Feasibility of using a combination of staphylococcal superantigen-like proteins 3, 7 and 11 in a fusion vaccine for Staphylococcus aureus.

Staphylococcus aureus is a significant bacterial pathogen in both community and hospital settings, and the escalation of antimicrobial-resistant strains is of immense global concern. Vaccination is an inviting long-term strategy to curb staphylococcal disease, but identification of an effective vaccine has proved to be challenging. Three well-characterized, ubiquitous, secreted immune evasion factors from the staphylococcal superantigen-like (SSL) protein family were selected for the development of a vaccine. Wild-type SSL3, 7 and 11, which inhibit signaling through Toll-like receptor 2, cleavage of complement component 5 and neutrophil function, respectively, were successfully combined into a stable, active fusion protein (PolySSL7311). Vaccination of mice with an attenuated form of the PolySSL7311 protein stimulated significantly elevated specific immunoglobulin G and splenocyte proliferation responses to each component relative to adjuvant-only controls. Vaccination with PolySSL7311, but not a mixture of the individual proteins, led to a > 102 reduction in S. aureus tissue burden compared with controls after peritoneal challenge. Comparable antibody responses were elicited after coadministration of the vaccine in either AddaVax (an analog of MF59) or an Alum-based adjuvant; but only AddaVax conferred a significant reduction in bacterial load, aligning with other studies that suggest both cellular and humoral immune responses are necessary for protective immunity to S. aureus. Anti-sera from mice immunized with PolySSL7311, but not individual proteins, partially neutralized the functional activities of SSL7. This study confirms the importance of these SSLs for the survival of S. aureus in vivo and suggests that PolySSL7311 is a promising vaccine candidate.

The Development of Toxoplasma gondii Recombinant Trivalent Chimeric Proteins as an Alternative to Toxoplasma Lysate Antigen (TLA) in Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Immunoglobulin G (IgG) in Small Ruminants.

This study presents an evaluation of seventeen newly produced recombinant trivalent chimeric proteins (containing the same immunodominant fragment of SAG1 and SAG2 of Toxoplasma gondii antigens, and an additional immunodominant fragment of one of the parasite antigens, such as AMA1, GRA1, GRA2, GRA5, GRA6, GRA7, GRA9, LDH2, MAG1, MIC1, MIC3, P35, and ROP1) as a potential alternative to the whole-cell tachyzoite lysate (TLA) used in the detection of infection in small ruminants. These recombinant proteins, obtained by genetic engineering and molecular biology methods, were tested for their reactivity with specific anti-Toxoplasma IgG antibodies contained in serum samples of small ruminants (192 samples of sheep serum and 95 samples of goat serum) using an enzyme-linked immunosorbent assay (ELISA). The reactivity of six recombinant trivalent chimeric proteins (SAG1-SAG2-GRA5, SAG1-SAG2-GRA9, SAG1-SAG2-MIC1, SAG1-SAG2-MIC3, SAG1-SAG2-P35, and SAG1-SAG2-ROP1) with IgG antibodies generated during T. gondii invasion was comparable to the sensitivity of TLA-based IgG ELISA (100%). The obtained results show a strong correlation with the results obtained for TLA. This suggests that these protein preparations may be a potential alternative to TLA used in commercial tests and could be used to develop a cheaper test for the detection of parasite infection in small ruminants.

SARS-CoV-2 spike-FLIPr fusion protein plus lipidated FLIPr protects against various SARS-CoV-2 variants in hamsters.

