The effects of anti-lung cancer in nude mice by a fully human single-chain antibody against associated antigen Ts7TMR between A549 cells and Trichinella spiralis.

Lung cancer is a dangerous disease that is lacking in an ideal therapy. Here, we evaluated the anti-lung cancer effect in nude mice of a fully human single-chain antibody (scFv) against the associated antigen 7 transmembrane receptor (Ts7TMR), which is also called G protein-coupled receptor, between A549 cells and Trichinella spiralis (T. spiralis). Our data showed that anti-Ts7TMR scFv could inhibit lung cancer growth in a dose-dependent manner, with a tumour inhibition rate of 59.1%. HE staining did not reveal any obvious tissue damage. Mechanistically, immunohistochemical staining revealed that the scFv down-regulated the expression of PCNA and VEGF in tumour tissues. Overall, this study found that anti-Ts7TMR scFv could inhibit A549 lung cancer growth by suppressing cell proliferation and angiogenesis, which may provide a new strategy for treating lung cancer.

Preparation of Bispecific IgY-scFvs Inhibition Adherences of Enterotoxigenic Escherichia coli (K88 and F18) to Porcine IPEC-J2 Cell.

Enterotoxigenic Escherichia coli (ETEC) strains are significant contributors to postweaning diarrhea in piglets. Of the ETEC causing diarrhea, K88 and F18 accounted for 92.7%. Despite the prevalence of ETEC K88 and F18, there is currently no effective vaccine available due to the diversity of these strains. This study presents an innovative approach by isolating chicken-derived single-chain variable fragment antibodies (scFvs) specific to K88 and F18 fimbrial antigens from chickens immunized against these ETEC virulence factors. These scFvs effectively inhibited adhesion of K88 and F18 to porcine intestinal epithelial cells (IPEC-J2), with the inhibitory effect demonstrating a dose-dependent increase. Furthermore, a bispecific scFv was designed and expressed in Pichia pastoris. This engineered construct displayed remarkable potency; at a concentration of 25.08 µg, it significantly reduced the adhesion rate of ETEC strains to IPEC-J2 cells by 72.10% and 69.11% when challenged with either K88 or F18 alone. Even in the presence of both antigens, the adhesion rate was notably decreased by 57.92%. By targeting and impeding the initial adhesion step of ETEC pathogenesis, this antibody-based intervention holds promise as a potential alternative to antibiotics, thereby mitigating the risks associated with antibiotic resistance and residual drug contamination in livestock production. Overall, this study lays the groundwork for the development of innovative treatments against ETEC infections in piglets.

An RNA-hydrolyzing recombinant minibody prevents both influenza A virus and coronavirus in co-infection models.

With the lifting of COVID-19 non-pharmaceutical interventions, the resurgence of common viral respiratory infections was recorded in several countries worldwide. It facilitates viral co-infection, further burdens the already over-stretched healthcare systems. Racing to find co-infection-associated efficacy therapeutic agents need to be rapidly established. However, it has encountered numerous challenges that necessitate careful investigation. Here, we introduce a potential recombinant minibody-associated treatment, 3D8 single chain variable fragment (scFv), which has been developed as a broad-spectrum antiviral drug that acts via its nucleic acid catalytic and cell penetration abilities. In this research, we demonstrated that 3D8 scFv exerted antiviral activity simultaneously against both influenza A viruses (IAVs) and coronaviruses in three established co-infection models comprising two types of coronaviruses [beta coronavirus-human coronavirus OC43 (hCoV-OC43) and alpha coronavirus-porcine epidemic diarrhea virus (PEDV)] in Vero E6 cells, two IAVs [A/Puerto Rico/8/1934 H1N1 (H1N1/PR8) and A/X-31 (H3N2/X-31)] in MDCK cells, and a combination of coronavirus and IAV (hCoV-OC43 and adapted-H1N1) in Vero E6 cells by a statistically significant reduction in viral gene expression, proteins level, and approximately around 85%, 65%, and 80% of the progeny of 'hCoV-OC43-PEDV', 'H1N1/PR8-H3N2/X-31', and 'hCoV-OC43-adapted-H1N1', respectively, were decimated in the presence of 3D8 scFv. Taken together, we propose that 3D8 scFv is a promising broad-spectrum drug for treatment against RNA viruses in co-infection.

