Atypical Asparagine Deamidation of NW Motif Significantly Attenuates the Biological Activities of an Antibody Drug Conjugate.

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.

ChAdOx1 interacts with CAR and PF4 with implications for thrombosis with thrombocytopenia syndrome.

Vaccines derived from chimpanzee adenovirus Y25 (ChAdOx1), human adenovirus type 26 (HAdV-D26), and human adenovirus type 5 (HAdV-C5) are critical in combatting the severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic. As part of the largest vaccination campaign in history, ultrarare side effects not seen in phase 3 trials, including thrombosis with thrombocytopenia syndrome (TTS), a rare condition resembling heparin-induced thrombocytopenia (HIT), have been observed. This study demonstrates that all three adenoviruses deployed as vaccination vectors versus SARS-CoV-2 bind to platelet factor 4 (PF4), a protein implicated in the pathogenesis of HIT. We have determined the structure of the ChAdOx1 viral vector and used it in state-of-the-art computational simulations to demonstrate an electrostatic interaction mechanism with PF4, which was confirmed experimentally by surface plasmon resonance. These data confirm that PF4 is capable of forming stable complexes with clinically relevant adenoviruses, an important step in unraveling the mechanisms underlying TTS.

Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells.

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.

Phase 1 Safety and Immunogenicity Study of a Quadrivalent Seasonal Flu Vaccine Comprising Recombinant Hemagglutinin-Flagellin Fusion Proteins.

Background. We evaluated the safety and immunogenicity of VAX2012Q, a quadrivalent influenza vaccine comprising 4 hemagglutinin subunits fused to flagellin. Methods. In this dose-ranging, open-label study, healthy adults (18-40 years) were divided into 7 cohorts for evaluation of 5 dose levels and 3 component ratios. Dose levels were as follows: (1) 1 mcg per component of VAX128C (H1N1), VAX181 (H3N2), VAX173 (B-YAM), and VAX172 (B-VIC), respectively; (2) 2 mcg per component, respectively; (3) 2, 4, 4, and 4 mcg of each component, respectively; (4) 2, 4, 6, and 6 mcg of each component, respectively; and (5) 3 mcg per component, respectively. Tolerability and immunogenicity data were analyzed. Results. Three hundred sixteen subjects received VAX2012Q (309 per protocol). At all dose levels, 54% to 65% of subjects reported mild injection site pain, the most common local reaction. Moderate injection site pain increased at dose levels 2 through 5 (22%-42%, compared with 20% at dose level 1). Systemic symptoms were mostly mild to moderate with moderate symptoms increasing in dose levels 3 and 4. Three dose level 3 subjects (6%) reported severe, transient chills and or fever. Mean fold rises in hemagglutination inhibition titers ranged from 2.5 to 6.9 despite high baseline titers. Mean seroprotection rates were ≥90% and mean seroconversion rates were ≥40% for all strains in all groups postvaccination. Conclusions. VAX2012Q elicited immune responses at all dose levels with no significant safety concerns. Doses of 2 or 3 mcg per component provided a favorable balance of tolerability and immunogenicity.

A rationally designed form of the TLR5 agonist, flagellin, supports superior immunogenicity of Influenza B globular head vaccines.

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.

Development of VAX128, a recombinant hemagglutinin (HA) influenza-flagellin fusion vaccine with improved safety and immune response.

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 20 µg. VAX128C was selected for second study performed in 100 subjects 18-64 years old comparing 1.25 and 2.5 µg 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 ≥2.5 µg in adults 18-49. In adults ≥65 years, the vaccines doses of ≥4 µg were required to induce a ≥4-fold rise in HAI titer, 50% seroconversion and 70% seroprotection. Based on safety, VAX128A was tested up to 8 µg, VAX128B to 16 µg and VAX128C to 20 µg. Dose escalation for VAX128A was stopped at 8 µg because one subject had temperature 101.6°F associated with a high CRP response, VAX128B was stopped at 16 µg because of a severe AE associated with a high CRP and IL-6 response. VAX128C was not stopped before reaching the 20 µg dose. In the second study VAX128C was well tolerated among 100 subjects who received 1.25 or 2.5 µg. 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.

