Evaluation and selection of potent fluorescent immunosensors by combining fluorescent peptide and nanobodies displayed on yeast surface

Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules rapidly and directly. However, because Q-bodies show different antigen responses depending on the antibody used, time-consuming optimization of the Q-body structure is often necessary, and a high-throughput screening method for discriminating and selecting good Q-bodies is required. Here, we aimed to develop a molecular display method of nanobody-based “mini Q-bodies” by combining yeast surface display and coiled-coil forming E4/K4 peptide-based fluorescence labeling. As a result, the yeast-displayed mini Q-body recognizing the anti-cancer agent methotrexate (MTX) showed significant quenching and MTX-dependent dequenching on cells. To demonstrate the applicability of the developed method to select highly responsive mini Q-bodies, a small nanobody library consisting of 30 variants that recognize human serum albumin was used as a model. The best variant, showing a 2.4-fold signal increase, was obtained through selection by flow cytometry. Furthermore, the same nanobody prepared from Escherichia coli also worked as a mini Q-body after dye labeling. The described approach will be applied to quickly obtain well-behaved Q-bodies and other fluorescent biosensors for various targets through directed evolutionary approaches.

Evaluation and Selection of Potent Fluorescent Immunosensors by Combining Fluorescent Peptide and Nanobodies Displayed on Yeast Surface

Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules rapidly and directly. However, because Q-bodies show different antigen responses depending on the antibody used, time-consuming optimization of the Q-body structure is often necessary, and a high-throughput screening method for discriminating and selecting good Q-bodies is required. Here, we aimed to develop a molecular display method of nanobody-based “mini Q-bodies” by combining yeast surface display and coiled-coil forming E4/K4 peptide-based fluorescence labeling. As a result, the yeast-displayed mini Q-body recognizing the anti-cancer agent methotrexate (MTX) showed significant quenching and MTX-dependent dequenching on cells. To demonstrate the applicability of the developed method to select highly responsive mini Q-bodies, a small nanobody library consisting of 30 variants that recognize human serum albumin was used as a model. The best variant, showing a 2.4-fold signal increase, was obtained through selection by flow cytometry. Furthermore, the same nanobody prepared from Escherichia coli also worked as a mini Q-body after dye labeling. The described approach will be applied to quickly obtain well-behaved Q-bodies and other fluorescent biosensors for various targets through directed evolutionary approaches.

Immunoglobulin Glycosylation – An Unexploited Potential for Immunomodulatory Strategies in Farm Animals

The function of antibodies, namely the identification and neutralization of pathogens, is mediated by their antigen binding site (Fab). In contrast, the subsequent signal transduction for activation of the immune system is mediated by the fragment crystallizable (Fc) region, which interacts with receptors or other components of the immune system, such as the complement system. This aspect of binding and interaction is more precise, readjusted by covalently attached glycan structures close to the hinge region of immunoglobulins (Ig). This fine-tuning of Ig and its actual state of knowledge is the topic of this review. It describes the function of glycosylation at Ig in general and the associated changes due to corresponding glycan structures. We discuss the functionality of IgG glycosylation during different physiological statuses, like aging, lactation and pathophysiological processes. Further, we point out what is known to date about Ig glycosylation in farm animals and how new achievements in vaccination may contribute to improved animal welfare.

Highly active engineered IgG3 antibodies against SARS-CoV-2

Monoclonal antibodies (mAbs) that efficiently neutralize SARS-CoV-2 have been developed at an unprecedented speed. Notwithstanding, there is a vague understanding of the various Ab functions induced beyond antigen binding by the heavy-chain constant domain. To explore the diverse roles of Abs in SARS-CoV-2 immunity, we expressed a SARS-CoV-2 spike protein (SP) binding mAb (H4) in the four IgG subclasses present in human serum (IgG1-4) using glyco-engineered Nicotiana benthamiana plants. All four subclasses, carrying the identical antigen-binding site, were fully assembled in planta and exhibited a largely homogeneous xylose- and fucose-free glycosylation profile. The Ab variants ligated to the SP with an up to fivefold increased binding activity of IgG3. Furthermore, all H4 subtypes were able to neutralize SARS-CoV-2. However, H4-IgG3 exhibited an up to 50-fold superior neutralization potency compared with the other subclasses. Our data point to a strong protective effect of IgG3 Abs in SARS-CoV-2 infection and suggest that superior neutralization might be a consequence of cross-linking the SP on the viral surface. This should be considered in therapy and vaccine development. In addition, we underscore the versatile use of plants for the rapid expression of complex proteins in emergency cases.

