Engineering and physicochemical characterization of a novel, stable, symmetric bispecific antibody with dual target-binding using a common light chain.

Bispecific antibodies (BsAbs) have emerged as a major class of antibody therapeutics owing to their substantial potential in disease treatment. While several BsAbs have been successfully approved in recent years, ongoing development efforts continue to focus on optimizing various BsAbs tailored to particular antigens and action mechanisms, aiming to achieve favorable physicochemical properties. BsAbs generally encounter challenges due to their unfavorable physicochemical characteristics and poor manufacturing efficiencies, highlighting the need for optimization to achieve reliable productivity and developability. Herein, we describe the development of a novel symmetric BsAb, REGULGENT™ (N-term/C-term), comprising two Fab domains, using a common light chain. The heavy chain fragment encoded two antigen-binding determinants in one chain. The design and production of REGULGENT™ (N-term/C-term) are simple owing to the use of the same light chain, which does not induce heavy and light chain mispairing, frequently observed with the asymmetric BsAb format. REGULGENT™ (N-term/C-term) exhibited high expression and low aggregation characteristics during cell culture and stress treatment under low pH conditions. Differential scanning calorimetric data indicated that REGULGENT™ molecules had high conformational stability, similar to that of stabilized monoclonal antibodies. Surface plasmon resonance data showed that REGULGENT™ (N-term/C-term) could bind to two antigens simultaneously and exhibited a high affinity for two antigens. In summary, the symmetric BsAb format of REGULGENT™ confers its desirable IgG-like physicochemical properties, thus making it an excellent candidate for commercial development. The findings demonstrate a novel BsAb with substantial development potential for clinical applications.

Engineering of pH-dependent antigen binding properties for toxin-targeting IgG1 antibodies using light-chain shuffling.

Immunoglobulin G (IgG) antibodies that bind their cognate antigen in a pH-dependent manner (acid-switched antibodies) can release their bound antigen for degradation in the acidic environment of endosomes, while the IgGs are rescued by the neonatal Fc receptor (FcRn). Thus, such IgGs can neutralize multiple antigens over time and therefore be used at lower doses than their non-pH-responsive counterparts. Here, we show that light-chain shuffling combined with phage display technology can be used to discover IgG1 antibodies with increased pH-dependent antigen binding properties, using the snake venom toxins, myotoxin II and α-cobratoxin, as examples. We reveal differences in how the selected IgG1s engage their antigens and human FcRn and show how these differences translate into distinct cellular handling properties related to their pH-dependent antigen binding phenotypes and Fc-engineering for improved FcRn binding. Our study showcases the complexity of engineering pH-dependent antigen binding IgG1s and demonstrates the effects on cellular antibody-antigen recycling.

[Cytoplasmic light-chain immunofluorescence combined with FISH in bone marrow smears to detect cytogenetic abnormalities in multiple myeloma].

Objective: To analyze the sensitivity of cytoplasmic light-chain immunofluorescence with fluorescence in situ hybridization in bone marrow smears (new FISH) for detecting cytogenetic abnormalities in multiple myeloma (MM) . Methods: 42 MM patients admitted to the First Affiliated Hospital of Nanjing Medical University from April 2022 to October 2023 were enrolled. The patients with MM were detected by new FISH and CD138 immunomagnetic bead sorting technology combined with FISH (MACS-FISH) or cytoplasmic immunoglobulin FISH (cIg-FISH) to analyze cytogenetic detection results using combination probes which included 1q21/1p32, p53, IgH, IgH/FGFR3 [t (4;14) ], and IgH/MAF [t (14;16) ]. Results: In 23 patients with MM, the abnormality detection rates of cIg-FISH and new FISH were 95.7% and 100.0%, respectively (P>0.05). The detection rates of 1q21+, 1p32-, p53 deletion, and IgH abnormalities by cIg-FISH and new FISH were consistent, which were 52.2%, 8.7%, 17.4%, and 65.2%, respectively. The results of the two methods further performed with t (4;14) and t (14;16) in patients with IgH abnormalities were identical. The positive rate of t (4;14) was 26.7%, whereas t (14;16) was not detected. In 19 patients with MM, the abnormality detection rates of MACS-FISH and new FISH were 73.7% and 63.2%, respectively (P>0.05). The positivity rate of 1q21+, 1p32- and IgH abnormalities detected by MACS-FISH were slightly higher than those detected by new FISH; however, the differences were not statistically significant (all P values >0.05) . Conclusion: The new FISH method has a higher detection rate of cytogenetic abnormalities in patients with MM and has good consistency with MACS-FISH and cIg-FISH.

