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The antibody‐drug conjugate (ADC) MORAb‐202, consisting of farletuzumab paired with a cathepsin B–cleavable linker and eribulin, targets folate receptor alpha (FRA), which is frequently overexpressed in various tumor types. MORAb‐202 was highly cytotoxic to FRA‐positive cells in vitro, with limited off‐target killing of FRA‐negative cells. Furthermore, MORAb‐202 showed a clear in vitro bystander cytotoxic effect in coculture with FRA‐positive/negative cells. In vivo antitumor efficacy studies of MORAb‐202 were conducted with a single administration of MORAb‐202 in triple‐negative breast cancer (TNBC) patient–derived xenograft (PDx) models expressing low and high levels of FRA. MORAb‐202 exhibited durable efficacy proportional to tumor FRA expression. Toxicology studies (Q3Wx2) in nonhuman primates suggested that the major observed toxicity of MORAb‐202 is hematologic toxicity. Overall, these findings support the concept that MORAb‐202 represents a promising investigational ADC for the treatment of TNBC patients. The antibody‐drug conjugate (ADC) MORAb‐202, consisting of farletuzumab with cleavable linker, is the first ADC that carries clinically validated eribulin as a payload and exhibits direct antitumor efficacy and a strong bystander effect. As a result, the therapeutic window of MORAb‐202 is much wider than other nonvalidated payloads. Importantly, its well validated safety profile is minimizes risk in the clinic. Thus, MORAb‐202 represents promising investigational therapeutics for TNBC patients.

Tau deposition in the brain is a pathological hallmark of many neurodegenerative disorders, including Alzheimer’s disease (AD). During the course of these tauopathies, tau spreads throughout the brain via synaptically-connected pathways. Such propagation of pathology is thought to be mediated by tau species (“seeds”) containing the microtubule binding region (MTBR) composed of either three repeat (3R) or four repeat (4R) isoforms. The tau MTBR also forms the core of the neuropathological filaments identified in AD brain and other tauopathies. Multiple approaches are being taken to limit tau pathology, including immunotherapy with anti-tau antibodies. Given its key structural role within fibrils, specifically targetting the MTBR with a therapeutic antibody to inhibit tau seeding and aggregation may be a promising strategy to provide disease-modifying treatment for AD and other tauopathies. Therefore, a monoclonal antibody generating campaign was initiated with focus on the MTBR. Herein we describe the pre-clinical generation and characterisation of E2814, a humanised, high affinity, IgG 1 antibody recognising the tau MTBR. E2814 and its murine precursor, 7G6, as revealed by epitope mapping, are antibodies bi-epitopic for 4R and mono-epitopic for 3R tau isoforms because they bind to sequence motif HVPGG. Functionally, both antibodies inhibited tau aggregation in vitro . They also immunodepleted a variety of MTBR-containing tau protein species. In an in vivo model of tau seeding and transmission, attenuation of deposition of sarkosyl-insoluble tau in brain could also be observed in response to antibody treatment. In AD brain, E2814 bound different types of tau filaments as shown by immunogold labelling and recognised pathological tau structures by immunohistochemical staining. Tau fragments containing HVPGG epitopes were also found to be elevated in AD brain compared to PSP or control. Taken together, the data reported here have led to E2814 being proposed for clinical development.

Background E2814 is a humanized monoclonal antibody that recognizes the microtubule‐binding region (MTBR) of tau, a region of the protein essential for filament formation and propagation in neurodegenerative diseases. Epitope mapping showed that E2814 binds to a specific sequence motif HVPGG in the MTBR. To elucidate the atomic interactions of E2814‐tau binding, we performed X‐ray crystallography studies with E2814 and various tau peptides containing the HVPGG motif. Method The Fab fragment of E2814 was incubated with 5 different tau peptides containing the 299‐HVPGG‐303 epitope at a 1:10 molar ratio prior to co‐crystallization screening. Co‐crystals of E2814/tau peptides were grown by sitting drop vapor diffusion, and the diffraction data were collected at synchrotron. Structures of E2814 bound to tau peptides were determined by molecular replacement. Result Crystal structures of E2814 Fab bound to five tau peptides containing 299‐HVPGG‐303 epitope were determined at 1.44 –1.64 Å resolution. The comparison of the five crystal structures revealed the sequence 299‐HVPG‐302 of tau presents the core region mediating recognition by E2814, which is also facilitated by I297 in the flanking region. The tau peptides are accommodated in the recognition groove formed by CDR regions from the VH and VL domains, and exhibited a U‐shaped conformation with close‐to quarter turns when bound to E2814. Both H299 and P301 at the turns mediate major interactions with E2814. Conclusion Our results reveal the structural details of the key interactions between E2814 and the tau HVPGG motif, which adopts a U‐shaped conformation upon binding to E2814. These findings provide atomic insights into the mechanism of E2814‐mediated inhibition of tau aggregation and propagation.

