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Background: Inflammatory bowel diseases (IBDs) pose significant challenges in terms of treatment non‐response, necessitating the development of novel therapeutic approaches. Although biological medicines that target TNF‐α (tumour necrosis factor‐α) have shown clinical success in some IBD patients, a substantial proportion still fails to respond. Methods: We designed bispecific nanobodies (BsNbs) with the ability to simultaneously target human macrophage‐expressed membrane TNF‐α (hmTNF‐α) and IL‐23. Additionally, we fused the constant region of human IgG1 Fc (hIgG1 Fc) to BsNb to create BsNb‐Fc. Our study encompassed in vitro and in vivo characterization of BsNb and BsNb‐Fc. Results: BsNb‐Fc exhibited an improved serum half‐life, targeting capability and effector function than BsNb. It's demonstrated that BsNb‐Fc exhibited superior anti‐inflammatory effects compared to the anti‐TNF‐α mAb (infliximab, IFX) combined with anti‐IL‐12/IL‐23p40 mAb (ustekinumab, UST) by Transwell co‐culture assays. Notably, in murine models of acute colitis brought on by 2,4,6‐trinitrobenzene sulfonic acid（TNBS) and dextran sulphate sodium (DSS), BsNb‐Fc effectively alleviated colitis severity. Additionally, BsNb‐Fc outperformed the IFX&UST combination in TNBS‐induced colitis, significantly reducing colon inflammation in mice with colitis produced by TNBS and DSS. Conclusion: These findings highlight an enhanced efficacy and improved biostability of BsNb‐Fc, suggesting its potential as a promising therapeutic option for IBD patients with insufficient response to TNF‐α inhibition. Key points: A bispecific nanobody (BsNb) was created to target TNF‐α and IL‐23p19, exhibiting high affinity and remarkable stability.BsNb‐Fc inhibited the release of cytokines in CD4+T cells during co‐culture experiments.BsNb‐Fc effectively alleviated colitis severity in mouse model with acute colitis induced by DSS or TNBS, outperforming the IFX&UST combination. [ABSTRACT FROM AUTHOR]

Abstract Background Inflammatory bowel diseases (IBDs) pose significant challenges in terms of treatment non‐response, necessitating the development of novel therapeutic approaches. Although biological medicines that target TNF‐α (tumour necrosis factor‐α) have shown clinical success in some IBD patients, a substantial proportion still fails to respond. Methods We designed bispecific nanobodies (BsNbs) with the ability to simultaneously target human macrophage‐expressed membrane TNF‐α (hmTNF‐α) and IL‐23. Additionally, we fused the constant region of human IgG1 Fc (hIgG1 Fc) to BsNb to create BsNb‐Fc. Our study encompassed in vitro and in vivo characterization of BsNb and BsNb‐Fc. Results BsNb‐Fc exhibited an improved serum half‐life, targeting capability and effector function than BsNb. It's demonstrated that BsNb‐Fc exhibited superior anti‐inflammatory effects compared to the anti‐TNF‐α mAb (infliximab, IFX) combined with anti‐IL‐12/IL‐23p40 mAb (ustekinumab, UST) by Transwell co‐culture assays. Notably, in murine models of acute colitis brought on by 2,4,6‐trinitrobenzene sulfonic acid（TNBS) and dextran sulphate sodium (DSS), BsNb‐Fc effectively alleviated colitis severity. Additionally, BsNb‐Fc outperformed the IFX&UST combination in TNBS‐induced colitis, significantly reducing colon inflammation in mice with colitis produced by TNBS and DSS. Conclusion These findings highlight an enhanced efficacy and improved biostability of BsNb‐Fc, suggesting its potential as a promising therapeutic option for IBD patients with insufficient response to TNF‐α inhibition. Key points A bispecific nanobody (BsNb) was created to target TNF‐α and IL‐23p19, exhibiting high affinity and remarkable stability. BsNb‐Fc inhibited the release of cytokines in CD4+T cells during co‐culture experiments. BsNb‐Fc effectively alleviated colitis severity in mouse model with acute colitis induced by DSS or TNBS, outperforming the IFX&UST combination.