Vaccine-induced mucosal immunity and broad protective capacity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remain inadequate. Formyl peptide receptor-like 1 inhibitory protein (FLIPr), produced by Staphylococcus aureus, can bind to various Fcγ receptor subclasses. Recombinant lipidated FLIPr (rLF) was previously found to be an effective adjuvant. In this study, we developed a vaccine candidate, the recombinant Delta SARS-CoV-2 spike (rDS)-FLIPr fusion protein (rDS-F), which employs the property of FLIPr binding to various Fcγ receptors. Our study shows that rDS-F plus rLF promotes rDS capture by dendritic cells. Intranasal vaccination of mice with rDS-F plus rLF increases persistent systemic and mucosal antibody responses and CD4/CD8 T-cell responses. Importantly, antibodies induced by rDS-F plus rLF vaccination neutralize Delta, Wuhan, Alpha, Beta, and Omicron strains. Additionally, rDS-F plus rLF provides protective effects against various SARS-CoV-2 variants in hamsters by reducing inflammation and viral loads in the lung. Therefore, rDS-F plus rLF is a potential vaccine candidate to induce broad protective responses against various SARS-CoV-2 variants.IMPORTANCEMucosal immunity is vital for combating pathogens, especially in the context of respiratory diseases like COVID-19. Despite this, most approved vaccines are administered via injection, providing systemic but limited mucosal protection. Developing vaccines that stimulate both mucosal and systemic immunity to address future coronavirus mutations is a growing trend. However, eliciting strong mucosal immune responses without adjuvants remains a challenge. In our study, we have demonstrated that using a recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-formyl peptide receptor-like 1 inhibitory protein (FLIPr) fusion protein as an antigen, in combination with recombinant lipidated FLIPr as an effective adjuvant, induced simultaneous systemic and mucosal immune responses through intranasal immunization in mice and hamster models. This approach offered protection against various SARS-CoV-2 strains, making it a promising vaccine candidate for broad protection. This finding is pivotal for future broad-spectrum vaccine development.

Lipid-Encapsulated mRNAs Encoding Complex Fusion Proteins Potentiate Antitumor Immune Responses.

Lipid nanoparticle (LNP)-encapsulated mRNA has been used for in vivo production of several secreted protein classes, such as IgG, and has enabled the development of personalized vaccines in oncology. Establishing the feasibility of delivering complex multispecific modalities that require higher-order structures important for their function could help expand the use of mRNA/LNP biologic formulations. Here, we evaluated whether in vivo administration of mRNA/LNP formulations of SIRPα-Fc-CD40L and TIGIT-Fc-LIGHT could achieve oligomerization and extend exposure, on-target activity, and antitumor responses comparable with that of the corresponding recombinant fusion proteins. Intravenous infusion of the formulated LNP-encapsulated mRNAs led to rapid and sustained production of functional hexameric proteins in vivo, which increased the overall exposure relative to the recombinant protein controls by ∼28 to 140 fold over 96 hours. High concentrations of the mRNA-encoded proteins were also observed in secondary lymphoid organs and within implanted tumors, with protein concentrations in tumors up to 134-fold greater than with the recombinant protein controls 24 hours after treatment. In addition, SIRPα-Fc-CD40L and TIGIT-Fc-LIGHT mRNAs induced a greater increase in antigen-specific CD8+ T cells in the tumors. These mRNA/LNP formulations were well tolerated and led to a rapid increase in serum and intratumoral IL2, delayed tumor growth, extended survival, and outperformed the activities of benchmark mAb controls. Furthermore, the mRNA/LNPs demonstrated improved efficacy in combination with anti-PD-L1 relative to the recombinant fusion proteins. These data support the delivery of complex oligomeric biologics as mRNA/LNP formulations, where high therapeutic expression and exposure could translate into improved patient outcomes. SIGNIFICANCE: Lipid nanoparticle-encapsulated mRNA can efficiently encode complex fusion proteins encompassing immune checkpoint blockers and costimulators that functionally oligomerize in vivo with extended pharmacokinetics and durable exposure to induce potent antitumor immunity.

Facts and Hopes on Chimeric Cytokine Agents for Cancer Immunotherapy.

