Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion.

Gastro-intestinal helminth infections trigger the release of interleukin-33 (IL-33), which induces type-2 helper T cells (Th2 cells) at the site of infection to produce IL-13, thereby contributing to host resistance in a T cell receptor (TCR)-independent manner. Here, we show that, as a prerequisite for IL-33-induced IL-13 secretion, Th2 cells required the expression of the epidermal growth factor receptor (EGFR) and of its ligand, amphiregulin, for the formation of a signaling complex between T1/ST2 (the IL-33R) and EGFR. This shared signaling complex allowed IL-33 to induce the EGFR-mediated activation of the MAP-kinase signaling pathway and consequently the expression of IL-13. Lack of EGFR expression on T cells abrogated IL-13 expression in infected tissues and impaired host resistance. EGFR expression on Th2 cells was TCR-signaling dependent, and therefore, our data reveal a mechanism by which antigen presentation controls the innate effector function of Th2 cells at the site of inflammation.

Stabilization of the transcription factor Foxp3 by the deubiquitinase USP7 increases Treg-cell-suppressive capacity.

Stable Foxp3 expression is required for the development of functional regulatory T (Treg) cells. Here, we demonstrate that the expression of the transcription factor Foxp3 can be regulated through the polyubiquitination of multiple lysine residues, resulting in proteasome-mediated degradation. Expression of the deubiquitinase (DUB) USP7 was found to be upregulated and active in Treg cells, being associated with Foxp3 in the nucleus. Ectopic expression of USP7 decreased Foxp3 polyubiquitination and increased Foxp3 expression. Conversely, either treatment with DUB inhibitor or USP7 knockdown decreased endogenous Foxp3 protein expression and decreased Treg-cell-mediated suppression in vitro. Furthermore, in a murine adoptive-transfer-induced colitis model, either inhibition of DUB activity or USP7 knockdown in Treg cells abrogated their ability to resolve inflammation in vivo. Our data reveal a molecular mechanism in which rapid temporal control of Foxp3 expression in Treg cells can be regulated by USP7, thereby modulating Treg cell numbers and function.

Amphiregulin enhances regulatory T cell-suppressive function via the epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR) is known to be critically involved in tissue development and homeostasis as well as in the pathogenesis of cancer. Here we showed that Foxp3(+) regulatory T (Treg) cells express EGFR under inflammatory conditions. Stimulation with the EGF-like growth factor Amphiregulin (AREG) markedly enhanced Treg cell function in vitro, and in a colitis and tumor vaccination model we showed that AREG was critical for efficient Treg cell function in vivo. In addition, mast cell-derived AREG fully restored optimal Treg cell function. These findings reveal EGFR as a component in the regulation of local immune responses and establish a link between mast cells and Treg cells. Targeting of this immune regulatory mechanism may contribute to the therapeutic successes of EGFR-targeting treatments in cancer patients.

Canonical Wnt signaling negatively modulates regulatory T cell function.

Foxp3 is crucial for both the development and function of regulatory T (Treg) cells; however, the posttranslational mechanisms regulating Foxp3 transcriptional output remain poorly defined. Here, we demonstrate that T cell factor 1 (TCF1) and Foxp3 associates in Treg cells and that active Wnt signaling disrupts Foxp3 transcriptional activity. A global chromatin immunoprecipitation sequencing comparison in Treg cells revealed considerable overlap between Foxp3 and Wnt target genes. The activation of Wnt signaling reduced Treg-mediated suppression both in vitro and in vivo, whereas disruption of Wnt signaling in Treg cells enhanced their suppressive capacity. The activation of effector T cells increased Wnt3a production, and Wnt3a levels were found to be greatly increased in mononuclear cells isolated from synovial fluid versus peripheral blood of arthritis patients. We propose a model in which Wnt produced under inflammatory conditions represses Treg cell function, allowing a productive immune response, but, if uncontrolled, could lead to the development of autoimmunity.

Hsp70 and NF-kB Mediated Control of Innate Inflammatory Responses in a Canine Macrophage Cell Line.