Antibodies utilizing VL6-57 light chains target a convergent cryptic epitope on SARS-CoV-2 spike protein and potentially drive the genesis of Omicron variants.

Continued evolution of SARS-CoV-2 generates variants to challenge antibody immunity established by infection and vaccination. A connection between population immunity and genesis of virus variants has long been suggested but its molecular basis remains poorly understood. Here, we identify a class of SARS-CoV-2 neutralizing public antibodies defined by their shared usage of VL6-57 light chains. Although heavy chains of diverse genotypes are utilized, convergent HCDR3 rearrangements have been observed among these public antibodies to cooperate with germline VL6-57 LCDRs to target a convergent epitope defined by RBD residues S371-S373-S375. Antibody repertoire analysis identifies that this class of VL6-57 antibodies is present in SARS-CoV-2-naive individuals and is clonally expanded in most COVID-19 patients. We confirm that Omicron-specific substitutions at S371, S373 and S375 mediate escape of antibodies of the VL6-57 class. These findings support that this class of public antibodies constitutes a potential immune pressure promoting the introduction of S371L/F-S373P-S375F in Omicron variants. The results provide further molecular evidence to support that antigenic evolution of SARS-CoV-2 is driven by antibody mediated population immunity.

UPF1 plays critical roles in early B cell development.

The ATP-dependent RNA helicase UPF1 plays a crucial role in various mRNA degradation pathways, most importantly in nonsense-mediated mRNA decay (NMD). Here, we show that UPF1 is upregulated during the early stages of B cell development and is important for early B cell development in the bone marrow. B-cell-specific Upf1 deletion in mice severely impedes the early to late LPre-B cell transition, in which VH-DHJH recombination occurs at the Igh gene. Furthermore, UPF1 is indispensable for VH-DHJH recombination, without affecting DH-JH recombination. Intriguingly, the genetic pre-arrangement of the Igh gene rescues the differentiation defect in early LPre-B cells under Upf1 deficient conditions. However, differentiation is blocked again following Ig light chain recombination, leading to a failure in development into immature B cells. Notably, UPF1 interacts with and regulates the expression of genes involved in immune responses, cell cycle control, NMD, and the unfolded protein response in B cells. Collectively, our findings underscore the critical roles of UPF1 during the early LPre-B cell stage and beyond, thus orchestrating B cell development.

The impact of exchanging the light and heavy chains on the structures of bovine ultralong antibodies.

The third complementary-determining regions of the heavy-chain (CDR3H) variable regions (VH) of some cattle antibodies are highly extended, consisting of 48 or more residues. These `ultralong' CDR3Hs form ß-ribbon stalks that protrude from the surface of the antibody with a disulfide cross-linked knob region at their apex that dominates antigen interactions over the other CDR loops. The structure of the Fab fragment of a naturally paired bovine ultralong antibody (D08), identified by single B-cell sequencing, has been determined to 1.6 Šresolution. By swapping the D08 native light chain with that of an unrelated antigen-unknown ultralong antibody, it is shown that interactions between the CDR3s of the variable domains potentially affect the fine positioning of the ultralong CDR3H; however, comparison with other crystallographic structures shows that crystalline packing is also a major contributor. It is concluded that, on balance, the exact positioning of ultralong CDR3H loops is most likely to be due to the constraints of crystal packing.

Light chain mutations contribute to defining the fibril morphology in systemic AL amyloidosis.

