Therapeutic bioconjugates are emerging as an essential tool to combat human disease. Site-specific conjugation technologies are widely recognized as the optimal approach for producing homogeneous drug products. Non-natural amino acid (nnAA) incorporation allows the introduction of bioconjugation handles at genetically defined locations. Escherichia coli (E. coli) is a facile host for therapeutic nnAA protein synthesis because it can stably replicate plasmids encoding genes for product and nnAA incorporation. Here, we demonstrate that by engineering E. coli to incorporate high levels of nnAAs, it is feasible to produce nnAA-containing antibody fragments and full-length immunoglobulin Gs (IgGs) in the cytoplasm of E. coli. Using high-density fermentation, it was possible to produce both of these types of molecules with site-specifically incorporated nnAAs at titers > 1 g/L. We anticipate this strategy will help simplify the production and manufacture of promising antibody therapeutics.

We describe the 'Crescendo Mouse', a human VH transgenic platform combining an engineered heavy chain locus with diverse human heavy chain V, D and J genes, a modified mouse Cγ1 gene and complete 3' regulatory region, in a triple knock-out (TKO) mouse background devoid of endogenous immunoglobulin expression. The addition of the engineered heavy chain locus to the TKO mouse restored B cell development, giving rise to functional B cells that responded to immunization with a diverse response that comprised entirely 'heavy chain only' antibodies. Heavy chain variable (VH) domain libraries were rapidly mined using phage display technology, yielding diverse high-affinity human VH that had undergone somatic hypermutation, lacked aggregation and showed enhanced expression in E. coli. The Crescendo Mouse produces human VH fragments, or Humabody® VH, with excellent bio-therapeutic potential, as exemplified here by the generation of antagonistic Humabody® VH specific for human IL17A and IL17RA.

About 15% of immunoglobulin G (IgG) molecules contain glycans linked to the antigen-binding fragments (Fab arms) in addition to the glycans linked to the crystallizable fragment (Fc tail) of all IgGs. Fab glycosylation appears to be an important feature of antibodies, for example by influencing antigen binding and antibody stability. The reliable generation of antibodies that either have or lack Fab glycans would be very helpful to study the role of Fab glycans in more detail. In this study, we set out to remove Fab glycans by treating polyclonal and monoclonal human IgG antibodies with two commonly used glycosidases and an improved version of one of the two (Endo F3, PNGase F, and Rapid™ PNGase F). Fc glycans can be removed using PNGase F and Rapid™ PNGase F, but not with Endo F3. For most antibody clones, Endo F3 partially cleaved off the Fab glycans. In contrast, PNGase F left the Fab glycans of most clones unaffected, but could remove glycans of some clones. Rapid™ PNGase F showed a higher glycosidase efficacy than PNGase F, and more clones could be deglycosylated using this enzyme. In summary, not all Fab glycans can be cleaved off by the tested glycosidases (under non-denaturing conditions), suggesting that Fab glycans are exposed to different degrees.

Interactions with cell surface receptors enhance the therapeutic properties of many important antibodies, including the low-affinity Fc γ Receptors (FcγRs). These interactions require proper processing of the immunoglobulin G Fc N-glycan, and eliminating the N-glycan abolishes binding, restricting antibody production to mammalian expression platforms. Yeasts, for example, generate extensively mannosylated N-glycans that are unsuitable for therapeutics. However, Fc with a specifically truncated N-glycan still engages receptors with considerable affinity. Here we describe the creation and applications of a novel Saccharomyces cerevisiae strain that specifically modifies the IgG1 Fc domain with an N-glycan consisting of a single N-acetylglucosamine residue. This strain displayed glycoengineered Fc on its surface for screening yeast surface display libraries and also served as an alternative platform to produce glycoengineered Rituximab. An IgG-specific endoglycosidase (EndoS2) truncates the IgG1 Fc N-glycan. EndoS2 was targeted to the yeast ER using the signal peptide from the yeast protein disulfide isomerase (PDI) and a yeast ER retention signal (HDEL). Furthermore, >99% of the yeast expressed Rituximab displayed the truncated glycoform as determined by SDS-PAGE and ESI-MS analyses. Lastly, the yeast expressed Rituximab engaged the FcγRIIIa with the expected affinity (KD = 2.0 ± 0.5 μM) and bound CD20 on Raji B cells.

