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During the drug development process, testing potency plays an important role in the quality assessment required for the manufacturing and marketing of biologics. Due to multiple operational and biological factors, higher variability is usually observed in bioassays compared with physicochemical methods. In this paper, we discuss different sources of bioassay variability and how this variability can be statistically estimated. In addition, we propose an algorithm to estimate the variability of reportable results associated with different numbers of runs and their corresponding OOS rates under a given specification. Numerical experiments are conducted on multiple assay formats to elucidate the empirical distribution of bioassay variability. [ABSTRACT FROM AUTHOR]

Asparagine deamidation is a post-translational modification (PTM) that converts asparagine residues into iso-aspartate and/or aspartate. Non-enzymatic asparagine deamidation is observed frequently during the manufacturing, processing, and/or storage of biotherapeutic proteins. Depending on the site of deamidation, this PTM can significantly impact the therapeutic's potency, stability, and/or immunogenicity. Thus, deamidation is routinely monitored as a potential critical quality attribute. The initial evaluation of an asparagine's potential to deamidate begins with identifying sequence liabilities, in which the n + 1 amino acid is of particular interest. NW is one motif that occurs frequently within the complementarity-determining region (CDR) of therapeutic antibodies, but according to the published literature, has a very low risk of deamidating. Here we report an unusual case of this NW motif readily deamidating within the CDR of an antibody drug conjugate (ADC), which greatly impacts the ADC's biological activities. Furthermore, this NW motif solely deamidates into iso-aspartate, rather than the typical mixture of iso-aspartate and aspartate. Interestingly, biological activities are more severely impacted by the conversion of asparagine into iso-aspartate via deamidation than by conversion into aspartate via mutagenesis. Here, we detail the discovery of this unusual NW deamidation occurrence, characterize its impact on biological activities, and utilize structural data and modeling to explain why conversion to iso-aspartate is favored and impacts biological activities more severely. [ABSTRACT FROM AUTHOR]

The article presents a study that explores the Vaccines derived from chimpanzee adenovirus Y25 (ChAdOx1) which interacts with CAR and PF4 with implications for thrombosis with thrombocytopenia syndrome. It mentions that thrombosis with thrombocytopenia syndrome (TTS), a rare condition resembling heparin-induced thrombocytopenia.

Novel phenol bis-Mannich adducts were identified as IL-2 expression inhibitors in a T cell proliferation screening assay. Analogues of the lead compound were prepared through parallel synthesis and a highly selective IL-2 inhibitor was discovered that provided a suitable compound for further optimization. [Copyright &y& Elsevier]

Bispecific antibodies (BsAbs) or fusion proteins (BsAbFPs) present a promising strategy for cancer immunotherapy. Numerous BsAbs targeting coinhibitory and costimulatory pathways have been developed for retargeting T cells and antigen presenting cells (APCs). It is challenging to assess the potency of BsAb that engages two different signaling pathways simultaneously in a single assay format, especially when the two antigen targets are expressed on different cells. To explore the potency of anti-PD-L1/CD40L BsAbFP, a fusion protein that binds to human CD40 and PD-L1, we engineered CHO cells as surrogate APCs that express T cell receptor activator and PD-L1, Jurkat cells with PD-1 and NFAT-luciferase reporter as effector T cells, and Raji cell with NFkB-luciferase that endogenously expresses CD40 as accessory B cells. A novel reporter gene bioassay was developed using these cell lines that allows anti-PD-L1/CD40L BsAbFP to engages both PD-1/PD-L1 and CD40/CD40L signaling pathways in one assay. As both reporters use firefly luciferase, the effects of activating both signaling pathways is observed as an increase in luminescence, either as a higher upper asymptote, a lower EC50, or both. This dual target reporter gene bioassay system reflects potential mechanism of action and demonstrated the ability of anti-PD-L1/CD40L BsAbFP to synergistically induce biological response compared to the combination of anti-PD-L1 monovalent monoclonal antibody and agonist CD40L fusion protein, or either treatment alone. The results also showed a strong correlation between the drug dose and biological response within the tested potency range with good linearity, accuracy, precision, specificity and stability indicating properties, suggesting that this "three-cell-in-one" dual target reporter gene bioassay is suitable for assessing potency, structure-function and critical quality attributes of anti-PD-L1/CD40L BsAbFP. This approach could be used for developing dual target bioassays for other BsAbs and antibodies used for combination therapy. [ABSTRACT FROM AUTHOR]

Previously, we demonstrated that for H1N1 and H5N1 influenza strains, the globular head of the hemagglutinin (HA) antigen fused to flagellin of Salmonella typhimurium fljB (STF2) is highly immunogenic in preclinical models and man (Song et al. (2008) [13]; Song et al. (2009) [14]; Taylor et al. (2012) [12]). Further we showed that the vaccine format, or point of attachment of the vaccine antigen to flagellin, can dramatically affect the immunogenicity and safety profile of the vaccine. However, Influenza B vaccines based on these formats are poor triggers of TLR5 and consequently are poorly immunogenic. Through rational design, here we show that we have identified a fusion position within domain 3 of flagellin that improves TLR5 signaling and consequently, immunogenicity of multiple influenza B vaccines. Our results demonstrate that, similar to influenza A strains, the protective subunit of the influenza B HA can be fused to flagellin and produced in a standard prokaryotic expression system thereby allowing for cost and time efficient production of multivalent seasonal influenza vaccines. [ABSTRACT FROM AUTHOR]