Engineering an Antibody V Gene-Selective Vaccine

The ligand-binding surface of the B cell receptor (BCR) is formed by encoded and non-encoded antigen complementarity determining regions (CDRs). Genetically reproducible or ‘public’ antibodies can arise when the encoded CDRs play deterministic roles in antigen recognition, notably within human broadly neutralizing antibodies against HIV and influenza virus. We sought to exploit this by engineering virus-like-particle (VLP) vaccines that harbor multivalent affinity against gene-encoded moieties of the BCR antigen binding site. As proof of concept, we deployed a library of RNA bacteriophage VLPs displaying random peptides to identify a multivalent antigen that selectively triggered germline BCRs using the human VH gene IGVH1-2*02. This VLP selectively primed IGHV1-2*02 BCRs that were present within a highly diversified germline antibody repertoire within humanized mice. Our approach thus provides methodology to generate antigens that engage specific BCR configurations of interest, in the absence of structure-based information.

Insertion of atypical glycans into the tumor antigen-binding site identifies DLBCLs with distinct origin and behavior

Glycosylation of the surface immunoglobulin variable region is a remarkable follicular lymphoma-associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCL) which acquire N-glycosylation sites selectively in the immunoglobulin (Ig) complementary-determining-regions (CDR) of the antigen-binding sites. Mass-spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures due to burial in the CDR loops. Acquisition of sites occurs in ~50% of germinal center B-cell-like DLBCL, mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell-like DLBCL Ig, which rarely has sites in the CDR, and appears not to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites associate with more rapid disease progression and with significant gene-set enrichment of the B-cell receptor, PI3K/AKT/MTORC1, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin DC-SIGN, mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate-recognition-domain. Oligomannosylation of the tumor immunoglobulin is a post-translational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN-expressing M2-polarized macrophages.

Contribution of HLA Class II Genes, DRB4*01:01, DRB1*07:01, and DQB1*03:03:2 to Clinical Features of Vitiligo Disease in Iranian Population

Background: Vitiligo is a multifactorial depigmentation condition, which is due to skin melanocyte destruction. The increased expression of HLA class II genes in patients with pre-lesions of Vitiligo suggests an important role for the participation of immune response in the Vitiligo development. Recent studies progressively focused on HLA-DRB1 and DQB1 genes. In this study, we have evaluated the association and role of HLA-DRB4*01:01, -DRB1*07:01, and -DQB1*03:03:2 genes in different clinical subtypes of Vitiligo in the Iranian population.Methods: First, Genomic DNA from peripheral blood of 125 unrelated Vitiligo patients and 100 unrelated healthy controls were extracted through salting-out method. Then, HLA CLASS II genotyping were performed using sequence-specific primer PCR method. Finally, the clinical relevance of the testing for these genotypes were evaluated by applying the PcPPV (prevalence-corrected positive predictive value) formula.Results: Our results indicated the positive associations of DRB4*01:01 and DRB1*07:01 allelic genes with early-onset Vitiligo (P= 0.024 and 0.022, respectively). The DRB4*01:01 also showed a strong protection against late-onset Vitiligo (P= 0.0016, RR=0.360). Moreover, our data revealed that the DRB1*07:01 increases the susceptibility to Sporadic Vitiligo (P=0.030, RR=1.702). Furthermore, our findings proposed that elevated vulnerability of Vitiligo patients due to DRB4*01:01 and DRB1*07:01 alleles may be is correlated with the presence of amino acid Arginine at position 71 at pocket 4 on the antigen-binding site of the HLA-DRB1 receptor.Conclusion: Our findings on different subtypes of Vitiligo suggest that, despite a more apparent autoimmune involvement, a non-autoimmune nature for the etiology of Vitiligo could also be considered.