The pathogenesis of many inflammatory diseases is associated with the uncontrolled activation of nuclear factor kappa B (NF-κB) in macrophages. Previous studies have shown that in various cell types, heat shock protein 70 (Hsp70) plays a crucial role in controlling NF-κB activity. So far, little is known about the role of Hsp70 in canine inflammatory processes. In this study we investigated the potential anti-inflammatory effects of Hsp70 in canine macrophages as well as the mechanisms underlying these effects. To this end, a canine macrophage cell line was stressed with arsenite, a chemical stressor, which upregulated Hsp70 expression as detected by flow cytometry and qPCR. A gene-edited version of this macrophage cell line lacking inducible Hsp70 was generated using CRISPR-Cas9 technology. To determine the effects of Hsp70 on macrophage inflammatory properties, arsenite-stressed wild-type and Hsp70 knockout macrophages were exposed to lipopolysaccharide (LPS), and the expression of the inflammatory cytokines IL-6, IL-1ß and tumor necrosis factor-α (TNF-α) and levels of phosphorylated NF-κB were determined by qPCR and Western Blotting, respectively. Our results show that non-toxic concentrations of arsenite induced Hsp70 expression in canine macrophages; Hsp70 upregulation significantly inhibited the LPS-induced expression of the pro-inflammatory mediators TNF-α and IL-6, as well as NF-κB activation in canine macrophages. Furthermore, the gene editing of inducible Hsp70 by CRISPR-Cas9-mediated gene editing neutralized this inhibitory effect of cell stress on NF-κB activation and pro-inflammatory cytokine expression. Collectively, our study reveals that Hsp70 may regulate inflammatory responses through NF-κB activation and cytokine expression in canine macrophages.

CD8(+) T cells of Listeria monocytogenes-infected mice recognize both linear and spliced proteasome products.

CD8(+) T cells responding to infection recognize pathogen-derived epitopes presented by MHC class-I molecules. While most of such epitopes are generated by proteasome-mediated antigen cleavage, analysis of tumor antigen processing has revealed that epitopes may also derive from proteasome-catalyzed peptide splicing (PCPS). To determine whether PCPS contributes to epitope processing during infection, we analyzed the fragments produced by purified proteasomes from a Listeria monocytogenes polypeptide. Mass spectrometry identified a known H-2K(b) -presented linear epitope (LLO296-304 ) in the digests, as well as four spliced peptides that were trimmed by ERAP into peptides with in silico predicted H-2K(b) binding affinity. These spliced peptides, which displayed sequence similarity with LLO296-304 , bound to H-2K(b) molecules in cellular assays and one of the peptides was recognized by CD8(+) T cells of infected mice. This spliced epitope differed by one amino acid from LLO296-304 and double staining with LLO296-304 - and spliced peptide-folded MHC multimers showed that LLO296-304 and its spliced variant were recognized by the same CD8(+) T cells. Thus, PCPS multiplies the variety of peptides that is processed from an antigen and leads to the production of epitope variants that can be recognized by cross-reacting pathogen-specific CD8(+) T cells. Such mechanism may reduce the chances for pathogen immune evasion.

Conjugation of cell-penetrating peptides to parathyroid hormone affects its structure, potency, and transepithelial permeation.

Delivery of therapeutic peptides and proteins by the use of cell-penetrating peptides (CPPs) as carriers has been suggested as a feasible strategy. The aim of the present study was to investigate the effect of conjugating a series of well-known CPPs to the biologically active part of parathyroid hormone, i.e., PTH(1-34), and to evaluate the effect with regard to secondary structure, potency in Saos-2 cells, immunogenicity, safety, as well as the transepithelial permeation across monolayers by using the Caco-2 cell culture model. Further, co-administration of CPP and PTH(1-34) as an alternative to covalent conjugation was compared with regard to the transepithelial permeation. CPP-conjugated PTH(1-34) fusion peptides were successfully expressed in Escherichia coli and purified from inclusion bodies. No clear correlation between the degree of secondary structure of the CPP-conjugated PTH(1-34) fusion peptides and their potency was found, albeit a general decrease in permeation was observed for both N- and C-terminally CPP-conjugated PTH(1-34) as compared to native PTH(1-34). However, attachment of CPP to the N-terminus significantly increased permeation across Caco-2 cell monolayers as compared to the corresponding C-terminally CPP-conjugated PTH(1-34). In addition, the nonaarginine sequence proved to be the only CPP capable of increasing permeation when conjugated to PTH(1-34) as compared to co-administration of CPP and PTH(1-34). This enhancement effect was, however, associated with an unacceptably low level of cell viability. In conclusion, covalent conjugation of CPPs to PTH(1-34) influenced the secondary structure, potency, and transepithelial permeation efficiency of the resulting conjugate, and hence this approach appears not to be favorable as compared to co-administration when optimizing CPP-mediated permeation of PTH(1-34) across an intestinal epithelium.