Systemic AL amyloidosis is one of the most frequently diagnosed forms of systemic amyloidosis. It arises from mutational changes in immunoglobulin light chains. To explore whether these mutations may affect the structure of the formed fibrils, we determine and compare the fibril structures from several patients with cardiac AL amyloidosis. All patients are affected by light chains that contain an IGLV3-19 gene segment, and the deposited fibrils differ by the mutations within this common germ line background. Using cryo-electron microscopy, we here find different fibril structures in each patient. These data establish that the mutations of amyloidogenic light chains contribute to defining the fibril architecture and hence the structure of the pathogenic agent.

Cu(II) binding to the λ6aJL2-R24G antibody light chain protein associated with light chain amyloidosis disease: The role of histidines.

Light chain amyloidosis is a conformational disease caused by the abnormal proliferation and deposition of antibody light chains as amyloid fibers in organs and tissues. The effect of Cu(II) binding to the model recombinant protein 6aJL2-R24G was previously characterized in our group, and we found an acceleration of the aggregation kinetics of the protein. In this study, in order to confirm the Cu(II) binding sites, histidine variants of 6aJL2-R24G were prepared and the effects of their interaction with Cu(II) were analyzed by circular dichroism, fluorescence spectroscopy, isothermal calorimetry titrations, and molecular dynamics simulations. Confirming our earlier work, we found that His8 and His99 are the highest affinity Cu(II) binding sites, and that Cu(II) binding to both sites is a cooperative event.

Immunoglobulin repertoire sequencing and de novo sequencing - Powerful tools for identifying free light chains from patients with light chain cast nephropathy.

In patients with light chain cast nephropathy (LCCN), abundantly produced monoclonal immunoglobulin free light chains (FLCs) play a vital role in pathogenesis. Determining the precise sequences of patient-derived FLCs is therefore highly desirable. Although immunoglobulin repertoire sequencing (5' RACE-seq) has been proven to be sensitive enough to provide full-length V(D)J region (variable, diversity and joining genes) of FLCs using bone marrow samples, an invasive and bone marrow independent method is still in demand. Here a de novo sequencing workflow based on the bottom-up proteomics for patient-derived FLCs was established. PEAKS software was used for the de novo sequencing of peptides that were further assembled into full-length FLC sequences. This de novo protein sequencing method can obtain the full-length amino acid sequences of FLCs, and had been shown to be as reliable as 5' RACE-seq. The two LCCN sequences derived from above the two methods were identical, and they possessed more hydrophobic or nonpolar amino acids compared with the corresponding germline, which may be associated with the pathogenesis.

Enzymatization of mouse monoclonal antibodies to the corresponding catalytic antibodies.

Catalytic antibodies possess a dual function that enables both antigen recognition and degradation. However, their time-consuming preparation is a significant drawback. This study developed a new method for quickly converting mice monoclonal antibodies into catalytic antibodies using site-directed mutagenesis. Three mice type monoclonal antibodies targeting hemagglutinin molecule of influenza A virus could be transformed into the catalytic antibodies by deleting Pro95 in CDR-3 of the light chain. No catalytic activity was observed for monoclonal antibodies and light chains. In contrast, the Pro95-deleted light chains exhibited a catalytic activity to cleave the antigenic peptide including the portion of conserved region of hemagglutinin molecule. The affinity of the Pro95-deleted light chains to the antigen increased approximately 100-fold compared to the wild-type light chains. In the mutants, three residues (Asp1, Ser92, and His93) come closer to the appropriate position to create the catalytic site and contributing to the enhancement of both catalytic function and immunoreactivity. Notably, the Pro95-deleted catalytic light chains could suppress influenza virus infection in vitro assay, whereas the parent antibody and the light chain did not. This strategy offers a rapid and efficient way to create catalytic antibodies from existing antibodies, accelerating the development for various applications in diagnostic and therapeutic applications.

Inferring B Cell Phylogenies from Paired H and L Chain BCR Sequences with Dowser.