Background: Fibroblast activation protein-α (FAPα) is selectively overexpressed in tumor-associated fibroblasts in more than 90% of epithelial tumors, and may be a good target for anticancer treatment, for example, using an anti-FAPα recombinant antibody (rAb) labeled with radionuclides. In the present report, the radiolabeling and preclinical evaluation of novel anti-FAPα rAbs were investigated. Materials and Methods: Two novel anti-FAPα VHHs (AMS002-1 and AMS002-2) with high binding affinity to FAPα were selected from an antibody phage library. The anti-FAPα VHHs were then fused with the Fc fragment of human IgG4 to create two VHH-Fc rAbs. The VHH-Fc rAbs were radiolabeled with 89Zr and 177Lu. The radiolabeled products were evaluated by radioligand-binding assays using FAPα-expressing cells. The biodistribution and tumor-targeting properties were investigated by small-animal PET/CT. AMS002-1-Fc, which showed promising tumor-targeting properties in 89Zr-microPET imaging, was radiolabeled with 177Lu for efficacy study on HT1080 tumor-bearing mice and monitored with SPECT/CT imaging. Results: The two VHH-Fc rAbs with good affinity with KD values in low nanomolar range were identified. Both PET/CT imaging with 89Zr-AMS002-1-Fc rAb and SPECT/CT imaging with 177Lu-AMS002-1-Fc rAb demonstrated highest tumor uptakes at 72 h p.i. and long tumor retention in the preclinical models. Furthermore, ex vivo biodistribution analysis revealed high tumor uptake of 89Zr-AMS002-1-Fc at 48 h p.i. with the value of 6.91% ± 2.08% ID/g. Finally, radioimmunotherapy with 177Lu-AMS002-1-Fc rAb delayed the tumor growth without significant weight loss in mice with HT1080 xenografts. The tumor size of untreated control group was 2.59 times larger compared with the treatment group with 177Lu-AMS002-1-Fc at day 29. Conclusion:89Zr/177Lu-AMS002-1-Fc represent a pair of promising radiopharmaceuticals for theranostics on FAPα-expressing tumors. [ABSTRACT FROM AUTHOR]

Abstract Background The advent of proprotein convertase subtilisin/kexin type 9 (PCSK9)–inhibiting drugs have provided an effective, but extremely expensive treatment for the management of low density lipoprotein (LDL). Our aim was to explore a cost-effective application of camelid anti-PCSK9 single domain antibodies (sdAbs), which are high variable regions of the camelid heavy chain antibodies (VHHs), as a human PCSK9 (hPCSK9) inhibitor. One female llama was immunized with hPCSK9. Screening of high affinity anti-PCSK9 VHHs was carried out based on surface plasmon resonance (SPR) technology. We reported a lysate kinetic analysis method improving the screening efficiency. To increase the serum half-life and targeting properties, the constant region fragment of the human immunoglobulin gamma sub-type 4 (IgG4 Fc) was incorporated to form a novel llama-human chimeric molecule (VHH-hFc). Results The PCSK9 inhibiting effects of the VHH proteins were analyzed in two human liver hepatocellular cells (HepG2 and Huh7) and in the hPCSK9 transgenic Sprague–Dawley (SD) rat model. The hPCSK9 antagonistic potency of the bivalent VHH-hFc exceeded the monovalent VHH (P

Monoclonal antibodies have contributed to improving the treatment of several diseases. However, limitations related to pharmacokinetic parameters and production costs have instigated the search for alternative products. Camelids produce functional immunoglobulins G devoid of light chains and CH1 domains, in which the antigenic recognition site is formed by a single domain called VHH or nanobody. VHHs' small size and similarity to the human VH domain contribute to high tissue penetration and low immunogenicity. In addition, VHHs provide superior antigen recognition compared to human antibodies, better solubility and stability. Due to these characteristics and the possibility of obtaining gene-encoding VHHs, applications of this biological tool, whether as a monomer or in related recombinant constructs, have been reported. To ensure antibody efficacy and cost-effectiveness, strategies for their expression, either using prokaryotic or eukaryotic systems, have been utilized. Plant-based expression systems are useful for VHH related constructs that require post-translational modifications. This system has exhibited versatility, low-cost upstream production, and safety. This article presents the main advances associated to the heterologous expression of VHHs in plant systems. Besides, we show insights related to the use of VHHs as a strategy for plant pathogen control and a tool for genomic manipulation in plant systems. [ABSTRACT FROM AUTHOR]