A single chain variable fragment antibody (Tn 64) cognate to fibronectin type III repeats promotes corneal wound healing by inhibiting fibrosis.

Corneal wound healing requires epithelial reorganization and stromal extracellular matrix (ECM) remodeling, with ECM proteins such as Tenascin C (TnC) regulating and maintaining corneal homeostasis. The N-terminal globular domain and C-terminal fibrinogen-related domains of TnC are separated by epidermal growth factor (EGF)-like repeats, and upto fifteen fibronectin type III domains (Tn fn). Overexpression of Tn fn 1-5 and its splice variants occurs in varied pathologies. We have previously used Tn64 (a single chain variable fragment antibody cognate to Tn fn 1-5) to establish roles of Tn fn 1-5 in fibrotic pathologies such as rheumatoid arthritis and posterior capsular opacification. Here, we show that Tn64 binds to Tn fn repeats 3-5 (which constitute the major site for binding of soluble fibronectin within TnC). Unlike other Tn fn domains, Tn fn 3-5 displays no inhibition of fibronectin matrix assembly. Rather, the Tn fn 3-5 construct is pro-fibrotic and elicits increased expression of fibronectin. We examined corneal epithelial as well as stromal wound healing through Tn64 binding to Tn fn 3-5, using a human corneal epithelial cell (HCEC) line, primary cultures of human corneal fibroblasts (HCFs), and an ex-vivo corneal organ culture model. Tn64 enhanced proliferation and adhesion of corneal epithelial cells, while inhibiting the migration of corneal fibroblasts and myofibroblasts. Tn64 appears to attenuate inflammation through downregulation of TNF-α, prevent corneal fibrosis by limiting fibronectin polymerization, and promote regeneration of corneal epithelia and stroma, suggesting that it could be developed as a therapeutic agent for effective anti-fibrotic corneal wound healing.

Facile generation of biepitopic antibodies with intrinsic agonism for activating tumor necrosis factor receptors.

Agonist antibodies are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their activation, which can be achieved using antibodies that recognize two unique epitopes. However, the generation of biepitopic (i.e., biparatopic) antibodies typically requires animal immunization and is laborious and unpredictable. Here, we report a simple method for identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses existing, receptor-specific IgGs, which lack intrinsic agonist activity, to block their corresponding epitopes, then selects single-chain antibodies that bind accessible epitopes. The selected antibodies are fused to the light chains of IgGs to generate human tetravalent antibodies. We highlight the broad utility of this approach by converting several clinical-stage antibodies against OX40 and CD137 (4-1BB) into biepitopic antibodies with potent agonist activity.

Design and Evaluation of a Humanized Anti-EGFR huscFv Antibody Fragment in Combination With Metformin in A549 Cells In Vitro.

BACKGROUND/AIM: The epidermal growth factor receptor (EGFR) is over-expressed in several types of cancer, and monoclonal antibody therapy has been the strategy that has shown the best results. This study focused on the construction of a humanized single chain antibody (huscFv) directed against EGFR (HER1). MATERIALS AND METHODS: The CDR grafting method was used to incorporate murine complementarity determining regions (CDRs) of cetuximab into human sequences. A dot blot assay was used to examine the affinity of the huscFv secreted by HEK293T for EGFR. The inhibitory effect on the viability of A549 cells was evaluated using the WST-1 assay. RESULTS: The incorporation of murine CDRs of cetuximab into human sequences increased the degree of humanness by 16.4%. The increase in the humanization of scFv did not affect the affinity for EGFR. Metformin had a dose-dependent effect, with an IC50 of 46 mM, and in combination with huscFv, the cell viability decreased by 45% compared to the 15% demonstrated by huscFv alone. CONCLUSION: The CDR grafting technique is efficient for the humanization of scFv, maintaining its affinity for EGFR and demonstrating its inhibitory effect when combined with metformin in A549 cells.

A Cell-Penetrating Peptide Improves Anti-HER2 Single-Chain Variable Fragment Internalization and Antitumor Activity against HER2-Positive Breast Cancer In Vitro and In Vivo.