Abstract Background: Several HER2-targeting antibody–drug conjugates (ADC) have gained market approval for the treatment of HER2-expressing metastasis. Promising responses have been reported with the new generation of ADCs in patients who do not respond well to other HER2-targeting therapeutics. However, these ADCs still face challenges of resistance and/or severe adverse effects associated with their particular payload toxins. Eribulin, a therapeutic agent for the treatment of metastatic breast cancer and liposarcoma, is a new choice of ADC payload with a distinct mechanism of action and safety profile. Methods: We’ve generated a novel HER2-tageting eribulin-containing ADC, BB-1701. The potency of BB-1701 was tested in vitro and in vivo against cancer cells where HER2-expressing levels vary in a large range. Bystander killing effect and toxin-induced immunogenic cell death (ICD) of BB-1701 were also tested. Results: In comparison with HER2-targeting ADCs with DM1 and Dxd payload, eribulin-containing ADC demonstrated higher in vitro cytotoxicity in HER2-low cancer cell lines. BB-1701 also effectively suppressed tumors in models resistant to DM1 or Dxd containing ADCs. Mode of action studies showed that BB-1701 had a significant bystander effect on HER2-null cells adjacent to HER2-high cells. In addition, BB-1701 treatment induced ICD. Repeated doses of BB-1701 in nonhuman primates showed favorable pharmacokinetics and safety profiles at the intended clinical dosage, route of administration, and schedule. Conclusions: The preclinical data support the test of BB-1701 in patients with various HER2-expressing cancers, including those resistant to other HER2-targeting ADCs. A phase I clinical trial of BB-1701 (NCT04257110) in patients is currently underway. Statement of Significance: A HER2-targeting eribulin-containing ADC had exhibited good plasma stability; BB-1701 had potent antitumor activities against multiple HER2-expressing cancers, especially those insensitive to other HER2-targeting ADCs; BB-1701 had bystander killing effect and induced immunogenic cell death; BB-1701 also showed favorable pharmacokinetics and safety profiles in nonhuman primates.

776.1 is a murine IgG1 monoclonal antibody to the human ovarian cancer antigen CA 125 that has the unique property of having a clear preference for binding to the cell-associated form of the antigen. We have examined the tumor localization properties and efficacy of 776.1 in a subcutaneous OVCAR-3 xenograft mouse model of human ovarian cancer. Biodistribution experiments using 125I-labeled 776.1 demonstrated a peak uptake in tumors at 72 hours postinjection, with an average of 17.7% of injected dose per gram localized to the tumor. Little uptake in other organs was observed. Further experiments using CA 125-transfected syngeneic tumors, as well as an immunoprecipitation assay using human chimeric 776.1, both clearly demonstrated that 776.1 localizes to the tumor in a CA 125-dependent manner. DOTA-776.1 (1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″ tetraacetic acid-conjugated 776.1) was labeled with 90Y and used in efficacy studies. [90Y-DOTA]776.1 at a single dose of 150 µCi was able to mediate efficient reduction of tumor growth, with regression observed in a subset of animals for a period ranging from 3 to 48 days, equivalent to 3 weekly administrations of cisplatin at 6 mg/kg. No significant regression was observed in groups receiving [90Y-DOTA]MOPC-21 control antibody at any dose. These results suggest that 776.1 may be a promising radioimmunotherapeutic agent for the treatment of human ovarian cancer.

Objective: Antibodies to CA 125 have been used to predict relapse of ovarian cancer, but have performed poorly as therapeutic agents. One rationale for this is antibody binding to circulating shed antigen. Our aim in this study was to develop antibodies to human CA 125 that have enhanced selectivity for the cell-associated form of the antigen. Methods: Monoclonal antibodies were raised to a recombinant fragment of CA 125 that included sequence proximal to the putative membrane attachment site. Antibodies were characterized in terms of their binding site, affinity and selectivity for cell-associated CA 125. Results: In assays using patient-derived CA 125, a subset of high-affinity (K D <5 nM) monoclonal antibodies demonstrated a 10- to greater than 200-fold increase in selectivity for cell-associated CA 125 when compared with controls. Based on mapping of the various monoclonal antibodies obtained, it was determined that shedding of CA 125 most likely occurs in the most C-terminal repeat domain. Conclusion: Results from competition analysis using patient-derived shed antigen predict that the antibodies described in this study may have significantly enhanced tumor-targeting properties when compared with existing antibodies to CA 125 in a tumor environment having high concentrations (>10,000 CA 125 units) of shed CA 125.