Cytokines are key mediators of immune responses that can modulate the antitumor activity of immune cells. Cytokines have been explored as a promising cancer immunotherapy. However, there are several challenges to cytokine therapy, especially a lack of tumor targeting, resulting in high toxicity and limited efficacy. To overcome these limitations, novel approaches have been developed to engineer cytokines with improved properties, such as chimeric cytokines. Chimeric cytokines are fusion proteins that combine different cytokine domains or link cytokines to antibodies (immunocytokines) or other molecules that can target specific receptors or cells. Chimeric cytokines can enhance the selectivity and stability of cytokines, leading to reduced toxicity and improved efficacy. In this review, we focus on two promising cytokines, IL2 and IL15, and summarize the current advances and challenges of chimeric cytokine design and application for cancer immunotherapy. Most of the current approaches focus on increasing the potency of cytokines, but another important goal is to reduce toxicity. Cytokine engineering is promising for cancer immunotherapy as it can enhance tumor targeting while minimizing adverse effects.

Bioinformatics-based design of a fusion vaccine with CTLA-4 variable region to combat Brucella.

Brucellosis has become a global zoonotic disease, seriously endangering the health of people all over the world. Vaccination is an effective strategy for protection against Brucella infection in livestock in developed countries. However, current vaccines are pathogenic to humans and pregnant animals, which limits their use. Therefore, it is very important to improve the safety and immune protection of Brucella vaccine. In this study, different bioinformatics approaches were carried out to predict the physicochemical properties, T/B epitope, and tertiary structure of Omp2b and Omp31. Then, these two proteins were sequentially linked, and the Cytotoxic T lymphocyte associated antigen-4 (CTLA-4) variable region was fused to the N-terminal of the epitope sequence. In addition, molecular docking was performed to show that the structure of the fusion protein vaccine had strong affinity with B7 (B7-1, B7-2). This study showed that the designed vaccine containing CTLA-4 had high potency against Brucella, which could provide a reference for the future development of efficient brucellosis vaccines.

Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands.

Protein A affinity chromatography is widely used for the large-scale purification of antibodies because of its high yield, selectivity, and compatibility with NaOH sanitation. A general platform to produce robust affinity capture ligands for proteins beyond antibodies would improve bioprocessing efficiency. We previously developed nanoCLAMPs (nano Clostridial Antibody Mimetic Proteins), a class of antibody mimetic proteins useful as lab-scale affinity capture reagents. This work describes a protein engineering campaign to develop a more robust nanoCLAMP scaffold compatible with harsh bioprocessing conditions. The campaign generated an improved scaffold with dramatically improved resistance to heat, proteases, and NaOH. To isolate additional nanoCLAMPs based on this scaffold, we constructed a randomized library of 1 × 1010 clones and isolated binders to several targets. We then performed an in-depth characterization of nanoCLAMPs recognizing yeast SUMO, a fusion partner used for the purification of recombinant proteins. These second-generation nanoCLAMPs typically had a Kd of <80 nM, a Tm of >70 °C, and a t1/2 in 0.1 mg/ml trypsin of >20 h. Affinity chromatography resins bearing these next-generation nanoCLAMPs enabled single-step purifications of SUMO fusions. Bound target proteins could be eluted at neutral or acidic pH. These affinity resins maintained binding capacity and selectivity over 20 purification cycles, each including 10 min of cleaning-in-place with 0.1 M NaOH, and remained functional after exposure to 100% DMF and autoclaving. The improved nanoCLAMP scaffold will enable the development of robust, high-performance affinity chromatography resins against a wide range of protein targets.

A Protease Activatable Interleukin-2 Fusion Protein Engenders Antitumor Immune Responses by Interferon Gamma-Dependent and Interferon Gamma-Independent Mechanisms.

