A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens

Bispecific antibodies based on full-length antibody structures are more optimal than fragment-based formats because they benefit from the favorable properties of the Fc region. However, the homodimeric nature of Fc effectively imposes bivalent binding on all current full-length bispecific antibodies, an attribute that can result in nonspecific activation of cross-linked receptors. We engineered a novel bispecific format, referred to as mAb-Fv, that utilizes a heterodimeric Fc region to enable monovalent co-engagement of a second target antigen in a full-length context. mAb-Fv constructs co-targeting CD16 and CD3 were expressed and purified as heterodimeric species, bound selectively to their co-target antigens and mediated potent cytotoxic activity by NK cells and T cells, respectively. The capacity to co-engage distinct target antigens simultaneously with different valencies is an improved feature for bispecific antibodies with promising therapeutic implications.Key words: bispecific, mAb-Fv, Fc, heterodimer, CD16, CD3, HER2, HM1.24, anti-tumor, cancerDespite the enormous success of antibody-based therapeutics for the treatment of a variety of diseases, research efforts to improve their clinical efficacy continue. One avenue being explored is the engineering of new antigen binding sites to permit co-engagement of two distinct targets. Such engineered antibodies are commonly referred to as bispecifics, and a wide variety of formats have been described in references ¹ and ². Co-target antigens can include two targets believed to be causal in the pathology of a particular disease, e.g., two cytokines or growth factors.^3–5 Alternatively, the co-target pair may be a cell surface antigen and an immune receptor such that a novel “effector” mechanism can be built into the antibody, beyond those mediated naturally by the Fc region.²In the 1980s, bispecific antibodies were made by fusing two cell lines that each produced a single monoclonal antibody (mAb).⁶ Although the resulting hybrid hybridoma or quadroma did produce bispecifics, they were only a minor population and extensive purification was required to isolate the desired antibody. Antibody fragments provided an engineering solution to this problem; because they lack the complex quaternary structure of a full-length antibody, multiple variable regions can be linked in single genetic constructs. Antibody fragments of many different forms have been generated, including diabodies, single chain diabodies, tandem scFvs and F(ab'')₂ bispecifics.^2,7 While these formats can be expressed at high levels in bacteria and, arguably, may have benefits due to their small size, they suffer from poor half-life in vivo and can present manufacturing challenges related to their production and stability. For example, the rapid clearance of some fragment-based bispecifics requires that they be infused continuously via a portable pump over one to two months.⁸ The principal source of these limitations for fragment formats is the lack of an antibody Fc region with its associated structural and functional benefits, including large size that precludes renal filtration; high stability; binding to various Fc ligands, one of which maintains serum persistence (the neonatal Fc receptor FcRn) and binding to proteins A and G, which facilitates large scale purification.Recent work has attempted to address the shortcomings of fragment-based bispecifics by engineering a second antigen binding site into full-length antibody-like formats.^5,9–12 The presence of an Fc region in theory provides these formats with the developability and pharmacokinetic properties of standard IgG mAbs. However, because these constructs build new antigen binding sites on top of a homodimeric constant chain, binding to the new antigen is always bivalent. This consequence may pose a constraint depending on the co-targeting goal.For many immune receptors, cellular activation is accomplished by cross-linking of a monovalent binding interaction. The mechanism of cross-linking is typically mediated by antibody/antigen immune complexes, or via effector cell to target cell engagement. For example, the low affinity activating Fc gamma receptors (FcγRs) such as CD16 (FcγRIIIa) and CD32a (FcγRIIa) that mediate cellular killing bind monovalently to the antibody Fc region. While monovalent binding does not result in cellular signaling, upon effector cell engagement with the target cell, receptors are cross-linked and clustered on the cell surface, leading to activation.¹³ On T cells, CD3 activation occurs when its associated T-cell receptor (TCR) engages antigen-loaded major histocompatibility complex (MHC) on antigen-presenting cells in an avid cell-to-cell synapse.¹⁴ Bivalent antibodies targeting CD3 can elicit massive cytokine release, and the consequent toxicity has presented challenges for the development of anti-CD3 antibodies as drugs;^15,16 in contrast, monovalent binding of CD3 in Fab^17,18 and bispecific¹⁹ formats generates much lower levels of T-cell activation. For bispecifics, a consequence of this biology is that bivalent cross-linking of receptors can lead to non-specific activation of an effector cell in the absence of target cell.Thus, when the therapeutic goal is the co-engagement of an immune receptor, the desired binding may be monovalent rather than bivalent. This mode is incompatible with the majority of current full-length bispecifics. We describe an engineering solution to this problem that utilizes a heterodimeric Fc region to enable a single additional variable region to be built monomerically onto an antibody. Our new bispecific format, which we refer to as mAb-Fv, enables the simultaneous bivalent and monovalent co-engagement of distinct target antigens.     

