No abstract

The tubular gland of the chicken oviduct is an attractive system for protein expression as large quantities of proteins are deposited in the egg, the production of eggs is easily scalable and good manufacturing practices for therapeutics from eggs have been established. Here we examined the ability of upstream and downstream DNA sequences of ovalbumin, a protein produced exclusively in very high quantities in chicken egg white, to drive tissue-specific expression of human mAb in chicken eggs. To accommodate these large regulatory regions, we established and transfected lines of chicken embryonic stem (cES) cells and formed chimeras that express mAb from cES cell-derived tubular gland cells. Eggs from high-grade chimeras contained up to 3 mg of mAb that possesses enhanced antibody-dependent cellular cytotoxicity (ADCC), nonantigenic glycosylation, acceptable half-life, excellent antigen recognition and good rates of internalization.

Rational modulation of the immune response with biologics represents one of the most promising and active areas for the realization of new therapeutic strategies. In particular, the use of function blocking monoclonal antibodies targeting checkpoint inhibitors such as CTLA-4 and PD-1 have proven to be highly effective for the systemic activation of the human immune system to treat a wide range of cancers. Ipilimumab is a fully human antibody targeting CTLA-4 that received FDA approval for the treatment of metastatic melanoma in 2011. Ipilimumab is the first-in-class immunotherapeutic for blockade of CTLA-4 and significantly benefits overall survival of patients with metastatic melanoma. Understanding the chemical and physical determinants recognized by these mAbs provides direct insight into the mechanisms of pathway blockade, the organization of the antigen-antibody complexes at the cell surface, and opportunities to further engineer affinity and selectivity. Here, we report the 3.0 Å resolution X-ray crystal structure of the complex formed by ipilimumab with its human CTLA-4 target. This structure reveals that ipilimumab contacts the front β-sheet of CTLA-4 and intersects with the CTLA-4:Β7 recognition surface, indicating that direct steric overlap between ipilimumab and the B7 ligands is a major mechanistic contributor to ipilimumab function. The crystallographically observed binding interface was confirmed by a comprehensive cell-based binding assay against a library of CTLA-4 mutants and by direct biochemical approaches. This structure also highlights determinants responsible for the selectivity exhibited by ipilimumab toward CTLA-4 relative to the homologous and functionally related CD28.immunotherapy | X-ray crystallography | CTLA-4 | ipilimumab | cancer

No abstract

Autoimmune diseases like multiple sclerosis (MS) and insulin-dependent diabetes (IDD) are believed to be mediated by pathogenic CD4+ autoreactive T cells which mediate selective destruction of specific host cells. Interrupting the trafficking of such T cells from host circulation to the sites of pathology, such as the central nervous system in the case of MS and the pancreas in the case of IDD, potentially offers a novel opportunity for therapeutic intervention in these diseases. The following summarizes our evolving thoughts on the role of the chemokine network in MS and IDD, and focuses on the chemokine receptor CXCR3 as a potential target for impeding T-cell-mediated destruction in these disease settings.

The ganglioside fucosyl-GM1 (FucGM1) is a tumor-associated antigen expressed in a large percentage of human small cell lung cancer (SCLC) tumors, but absent in most normal adult tissues, making it a promising target in immuno-oncology. This study was undertaken to evaluate the preclinical efficacy of BMS-986012, a novel, nonfucosylated, fully human IgG1 antibody that binds specifically to FucGM1. The antitumor activity of BMS-986012 was evaluated in assays using SCLC cells and in mouse xenograft and syngeneic tumor models, with and without chemotherapeutic agents and checkpoint inhibitors. BMS-986012 showed a high binding affinity for FcγRIIIa (CD16), which resulted in enhanced antibody-dependent cellular cytotoxicity (ADCC) against FucGM1-expressing tumor cell lines. BMS-986012-mediated tumor cell killing was also observed in complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) assays. In several mouse SCLC models, BMS-986012 demonstrated efficacy and was well tolerated. In the DMS79 xenograft model, tumor regression was achieved with BMS-986012 doses of 0.3 mg/kg and greater; antitumor activity was enhanced when BMS-986012 was combined with standard-of-care cisplatin or etoposide. In a syngeneic model, tumors derived from a genetically engineered model of SCLC were treated with BMS-986012 or anti-FucGM1 with a mouse IgG2a Fc and their responses evaluated; when BMS-986012 was combined with anti-PD-1 or anti-CD137 antibody, therapeutic responses significantly improved. Single-agent BMS-986012 demonstrated robust antitumor activity, with the addition of chemotherapeutic or immunomodulatory agents further inhibiting SCLC growth in the same models. These preclinical data supported evaluation of BMS-986012 in a phase I clinical trial of patients with relapsed, refractory SCLC. .

IFN-γ-inducible protein 10 (CXCL10), a chemokine that is abundantly secreted in response to inflammatory stimuli, has been implicated in the pathogenesis of multiple inflammatory diseases, such as inflammatory bowel disease. Whereas CXCL10 is traditionally recognized for recruiting pathogenic T cells to inflamed sites, its nonchemotactic role during inflammation remains poorly defined. In this report, we identified a novel function of CXCL10 in the regulation of the inflammatory potential of human monocytes to produce cytokines. We found that CXCL10 was necessary and sufficient for IFN-γ-primed human monocytes to induce a robust production of proinflammatory cytokines, such as IL-12 and IL-23. CXCL10-induced monocyte production of these cytokines depended on CXCR3 receptor engagement as well as on the Iκ B kinase and p38 MAPK signaling pathways. By using an innate-mediated murine colitis model, we demonstrated that anti-CXCL10 Ab treatment robustly suppressed the local production of myeloid-derived inflammatory cytokines and intestinal tissue damage. Together, our data unravel a previously unappreciated role of CXCL10 in the amplification of myeloid cell-mediated inflammatory responses. Targeting CXCL10 is therefore an attractive approach to treating inflammatory diseases that are driven by innate and adaptive immunity.

Antibody drug conjugates (ADCs) can undergo in vivo biotransformation (e.g., payload metabolism, deconjugation) leading to reduced or complete loss of activity. The location/site of conjugation of payload-linker can have an effect on ADC stability and hence needs to be carefully optimized. Affinity capture LC–MS of intact ADCs or ADC subfragments has been extensively used to evaluate ADC biotransformation. However, the current methods have certain limitations such as the requirement of specific capture reagents, limited mass resolution of low mass change metabolites, low sensitivity, and use of capillary or nanoflow LC–MS. To address these challenges, we developed a generic affinity capture LC–MS assay that can be utilized to evaluate the biotransformation of any site-specific ADC independent of antibody type and site of conjugation (Fab and Fc) in preclinical studies. The method involves a combination of some or all of these steps: (1) “mono capture” or “dual capture” of ADCs from serum with streptavidin magnetic beads coated with a generic biotinylated antihuman capture reagent, (2) “on-bead” digestion with IdeS and/or PNGase F, and (3) reduction of interchain disulfide bonds to generate ∼25 kDa ADC subfragments, which are finally analyzed by LC–HRMS on a TOF mass spectrometer. The advantages of this method are that it can be performed using commercially available generic reagents and requires sample preparation time of less than 7 h. Furthermore, by reducing the size of intact ADC (∼150 kDa) to subfragments (∼25 kDa), the identification of conjugated payload and its metabolites can be achieved with excellent sensitivity and resolution (hydrolysis and other small mass change metabolites). This method was successfully applied to evaluate the in vitro and in vivo biotransformation of ADCs conjugated at different sites (LC, HC-Fab, and HC-Fc) with various classes of payload-linkers.