Cell-penetrating peptides (CPPs) are invaluable tools for delivering various substances into cells by crossing biological membranes. However, the effects of cell-penetrating peptide fusion proteins on the biological activity of antibodies remain to be fully understood. Here, we engineered a recombinant protein, LP-scFv, which combines the single-chain variable region of anti-human epidermal growth factor receptor-2 with a novel and non-oxic cell-penetrating peptide as a leader peptide. The introduction of this leader peptide led to a more than twofold increase in the internalization efficiency of the single-chain antibody, as confirmed using microscopic analysis and flow cytometry. The effects of the single-chain antibodies and LP-scFv on cell viability were evaluated using the MTT assay. Both the single-chain antibodies and LP-scFv reduced the viability of BT474 and NCI-N87 cells in a dose-dependent manner while exhibiting minimal toxicity towards MCF-7 and MCF-10A cells. Further investigation into LP-scFv's mechanism revealed that the induced leader peptide does not alter the MAPK-ERK1/2 and PI3K/AKT pathways of single-chain antibodies. An enhanced antitumor activity was also confirmed in an NCI-N87 tumor xenograft model in mice with a reduction of 45.2% in tumor growth inhibition (vs. 23.1% for scFv) with a 50 mg/kg dose after orthotopic injection administration, which was equivalent to that of trastuzumab (vs. 55.7% for trastuzumab). Overall, these results indicate that LP-scFv exhibits significant permeation activity in HER2-positive cells to enhance the intracellular dose effect on antitumor activity in vitro and in vivo. This research lays the foundation for designing novel antibody-based therapies for cancer.

Fully human anti-B7-H3 recombinant antibodies inhibited tumor growth by increasing T cell infiltration.

Mortality due to malignant tumors is one of the major factors affecting the life expectancy of the global population. Therapeutic antibodies are a cutting-edge treatment method for restricting tumor growth. B7-H3 is highly expressed in tumor tissues, but rarely in normal tissues. B7-H3 is closely associated with poor prognosis in patients with tumors. B7-H3 is an important target for antitumor therapy. In this study, the fully human anti-B7H3 single-chain antibodies (scFvs) were isolated and screened from the fully human phage immune library with B7H3 as the target. The antibodies screened from a fully human phage library had low immunogenicity and high affinity, which was more beneficial for clinical application. Leveraging B7-H3 scFvs as a foundation, we constructed two distinct recombinant antibody formats, scFv-Fc and IgG1, characterized by elevated affinity and a prolonged half-life. The results demonstrated that the recombinant antibodies had high specificity and affinity for the B7-H3 antigen and inhibited tumor cell growth by enhancing the ADCC. After treatment with anti-B7H3 recombinant antibody, the number of infiltrating T cells in the tumor increased and the secretion of IFN- γ by infiltrating T cells increased in vivo. Additionally, the use of pleural fluid samples obtained from tumor-afflicted patients revealed the ability of anti-B7-H3 recombinant antibodies to reverse CD8+ T cell exhaustion. In summary, we screened the fully human anti-B7H3 recombinant antibodies with specificity and high affinity that increase immune cell infiltration and IFN-γ secretion, thereby inhibiting tumor cell growth to a certain extent. This finding provides a theoretical basis for the development of therapeutic tumor antibodies and could help promote further development of antibody-based drugs.

Anti-Acinetobacter Baumannii single-chain variable fragments provide therapeutic efficacy in an immunocompromised mouse pneumonia model.

BACKGROUND: The emergence of carbapenem-resistant and extensively drug-resistant (XDR) Acinetobacter baumannii as well as inadequate effective antibiotics calls for an urgent effort to find new antibacterial agents. The therapeutic efficacy of two human scFvs, EB211 and EB279, showing growth inhibitory activity against A. baumannii in vitro, was investigated in immunocompromised mice with A. baumannii pneumonia. RESULTS: The data revealed that infected mice treated with EB211, EB279, and a combination of the two scFvs showed better survival, reduced bacterial load in the lungs, and no marked pathological abnormalities in the kidneys, liver, and lungs when compared to the control groups receiving normal saline or an irrelevant scFv. CONCLUSIONS: The results from this study suggest that the scFvs with direct growth inhibitory activity could offer promising results in the treatment of pneumonia caused by XDR A. baumannii.

An immunocytokine consisting of a TNFR2 agonist and TNFR2 scFv enhances the expansion of regulatory T cells through TNFR2 clustering.