The bone marrow functions as the central site of proliferation for long-lived NK cells.

NK cells play an important role in the early defense against invading pathogens. Although it is well established that infection leads to a substantial, local increase in NK cell numbers, little is known about the mechanisms that trigger their proliferation and migration. In this study, we investigated the dynamics of NK cell responses after intranasal respiratory virus infection. We show that NK cell numbers increased in the airways after influenza virus infection but find no evidence of proliferation either at the site of infection or in the draining lymph nodes. Instead, we find that the bone marrow (BM) is the primary site of proliferation of both immature and mature NK cells during infection. Using an adoptive transfer model, we demonstrate that peripheral, long-lived and phenotypically mature NK cells migrate back to the BM and proliferate there, both homeostatically and in response to infection. Thus, the BM is not only a site of NK cell development but also an important site for proliferation of long-lived mature NK cells.

Intranasal delivery of Salmonella OMVs decorated with Chlamydia trachomatis antigens induces specific local and systemic immune responses.

Chlamydia trachomatis is an obligate intracellular pathogen responsible for the most prevalent bacterial sexually transmitted disease globally. The high prevalence of chlamydial infections underscores the urgent need for licensed and effective vaccines to prevent transmission in populations. Bacterial outer membrane vesicles (OMVs) have emerged as promising mucosal vaccine carriers due to their inherent adjuvant properties and the ability to display heterologous antigens. In this proof-of-concept study, we evaluated the immunogenicity of Salmonella OMVs decorated with C. trachomatis MOMP-derived CTH522 or HtrA antigens in mice. Following a prime-boost intranasal vaccination approach, two OMV-based C. trachomatis vaccines elicited significant humoral responses specific to the antigens in both systemic and vaginal compartments. Furthermore, we demonstrated strong antigen-specific IFN-γ and IL17a responses in splenocytes and cervical lymph node cells of vaccinated mice, indicating CD4+ Th1 and Th17 biased immune responses. Notably, the OMV-CTH522 vaccine also induced the production of spleen-derived CD8+ T cells expressing IFN-γ. In conclusion, these results highlight the potential of OMV-based C. trachomatis vaccines for successful use in future challenge studies and demonstrate the suitability of our modular OMV platform for intranasal vaccine applications.

Proteasome immunosubunits protect against the development of CD8 T cell-mediated autoimmune diseases.

Exposure of cells to inflammatory cytokines induces the expression of three proteasome immunosubunits, two of which are encoded in the MHC class II region. The induced subunits replace their constitutive homologs in newly formed "so-called" immunoproteasomes. Immunosubunit incorporation enhances the proteasome's proteolytic activity and modifies the proteasome's cleavage-site preferences, which improves the generation of many MHC class I-presented peptides and shapes the fine specificity of pathogen-specific CD8 T cell responses. In this article, we report on a second effect of immunoproteasome formation on CD8 T cell responses. We show that mice deficient for the immunosubunits ß5i/low molecular mass polypeptide (LMP7) and ß2i/multicatalytic endopeptidase complex-like-1 develop early-stage multiorgan autoimmunity following irradiation and bone marrow transplantation. Disease symptoms are caused by CD8 T cells and are transferable into immunosubunit-deficient, RAG1-deficient mice. Moreover, using the human Type 1 Diabetes Genetics Consortium MHC dataset, we identified two single nucleotide polymorphisms within the ß5i/LMP7-encoding gene sequences, which were in strong linkage disequilibrium, as independent genetic risk factors for type 1 diabetes development in humans. Strikingly, these single nucleotide polymorphisms significantly enhanced the risk conferred by HLA haplotypes that were previously shown to predispose for type 1 diabetes. These data suggested that inflammation-induced immunosubunit expression in peripheral tissues constitutes a mechanism that prevents the development of CD8 T cell-mediated autoimmune diseases.

Mouse IgG2a Isotype Therapeutic Antibodies Elicit Superior Tumor Growth Control Compared with mIgG1 or mIgE.