Cytokines are powerful mediators of immune responses and some, such as interleukin-2 (IL-2), have achieved dramatic responses as cancer immunotherapies. Unfortunately, systemic administration often results in deleterious side effects, prompting exploration of strategies to localize cytokine activity to the tumor microenvironment (TME). To this end, we constructed an IL-2/IL2Ra fusion protein (IL-2FP) with an MMP2/9-specific cleavage site, designed to exploit the dysregulated protease activity in the TME to selectively activate IL-2 in the tumor. To determine if TME protease activity is sufficient to cleave the FP and if FP activity is due to specific cleavage, we created Colon 38 tumor cell lines expressing similar levels of IL-2FPs with either a functional cleavage site [H11(cs-1FP)] or a scrambled, noncleavable sequence [H2(scramFP)]. H11(cs-1FP) tumors demonstrated reduced tumor growth, characterized by regressions not observed in H2(scramFP) tumors. Analysis through qRT-PCR, flow cytometry, and immunohistochemistry indicate robust CD8 responses in the H11(cs-1FP) tumors. Interferon gamma (IFNg) knockout mice revealed that the immune effects of the cleavable FP are mediated through both IFNg-dependent and IFNg-independent mechanisms. Collectively, these data suggest that matrix metalloproteinases (MMPs) in the TME can cleave the IL-2FP specifically, thus enhancing an antitumor response, and provide a rationale for further developing this approach.

Potentiating adoptive cell therapy using synthetic IL-9 receptors.

Synthetic receptor signalling has the potential to endow adoptively transferred T cells with new functions that overcome major barriers in the treatment of solid tumours, including the need for conditioning chemotherapy1,2. Here we designed chimeric receptors that have an orthogonal IL-2 receptor extracellular domain (ECD) fused with the intracellular domain (ICD) of receptors for common γ-chain (γc) cytokines IL-4, IL-7, IL-9 and IL-21 such that the orthogonal IL-2 cytokine elicits the corresponding γc cytokine signal. Of these, T cells that signal through the chimeric orthogonal IL-2Rß-ECD-IL-9R-ICD (o9R) are distinguished by the concomitant activation of STAT1, STAT3 and STAT5 and assume characteristics of stem cell memory and effector T cells. Compared to o2R T cells, o9R T cells have superior anti-tumour efficacy in two recalcitrant syngeneic mouse solid tumour models of melanoma and pancreatic cancer and are effective even in the absence of conditioning lymphodepletion. Therefore, by repurposing IL-9R signalling using a chimeric orthogonal cytokine receptor, T cells gain new functions, and this results in improved anti-tumour activity for hard-to-treat solid tumours.

The Natural Cytotoxicity Receptor NKp44 (NCR2, CD336) Is Expressed on the Majority of Porcine NK Cells Ex Vivo Without Stimulation.

Natural killer (NK) cells have been studied extensively in humans and mice for their vital role in the vertebrate innate immune system. They are known to rapidly eliminate tumors or virus infected cells in an immune response utilizing their lytic properties. The natural cytotoxicity receptors (NCRs) NKp30 (NCR3), NKp44 (NCR2), and NKp46 (NCR1) are important mediators of NK-cell cytotoxicity. NKp44 expression was reported for NK cells in humans as well as in some non-human primates and found exclusively on activated NK cells. Previously, no information was available on NKp44 protein expression and its role in porcine lymphocytes due to the lack of species-specific monoclonal antibodies (mAbs). For this study, porcine-specific anti-NKp44 mAbs were generated and their reactivity was tested on blood and tissue derived NK cells in pigs of different age classes. Interestingly, NKp44 expression was detected ex vivo already on resting NK cells; moreover, the frequency of NKp44+ NK cells was higher than that of NKp46+ NK cells in most animals analyzed. Upon in vitro stimulation with IL-2 or IL-15, the frequency of NKp44+ NK cells, as well as the intensity of NKp44 expression at the single cell level, were increased. Since little is known about swine NK cells, the generation of a mAb (clone 54-1) against NKp44 will greatly aid in elucidating the mechanisms underlying the differentiation, functionality, and activation of porcine NK cells.