Chronic idiopathic neutropenia (CIN) is a granulocytic disorder associated with presence of activated, myelosuppressive T-lymphocytes. In the present study we have evaluated constituents of humoral immunity in CIN patients (n=48) compared to healthy controls (n=52). CIN patients displayed lower serum IgG levels due to a reduction in IgG1, IgG3, IgG4 but not IgG2, lower IgA and increased IgM levels compared to controls. The proportion of CD19+ cells did not differ between patients and controls; however the proportion of the naïve IgD+/CD27- B-cells was increased and the proportion of class-switched memory IgD-/CD27+ B-cells was decreased in the patients. The percentage of CD40+ B-cells did not differ between patients and controls and no aberrations in the CD40-meadiated signal transduction pathway or in CD40-gene polymorphisms were identified. These data provide further evidence that immune disturbances are associated with the pathophysiology of CIN and point out for the first time the implication of the B-cell system.

Applications of the CRISPR-Cas9 system to edit the genome have widely expanded to include DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. Here we show the application of CRISPR-Cas9 technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas9 and guide-RNA (gRNA) with retro- or lenti-virus to IgM(+) mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. Similarly, we induce CSR in all human B cell lines tested with high efficiency to targeted IgH subclass. Finally, we engineer mouse hybridomas to secrete Fab' fragments instead of the whole Ig. Our results indicate that Ig genes in mouse and human cells can be edited to obtain any desired IgH switching helpful to study the biology of normal and lymphoma B cells. We also propose applications that could transform the technology of antibody production.

Plasmodium falciparum causes the most severe malaria in humans. Immunoglobulin M (IgM) serves as the first line of humoral defense against infection and potently activates the complement pathway to facilitate P. falciparum clearance. A number of P. falciparum proteins bind IgM, leading to immune evasion and severe disease. However, the underlying molecular mechanisms remain unknown. Here, using high-resolution cryo-electron microscopy, we delineate how P. falciparum proteins VAR2CSA, TM284VAR1, DBLMSP, and DBLMSP2 target IgM. Each protein binds IgM in a different manner, and together they present a variety of Duffy-binding-like domain-IgM interaction modes. We further show that these proteins interfere directly with IgM-mediated complement activation in vitro, with VAR2CSA exhibiting the most potent inhibitory effect. These results underscore the importance of IgM for human adaptation of P. falciparum and provide critical insights into its immune evasion mechanism.

DNA double-strand breaks (DSBs) introduced in the switch (S) regions are intermediates during immunoglobulin class switch recombination (CSR). These breaks are subsequently recognized, processed, and joined, leading to recombination of the two S regions. Nonhomologous end-joining (NHEJ) is believed to be the principle mechanism involved in DSB repair during CSR. One important component in NHEJ, Artemis, has however been considered to be dispensable for efficient CSR. In this study, we have characterized the S recombinational junctions from Artemis-deficient human B cells. Smu-Salpha junctions could be amplified from all patients tested and were characterized by a complete lack of "direct" end-joining and a remarkable shift in the use of an alternative, microhomology-based end-joining pathway. Smu-Sgamma junctions could only be amplified from one patient who carries "hypomorphic" mutations. Although these Smu-Sgamma junctions appear to be normal, a significant increase of an unusual type of sequential switching from immunoglobulin (Ig)M, through one IgG subclass, to a different IgG subclass was observed, and the Sgamma-Sgamma junctions showed long microhomologies. Thus, when the function of Artemis is impaired, varying modes of CSR junction resolution may be used for different S regions. Our findings strongly link Artemis to the predominant NHEJ pathway during CSR.

