Therapeutic Antibody Targeting Research Articles

More Is Always Better Than One: The N-Terminal Domain of the Spike Protein as Another Emerging Target for Hampering the SARS-CoV-2 Attachment to Host Cells.

Although the approved vaccines are proving to be of utmost importance in containing the Coronavirus disease 2019 (COVID-19) threat, they will hardly be resolutive as new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, a single-stranded RNA virus) variants might be insensitive to the immune response they induce. In this scenario, developing an effective therapy is still a dire need. Different targets for therapeutic antibodies and diagnostics have been identified, among which the SARS-CoV-2 spike (S) glycoprotein, particularly its receptor-binding domain, has been defined as crucial. In this context, we aim to focus attention also on the role played by the S N-terminal domain (S1-NTD) in the virus attachment, already recognized as a valuable target for neutralizing antibodies, in particular, building on a cavity mapping indicating the presence of two druggable pockets and on the recent literature hypothesizing the presence of a ganglioside-binding domain. In this perspective, we aim at proposing S1-NTD as a putative target for designing small molecules hopefully able to hamper the SARS-CoV-2 attachment to host cells.

Early administration of amatuximab, a chimeric high-affinity anti-mesothelin monoclonal antibody, suppresses liver metastasis of mesothelin-expressing pancreatic cancer cells and enhances gemcitabine sensitivity in a xenograft mouse model.

Amatuximab is a promising therapeutic antibody targeting mesothelin, a 40-kDa glycoprotein that is highly expressed in pancreatic cancer. We investigated the effectiveness of early amatuximab treatment, imitating an adjuvant chemotherapy setting, and combination therapy with amatuximab and gemcitabine in liver metastasis of pancreatic cancer. Liver metastasis mouse models were established in 8-week-old male BALB/c nu/nu mice using the hemisplenic injection method. Tridaily amatuximab monotherapy or combination with gemcitabine was administered to the liver metastasis mouse model before metastatic lesions had formed huge masses. Gaussia luciferase-transfected AsPC-1 was used as a mesothelin-overexpressing pancreatic cancer cell line. The amount of liver metastases and the serum luciferase activity were significantly lower in the treatment groups than those in the control IgG group. Notably, the anti-tumor activity of gemcitabine was synergically enhanced by combination therapy with amatuximab. Furthermore, western blotting revealed that the high expression of phosphorylated c-Met and AKT in liver metastatic lesions treated with gemcitabine monotherapy was canceled by its combination with amatuximab. This result indicated that the observed synergic therapeutic effect may have occurred as a result of the inhibitory effect of amatuximab on the phosphorylation of c-Met and AKT, which were promoted by exposure to GEM. In conclusion, our study revealed that early administration of amatuximab alone or in combination with GEM significantly suppressed the liver metastases of mesothelin-expressing pancreatic cancer cells. A phase II clinical trial of amatuximab as part of an adjuvant chemotherapy regimen for resected pancreatic cancer is expected.

COM902, a novel therapeutic antibody targeting TIGIT augments anti-tumor T cell function in combination with PVRIG or PD-1 pathway blockade.

Immune checkpoint inhibitors (ICIs) have emerged as promising therapies for the treatment of cancer. However, existing ICIs, namely PD-(L)1 and CTLA-4 inhibitors, generate durable responses only in a subset of patients. TIGIT is a co-inhibitory receptor and member of the DNAM-1 family of immune modulating proteins. We evaluated the prevalence of TIGIT and its cognate ligand, PVR (CD155), in human cancers by assessing their expression in a large set of solid tumors. TIGIT is expressed on CD4+ and CD8+ TILs and is upregulated in tumors compared to normal tissues. PVR is expressed on tumor cells and tumor-associated macrophages from multiple solid tumors. We explored the therapeutic potential of targeting TIGIT by generating COM902, a fully human anti-TIGIT hinge-stabilized IgG4 monoclonal antibody that binds specifically to human, cynomolgus monkey, and mouse TIGIT, and disrupts the binding of TIGIT with PVR. COM902, either alone or in combination with a PVRIG (COM701) or PD-1 inhibitor, enhances antigen-specific human T cell responses in-vitro. In-vivo, a mouse chimeric version of COM902 in combination with an anti-PVRIG or anti-PD-L1 antibody inhibited tumor growth and increased survival in two syngeneic mouse tumor models. In summary, COM902 enhances anti-tumor immune responses and is a promising candidate for the treatment of advanced malignancies.

Bisecting-GlcNAc on Asn388 is characteristic to ERC/mesothelin expressed on epithelioid mesothelioma cells.

