litwiniuk, Yaroslav V Shparyk, Gwo Fuang Ho, Nikolay V Kislov, Marek Wojtukiewicz, Tomasz Sarosiek, Yee Soo Chae, Jin Seok Ahn, Hyerin Jang, Sujung Kim, Jiwon Lee, Soo Young Lee, Ye Chan Yoon. Final survival analysis of a phase 3 study comparing SB3 (trastuzumab biosimilar) and reference trastuzumab in HER2-positive early or locally advanced breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-04.

A bispecific antibody targeting HER2 and PD-L1 inhibits tumor growth with superior efficacy

Activation of the programmed cell death protein 1 and programmed cell death ligand 1 (PD-1/PD-L1) signaling axis plays important roles in intrinsic or acquired resistance to human epidermal growth factor receptor 2 (HER2)-directed therapies in the clinic. Therefore, therapies simultaneously targeting both HER2 and PD-1/PD-L1 signaling pathways are of great significance. Here, aiming to direct the anti-PD-L1 responses toward HER2-expressing tumor cells, we constructed a humanized bispecific IgG1 subclass antibody targeting both HER2 and PD-L1 (HER2/PD-L1; BsAb), which displayed satisfactory purity, thermostability, and serum stability. We found that BsAb showed enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitro. In the late phase of peripheral blood mononuclear cell (PBMC)-humanized HER2+ tumor xenograft models, BsAb showed superior therapeutic efficacies as compared with monoclonal antibodies (mAbs) or combination treatment strategies. In cynomolgus monkeys, BsAb showed favorable pharmacokinetics and toxicity profiles when administered at a 10 mg/kg dosage. Thus, HER2/PD-L1 BsAb was demonstrated as a potentially effective option for managing HER2+ and trastuzumab-resistant tumors in the clinic. We propose that the enhanced antitumor activities of BsAb in vivo may be due to direct inhibition of HER2 signaling or activation of T cells.

A Pilot Phase I Trial of IL-21 Expanded Ideal-Donor Natural Killer (NK) Cells in Combination with Mogamulizumab in Patients with Cutaneous T-Cell Lymphomas (CTCL) or Adult T-Cell Leukemia/Lymphomas (ATLL)

A Pilot Phase I Trial of IL-21 Expanded Ideal-Donor Natural Killer (NK) Cells in Combination with Mogamulizumab in Patients with Cutaneous T-Cell Lymphomas (CTCL) or Adult T-Cell Leukemia/Lymphomas (ATLL)