Phage display technology in combination with next-generation sequencing (NGS) currently is a state-of-the-art method for the enrichment and isolation of monoclonal antibodies from diverse libraries. However, the current NGS methods employed for sequencing phage display libraries are limited by the short contiguous read lengths associated with second-generation sequencing platforms. Consequently, the identification of antibody sequences has conventionally been restricted to individual antibody domains or to the analysis of single domain binding moieties such as camelid VHH or cartilaginous fish IgNAR antibodies. In this study, we report the application of third-generation sequencing to address this limitation. We used single molecule real time (SMRT) sequencing coupled with hairpin adaptor loop ligation to facilitate the accurate interrogation of full-length single-chain Fv (scFv) libraries. Our method facilitated the rapid isolation and testing of scFv antibodies enriched from phage display libraries within days following panning. Two libraries against CD160 and CD123 were panned and monitored by NGS. Analysis of NGS antibody data sets led to the isolation of several functional scFv antibodies that were not identified by conventional panning and screening strategies. Our approach, which combines phage display selection of immune libraries with the full-length interrogation of scFv fragments, is an easy method to discover functional antibodies, with a range of affinities and biophysical characteristics.

Aggregation of the multifunctional RNA-binding protein TDP-43 defines large subgroups of amyotrophic lateral sclerosis and frontotemporal dementia and correlates with neurodegeneration in both diseases. In disease, characteristic C-terminal fragments of ~25 kDa ("TDP-25") accumulate in cytoplasmic inclusions. Here, we analyze gain-of-function mechanisms of TDP-25 combining cryo-electron tomography, proteomics, and functional assays. In neurons, cytoplasmic TDP-25 inclusions are amorphous, and photobleaching experiments reveal gel-like biophysical properties that are less dynamic than nuclear TDP-43. Compared with full-length TDP-43, the TDP-25 interactome is depleted of low-complexity domain proteins. TDP-25 inclusions are enriched in 26S proteasomes adopting exclusively substrate-processing conformations, suggesting that inclusions sequester proteasomes, which are largely stalled and no longer undergo the cyclic conformational changes required for proteolytic activity. Reporter assays confirm that TDP-25 impairs proteostasis, and this inhibitory function is enhanced by ALS-causing TDP-43 mutations. These findings support a patho-physiological relevance of proteasome dysfunction in ALS/FTD.

Human immunoglobulin G (IgG) molecules, IgG1, IgG2 and IgG3, exhibit substantial inter-individual variation in their constant heavy chain regions, as discovered by serological methods. This polymorphism is encoded by the IGHG1, IGHG2, and IGHG3 genes and may influence antibody function. We sequenced the coding fragments of these genes in 95 European Americans, 94 African Americans, and 94 Black South Africans. Striking differences were observed between the population groups, including extremely low amino acid sequence variation in IGHG1 among South Africans, and higher IGHG2 and IGHG3 diversity in individuals of African descent compared to individuals of European descent. Molecular definition of the loci illustrates a greater level of allelic polymorphism than previously described, including the presence of common IGHG2 and IGHG3 variants that were indistinguishable serologically. Comparison of our data with the 1000 Genome Project sequences indicates overall agreement between the datasets, although some inaccuracies in the 1000 Genomes Project are likely. These data represent the most comprehensive analysis of IGHG polymorphisms across major populations, which can now be applied to deciphering their functional impact.

Single-chain variable fragments (scFvs), composed of variable domains of heavy and light chains of an antibody joined by a linker, share antigen binding capacity with their parental antibody. Due to intrinsically low solubility and stability, only two Escherichia coli-produced scFvs have been approved for therapy. Here we report that a 33-residue peptide, termed P17 tag, increases the solubility of multiple scFvs produced in Escherichia coli SHuffle strain by up to 11.6 fold. Hydrophilic sequence, especially charged residues, but not the predicted α-helical secondary structure of P17 tag, contribute to the solubility enhancement. Notably, the P17 tag elevates the thermostability of scFv as efficiently as intra-domain disulfide bonds. Moreover, a P17-tagged scFv targeting hepatitis B virus surface proteins shows over two-fold higher antigen-binding affinity and virus-neutralizing activity than the untagged version. These data strongly suggest a type I intramolecular chaperone-like activity of the P17 tag. Hence, the P17 tag could benefit the research, production, and application of scFv.