Mesothelioma is a highly aggressive tumour associated with asbestos exposure and is histologically classified into three types: epithelioid-type, sarcomatoid-type and biphasic-type. The prognosis of mesothelioma patients is poor and there is no effective molecular-targeting therapy as yet. ERC/mesothelin is a glycoprotein that is highly expressed on several types of cancers including epithelioid mesothelioma, but also expressed on normal mesothelial cells. This is a predicted reason why there is no clinically approved therapeutic antibody targeting ERC/mesothelin. In the present study, we focussed on the differential glycosylation between ERC/mesothelin present on epithelioid mesothelioma and that on normal mesothelial cells and aimed to reveal a distinct feature of epithelioid mesothelioma cells. Lectin microarray analysis of ERC/mesothelin using cells and patient specimens showed significantly stronger binding of PHA-E4 lectin, which recognizes complex-type N-glycans having a so-called bisecting-GlcNAc structure, to ERC/mesothelin from epithelioid mesothelioma cells than that from normal mesothelial cells. Further, liquid chromatography/mass spectrometry analysis on ERC/mesothelin from epithelioid mesothelioma cells confirmed the presence of a bisecting-GlcNAc attached to Asn388 of ERC/mesothelin. These results suggest that this glycoproteome could serve as a potential target for the generation of a highly selective and safe therapeutic antibody for epithelioid mesothelioma.

Anti-LYPD1/CD3 T-Cell-Dependent Bispecific Antibody for the Treatment of Ovarian Cancer.

Ovarian cancer is a diverse class of tumors with very few effective treatment options and suboptimal response rates in early clinical studies using immunotherapies. Here we describe LY6/PLAUR domain containing 1 (LYPD1) as a novel target for therapeutic antibodies for the treatment of ovarian cancer. LYPD1 is broadly expressed in both primary and metastatic ovarian cancer with ∼70% prevalence in the serous cancer subset. Bispecific antibodies targeting CD3 on T cells and a tumor antigen on cancer cells have demonstrated significant clinical activity in hematologic cancers. We have developed an anti-LYPD1/CD3 T-cell-dependent bispecific antibody (TDB) to redirect T-cell responses to LYPD1 expressing ovarian cancer. Here we characterize the nonclinical pharmacology of anti-LYPD1/CD3 TDB and show induction of a robust polyclonal T-cell activation and target dependent killing of LYPD1 expressing ovarian cancer cells resulting in efficient in vivo antitumor responses in PBMC reconstituted immune-deficient mice and human CD3 transgenic mouse models. Anti-LYPD1/CD3 TDB is generally well tolerated at high-dose levels in mice, a pharmacologically relevant species, and showed no evidence of toxicity or damage to LYPD1 expressing tissues.

Refinement of the Transmembrane Domain in Constructing the Membrane-Embedded SARS-COV-2 Spike Protein Model

The current widespread coronavirus pandemic has caused great concern and drawn research attention to the virus all over the world. The SARS-COV-2 spike glycoprotein, which binds to the human ACE2 receptor and mediates the membrane fusion and virus entry, is the main target for therapeutic antibodies. However, in recently resolved structures of the protein, some structural regions are still missing, for example, the hairpin repeat 2 (HR2) and the transmembrane C terminal domain (TMD), with the latter even lacking suitable modeling templates from its homologs.

A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein

Vaccines and therapeutics are urgently needed for the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we screen human monoclonal antibodies (mAb) targeting the receptor binding domain (RBD) of the viral spike protein via antibody library constructed from peripheral blood mononuclear cells of a convalescent patient. The CT-P59 mAb potently neutralizes SARS-CoV-2 isolates including the D614G variant without antibody-dependent enhancement effect. Complex crystal structure of CT-P59 Fab/RBD shows that CT-P59 blocks interaction regions of RBD for angiotensin converting enzyme 2 (ACE2) receptor with an orientation that is notably different from previously reported RBD-targeting mAbs. Furthermore, therapeutic effects of CT-P59 are evaluated in three animal models (ferret, hamster, and rhesus monkey), demonstrating a substantial reduction in viral titer along with alleviation of clinical symptoms. Therefore, CT-P59 may be a promising therapeutic candidate for COVID-19.

A broadly protective antibody that targets the flavivirus NS1 protein.