Multiple interferon stimulated genes synergize with the zinc finger antiviral protein to mediate anti-alphavirus activity

The zinc finger antiviral protein (ZAP) is a host factor that mediates inhibition of viruses in the Filoviridae, Retroviridae and Togaviridae families. We previously demonstrated that ZAP blocks replication of Sindbis virus (SINV), the prototype Alphavirus in the Togaviridae family at an early step prior to translation of the incoming genome and that synergy between ZAP and one or more interferon stimulated genes (ISGs) resulted in maximal inhibitory activity. The present study aimed to identify those ISGs that synergize with ZAP to mediate Alphavirus inhibition. Using a library of lentiviruses individually expressing more than 350 ISGs, we screened for inhibitory activity in interferon defective cells with or without ZAP overexpression. Confirmatory tests of the 23 ISGs demonstrating the largest infection reduction in combination with ZAP revealed that 16 were synergistic. Confirmatory tests of all potentially synergistic ISGs revealed 15 additional ISGs with a statistically significant synergistic effect in combination with ZAP. These 31 ISGs are candidates for further mechanistic studies. The number and diversity of the identified ZAP-synergistic ISGs lead us to speculate that ZAP may play an important role in priming the cell for optimal ISG function.     

2,4-Diaryl-5,6-dihydro-1,10-phenanthroline and 2,4-diaryl-5,6-dihydrothieno[2,3-h] quinoline derivatives for topoisomerase I and II inhibitory activity, cytotoxicity, and structure-activity relationship study

Designed and synthesized thirty-two 2,4-diaryl-5,6-dihydro-1,10-phenanthroline and 2,4-diaryl-5,6-dihydrothieno[2,3-h] quinoline derivatives as rigid analogs of 2,4,6-trisubstituted pyridines were evaluated for topoisomerase I and II inhibitory activities as well as cytotoxicities against several human cancer cell lines. Structure-activity relationship study showed that [2,2';6',2"]-terpyridine skeleton is important for the cytotoxicity against several human cancer cell lines.     

Azido-containing aryl beta-diketo acid HIV-1 integrase inhibitors

Aryl beta-diketo acids (ADK) comprise a general class of potent HIV-1 integrase (IN) inhibitors, which can exhibit selective inhibition of strand transfer reactions in extracellular recombinant IN assays and provide potent antiviral effects in HIV-infected cells. Recent studies have shown that polycyclic aryl or aryl rings bearing aryl-containing substituents are components of potent members of this class. Reported herein is the first use of azido functionality as an aryl replacement in beta-diketo acid IN inhibitors. The ability of azido-containing inhibitors to exhibit potent inhibition of IN and antiviral protection in HIV-infected cells, renders the azide group of potential value in the further development of ADK-based IN inhibitors.     