Regulatory T cells (Tregs) are lymphocytes that play a central role in peripheral immune tolerance. Tregs are promising targets for the prevention and suppression of autoimmune diseases, allergies, and graft-versus-host disease, and treatments aimed at regulating their functions are being developed. In this study, we created a new modality consisting of a protein molecule that suppressed excessive immune responses by effectively and preferentially expanding Tregs. Recent studies reported that tumor necrosis factor receptor type 2 (TNFR2) expressed on Tregs is involved in the proliferation and activation of Tregs. Therefore, we created a functional immunocytokine, named TNFR2-ICK-Ig, consisting of a fusion protein of an anti-TNFR2 single-chain Fv (scFv) and a TNFR2 agonist TNF-α mutant protein, as a new modality that strongly enhances TNFR2 signaling. The formation of agonist-receptor multimerization (TNFR2 cluster) is effective for the induction of a strong TNFR2 signal, similar to the TNFR2 signaling mechanism exhibited by membrane-bound TNF. TNFR2-ICK-Ig improved the TNFR2 signaling activity and promoted TNFR2 cluster formation compared to a TNFR2 agonist TNF-α mutant protein that did not have an immunocytokine structure. Furthermore, the Treg expansion efficiency was enhanced. TNFR2-ICK-Ig promotes its effects via scFv, which crosslinks receptors whereas the agonists transmit stimulatory signals. Therefore, this novel molecule expands Tregs via strong TNFR2 signaling by the formation of TNFR2 clustering.

Canine distemper virus (CDV)-neutralizing activities of an anti-CDV canine-derived single-chain variable antibody fragment 4-15 (scFv 4-15) screened by phage display technology.

Canine distemper virus (CDV) is a highly contagious pathogen that causes severe diarrhea, fever and vomiting in domestic dogs, posing a serious threat to the dog breeding industry. Currently, there are no effective therapeutic agents for emergency treatment despite the availability of vaccines against CDV infection. Single-chain fragment variable (scFv) antibody has been demonstrated to effectively inhibit virus infections, suggesting a potential candidate as a therapeutic agent for canine distemper. In this study, a phage-displayed scFv library was constructed from the peripheral blood lymphocytes of dog immunized intramuscularly with live-attenuated CDV vaccine, and was subjected to four rounds of pannings against CDV. Subsequent indirect enzyme-linked immunosorbent assay screening revealed high-affinity scFv antibodies specific to CDV, and indirect immunofluorescence assay screening revealed CDV-neutralizing activity of scFv antibodies. Our results showed that a scFv antibody 4-15 (scFv 4-15) with high-affinity binding to CDV and neutralizing activity against CDV was obtained, which displayed effective therapeutic potential in vivo for dogs challenged with a lethal dose of CDV. Conclusively, the scFv 4-15 with high-affinity binding and neutralizing activity to CDV that was obtained by phage display technology provides a promising candidate for the therapeutic agents against CDV infection.

Engineered CD4 T cells expressing a membrane anchored viral inhibitor restrict HIV-1 through cis and trans mechanisms.

HIV-1 infection of target cells can occur through either cell-free virions or cell-cell transmission in a virological synapse, with the latter mechanism of infection reported to be 100- to 1,000-fold more efficient. Neutralizing antibodies and entry inhibitors effectively block cell-free HIV-1, but with few exceptions, they display much less inhibitory activity against cell-mediated HIV-1 transmission. Previously, we showed that engineering HIV-1 target cells by genetically linking single-chain variable fragments (scFvs) of antibodies to glycosyl phosphatidylinositol (GPI) potently blocks infection by cell-free virions and cell-mediated infection by immature dendritic cell (iDC)-captured HIV-1. Expression of scFvs on CD4+ cell lines by transduction with X5 derived anti-HIV-1 Env antibody linked to a GPI attachment signal directs GPI-anchored scFvs into lipid rafts of the plasma membrane. In this study, we further characterize the effect of GPI-scFv X5 on cell-cell HIV-1 transmission from DCs to target cells. We report that expression of GPI-scFv X5 in transduced CD4+ cell lines and human primary CD4+ T cells potently restricts viral replication in iDC- or mDC-captured HIV-1 in trans. Using live-cell imaging, we observed that when GPI-GFP or GPI-scFv X5 transduced T cells are co-cultured with iDCs, GPI-anchored proteins enrich in contact zones and subsequently migrate from T cells into DCs, suggesting that transferred GPI-scFv X5 interferes with HIV-1 infection of iDCs. We conclude that GPI-scFv X5 on the surface of transduced CD4+ T cells not only potently blocks cell-mediated infection by DCs, but it transfers from transduced cells to the surface of iDCs and neutralizes HIV-1 replication in iDCs. Our findings have important implications for HIV-1 antibody-based immunotherapies as they demonstrate a viral inhibitory effect that extends beyond the transduced CD4+ T cells to iDCs which can enhance HIV-1 replication.