The bacterium Clostridium botulinum is the causative agent of botulism-a severe intoxication caused by botulinum neurotoxin (BoNT) and characterized by damage to the nervous system. In an effort to develop novel C. botulinum immunotherapeutics, camelid single-domain antibodies (sdAbs, VHHs, or nanobodies) could be used due to their unique structure and characteristics. In this study, VHHs were produced using phage display technology. A total of 15 different monoclonal VHHs were selected based on their comlementarity-determining region 3 (CDR3) sequences. Different toxin lethal dose (LD50) challenges with each selected phage clone were conducted in vivo to check their neutralizing potency. We demonstrated that modification of neutralizing VHHs with a human immunoglobulin G (IgG)1 Fc (fragment crystallizable) fragment (fusionbody, VHH-Fc) significantly increased the circulation time in the blood (up to 14 days). At the same time, VHH-Fc showed the protective activity 1000 times higher than monomeric form when challenged with 5 LD50. Moreover, VHH-Fcs remained protective even 14 days after antibody administration. These results indicate that this VHH-Fc could be used as an effective long term antitoxin protection against botulinum type A.

The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro,the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)2. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.

West Nile virus (WNV) is prevalent in Africa, Europe, the Middle East, West Asia, and North America, and causes epidemic encephalitis. To date, no effective therapy for WNV infection has been developed; therefore, there is urgent need to find an efficient method to prevent WNV disease. In this study, we prepared and evaluated the protective efficacy of immune serum IgG and pepsin-digested F(ab')₂ fragments from horses immunized with the WNV virus-like particles (VLP) expressing the WNV M and E proteins. Immune equine F(ab')₂ fragments and immune horse sera efficiently neutralized WNV infection in tissue culture. The passive transfer of equine immune antibodies significantly accelerated the virus clearance in the spleens and brains of WNV infected mice, and reduced mortality. Thus, equine immunoglobulin or equine neutralizing F(ab')₂ passive immunotherapy is a potential strategy for the prophylactic or therapeutic treatment of patients infected with WNV.

In vivo phage display selection is a powerful strategy for directly identifying agents that target the vasculature of normal or diseased tissues in living animals. We describe here a new in vivo biopanning strategy in which a human phage single-chain antibody (scFv) library was injected into high-fat diet-fed ApoE(-/-) mice. Extracellular and internalized phage scFvs were selectively recovered from atherosclerotic vascular endothelium and subjacent tissues. After three successive biopanning rounds, a panel of six clones with distinct gene sequences was isolated. Four scFvs produced and purified in soluble form were shown to interact in vitro with a rabbit atheromatous protein extract by time-resolved fluorescence resonance energy transfer and to target the endothelial cell surface and inflamed intima-related regions of rabbit and human tissue sections ex vivo. These new scFvs selected in a mouse model recognized both rabbit and human tissue, underlying the interspecies similarities of the recognized epitopes. By combining immunoprecipitation and mass spectrometry, one of the selected scFvs was shown to recognize carbonic anhydrase II, an up-regulated enzyme involved in resorption of ectopic calcification. These results show that in vivo biopanning selection in hypercholesterolemic animals makes it possible to identify both scFvs homing to atherosclerotic endothelial and subendothelial tissues, and lesion-associated biomarkers. Such scFvs offer promising opportunities in the field of molecular targeting for the treatment of atherosclerosis.

COVID-19, which is caused by the emerging human coronavirus SARS-CoV-2, has become a global pandemic that poses a serious threat to human health. To date, no vaccines or specific antiviral drugs have been approved for the treatment of this disease in clinic. Herein, therapeutic antibodies for SARS-CoV-2 were obtained from hyperimmune equine plasma. First, a recombinant SARS-CoV-2 spike protein receptor-binding domain (RBD) was obtained in gram-level quantities through high-cell density fermentation of Chinese hamster ovary cells. Then, the binding of the RBD to the SARS-CoV-2 receptor, human angiotensin-converting enzyme 2, was verified by several biochemical methods. The efficacy of the RBD in triggering antibody response in vivo was subsequently tested in both mice and equines, and the results showed that the RBD triggered high-titer neutralizing antibody production in vivo. Immunoglobulin F(ab')2 fragments were prepared from equine antisera via removal of the Fc region from the immunoglobulins. Finally, a neutralization test with live virus demonstrated that RBD-specific F(ab')2 inhibited SARS-CoV-2 with an EC50 of 0.07 μg/ml and an EC80 of 0.18 μg/ml, showing a potent inhibitory effect on SARS-CoV-2. These results highlight RBD-specific equine immunoglobulin F(ab')2 fragment as a candidate for the treatment of SARS-CoV-2.