PLAC-24 is a cytoplasmic dynein-binding protein that is recruited to sites of cell-cell contact

We screened for polypeptides that interact specifically with dynein and identified a novel 24-kDa protein (PLAC-24) that binds directly to dynein intermediate chain (DIC). PLAC-24 is not a dynactin subunit, and the binding of PLAC-24 to the dynein intermediate chain is independent of the association between dynein and dynactin. Immunocytochemistry using PLAC-24-specific polyclonal antibodies revealed a punctate perinuclear distribution of the polypeptide in fibroblasts and isolated epithelial cells. However, as epithelial cells in culture make contact with adjacent cells, PLAC-24 is specifically recruited to the cortex at sites of contact, where the protein colocalizes with components of the adherens junction. Disruption of the cellular cytoskeleton with latrunculin or nocodazole indicates that the localization of PLAC-24 to the cortex is dependent on intact actin filaments but not on microtubules. Overexpression of beta-catenin also leads to a loss of PLAC-24 from sites of cell-cell contact. On the basis of these data and the recent observation that cytoplasmic dynein is also localized to sites of cell-cell contact in epithelial cells, we propose that PLAC-24 is part of a multiprotein complex localized to sites of intercellular contact that may function to tether microtubule plus ends to the actin-rich cellular cortex.     

Synthesis of Novel Pyrazole-Oxindole Conjugates with Cytotoxicity in Human Cancer Cells via Apoptosis

A novel series of pyrazole-oxindole conjugates were prepared and characterized as potential cytotoxic agents by FT-IR, NMR and HR-MS. The cytotoxic activity of these compounds was tested in the Jurkat acute T cell leukemia, CEM acute lymphoblastic leukemia, MCF10 A mammary epithelial and MDA-MB 231 triple negative breast cancer cell lines. Among the tested conjugates, 5-methyl-3-((3-(1-phenyl)-3-(p-tolyl)-1H-pyrazol-4-yl)methylene)indolin-2-one 6h emerged as the most cytotoxic with a CC₅₀ of 4.36+/−0.2 μM against Jurkat cells. The mechanism of cell death induced by 6h was investigated through the Annexin V-FITC assay via flow cytometry. Reactive oxygen species (ROS) accumulation, mitochondrial health and the cell cycle progression were also evaluated in cells exposed to 6h . Results demonstrated that 6h induces apoptosis in a dose-response manner, without generating ROS and/or altering mitochondrial health. In addition, 6h disrupted the cell cycle distribution causing an increase in DNA fragmentation (Sub G0-G1), and an arrest in the G0-G1 phase. Taken together, the 6h compound revealed a strong potential as an antineoplastic agent evidenced by its cytotoxicity in leukemia cells, the activation of apoptosis and restriction of the cell cycle progression.     

Bandgap Narrowing in Non‐Fullerene Acceptors: Single Atom Substitution Leads to High Optoelectronic Response Beyond 1000 nm

Two narrow bandgap non‐fullerene acceptors (NBG‐NFAs), namely, COTIC‐4F and SiOTIC‐4F, are designed and synthesized for the fabrication of efficient near‐infrared organic solar cells (OSCs). The chemical structures of the NBG‐NFAs contain a D′‐D‐D′ electron‐rich internal core based on a cyclopentadithiophene (or dithienosilole) (D) and alkoxythienyl (D′) core, end‐capped with the highly electron‐deficient unit 2‐(5,6‐difluoro‐3‐oxo‐2,3‐dihydro‐1H‐inden‐1‐ylidene)malononitrile (A), ultimately providing a A‐D′‐D‐D′‐A molecular configuration that enhances the intramolecular charge transfer characteristics of the excited states. One can thereby reduce the optical bandgap (E_g^opt) to as low as ≈1.10 eV, one of the smallest values for NFAs reported to date. In bulk‐heterojunction (BHJ) OSCs, NBG‐NFA blends with the polymer donor PTB7‐Th yield power conversion efficiencies (PCE) of up to 9.0%, which is particularly high when compared against a range of NBG BHJ blends. Most significantly, it is found that, despite the small energy loss (E_g^opt ? eV_OC) of 0.52 eV, the PTB7‐Th/NBG‐NFA bulk heterojunction blends can yield short‐circuit current densities of up to 22.8 mA cm^?2, suggesting that the design and application of NBG‐NFA materials have substantial potential to further improve the PCE of OSCs.     