In the last decades, antibody-based tumor therapy has fundamentally improved the efficacy of treatment for patients with cancer. Currently, almost all tumor antigen-targeting antibodies approved for clinical application are of IgG1 Fc isotype. Similarly, the mouse homolog mIgG2a is the most commonly used in tumor mouse models. However, in mice, the efficacy of antibody-based tumor therapy is largely restricted to a prophylactic application. Direct isotype comparison studies in mice in a therapeutic setting are scarce. In this study, we assessed the efficacy of mouse tumor-targeting antibodies of different isotypes in a therapeutic setting using a highly systematic approach. To this end, we engineered and expressed antibodies of the same specificity but different isotypes, targeting the artificial tumor antigen CD90.1/Thy1.1 expressed by B16 melanoma cells. Our experiments revealed that in a therapeutic setting mIgG2a was superior to both mIgE and mIgG1 in controlling tumor growth. Furthermore, the observed mIgG2a antitumor effect was entirely Fc mediated as the protection was lost when an Fc-silenced mIgG2a isotype (LALA-PG mutations) was used. These data confirm mIgG2a superiority in a therapeutic tumor model. Significance: Direct comparisons of different antibody isotypes of the same specificity in cancer settings are still scarce. Here, it is shown that mIgG2a has a greater effect compared with mIgG1 and mIgE in controlling tumor growth in a therapeutic setting.

Characterization of polarization states of canine monocyte derived macrophages.

Macrophages can reversibly polarize into multiple functional subsets depending on their micro-environment. Identification and understanding the functionality of these subsets is relevant for the study of immune­related diseases. However, knowledge about canine macrophage polarization is still in its infancy. In this study, we polarized canine monocytes using GM-CSF/IFN- γ and LPS towards M1 macrophages or M-CSF and IL-4 towards M2 macrophages and compared them to undifferentiated monocytes (M0). Polarized M1 and M2 macrophages were thoroughly characterized for morphology, surface marker features, gene profiles and functional properties. Our results showed that canine M1-polarized macrophages obtained a characteristic large, roundish, or amoeboid shape, while M2-polarized macrophages were smaller and adopted an elongated spindle-like morphology. Phenotypically, all macrophage subsets expressed the pan-macrophage markers CD14 and CD11b. M1-polarized macrophages expressed increased levels of CD40, CD80 CD86 and MHC II, while a significant increase in the expression levels of CD206, CD209, and CD163 was observed in M2-polarized macrophages. RNAseq of the three macrophage subsets showed distinct gene expression profiles, which are closely associated with immune responsiveness, cell differentiation and phagocytosis. However, the complexity of the gene expression patterns makes it difficult to assign clear new polarization markers. Functionally, undifferentiated -monocytes, and M1- and M2- like subsets of canine macrophages can all phagocytose latex beads. M2-polarized macrophages exhibited the strongest phagocytic capacity compared to undifferentiated monocytes- and M1-polarized cells. Taken together, this study showed that canine M1 and M2-like macrophages have distinct features largely in parallel to those of well-studied species, such as human, mouse and pig. These findings enable future use of monocyte derived polarized macrophages particularly in studies of immune related diseases in dogs.

PA28 and the proteasome immunosubunits play a central and independent role in the production of MHC class I-binding peptides in vivo.

Proteasomes play a fundamental role in the processing of intracellular antigens into peptides that bind to MHC class I molecules for the presentation of CD8(+) T cells. Three IFN-γ-inducible catalytic proteasome (immuno)subunits as well as the IFN-γ-inducible proteasome activator PA28 dramatically accelerate the generation of a subset of MHC class I-presented antigenic peptides. To determine whether these IFN-γ-inducible proteasome components play a compounded role in antigen processing, we generated mice lacking both PA28 and immunosubunits ß5i/LMP7 and ß2i/MECL-1. Analyses of MHC class I cell-surface levels ex vivo demonstrated that PA28 deficiency reduced the production of MHC class I-binding peptides both in cells with and without immunosubunits, in the latter cells further decreasing an already diminished production of MHC ligands in the absence of immunoproteasomes. In contrast, the immunosubunits but not PA28 appeared to be of critical importance for the induction of CD8(+) T-cell responses to multiple dominant Influenza and Listeria-derived epitopes. Taken together, our data demonstrate that PA28 and the proteasome immunosubunits use fundamentally different mechanisms to enhance the supply of MHC class I-binding peptides; however, only the immunosubunit-imposed effects on proteolytic epitope processing appear to have substantial influence on the specificity of pathogen-specific CD8(+) T-cell responses.