OX40-Fc Fusion Protein Alleviates PD-1-Fc-Aggravated Rheumatoid Arthritis by Inhibiting Inflammatory Response.

BACKGROUND: Researches have confirmed that the abnormal signals of OX40 and PD-1 lead to the changes of T cell biological behavior, thus participating the immunopathological process of RA. However, the pathogenesis of RA immunopathological process has not been clarified yet. METHODS: 30 DBA/1 mice were randomly divided into 5 groups (6 mice per group): control group, collagen-induced arthritis (CIA) group, PD-1-Fc/CIA group, OX40-Fc/CIA group, and PD-1-Fc + OX40-Fc/CIA group. The pathological changes in mice joints were observed by H&E staining. The proportion of CD4+ T, CD8+ T, CD28+, and CD19+ cells in peripheral blood mononuclear cells (PBMCs) was detected by flow cytometry. Serum inflammatory factors (CRP, IL-2, IL-4, IL-1ß, INF-γ) and bone metabolism-related genes (CTX-I, TRACP-5b, BALP) were detected by ELISA assay. Western blotting was applied to measure the NF-κB signaling pathway-related protein (p-IKKß, p-IκBα, p50) expression in synovial tissue of mice joint. RESULTS: Compared with the control group, CIA mice showed significant increases in arthritis score and pathological score. In the CIA group, a marked decrease was identified in the proportion of CD8+ T, CD19+, and CD68+ cells. Additionally, the CIA group was associated with upregulation of secretion of inflammatory factors in serum and expression of bone metabolism-related genes and NF-κB pathway-related proteins. Compared with the CIA group, the same indexes above showed a further aggravation in the PD-1-Fc group while all indexes improved in the OX40-Fc group. Besides, OX40-Fc fusion protein slowed down significantly the further deterioration of CIA mouse pathological process caused by PD-1-Fc fusion protein. CONCLUSION: OX40-Fc fusion protein alleviates PD-1-Fc-aggravated RA by inhibiting inflammatory response. This research provides biological markers with clinical significance for diagnosis and prognosis of RA, as well as offers theoretical and experimental foundation to the new targets for immune intervention.

Escherichia coli recombinant expression of SARS-CoV-2 protein fragments.

We have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli. Our approach uses the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline-threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All of the fusion proteins were highly expressed in E. coli and the CBM9-ID-H1 fusion protein was shown to yield 122 mg/L of purified product. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibodies directed to the appropriate SARS-CoV-2 antigenic regions. The largest intact CBM9 fusion protein, CBM9-ID-H1, incorporates spike protein amino acids 540-588, which is a conserved region overlapping and C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.

Computational design of a neutralizing antibody with picomolar binding affinity for all concerning SARS-CoV-2 variants.

Coronavirus disease 2019, caused by SARS-CoV-2, remains an on-going pandemic, partly due to the emergence of variant viruses that can "break-through" the protection of the current vaccines and neutralizing antibodies (nAbs), highlighting the needs for broadly nAbs and next-generation vaccines. We report an antibody that exhibits breadth and potency in binding the receptor-binding domain (RBD) of the virus spike glycoprotein across SARS coronaviruses. Initially, a lead antibody was computationally discovered and crystallographically validated that binds to a highly conserved surface of the RBD of wild-type SARS-CoV-2. Subsequently, through experimental affinity enhancement and computational affinity maturation, it was further developed to bind the RBD of all concerning SARS-CoV-2 variants, SARS-CoV-1 and pangolin coronavirus with pico-molar binding affinities, consistently exhibited strong neutralization activity against wild-type SARS-CoV-2 and the Alpha and Delta variants. These results identify a vulnerable target site on coronaviruses for development of pan-sarbecovirus nAbs and vaccines.

Non-targeted characterization of attributes affecting antibody-FcγRIIIa V158 (CD16a) binding via online affinity chromatography-mass spectrometry.