publisher‐imprint‐name Springer volume‐issue‐count 1 issue‐article‐count 16 issue‐toc‐levels 0 issue‐pricelist‐year 2020 issue‐copyright‐holder The Author(s) issue‐copyright‐year 2020 article‐contains‐esm Yes article‐numbering‐style Unnumbered article‐registration‐date‐year 2020 article‐registration‐date‐month 1 article‐registration‐date‐day 21 article‐toc‐levels 0 toc‐levels 0 volume‐type Regular journal‐product ArchiveJournal numbering‐style Unnumbered article‐grants‐type OpenChoice metadata‐grant OpenAccess abstract‐grant OpenAccess bodypdf‐grant OpenAccess bodyhtml‐grant OpenAccess bibliography‐grant OpenAccess esm‐grant OpenAccess online‐first false pdf‐file‐reference BodyRef/PDF/40169_2020_Article_265.pdf pdf‐type Typeset target‐type OnlinePDF issue‐type Regular article‐type OriginalPaper journal‐subject‐primary Medicine & Public Health journal‐subject‐secondary Medicine/Public Health, general journal‐subject‐collection Medicine open‐access true --> Background: The advent of proprotein convertase subtilisin/kexin type 9 (PCSK9)–inhibiting drugs have provided an effective, but extremely expensive treatment for the management of low density lipoprotein (LDL). Our aim was to explore a cost‐effective application of camelid anti‐PCSK9 single domain antibodies (sdAbs), which are high variable regions of the camelid heavy chain antibodies (VHHs), as a human PCSK9 (hPCSK9) inhibitor. One female llama was immunized with hPCSK9. Screening of high affinity anti‐PCSK9 VHHs was carried out based on surface plasmon resonance (SPR) technology. We reported a lysate kinetic analysis method improving the screening efficiency. To increase the serum half‐life and targeting properties, the constant region fragment of the human immunoglobulin gamma sub‐type 4 (IgG4 Fc) was incorporated to form a novel llama‐human chimeric molecule (VHH‐hFc). Results: The PCSK9 inhibiting effects of the VHH proteins were analyzed in two human liver hepatocellular cells (HepG2 and Huh7) and in the hPCSK9 transgenic Sprague–Dawley (SD) rat model. The hPCSK9 antagonistic potency of the bivalent VHH‐hFc exceeded the monovalent VHH (P < 0.001) in hepatocarcinoma cells. Furthermore, the llama‐human chimeric VHH‐Fc protein had a similar reduction (~ 40%) of the LDL‐c and total cholesterol when compared to the approved evolocumab in transgenic SD rat model, but with low cost. More surprisingly, the chimeric heavy chain antibodies could be persevered for 3 months at room temperature with little loss of the affinity. Conclusions: Due to the high yield and low cost of Pichia pastoris, lipid‐lowering effect and strong stability, the llama‐human chimeric antibody (VHH‐Fc) offers a potent therapeutic candidate for the control of the serum lipid level. [ABSTRACT FROM AUTHOR]

Targeting a vaccine to the mucosal surface has recently been recognized as a promising approach to efficiently induce mucosal immune responses against enteric pathogens. However, poor uptake and inefficient transport of orally delivered subunit vaccines across the intestinal epithelium combined with weak immune responses still present important bottlenecks for mucosal vaccination. A possible strategy suggested to surmount these hurdles is to target the selected antigen to transcytotic receptors, such as aminopeptidase N (APN) present on enterocytes and antigen-presenting cells (APCs). Therefore, we aimed to identify potent and selective VHHs against porcine aminopeptidase N (pAPN), that were fused to the fragment crystallizable (Fc) domain of the murine IgG2a, resulting in dimeric VHH-MG fusions. Out of a library of 30 VHH-MG fusion candidates, two fusions displaying the best binding on pAPN-expressing cells were selected and showed in vivo internalization across the porcine gut epithelium. One of these fusions triggered systemic and intestinal IgA responses upon oral administration. Our results demonstrate the potential of bivalent VHH-MG fusions as delivery vehicles for vaccine antigens. VHH-mediated targeting of antigens to APN to generate protective immunity at the mucosal surface remains to be further validated. Unlabelled Image [ABSTRACT FROM AUTHOR]