Opportunities and challenges of bi-specific antibodies.

The recent clinical approval of different Bi-specific antibodies (BsAbs) has revealed the great therapeutic potential of this novel class of biologicals. For example, the bispecific T-cell engager (BiTE), Blinatumomab, demonstrated the unique capacity of BsAbs to link T-cells with tumor cells, inducing targeted tumor cell removal. Additionally, Amivantamab, recognizing the EGFR and cMet in cis, revealed a substantial improvement of therapeutic efficacy by concomitantly targeting two tumor antigens. Cis-targeting BsAbs furthermore allow discerning cell populations which concurrently express two antigens, for which each antigen expression pattern in itself might not be selective. In this way, BsAbs harbor the great prospect of being more specific and showing fewer side effects than monoclonal antibodies. Nevertheless, BsAbs have also faced major obstacles, for instance, in ensuring reliable assembly and clinical-grade purification. In this review, we summarize the different available antibody platforms currently used for the generation of IgG-like and non-IgG-like BsAbs and explain which approaches have been used to assemble those BsAbs which are currently approved for clinical application. By focusing on the example of regulatory T-cells (Tregs) and the different, ongoing approaches to develop BsAbs specifically targeting Tregs within the tumor microenvironment, our review highlights the huge potential as well as the pitfalls BsAb face in order to emerge as one of the most effective therapeutic biologicals targeting desired cell populations in a highly selective way. Such BsAb may improve treatment efficacy and reduce side effects, thereby opening novel treatment opportunities for a range of different diseases, such as cancer or autoimmune diseases.

Shortened Hinge Design of Fab x sdAb-Fc Bispecific Antibodies Enhances Redirected T-Cell Killing of Tumor Cells.

T cell engager (TCE) antibodies have emerged as promising cancer therapeutics that link cytotoxic T-cells to tumor cells by simultaneously binding to CD3E on T-cells and to a tumor-associated antigen (TAA) expressed by tumor cells. We previously reported a novel bispecific format, the IgG-like Fab x sdAb-Fc (also known as half-IG_VH-h-CH2-CH3), combining a conventional antigen-binding fragment (Fab) with a single domain antibody (sdAb). Here, we evaluated this Fab x sdAb-Fc format as a T-cell redirecting bispecific antibody (TbsAbs) by targeting mEGFR on tumor cells and mCD3E on T cells. We focused our attention specifically on the hinge design of the sdAb arm of the bispecific antibody. Our data show that a TbsAb with a shorter hinge of 23 amino acids (TbsAb.short) showed a significantly better T cell redirected tumor cell elimination than the TbsAb with a longer, classical antibody hinge of 39 amino acids (TbsAb.long). Moreover, the TbsAb.short form mediated better T cell-tumor cell aggregation and increased CD69 and CD25 expression levels on T cells more than the TbsAb.long form. Taken together, our results indicate that already minor changes in the hinge design of TbsAbs can have significant impact on the anti-tumor activity of TbsAbs and may provide a new means to improve their potency.

The Function of Immunoproteasomes-An Immunologists' Perspective.

Proteasomes are responsible for intracellular proteolysis and play an important role in cellular protein homeostasis. Cells of the immune system assemble a specialized form of proteasomes, known as immunoproteasomes, in which the constitutive catalytic sites are replaced for cytokine-inducible homologues. While immunoproteasomes may fulfill all standard proteasome' functions, they seem specially adapted for a role in MHC class I antigen processing and CD8+ T-cell activation. In this way, they may contribute to CD8+ T-cell-mediated control of intracellular infections, but also to the immunopathogenesis of autoimmune diseases. Starting at the discovery of its catalytic subunits in the genome, here, we review the observations shaping our current understanding of immunoproteasome function, and the consequential novel opportunities for immune intervention.

Generation and characterization of novel co-stimulatory anti-mouse TNFR2 antibodies.