Antibodies facilitate targeted cell killing by engaging with immune cells such as natural killer cells through weak binding interactions with Fcγ receptors on the cell surface. Here, we evaluate the binding affinity of the receptor FcγRIIIa V158 (CD16a) for several therapeutic antibody classes, isoforms, and Fc-fusion proteins using an immobilized receptor affinity liquid chromatography (LC) approach coupled with online mass spectrometry (MS) detection. Aglycosylated FcγRIIIa was used in the affinity chromatography and compared with published affinities using glycosylated receptors. Affinity LC-MS differentiated the IgG1 antibodies primarily according to their Fc glycosylation patterns, with highly galactosylated species having greater affinity for the immobilized receptors and thus eluting later from the column (M5< G0F < G0 afucosylated ≅ G1F < G2F). Sialylated species bound weaker to their asialylated counterparts as reported previously. High mannose glycoforms bound weaker than G0F, contrary to previously published studies using glycosylated receptors. Also, increased receptor binding affinity associated with afucosylated antibodies was not observed with the aglycosylated FcγRIIIa. This apparent difference from previous findings highlighted the importance of the glycans on the receptors for mediating stronger binding interactions. Characterization of temperature-stressed samples by LC-MS peptide mapping revealed over 200 chemical and post-translational modifications, but only the Fc glycans, deamidation of EU N325, and an unknown modification to either proline or cysteine residues of the hinge region were found to have a statistically significant impact on binding.Abbreviations: Antibody-dependent cell-mediated cytotoxicity (ADCC), chimeric antigen receptor (CAR), Chinese hamster ovary (CHO), dithiothreitol (DTT), electrospray ionization (ESI), hydrogen-deuterium exchange (HDX), filter aided-sample preparation (FASP), Fcγ receptor (FcγR), fragment crystallizable (Fc), high-pressure liquid chromatography (HPLC), immunoglobulin G (IgG), liquid chromatography (LC), monoclonal antibody (mAb), mass spectrometry (MS), natural killer (NK), N-glycolylneuraminic acid (NGNA), N-acetylneuraminic acid (NANA), principal component analysis (PCA), surface plasmon resonance (SPR), trifluoroacetic acid (TFA), and extracted mass chromatogram (XMC).

Maximizing response to intratumoral immunotherapy in mice by tuning local retention.

Direct injection of therapies into tumors has emerged as an administration route capable of achieving high local drug exposure and strong anti-tumor response. A diverse array of immune agonists ranging in size and target are under development as local immunotherapies. However, due to the relatively recent adoption of intratumoral administration, the pharmacokinetics of locally-injected biologics remains poorly defined, limiting rational design of tumor-localized immunotherapies. Here we define a pharmacokinetic framework for biologics injected intratumorally that can predict tumor exposure and effectiveness. We find empirically and computationally that extending the tumor exposure of locally-injected interleukin-2 by increasing molecular size and/or improving matrix-targeting affinity improves therapeutic efficacy in mice. By tracking the distribution of intratumorally-injected proteins using positron emission tomography, we observe size-dependent enhancement in tumor exposure occurs by slowing the rate of diffusive escape from the tumor and by increasing partitioning to an apparent viscous region of the tumor. In elucidating how molecular weight and matrix binding interplay to determine tumor exposure, our model can aid in the design of intratumoral therapies to exert maximal therapeutic effect.

Preparation and characterization of a high-affinity monoclonal antibody against nerve growth factor.