Single-chain fragment antibody disrupting the EphA4 function as a therapeutic drug for gastric cancer.

Studies have shown that the high expression of EphA4 in gastric cancer tissues may correlate with unfavorable clinical pathological characteristics. Therefore, EphA4 may be an effective target for treating gastric cancer in addition to HER-2/neu. In this study, generated scFv S3 can bind endogenous EphA4 of gastric cancer cells and has significant membrane staining. Additionally, scFv S3 binding to EphA4 inhibits the growth and migration of cancer cells and the growth induction that ephrinA1 generates in gastric cancer cells. We found that EphA4 molecules may degrade through antibody treatment of cells, and the increase in LAMP1 and LAMP2 indicates that lysosome is involved in the degradation. The scFv S3 administration leads to the signals pAKT, pERK, and pSTAT3 decrease in cancer cells. The xenograft model of HER-2/neu low expressing gastric cancer cell SNU-16 exhibits better therapeutic effects by scFv S3 than trastuzumab scFv. The scFv S3 administration in vivo can degrade EphA4 molecules in tumor tissues, decreasing Ki67 and increasing cleaved C3 molecule expression. Furthermore, we identified and validated that scFv S3 generates essential ionic bonding with R162 on EphA4. The antibody may provide effective treatment for patients with gastric cancer and abnormal activation or overexpression of EphA4 signaling.

CSPG4 as a target for the specific killing of triple-negative breast cancer cells by a recombinant SNAP-tag-based antibody-auristatin F drug conjugate.

PURPOSE: Triple-negative breast cancer (TNBC) is phenotypic of breast tumors lacking expression of the estrogen receptor (ER), the progesterone receptor (PgR), and the human epidermal growth factor receptor 2 (HER2). The paucity of well-defined molecular targets in TNBC, coupled with the increasing burden of breast cancer-related mortality, emphasizes the need to develop targeted diagnostics and therapeutics. While antibody-drug conjugates (ADCs) have emerged as revolutionary tools in the selective delivery of drugs to malignant cells, their widespread clinical use has been hampered by traditional strategies which often give rise to heterogeneous mixtures of ADC products. METHODS: Utilizing SNAP-tag technology as a cutting-edge site-specific conjugation method, a chondroitin sulfate proteoglycan 4 (CSPG4)-targeting ADC was engineered, encompassing a single-chain antibody fragment (scFv) conjugated to auristatin F (AURIF) via a click chemistry strategy. RESULTS: After showcasing the self-labeling potential of the SNAP-tag component, surface binding and internalization of the fluorescently labeled product were demonstrated on CSPG4-positive TNBC cell lines through confocal microscopy and flow cytometry. The cell-killing ability of the novel AURIF-based recombinant ADC was illustrated by the induction of a 50% reduction in cell viability at nanomolar to micromolar concentrations on target cell lines. CONCLUSION: This research underscores the applicability of SNAP-tag in the unambiguous generation of homogeneous and pharmaceutically relevant immunoconjugates that could potentially be instrumental in the management of a daunting disease like TNBC.

Cell-penetrating porcine single-chain antibodies (transbodies) against nonstructural protein 1ß (NSP1ß) of porcine reproductive and respiratory syndrome virus inhibit virus replication.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS) worldwide, especially in domestic pigs, with an enormous economic impact, estimated at $664 million in losses every year to the pig industry. Current vaccines confer limited protection, and no direct-acting anti-PRRS treatment is available. Non-structural protein (NSP) 1ß, a cysteine-like protease (CLPro) of PRRSV plays an essential role in viral polyprotein processing, subgenomic RNA synthesis, and evasion of host innate immunity. Therefore, agents that interfere with the bioactivity of NSP1ß would be expected to inhibit virus replication. In this study, a porcine single-chain antibody (scFv)-phage display library was constructed and used as a tool for production of NSP1ß-specific porcine scFvs (pscFvs). The pscFvs to NSP1ß were linked to a cell-penetrating peptide to form cell-penetrating pscFvs (transbodies), which could be internalized and inhibit PRRSV replication in infected cells. A computer simulation indicated that the effective pscFvs used several residues in multiple complementarity determining regions (CDRs) to interact with multiple residues in the CLPro and C-terminal motifs, which might explain the mechanism of pscFv-mediated inhibition of virus replication. Although experiments are needed to determine the antiviral mechanism of the transbodies, the current data indicate that transbodies can potentially be applied for treatment and prevention of PRRSV infection.