There are no approved flaviviral therapies and the development of vaccines against flaviruses has the potential of being undermined by antibody-dependent enhancement (ADE). The flavivirus nonstructural protein 1 (NS1) is a promising vaccine antigen with low ADE risk but has yet to be explored as a broad-spectrum therapeutic antibody target. Here, we provide the structural basis of NS1 antibody cross-reactivity through cocrystallization of the antibody 1G5.3 with NS1 proteins from dengue and Zika viruses. The 1G5.3 antibody blocks multi-flavivirus NS1-mediated cell permeability in disease-relevant cell lines, and therapeutic application of 1G5.3 reduces viremia and improves survival in dengue, Zika, and West Nile virus murine models. Finally, we demonstrate that 1G5.3 protection is independent of effector function, identifying the 1G5.3 epitope as a key site for broad-spectrum antiviral development.

Therapeutic monoclonal antibody targeting of neuronal pentraxin receptor to control metastasis in gastric cancer

BackgroundControlling metastasis is essential for improving the prognosis of patients with gastric cancer (GC). Here, we aimed to identify a molecule required for GC metastasis and to investigate its potential utility as a target for the development of therapeutic antibodies (Abs).MethodsTranscriptome and bioinformatics analyses of human GC cell lines identified the neuronal pentraxin receptor (NPTXR) as a candidate molecule. NPTXR function was probed by modulating its expression in GC cells and assessing the effects on intracellular signaling and malignant behaviors in vitro and in mouse xenograft models. We also generated anti-NPTXR Abs and Nptxr−/− mice, and assessed the clinical significance of NPTXR expression in GC specimens.ResultsNPTXR mRNA expression in clinical specimens was associated with disease progression and was significantly higher in tissues from GC patients with distant metastasis compared with those without. NPTXR regulated expression of genes involved in metastatic behaviors as well as activation of the PI3K–AKT–mTOR, FAK–JNK, and YAP signaling pathways. NPTXR silencing promoted caspase-mediated apoptosis and attenuated GC cell proliferation, cell cycle progression, migration, invasion, adhesion, stem cell-like properties, and resistance to 5-fluorouracil in vitro, and also inhibited the tumorigenicity of GC cells in vivo. Anti-NPTXR Abs inhibited GC peritoneal metastasis in mice. Nptxr−/− mice showed no abnormalities in reproduction, development, metabolism, or motor function.ConclusionsNPTXR plays an essential role in controlling the malignant behavior of GC cells in vitro and in vivo. NPTXR-targeting Abs may thus have utility as novel diagnostic tools and/or treatment modalities for GC.

Hitting SARS-CoV-2 in a new spot

Coronavirus A key target for therapeutic antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the spike protein, a trimeric protein complex with each monomer comprising an S1 and an S2 domain that mediate binding to host cells and membrane fusion, respectively. In addition to the receptor binding domain (RBD), S1 has an N-terminal domain (NTD). In searching for neutralizing antibodies, there has been a focus on the RBD. Chi et al. isolated antibodies from 10 convalescent patients and identified an antibody that potently neutralizes the virus but does not bind the RBD. Cryo–electron microscopy revealed the epitope as the NTD. This NTD-targeting antibody may be useful to combine with RBD-targeting antibodies in therapeutic cocktails. Science , this issue p. [650][1] [1]: /lookup/doi/10.1126/science.abc6952

A Multi-Targeting Approach to Fight SARS-CoV-2 Attachment.

The public health has declared an international state of emergency due to the spread of a new coronavirus (SARS-CoV-2) representing a real pandemic threat so that to find potential therapeutic agents is a dire need. To this aim, the SARS-CoV-2 spike (S) glycoprotein represents a crucial target for vaccines, therapeutic antibodies, and diagnostics. Since virus binding to ACE-2 alone could not be sufficient to justify such severe infection, in order to facilitate medical countermeasure development and to search for new targets, two further regions of S protein have been taken into consideration here. One is represented by the recently identified ganglioside binding site, exactly localized in our study in the galectin-like domain, and the other one by the putative integrin binding sites contained in the RBD. We propose that a cooperating therapy using inhibitors against multiple targets altogether i.e., ACE2, integrins and sugars could be definitely more effective.

A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2.

Developing therapeutics against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be guided by the distribution of epitopes, not only on the receptor binding domain (RBD) of the Spike (S) protein but also across the full Spike (S) protein. We isolated and characterized monoclonal antibodies (mAbs) from 10 convalescent COVID-19 patients. Three mAbs showed neutralizing activities against authentic SARS-CoV-2. One mAb, named 4A8, exhibits high neutralization potency against both authentic and pseudotyped SARS-CoV-2 but does not bind the RBD. We defined the epitope of 4A8 as the N-terminal domain (NTD) of the S protein by determining with cryo-eletron microscopy its structure in complex with the S protein to an overall resolution of 3.1 angstroms and local resolution of 3.3 angstroms for the 4A8-NTD interface. This points to the NTD as a promising target for therapeutic mAbs against COVID-19.