Epidemiology,Impact and Control of Rabies in Nepal: A Systematic Review

Background

Rabies is a vaccine-preventable viral zoonosis belonging to the group of neglected tropical diseases. Exposure to a rabid animal may result in a fatal acute encephalitis if effective post-exposure prophylaxis is not provided. Rabies occurs worldwide, but its burden is disproportionately high in developing countries, including Nepal. We aimed to summarize current knowledge on the epidemiology, impact and control of rabies in Nepal.

Methods

We performed a systematic review of international and national scientific literature and searched grey literature through the World Health Organization Digital Library and the library of the National Zoonoses and Food Hygiene Research Centre, Nepal, and through searching Google and Google Scholar. Further data on animal and human rabies were obtained from the relevant Nepalese government agencies. Finally, we surveyed the archives of a Nepalese daily to obtain qualitative information on rabies in Nepal.

Findings

So far, only little original research has been conducted on the epidemiology and impact of rabies in Nepal. Per year, rabies is reported to kill about 100 livestock and 10–100 humans, while about 1,000 livestock and 35,000 humans are reported to receive rabies post-exposure prophylaxis. However, these estimates are very likely to be serious underestimations of the true rabies burden. Significant progress has been made in the production of cell culture-based anti-rabies vaccine and rabies immunoglobulin, but availability and supply remain a matter of concern, especially in remote areas. Different state and non-state actors have initiated rabies control activities over the years, but efforts typically remained focalized, of short duration and not harmonized. Communication and coordination between veterinary and human health authorities is limited at present, further complicating rabies control in Nepal. Important research gaps include the reporting biases for both human and animal rabies, the ecology of stray dog populations and the true contribution of the sylvatic cycle.

Interpretation

Better data are needed to unravel the true burden of animal and human rabies. More collaboration, both within the country and within the region, is needed to control rabies. To achieve these goals, high level political commitment is essential. We therefore propose to make rabies the model zoonosis for successful control in Nepal.     

Plk1 inhibition enhances the efficacy of gemcitabine in human pancreatic cancer

Gemcitabine is the standard-of-care for chemotherapy in patients with pancreatic adenocarcinoma and it can directly incorporate into DNA or inhibit ribonucleotide reductase to prevent DNA replication and, thus, tumor cell growth. Most pancreatic tumors, however, develop resistance to gemcitabine. Polo-like kinase 1 (Plk1), a critical regulator in many cell cycle events, is significantly elevated in human pancreatic cancer. In this study, we show that Plk1 is required for the G1/S transition and that inhibition of Plk1 significantly reduces the DNA synthesis rate in human pancreatic cancer cells. Furthermore, the combined effect of a specific Plk1 inhibitor GSK461364A with gemcitabine was examined. We show that inhibition of Plk1 significantly potentiates the anti-neoplastic activity of gemcitabine in both cultured pancreatic cancer cells and Panc1-derived orthotopic pancreatic cancer xenograft tumors. Overall, our study demonstrates that co-targeting Plk1 can significantly enhance the efficacy of gemcitabine, offering a promising new therapeutic option for the treatment of gemcitabine-resistant human pancreatic cancer.     

A dynactin subunit with a highly conserved cysteine-rich motif interacts directly with Arp1

Dynactin is a multisubunit complex and a required cofactor for most, or all, of the cellular processes powered by the microtubule-based motor cytoplasmic dynein. Using a dynein affinity column, the previously uncharacterized p62 subunit of dynactin was isolated and microsequenced. Two peptide sequences were used to clone human cDNAs encoding p62. Sequence analysis of the predicted human polypeptide of 53 kDa revealed a highly conserved pattern of eleven cysteine residues, eight of which fit the consensus sequence for a Zn(2+)-binding RING domain. We have characterized p62 as an integral component of 20 S dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments demonstrate that p62 binds directly to the Arp1 subunit of dynactin. Immunocytochemistry with antibodies to p62 demonstrates that this polypeptide has a punctate cytoplasmic distribution as well as centrosomal distribution typical of dynactin. In transfected cells, overexpression of p62 did not disrupt microtubule organization or the integrity of the Golgi but did cause both cytosolic and nuclear distribution of the protein, suggesting that this polypeptide may be targeted to the nucleus at very high expression levels.