Nerve growth factor (NGF) is produced and released in injured tissues or chronic pain tissues caused by other diseases. Studies have shown that monoclonal antibodies targeting NGF have a good efficacy in the treatment of osteoarthritis (OA), low back pain and chronic pain, which may be a promising therapy. In this study, DNA sequences of NGF-his and NGF-hFc were synthesized using eukaryotic expression system and subcloned into pTT5 expression vector. After that, NGF proteins were expressed by transient expression in HEK293E cells. We immunized mice with NGF-hFc protein and fused mouse spleen cells to prepare hybridomas. NGF-His protein was used to screen out the hybridoma supernatant that could directly bind to NGF. Antibodies were purified from hybridioma supernatant. Futhermore, via surface plasmon resonance (SPR) screening, six anti-NGF mAbs were screened to block the binding of NGF and TrkA receptor in the treatment of chronic pain. Among them, 58F10G10H showed high affinity (KD = 1.03 × 10-9 M) and even better than that of positive control antibody Tanezumab (KD = 1.53 × 10-9 M). Moreover, the specific reactivity of 58F10G10H was demonstrated by TF-1 cell proliferation activity experiments, competitive binding Enzyme-linked immunosorbent assay (ELISA) and the arthritis animal models in mice, respectively. In conclusion, in this study, a method for the preparation of high-yield NGF-HFC and NGF-His proteins was designed, and a high-affinity monoclonal antibody against NGF with potential for basic research and clinical application was prepared.

Optimization strategies for expression of a novel bifunctional anti-PD-L1/TGFBR2-ECD fusion protein.

The novel anti-PD-L1/TGFBR2-ECD fusion protein (BR102) comprises an anti-PD-L1 antibody (HS636) which is fused at the C terminus of the heavy chain to a TGF-ß1 receptor Ⅱ ectodomain (TGFBR2-ECD), and which can sequester the PD-1/PD-L1 pathway and TGF-ß bioactivity in the immunosuppressive tumor microenvironment. In the expression of TGFBR2-ECD wild-type fused protein (BR102-WT), a 50 kDa clipped species was confirmed to be induced by proteolytic cleavage at a "QKS" site located in the N-terminus of the ectodomain, which resulted in the formation of IgG-like clipping. The matrix metalloproteinase-9 was determined to be associated with BR102-WT digestion. In addition, it was observed that the N-glycosylation modifications of the fusion protein were tightly involved in regulating proteolytic activity and the levels of cleavage could be significantly suppressed by MMP-inhibitors. To avoid proteolytic degradation, eliminating protease-sensitive amino acid motifs and introducing potential glycosylation were performed. Three sensitive motifs were mutated, and the levels of clipping were strongly restrained. The mutant candidates exhibited similar binding affinities to hPD-L1 and hTGF-ß1 as well as highly purified BR102-WT2. Furthermore, the mutants displayed more significant proteolytic resistance than that of BR102-WT2 in the lysate incubation reaction and the plasma stability test. Moreover, the bifunctional candidate Mu3 showed an additive antitumor effect in MC38/hPD-L1 bearing models as compared to that of with anti-PD-L1 antibody alone. In conclusion, in this study, the protease-sensitive features of BR102-WT were well characterized and efficient optimization was performed. The candidate BR102-Mutants exhibited advanced druggability in drug stability and displayed desirable antitumor activity.

Passive immunization with anti- chimeric protein PilQ/PilA -DSL region IgY does not protect against mortality associated with Pseudomonas aeruginosa sepsis in a rabbit model.