Production of the Extracellular Part of the ErbB2 Receptor for the Study of Immunobiologicals

The extracellular part of the ErbB2 receptor (ecdErbB2), an oncological marker and a target for therapeutic antibodies, has been expressed in eukaryotic CHO cells. The extracellular part of the ErbB2 receptor has been isolated from a culture medium by metal affinity chromatography and characterized by biochemical and immunochemical methods. It has been shown that the receptor is produced in monomeric, dimeric, and trimeric forms. The oligomeric forms have been separated by gel filtration, and their ability to bind to Herceptin, one of the main drugs in the treatment of breast cancer, has been characterized by ELISA. The receptor produced can be used for in vitro immunochemical experiments.

Coronary inflammation: why searching, how to identify and treat it.

Inflammation plays an important role in the development of atherosclerotic lesions. A variety of stimuli promote atherosclerosis, including increased LDL cholesterol in blood, exposure to tobacco, diabetes mellitus, hypertension, or rheological stress. Inflammatory cells have an established role in the growth of atherosclerotic lesions. Macrophages recognize and internalise ox-LDL to eventually become lipid-laden foam cells, the hallmark cellular component of atheroma. Infiltrating CD4-T cells have a role too, by interacting with ox-LDL and other antigens. Cytokines secreted by inflammatory cells stimulate smooth muscle cells migration whilst macrophages produce metalloprotease that lead to fibrous cap rupture. The necrotic debris of died macrophages and smooth muscle cells help to continue the inflammatory process. The inflammatory response can also directly activate platelets and promote thrombus formation at the surface of complicated coronary plaques. The CANTOS trial can be waived as an innovative study promoting a novel approach of personalized medicine. In patients with previous myocardial infarction, high-sensitivity C-reactive protein level of 2 mg and normal LDL level (<70 mg/dL), canakinumab a therapeutic monoclonal antibody targeting interleukin-1β, at a dose of 150 mg every 3 months, led to a significant reduction of the primary efficacy end point: nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death at 48 months. Based on the CANTOS results, patients on statins and residual inflammatory risk as assessed by means of a high-sensitivity CRP >2 mg/l at baseline have a high risk of future cardiac events, comparable to that of statin-treated patients with suboptimal cholesterol LDL level. The inhibition of interleukin-1β by means of canakinumab, which is only one of many potential anti-inflammatory pathways, open new perspectives, showing that a selective inhibition of the inflammatory pathway may be beneficial in reducing cardiovascular risk. In a process of personalized medicine, there is need to accurately identify patients at high risk of events, to be treated with potent statins or anti-inflammatory drugs. Perhaps in the near future a more specific assessment of coronary inflammations, possibly obtained with imaging modalities (either invasive or non-invasive), will better select patients at risk of events. In this scenario, in the setting of secondary prevention, OCT may serve the scope of identifying vulnerable plaques with local aggregates of inflammatory cells. Future studies are needed to understand the clinical effectiveness of strategies based on invasive coronary assessment.

Clustering for the kill

Structural Biology Cluster of differentiation 20 (CD20) is a membrane protein that defines most B cell populations and is the target of therapeutic antibodies to treat malignancies and autoimmune disorders. Rouge et al. present the structure of CD20 bound to the antibody rituximab that activates the complement system to kill B cells. CD20 forms a dimer and each monomer binds one rituximab antigen-binding fragment (Fab) to give 2:2 stoichiometry. The compact packing between Fab arms and CD20 gives rise to circular assemblies with a diameter similar to that of antibody hexamers known to recruit the first component of the complement cascade. Science , this issue p. [1224][1] [1]: /lookup/doi/10.1126/science.aaz9356

A Novel T Cell-Engaging Bispecific Antibody for Treating Mesothelin-Positive Solid Tumors.