A small albumin-binding domain (ABD) of 46 amino acids derived from streptococcal protein G was employed for the directed attachment of recombinant immunoglobulin (Ig) fragments to microtitre plates that had been coated with human serum albumin (HSA). Generic vectors were constructed in order to produce the Fv or Fab fragments fused with the ABD in Escherichia coli. Using the anti-lysozyme antibody D1.3 as the capture antibody fragment it was possible to quantify the non-radioactively labelled antigen with high sensitivity in a sandwich ELISA. The new strategy avoids denaturation or an unfavourable orientation of the Ig fragment, which can occur during direct adsorption to the microtitre plate. The HSA that serves to complex the ABD ensures efficient saturation of reactive binding sites on the plastic surface as well so that no additional blocking steps are necessary and the assay can be quickly performed.

Heat shock protein 60 (HSP60) is an important autoantigen in atherosclerosis. The genetic structures and pathogenic roles of anti-HSP60 autoantibodies, however, have not been well elucidated. Here, we cloned nine monoclonal IgG Fabs against human HSP60 from peripheral blood lymphocytes of atherosclerosis patients. Analysis of the variable region sequences revealed that the antibodies used diverse members of V(H) gene families with different D(H) and J(H) segments. However, in V(L), KV3-20 gene family member along with KJ1 segment was used often. Similarities between the rearranged genes and the closest germline sequences were low. The sequences of V(H) were highly mutated and V(H)-CDR3 varied greatly in length and sequences. The ratios of R/S (replacement mutation to silent mutation) were remarkably high in CDRs in all V(H) regions except one clone. Furthermore, mutations to positively charged amino acids were frequent in all V(H) and most V(L). These results suggest that the occurrence of somatic hypermutation and antigenic selection is critical, not the usage of certain V(H) gene family members or segments, in producing affinity-matured anti-HSP60 autoantibodies in atherosclerosis. However, expression of the combined germline genes of KV3-20 with KJ1 might be important for the selection by HSP60 at the early stage of B cell development. Two of these anti-HSP60 Fabs inhibited the binding and uptake of human HSP60 by murine macrophage cells. One of them also reduced the release of the pro-inflammatory mediators and inhibited the activation of NF-κB in HSP60-stimulated macrophages. To elucidate the functional roles of anti-HSP60 autoantibodies in atherosclerosis and the potential use of these Fabs to treat atherosclerosis, further investigation is worthy to be performed.

For the expression of human intact IgG antibodies, we have constructed a set of baculovirus expression vectors designed to facilitate rapid insertion of heavy and light chain genes of Fab or scFv antibodies derived from phage display antibody libraries. By linking them to human constant or Fc regions, expression of complete human immunoglobulin molecules was achieved in insect cells by infection with recombinant baculovirus. The IgG expression cassette vectors are based on the backbone vector which contains two back to back polyhedron and p10 promoters. The IgG expression cassette elements, including the authentic IgG lambda or kappa and heavy chain signal sequences, as well as light chain (lambda or kappa) and heavy chain constant region genes are combined in a single vector and are controlled by the p10 and polyhedron promoter respectively. Either of VL or Fab-L and VH or Fab-Fd genes from common phage display systems can be directly inserted into one of the cassette vectors through in-frame cloning sites. This design of a single cassette vector combining heavy and light chain expression elements allowed rapid production and secretion of correctly processed and assembled intact immunoglobulins from recombinant baculovirus infected insect cells. The recombinant antibodies showed the expected molecular size of the H2L2 heterodimer in non reducing SDS-PAGE. No apparent differences were found between the expression level of heavy and light chains, and antigen binding function was preserved. For various antibodies, yields between 6 and 18 mg/l IgG were obtained.