Immunogenicity and efficacy of flagellin-fused vaccine candidates targeting 2009 pandemic H1N1 influenza in mice.

We have previously demonstrated that the globular head of the hemagglutinin (HA) antigen fused to flagellin of Salmonella typhimurium fljB (STF2, a TLR5 ligand) elicits protective immunity to H1N1 and H5N1 lethal influenza infections in mice (Song et al., 2008, PLoS ONE 3, e2257; Song et al., 2009, Vaccine 27, 5875-5888). These fusion proteins can be efficiently and economically manufactured in E. coli fermentation systems as next generation pandemic and seasonal influenza vaccines. Here we report immunogenicity and efficacy results of three vaccine candidates in which the HA globular head of A/California/07/2009 (H1N1) was fused to STF2 at the C-terminus (STF2.HA1), in replace of domain 3 (STF2R3.HA1), or in both positions (STF2R3.2xHA1). For all three vaccines, two subcutaneous immunizations of BALB/c mice with doses of either 0.3 or 3 µg elicit robust neutralizing (HAI) antibodies, that lead to > = 2 Log(10) unit reduction in day 4 lung virus titer and full protection against a lethal A/California/04/2009 challenge. Vaccination with doses as low as 0.03 µg results in partial to full protection. Each candidate, particularly the STF2R3.HA1 and STF2R3.2xHA1 candidates, elicits robust neutralizing antibody responses that last for at least 8 months. The STF2R3.HA1 candidate, which was intermediately protective in the challenge models, is more immunogenic than the H1N1 components of two commercially available trivalent inactivated influenza vaccines (TIVs) in mice. Taken together, the results demonstrate that all three vaccine candidates are highly immunogenic and efficacious in mice, and that the STF2R3.2xHA1 format is the most effective candidate vaccine format.

Superior efficacy of a recombinant flagellin:H5N1 HA globular head vaccine is determined by the placement of the globular head within flagellin.

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.

Efficacious recombinant influenza vaccines produced by high yield bacterial expression: a solution to global pandemic and seasonal needs.

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.

Potent immunogenicity and efficacy of a universal influenza vaccine candidate comprising a recombinant fusion protein linking influenza M2e to the TLR5 ligand flagellin.

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 0.3 microg 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.

Plasmid vectors encoding cholera toxin or the heat-labile enterotoxin from Escherichia coli are strong adjuvants for DNA vaccines.

Two plasmid vectors encoding the A and B subunits of cholera toxin (CT) and two additional vectors encoding the A and B subunits of the Escherichia coli heat-labile enterotoxin (LT) were evaluated for their ability to serve as genetic adjuvants for particle-mediated DNA vaccines administered to the epidermis of laboratory animals. Both the CT and the LT vectors strongly augmented Th1 cytokine responses (gamma interferon [IFN-gamma]) to multiple viral antigens when codelivered with DNA vaccines. In addition, Th2 cytokine responses (interleukin 4 [IL-4]) were also augmented by both sets of vectors, with the effects of the LT vectors on IL-4 responses being more antigen dependent. The activities of both sets of vectors on antibody responses were antigen dependent and ranged from no effect to sharp reductions in the immunoglobulin G1 (IgG1)-to-IgG2a ratios. Overall, the LT vectors exhibited stronger adjuvant effects in terms of T-cell responses than did the CT vectors, and this was correlated with the induction of greater levels of cyclic AMP by the LT vectors following vector transfection into cultured cells. The adjuvant effects observed in vivo were due to the biological effects of the encoded proteins and not due to CpG motifs in the bacterial genes. Interestingly, the individual LT A and B subunit vectors exhibited partial adjuvant activity that was strongly influenced by the presence or absence of signal peptide coding sequences directing the encoded subunit to either intracellular or extracellular locations. Particle-mediated delivery of either the CT or LT adjuvant vectors in rodents and domestic pigs was well tolerated, suggesting that bacterial toxin-based genetic adjuvants may be a safe and effective strategy to enhance the potency of both prophylactic and therapeutic DNA vaccines for the induction of strong cellular immunity.

The synthesis and selective IL-2 inhibitory activity of bis piperazine-phenol Mannich adducts.

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.