Development of a MET-targeted single-chain antibody fragment as an anti-oncogene targeted therapy for breast cancer.

The usage of monoclonal antibodies (mAbs) and antibody fragments, as a matter associated with the biopharmaceutical industry, is increasingly growing. Harmonious with this concept, we designed an exclusive modeled single-chain variable fragment (scFv) against mesenchymal-epithelial transition (MET) oncoprotein. This scFv was newly developed from Onartuzumab sequence by gene cloning, and expression using bacterial host. Herein, we examined its preclinical efficacy for the reduction of tumor growth, invasiveness and angiogenesis in vitro and in vivo. Expressed anti-MET scFv demonstrated high binding capacity (48.8%) toward MET-overexpressing cancer cells. The IC50 value of anti-MET scFv against MET-positive human breast cancer cell line (MDA-MB-435) was 8.4 µg/ml whereas this value was measured as 47.8 µg/ml in MET-negative cell line BT-483. Similar concentrations could also effectively induce apoptosis in MDA-MB-435 cancer cells. Moreover, this antibody fragment could reduce migration and invasion in MDA-MB-435 cells. Grafted breast tumors in Balb/c mice showed significant tumor growth suppression as well as reduction of blood-supply in response to recombinant anti-MET treatment. Histopathology and immunohistochemical assessments revealed higher rate of response to therapy. In our study, we designed and synthetized a novel anti-MET scFv which could effectively suppress MET-overexpressing breast cancer tumors.

A single-chain variable fragment-anticancer lytic peptide (scFv-ACLP) fusion protein for targeted cancer treatment.

Antibody-directed drugs for targeted cancer treatment have become a hot topic in new anticancer drug development; however, antibody-fused therapeutic peptides were rarely documented. Herein, we designed a fusion protein with a cetuximab-derived single-chain variable fragment targeting epidermal growth factor receptor (anti-EGFR scFv) and the anticancer lytic peptide (ACLP) ZXR2, connected by a linker (G4 S)3 and MMP2 cleavage site. The anti-EGFR scFv-ZXR2 recombinant protein showed specific anticancer activity on EGFR-overexpressed cancer cell lines in a concentration- and time-dependent manner, as it can bind to EGFR on cancer cell surfaces. This fusion protein caused cell membrane lysis as ZXR2, and showed improved stability in serum compared with ZXR2. These results suggest that scFv-ACLP fusion proteins may be potential anticancer drug candidates for targeted cancer treatment, which also provide a feasible idea for targeted drug design.

Adenovirus vector-mediated single chain variable fragments target the nucleocapsid protein of porcine epidemic diarrhea virus and protect against viral infection in piglets.