As mesothelin is overexpressed in various types of cancer, it is an attractive target for therapeutic antibodies. T-cell bispecific antibodies bind to target cells and engage T cells via binding to CD3, resulting in target cell killing by T-cell activation. However, the affinity of the CD3-binding arm may influence CD3-mediated plasma clearance or antibody trapping in T-cell-containing tissues. This may then affect the biodistribution of bispecific antibodies. In this study, we used scFab and knob-into-hole technologies to construct novel IgG-based 1 + 1 MG1122-A and 2 + 1 MG1122-B bispecific antibodies against mesothelin and CD3ε. MG1122-B was designed to be bivalent to mesothelin and monovalent to CD3ε, using a 2 + 1 head-to-tail format. Activities of the two antibodies were evaluated in mesothelin-positive tumor cells in vitro and xenograft models in vivo. Although both antibodies exhibited target cell killing efficacy and produced regression of xenograft tumors with CD8+ T-cell infiltration, the antitumor efficacy of MG1122-B was significantly higher. MG1122-B may improve tumor targeting because of its bivalency for tumor antigen. It may also reduce systemic toxicity by limiting the activation of circulating T cells. Thus, MG1122-B may be useful for treating mesothelin-positive solid tumors.

Monoclonal Antibody Targeting Sialyl-di-Lewisa-Containing Internalizing and Noninternalizing Glycoproteins with Cancer Immunotherapy Development Potential.

Tumor glycans constitute attractive targets for therapeutic antibodies. The sialylated glycocalyx plays a prominent role in cancer progression and immune evasion. Here, we describe the characterization of the mAb, FG129, which targets tumor-associated sialylated glycan, and demonstrate its potential for multimodal cancer therapy. FG129, obtained through BALB/c mouse immunizations with liposomes containing membrane glycan extracts from the colorectal cancer cell line LS180, is an mIgG1κ that targets sialyl-di-Lewisa-containing glycoproteins. FG129, as well as its chimeric human IgG1 variant, CH129, binds with nanomolar functional affinity to a range of colorectal, pancreatic, and gastric cancer cell lines. FG129 targets 74% (135/182) of pancreatic, 50% (46/92) of gastric, 36% (100/281) of colorectal, 27% (89/327) of ovarian, and 21% (42/201) of non-small cell lung cancers, by IHC. In our pancreatic cancer cohort, high FG129 glyco-epitope expression was significantly associated with poor prognosis (P = 0.004). Crucially, the glyco-epitope displays limited normal tissue distribution, with FG129 binding weakly to a small percentage of cells within gallbladder, ileum, liver, esophagus, pancreas, and thyroid tissues. Owing to glyco-epitope internalization, we validated payload delivery by CH129 through monomethyl auristatin E (MMAE) or maytansinoid (DM1 and DM4) conjugation. All three CH129 drug conjugates killed high-binding colorectal and pancreatic cancer cell lines with (sub)nanomolar potency, coinciding with significant in vivo xenograft tumor control by CH129-vcMMAE. CH129, with its restricted normal tissue distribution, avid tumor binding, and efficient payload delivery, is a promising candidate for the treatment of sialyl-di-Lewisa-expressing solid tumors, as an antibody-drug conjugate or as an alternative cancer immunotherapy modality.

Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab.

Cluster of differentiation 20 (CD20) is a B cell membrane protein that is targeted by monoclonal antibodies for the treatment of malignancies and autoimmune disorders but whose structure and function are unknown. Rituximab (RTX) has been in clinical use for two decades, but how it activates complement to kill B cells remains poorly understood. We obtained a structure of CD20 in complex with RTX, revealing CD20 as a compact double-barrel dimer bound by two RTX antigen-binding fragments (Fabs), each of which engages a composite epitope and an extensive homotypic Fab:Fab interface. Our data suggest that RTX cross-links CD20 into circular assemblies and lead to a structural model for complement recruitment. Our results further highlight the potential relevance of homotypic Fab:Fab interactions in targeting oligomeric cell-surface markers.

The terminal sialic acid of stage-specific embryonic antigen-4 has a crucial role in binding to a cancer-targeting antibody

The terminal sialic acid of stage-specific embryonic antigen-4 has a crucial role in binding to a cancer-targeting antibody

Programmable Bivalent Peptide-DNA Locks for pH-Based Control of Antibody Activity.

The ability to control antibody activity by pH has important applications in diagnostics, therapeutic antibody targeting, and antibody-guided imaging. Here, we report the rational design of bivalent peptide–DNA ligands that allow pH-dependent control of antibody activity. Our strategy uses a pH-responsive DNA triple helix to control switching from a tight-binding bivalent peptide–DNA lock into a weaker-binding monovalent ligand. Different designs are introduced that allow antibody activation at both basic and acidic pHs, either autonomously or in the presence of an additional oligonucleotide trigger. The pH of antibody activation could be precisely tuned by changing the DNA triple helix sequence. The peptide–DNA locks allowed pH-dependent antibody targeting of tumor cells both in bulk and for single cells confined in water-in-oil microdroplets. The latter approach enables high-throughput antibody-mediated detection of single tumor cells based on their distinctive metabolic activity.