Antitumor efficacy and reduced toxicity using an anti-CD137 Probody therapeutic

Costimulation via CD137 (4-1BB) enhances antitumor immunity mediated by cytotoxic T lymphocytes. Anti-CD137 agonist antibodies elicit mild liver inflammation in mice, and the maximum tolerated dose of Urelumab, an anti-human CD137 agonist monoclonal antibody, in the clinic was defined by liver inflammation–related side effects. A protease-activated prodrug form of the anti-mouse CD137 agonist antibody 1D8 (1D8 Probody therapeutic, Pb-Tx) was constructed and found to be selectively activated in the tumor microenvironment. This construct, which encompasses a protease-cleavable linker holding in place a peptide that masks the antigen binding site, exerted antitumor effects comparable to the unmodified antibody but did not result in liver inflammation. Moreover, it efficaciously synergized with both PD-1 blockade and adoptive T-cell therapy. Surprisingly, minimal active Pb-Tx reached tumor-draining lymph nodes, and regional lymphadenectomy did not abrogate antitumor efficacy. By contrast, S1P receptor–dependent recirculation of T cells was absolutely required for efficacy. The preferential cleavage of the anti-CD137 Pb-Tx by tumor proteases offers multiple therapeutic opportunities, including neoadjuvant therapy, as shown by experiments in which the Pb-Tx is given prior to surgery to avoid spontaneous metastases.

Predominant VH1-69 IgBCR Clones Show Higher Expression of CD5 in Heterogeneous Chronic Lymphocytic Leukemia Populations

The immunoglobulin B cell receptor (IgBCR) expressed by chronic lymphocytic leukemia (CLL) B cells plays a pivotal role in tumorigenesis, supporting neoplastic transformation, survival, and expansion of tumor clones. We demonstrated that in the same patient, two or more CLL clones could coexist, recognized by the expression of different variable regions of the heavy chain of IgBCR, composing the antigen-binding site. In this regard, phage display screening could be considered the easier and most advantageous methodology for the identification of small peptide molecules able to mimic the natural antigen of the tumor IgBCRs. These molecules, properly functionalized, could be used as a probe to specifically identify and isolate single CLL subpopulations, for a deeper analysis in terms of drug resistance, phenotype, and gene expression. Furthermore, CLL cells express another surface membrane receptor, the CD5, which is commonly expressed by normal T cells. Piece of evidence supports a possible contribution of CD5 to the selection and maintenance of autoreactivity in B cells and the constitutive expression of CD5 on CLL cells could induce pro-survival stimuli. In this brief research report, we describe a peptide-based single-cell sorting using as bait the IgBCR of tumor cells; in the next step, we performed a quantitative analysis of CD5 expression by qRT-PCR related to the expressed IgBCR. Our approach could open a new perspective for the identification, isolation, and investigation of all subsets of IgBCR-related CLL clones, with particular attention to the more aggressive clones.

Introduction on Monoclonal Antibodies

Monoclonal antibodies (mAbs) are a group of antibodies produced by identical clones of B lymphocytes against a particular antigen. mAbs are identical in several properties such as protein sequence, antigen-binding site region, binding affinity for their targets, and identical downstream functional effects. These characteristics of mAbs highlight their differences with the polyclonal antibodies which have heterogenous activities and recognize different epitopes on an antigen. Murine mAbs was the first generation of mAbs developed by hybridoma technology however, because of their murine origin, they can trigger the anti-mouse antibody response in the host which could accelerate mAb clearance and undesirable allergic reactions upon repeated administration. This issue was resolved by developing engineering methods toward producing less immunologic chimeric or humanized antibodies. mAbs applications have become a novel way of targeting antigens in a wide variety of diseases such as autoimmunity, malignancies, and asthma. In addition, high specificity and high affinity binding properties of mAbs make them effective biological reagents in immunodiagnostic assays. They can be used in diagnosis of infectious diseases and detection of certain antigens or in serological assessments for detection of antibodies against a certain antigen. This chapter summarizes the general properties of mAbs, their production processes, and their important diagnostic and therapeutic applications.