Porcine epidemic diarrhea virus (PEDV) mainly infects the intestinal epithelial cells of pigs, causing porcine epidemic diarrhea (PED). In particular, the virus causes severe diarrhea, dehydration, and death in neonatal piglets. Maternal immunity effectively protects neonatal piglets from PEDV infection; however, maternal antibodies can only prevent PEDV attachment and entry into target cells, but have no effects on intracellular viruses. Intracellular antibodies targeting virus-encoded proteins are effective in preventing viral infection. We previously identified four single chain variable fragments (scFvs), ZW1-16, ZW3-21, ZW1-41, and ZW4-16, which specifically targeted the PEDV N protein and significantly inhibited PEDV replication and up-regulated interferon-λ1 (IFN-λ1) expression in host cells. In our current study, the four scFvs were subcloned into replication-defective adenovirus vectors to generate recombinant adenoviruses rAdV-ZW1-16, rAdV-ZW3-21, rAdV-ZW1-41, and rAdV-ZW4-16. ScFvs were successfully expressed in Human Embryonic Kidney 293 (HEK293) cells and intestinal porcine epithelial cell line J2 (IPEC-J2) and were biosafe for piglets as indicated by body temperature and weight, scFv excretion in feces, IFN-γ and interleukin-4 (IL-4) expression in jejunum, and pathological changes in porcine tissue after oral administration. Western blotting, immunofluorescence, and immunohistochemical analyses showed that scFvs were expressed in porcine jejunum. The prophylactic effects of rAdV-ZW, a cocktail of the four rAdV-scFvs, on piglet diarrhea caused by PEDV was investigated. Clinical symptoms in piglets orally challenged with PEDV, following a two-time treatment with rAdV-ZW, were significantly reduced when compared with PEDV-infected piglets treated with phosphate buffered saline (PBS) or rAdV-wild-type. Also, no death and jejunal lesions were observed. ScFv co-localization with the PEDV N protein in vivo was also observed. Next, the expression of pro-inflammatory serum cytokines such as tumor necrosis factor-α (TNF-α), IL-6, IL-8, IL-12, and IFN-λ was assessed by enzyme-linked immunosorbent assay (ELISA), which showed that scFvs significantly suppressed PEDV-induced pro-inflammatory cytokine expression and restored PEDV-inhibited IFN-λ expression. Therefore, our study supported a promising role for intracellular scFvs targeting the PEDV N protein to prevent and treat diarrhea in PEDV-infected piglets.

Rational design of a Nivolumab-based ANTI-PD-1 single chain variable fragment that blocks the interaction between PD-1 expressed on T-CELLS and PD-L1 ON CHO cells.

Antibodies that block the interaction between PD-1 expressing T-cells and cancer cells expressing PD-L1 play a central role in contemporary immunotherapy regimes [1-3]. We previously reported the isolation of a single chain variable fragment (scFv) of the monoclonal anti-PD-1 antibody Nivolumab, that binds to purified PD-1 and blocked its interaction with PD-L1 [4]. This anti-PD-1 scFv did not, however, function in a cell-based assay designed to detect the disruption of the PD-1/PD-L1 interaction, a result likely due to its poor solubility in tissue culture media. Herein we report that following a series of structure-based rational design analyses, including Aggreescan3D, we have isolated a variant of the anti-PD-1 scFv having significantly improved solubility in tissue culture medium. Moreover, this soluble anti-PD-1 scFv variant disrupted the interaction between PD-1 expressed on Jurkat Cells and PD-L1 expressed on CHO cells. These findings are discussed in terms of the related observation that the residues mutated to form the anti-PD-1 variant are conserved in many other scFvs; thus, the properties of a range of scFvs will likely be enhanced by similar mutations of the conserved residues.

Synthesis of an Anti-CD7 Recombinant Immunotoxin Based on PE24 in CHO and E. coli Cell-Free Systems.

Recombinant immunotoxins (RITs) are an effective class of agents for targeted therapy in cancer treatment. In this article, we demonstrate the straight-forward production and testing of an anti-CD7 RIT based on PE24 in a prokaryotic and a eukaryotic cell-free system. The prokaryotic cell-free system was derived from Escherichia coli BL21 StarTM (DE3) cells transformed with a plasmid encoding the chaperones groEL/groES. The eukaryotic cell-free system was prepared from Chinese hamster ovary (CHO) cells that leave intact endoplasmic reticulum-derived microsomes in the cell-free reaction mix from which the RIT was extracted. The investigated RIT was built by fusing an anti-CD7 single-chain variable fragment (scFv) with the toxin domain PE24, a shortened variant of Pseudomonas Exotoxin A. The RIT was produced in both cell-free systems and tested for antigen binding against CD7 and cell killing on CD7-positive Jurkat, HSB-2, and ALL-SIL cells. CD7-positive cells were effectively killed by the anti-CD7 scFv-PE24 RIT with an IC50 value of 15 pM to 40 pM for CHO and 42 pM to 156 pM for E. coli cell-free-produced RIT. CD7-negative Raji cells were unaffected by the RIT. Toxin and antibody domain alone did not show cytotoxic effects on either CD7-positive or CD7-negative cells. To our knowledge, this report describes the production of an active RIT in E. coli and CHO cell-free systems for the first time. We provide the proof-of-concept that cell-free protein synthesis allows for on-demand testing of antibody−toxin conjugate activity in a time-efficient workflow without cell lysis or purification required.