Tumor necrosis factor receptor 2 (TNFR2) has gained much research interest in recent years because of its potential pivotal role in autoimmune disease and cancer. However, its function in regulating different immune cells is not well understood. There is a need for well-characterized reagents to selectively modulate TNFR2 function, thereby enabling definition of TNFR2-dependent biology in human and mouse surrogate models. Here, we describe the generation, production, purification, and characterization of a panel of novel antibodies targeting mouse TNFR2. The antibodies display functional differences in binding affinity and potency to block TNFα. Furthermore, epitope binding showed that the anti-mTNFR2 antibodies target different domains on the TNFR2 protein, associated with varying capacity to enhance CD8+ T-cell activation and costimulation. Moreover, the anti-TNFR2 antibodies demonstrate binding to isolated splenic mouse Tregs ex vivo and activated CD8+ cells, reinforcing their potential use to establish TNFR2-dependent immune modulation in translational models of autoimmunity and cancer.

Human milk extracellular vesicles target nodes in interconnected signalling pathways that enhance oral epithelial barrier function and dampen immune responses.

Maternal milk is nature's first functional food. It plays a crucial role in the development of the infant's gastrointestinal (GI) tract and the immune system. Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer enclosed vesicles released by cells for intercellular communication and are a component of milk. Recently, we discovered that human milk EVs contain a unique proteome compared to other milk components. Here, we show that physiological concentrations of milk EVs support epithelial barrier function by increasing cell migration via the p38 MAPK pathway. Additionally, milk EVs inhibit agonist-induced activation of endosomal Toll like receptors TLR3 and TLR9. Furthermore, milk EVs directly inhibit activation of CD4+ T cells by temporarily suppressing T cell activation without inducing tolerance. We show that milk EV proteins target key hotspots of signalling networks that can modulate cellular processes in various cell types of the GI tract.

Bispecific antibodies targeting dual tumor-associated antigens in cancer therapy.

PURPOSE: Bispecific antibodies (BsAbs) have emerged as a leading drug class for cancer therapy and are becoming increasingly of interest for therapeutic applications. As of April 2020, over 123 BsAbs are under clinical evaluation for use in oncology (including the two marketed BsAbs Blinatumomab and Catumaxomab). The majority (82 of 123) of BsAbs under clinical evaluation can be categorized as bispecific immune cell engager whereas a second less well-discussed subclass of BsAbs targets two tumor-associated antigens (TAAs). In this review, we summarize the clinical development of dual TAAs targeting BsAbs and provide an overview of critical considerations when designing dual TAA targeting BsAbs. METHODS: Herein the relevant literature and clinical trials published in English until April 1st 2020 were searched using PubMed and ClinicalTrials.gov database. BsAbs were considered to be active in clinic if their clinical trials were not terminated, withdrawn or completed before 2018 without reporting results. Data missed by searching ClinicalTrials.gov was manually curated. RESULTS: Dual TAAs targeting BsAbs offer several advantages including increased tumor selectivity, potential to concurrently modulate two functional pathways in the tumor cell and may yield improved payload delivery. CONCLUSIONS: Dual TAAs targeting BsAbs represent a valuable class of biologics and early stage clinical studies have demonstrated promising anti-tumor efficacy in both hematologic malignancies and solid tumors.

A novel efficient bispecific antibody format, combining a conventional antigen-binding fragment with a single domain antibody, avoids potential heavy-light chain mis-pairing.

Due to the technical innovations in generating bispecific antibodies (BsAbs) in recent years, BsAbs have become important reagents for diagnostic and therapeutic applications. However, the difficulty of producing a heterodimer consisting of two different arms with high yield and purity constituted a major limitation for their application in academic and clinical settings. Here, we describe a novel Fc-containing BsAb format (Fab × sdAb-Fc) composed of a conventional antigen-binding fragment (Fab), and a single domain antibody (sdAb), which avoids heavy-light chain mis-pairing during antibody assembly. In this study, the Fab x sdAb-Fc BsAbs were efficiently produced by three widely used heavy-heavy chain heterodimerization methods: Knobs-into-holes (KIH), Charge-pairs (CP) and controlled Fab-arm exchange (cFAE), respectively. The novel Fab x sdAb-Fc format provided a rapid and efficient strategy to generate BsAb with high purity and a unique possibility to further purify desired BsAbs from undesired antibodies based on molecular weight (MW). Compared to conventional BsAb formats, the advantages of Fab x sdAb-Fc format may thus provide a straightforward opportunity to apply bispecific antibody principles to research and development of novel targets and pathways in diseases such as cancer and autoimmunity.