Abstract: Background: We evaluated the safety and immunogenicity profiles of 3 novel influenza vaccine constructs consisting of the globular head of the HA1 domain of the Novel H1N1 genetically fused to the TLR5 ligand, flagellin. HA1 was fused to the C-terminus of flagellin in VAX128A, replaced the D3 domain of flagellin in VAX128B and was fused in both positions in VAX128C. Methods: In a dose escalation trial, 112 healthy subjects 18–49 and 100 adults ≥65 years old were enrolled in a double blind, placebo controlled clinical trial at two centers. Vaccines were administered IM at doses ranging from 0.5 to 20μg. VAX128C was selected for second study performed in 100 subjects 18–64 years old comparing 1.25 and 2.5μg doses. All subjects were followed for safety and sera collected pre- and post-vaccination were tested by hemagglutination-inhibition (HAI). Serum C-reactive protein and cytokine levels were also measured. Conclusions: In the first study high HAI titers and high seroconversion and seroprotection rates were observed at doses ≥2.5μg in adults 18–49. In adults ≥65 years, the vaccines doses of ≥4μg were required to induce a ≥4-fold rise in HAI titer, 50% seroconversion and 70% seroprotection. Based on safety, VAX128A was tested up to 8μg, VAX128B to 16μg and VAX128C to 20μg. Dose escalation for VAX128A was stopped at 8μg because one subject had temperature 101.6°F associated with a high CRP response, VAX128B was stopped at 16μg because of a severe AE associated with a high CRP and IL-6 response. VAX128C was not stopped before reaching the 20μg dose. In the second study VAX128C was well tolerated among 100 subjects who received 1.25 or 2.5μg. The peak GMT was 385 (95%CI 272–546), 79% (71–87%) seroconversion and 92% (84–96%) seroprotection. Discussion: Flagellin adjuvanted vaccines can be designed to minimize reactogenicity and retain immunogenicity, thereby representing a promising next generation vaccine technology. [Copyright &y& Elsevier]

Abstract: Transmission of highly pathogenic avian influenza (HPAI) between birds and humans is an ongoing threat that holds potential for the emergence of a pandemic influenza strain. A major barrier to an effective vaccine against avian influenza has been the generally poor immunopotency of many of the HPAI strains coupled with the manufacturing constraints employing conventional methodologies. Fusion of flagellin, a toll-like receptor-5 ligand, to vaccine antigens has been shown to enhance the immune response to the fused antigen in preclinical studies. Here, we have evaluated the immunogenicity and efficacy of a panel of flagellin-based hemagglutinin (HA) globular head fusion vaccines in inbred mice. The HA globular head of these vaccines is derived from the A/Vietnam/1203/04 (VN04; H5N1) HA molecule. We find that replacement of domain D3 of flagellin with the VN04 HA globular head creates a highly effective vaccine that elicits protective HAI titers which protect mice against disease and death in a lethal challenge model. [Copyright &y& Elsevier]

It is known that physical linkage of TLR ligands and vaccine antigens significantly enhances the immunopotency of the linked antigens. We have used this approach to generate novel influenza vaccines that fuse the globular head domain of the protective hemagglutinin (HA) antigen with the potent TLR5 ligand, flagellin. These fusion proteins are efficiently expressed in standard E. coli fermentation systems and the HA moiety can be faithfully refolded to take on the native conformation of the globular head. In mouse models of influenza infection, the vaccines elicit robust antibody responses that mitigate disease and protect mice from lethal challenge. These immunologically potent vaccines can be efficiently manufactured to support pandemic response, pre-pandemic and seasonal vaccines. [ABSTRACT FROM AUTHOR]

Summary: The recognition of specific pathogen associated molecular patterns (PAMPs) by members of the Toll-like receptor (TLR) family is critical for the activation of the adaptive immune response. Thus, incorporation of PAMPs into vaccines should result in more potent, protective antigen-specific responses in the absence of adjuvants or complex formulations. Here we describe an influenza A vaccine that is refractory to the genetic instability of hemagglutinin and neuraminidase and includes a trigger of the innate immune response to enhance immunogenicity and efficacy. A recombinant protein comprising the TLR5 ligand flagellin fused to four tandem copies of the ectodomain of the conserved influenza matrix protein M2 (M2e) was expressed in Escherichia coli and purified to homogeneity. This protein, STF2.4xM2e, retained TLR5 activity and displayed the protective epitope of M2e defined by a monoclonal antibody, 14C2. Mice immunized with STF2.4xM2e in aqueous buffer, without adjuvants or other formulation additives, developed potent M2e-specific antibody responses that were quantitatively and qualitatively superior to those observed with M2e peptide delivered in alum. The antibody response was dependent on the physical linkage of the antigen to flagellin and recognized the epitope defined by monoclonal antibody 14C2, which has been shown to protect mice from challenge with influenza A virus. Moreover, immunization with STF2.4xM2e at a dose of 0.3μg per mouse protected mice from a lethal challenge with influenza A virus, and significantly reduced weight loss and clinical symptoms. These data demonstrate that the linkage of specific TLR ligand with influenza M2e yields a vaccine candidate that offers significant promise for widespread protection against multiple influenza A virus strains. [Copyright &y& Elsevier]