Background Staphylococcal enterotoxins are considered potential biowarfare agents that can be spread through ingestion or inhalation. Staphylococcal enterotoxin B (SEB) is a widely studied superantigen that can directly stimulate T-cells to release a massive amount of proinflammatory cytokines by bridging the MHC II molecules on an antigen presenting cell (APC) and the Vβ chains of the T-cell receptor (TCR). This potentially can lead to toxic, debilitating and lethal effects. Currently, there are no preventative measures for SEB exposure, only supportive therapies. Methods To develop a potential therapeutic candidate to combat SEB exposure, we have generated three human B-cell hybridomas that produce human monoclonal antibodies (HuMAbs) to SEB. These HuMAbs were screened for specificity, affinity and the ability to block SEB activity in vitro as well as its lethal effect in vivo . Results The high-affinity HuMAbs, as determined by BiaCore analysis, were specific to SEB with minimal crossreactivity to related toxins by ELISA. In an immunoblotting experiment, our HuMAbs bound SEB mixed in a cell lysate and did not bind any of the lysate proteins. In an in vitro cell-based assay, these HuMAbs could inhibit SEB-induced secretion of the proinflammatory cytokines (INF-γ and TNF-α) by primary human lymphocytes with high potency. In an in vivo LPS-potentiated mouse model, our lead antibody, HuMAb-154, was capable of neutralizing up to 100 μg of SEB challenge equivalent to 500 times over the reported LD 50 (0.2 μg) , protecting mice from death. Extended survival was also observed when HuMAb-154 was administered after SEB challenge. Conclusion We have generated high-affinity SEB-specific antibodies capable of neutralizing SEB in vitro as well as in vivo in a mouse model. Taken together, these results suggest that our antibodies hold the potential as passive immunotherapies for both prophylactic and therapeutic countermeasures of SEB exposure.

Tissue factor (TF) is a type I transmembrane protein and the initiator of the extrinsic blood coagulation pathway. TF plays a critical role in tumor development and its overexpression is observed in many tumors. To understand the prevalence and relative level of TF expression in non–small-cell lung cancer(NSCLC), we analyzed 50 NSCLC tumors by immunohistochemical staining and found that 88% of human NSCLC tumors overexpressed TF. We then generated a high affinity anti-TF antibody, TF278, which specifically binds TF on the surface of cells and is internalized upon binding. An 111In-labeled TF278 demonstrated favorable tumor accumulation in an SW-900 xenograft tumor model with a maximum mean percent of injected dose per gram of tissue (%ID/g) of 73.1% at 96 hours postinjection. In addition, we labeled the antibody with 90Y and tested its ability to inhibit the growth of tumors in an SW-900 xenograft tumor model in immunocompromised mice. The 90Y-TF278 slowed the growth of SW-900 tumors at a 50µCi dose and completely regressed SW-900 tumors at a 150 µCi dose with little toxicity.

Mucins are secreted glycoproteins which aid to protect and hydrate the exposed epithelial surfaces of mucosal systems. Until recently, detailed sequence and structural analysis of the protein moiety was limited by their resistance to standard biochemical and analytical analysis. In order to understand better the structure of apomucins and how their synthesis and expression is regulated, a low molecular weight mucin from rat submandibular glands (RSMG) was purified using a combination of anion exchange, gel filtration, and high-performance liquid chromatography. The mucin was chemically deglycosylated and partial amino acid sequence was obtained by Edman degradation. The sequence information was used to amplify mucin-like sequences by polymerase chain reaction, which were then used to isolate full length cDNA clones. The conceptually translated apoprotein consists of a signal peptide, followed by a secreted protein consisting of three separate domains--a non-glycosylated N-terminal domain, a central repeat domain consisting of eleven tandem repeats of thirteen amino acids, and a non-repetitive C-terminal domain. The last two domains are presumably heavily O-glycosylated due to their large content of hydroxyamino acids. Their differing ratios of hydroxyamino acid to proline suggest that they may represent two distinctly different domains of O-glycosylation. Transcripts corresponding to the cDNAs were found to be limited in their expression to the rat submandibular gland. Southern blot analysis indicated that the gene showed limited allelic polymorphism in the number of tandem repeats, a common feature of mucin genes. A cDNA clone was used to isolate overlapping lambda phage genomic clones, which showed a gene structure of three exons of 106 bp, 69 bp, and 991 bp, respectively, separated by introns of 0.9 kb and 12.5 kb. Sequence analysis of 3.8 kb of 5 $\\sp\\prime$flanking DNA revealed no homology with any other mucin or salivary genes cloned to date. Analysis of expression of the gene by Northern blot analysis from rats chronically exposed to the$\\beta$ -andrenergic agonist isoproterenol revealed no differences between agonist-exposed rats and saline controls. This gene represents only the third mucin gene, and the first secreted mucin, cloned in its entirety. Accordingly, the gene is referred to as RSM-1.