BACKGROUND: Pseudomonas aeruginosa sepsis is associated with unacceptably high mortality and, for many of those who survive, long-term morbidity. The aims of this study were to production of IgY against chimeric protein pilQ-pilA-DSL region and killed- whole cell Pseudomonas aeruginosa O1 (PAO1) strain and their efficacy for immunoprophylaxis of sepsis caused by P. aeruginosa in a rabbit model. METHODS: Specific IgY was obtained by immunization of hens. The purity of IgY was determined by SDS-PAGE analysis. The effect of IgY on growth and hydrophobicity of P. aeruginosa were performed through time-kill assay and microbial adhesion to hydrocarbons test (MATH), respectively. The efficacy of specific IgYs was examined against P. aeruginosa sepsis in a rabbit model. The rabbits were monitored for 72 h to record physiological characters and survival. Hematologic factors, C-reactive protein, pro-inflammatory cytokines, and bacterial count from blood and solid organs were measured, periodically. RESULTS: We found that the growth inhibitory effect of the anti- killed whole cell IgY was higher than anti-pilQ-pilA IgY (P < 0.001). The hydrophobicity effect of PAO1 increased when bacteria were opsonized by anti- killed whole cell IgY while the hydrophobicity activity was decreased following incubation of PAO1 with anti-pilQ-pilA IgY in a broth medium (P < 0.001). Following intravenous (IV) administration of produced IgYs, no significant difference was observed in the survival, decrease in inflammatory mediators and clinical symptoms between the groups 48h post infection (P > 0.05). Moreover, no considerable decrease was observed in the bacterial load of blood, lungs and kidneys in rabbits treated with specific IgYs and control groups (P > 0.05). No bacteria were found in the spleen and liver samples from infected rabbits. CONCLUSION: Although produced IgYs had a good immunoreactivity, IV immunization of IgYs was not protective against P. aeruginosa sepsis in the rabbit model. Further studies are needed to assess the immune response and decreasing mortality rate using the rabbit sepsis model.

Refined immunoRNases for the efficient targeting and selective killing of tumour cells: A novel strategy.

In order to overcome limitations of conventional cancer therapy methods, immunotoxins with the capability of target-specific action have been designed and evaluated pre-clinically, and some of them are in clinical studies. Targeting cancer cells via antibodies specific for tumour-associated surface proteins is a new biomedical approach that could provide the selectivity that is lacking in conventional cancer therapy methods such as radiotherapy and chemotherapy. A successful example of an approved immunotoxin is represented by immunoRNases. ImmunoRNases are fusion proteins in which the toxin has been replaced by a ribonuclease. Conjugation of RNase molecule to monoclonal antibody or antibody fragment was shown to enhance specific cell-killing by several orders of magnitude, both in vitro and in animal models. There are several RNases obtained from different mammalian cells that are expected to be less immunogenic and systemically toxic. In fact, RNases are pro-toxins which become toxic only upon their internalization in target cells mediated by the antibody moiety. The structure and large size of the antibody molecules assembled with the immunoRNases have always been a challenge in the application of immunoRNases as an antitoxin. To overcome this obstacle, we have offered a new strategy for the application of immunoRNases as a promising approach for upgrading immunoRNAses with maximum affinity and high stability in the cell, which can ultimately act as an effective large-scale cancer treatment. In this review, we introduce the optimized antibody-like molecules with small size, approximately 10 kD, which are presumed to significantly enhance RNase activity and be a suitable agent with the potential for anti-cancer functionality. In addition, we also discuss new molecular entities such as monobody, anticalin, nonobody and affilin as refined versions in the development of immunoRNases. These small molecules express their functionality with the suitable small size as well as with low immunogenicity in the cell, as a part of immunoRNases.

Enhanced cross protection by hetero prime-boost vaccination with recombinant influenza viruses containing chimeric hemagglutinin-M2e epitopes.

Annual repeat influenza vaccination raises concerns about protective efficacy against mismatched viruses. We investigated the impact of heterologous prime-boost vaccination on inducing cross protection by designing recombinant influenza viruses with chimeric hemagglutinin (HA) carrying M2 extracellular domains (M2e-HA). Heterologous prime-boost vaccination of C57BL/6 mice with M2e-HA chimeric virus more effectively induced M2e and HA stalk specific IgG antibodies correlating with cross protection than homologous prime-boost vaccination. Induction of M2e and HA stalk specific IgG antibodies was compromised in 1-year old mice, indicating significant aging effects on priming subdominant M2e and HA stalk IgG antibody responses. This study demonstrates that a heterologous prime-boost strategy with recombinant influenza virus expressing extra M2e epitopes provides more effective cross protection than homologous vaccination.