Abs are vital to human immune responses and are composed of genetically variable H and L chains. These structures are initially expressed as BCRs. BCR diversity is shaped through somatic hypermutation and selection during immune responses. This evolutionary process produces B cell clones, cells that descend from a common ancestor but differ by mutations. Phylogenetic trees inferred from BCR sequences can reconstruct the history of mutations within a clone. Until recently, BCR sequencing technologies separated H and L chains, but advancements in single-cell sequencing now pair H and L chains from individual cells. However, it is unclear how these separate genes should be combined to infer B cell phylogenies. In this study, we investigated strategies for using paired H and L chain sequences to build phylogenetic trees. We found that incorporating L chains significantly improved tree accuracy and reproducibility across all methods tested. This improvement was greater than the difference between tree-building methods and persisted even when mixing bulk and single-cell sequencing data. However, we also found that many phylogenetic methods estimated significantly biased branch lengths when some L chains were missing, such as when mixing single-cell and bulk BCR data. This bias was eliminated using maximum likelihood methods with separate branch lengths for H and L chain gene partitions. Thus, we recommend using maximum likelihood methods with separate H and L chain partitions, especially when mixing data types. We implemented these methods in the R package Dowser: https://dowser.readthedocs.io.

Chickens with a Truncated Light Chain Transgene Express Single-Domain H Chain-Only Antibodies.

H chain-only Igs are naturally produced in camelids and sharks. Because these Abs lack the L chain, the Ag-binding domain is half the size of a traditional Ab, allowing this type of Ig to bind to targets in novel ways. Consequently, the H chain-only single-domain Ab (sdAb) structure has the potential to increase the repertoire and functional range of an active humoral immune system. The majority of vertebrates use the standard heterodimeric (both H and L chains) structure and do not produce sdAb format Igs. To investigate if other animals are able to support sdAb development and function, transgenic chickens (Gallus gallus) were designed to produce H chain-only Abs by omitting the L chain V region and maintaining only the LC region to serve as a chaperone for Ab secretion from the cell. These birds produced 30-50% normal B cell populations within PBMCs and readily expressed chicken sequence sdAbs. Interestingly, the H chains contained a spontaneous CH1 deletion. Although no isotype switching to IgY or IgA occurred, the IgM repertoire was diverse, and immunization with a variety of protein immunogens rapidly produced high and specific serum titers. mAbs of high affinity were efficiently recovered by single B cell screening. In in vitro functional assays, the sdAbs produced by birds immunized against SARS-CoV-2 were also able to strongly neutralize and prevent viral replication. These data suggest that the truncated L chain design successfully supported sdAb development and expression in chickens.

Loss of TET2 increases B-1 cell number and IgM production while limiting CDR3 diversity.

Recent studies have demonstrated a role for Ten-Eleven Translocation-2 (TET2), an epigenetic modulator, in regulating germinal center formation and plasma cell differentiation in B-2 cells, yet the role of TET2 in regulating B-1 cells is largely unknown. Here, B-1 cell subset numbers, IgM production, and gene expression were analyzed in mice with global knockout of TET2 compared to wildtype (WT) controls. Results revealed that TET2-KO mice had elevated numbers of B-1a and B-1b cells in their primary niche, the peritoneal cavity, as well as in the bone marrow (B-1a) and spleen (B-1b). Consistent with this finding, circulating IgM, but not IgG, was elevated in TET2-KO mice compared to WT. Analysis of bulk RNASeq of sort purified peritoneal B-1a and B-1b cells revealed reduced expression of heavy and light chain immunoglobulin genes, predominantly in B-1a cells from TET2-KO mice compared to WT controls. As expected, the expression of IgM transcripts was the most abundant isotype in B-1 cells. Yet, only in B-1a cells there was a significant increase in the proportion of IgM transcripts in TET2-KO mice compared to WT. Analysis of the CDR3 of the BCR revealed an increased abundance of replicated CDR3 sequences in B-1 cells from TET2-KO mice, which was more clearly pronounced in B-1a compared to B-1b cells. V-D-J usage and circos plot analysis of V-J combinations showed enhanced usage of VH11 and VH12 pairings. Taken together, our study is the first to demonstrate that global loss of TET2 increases B-1 cell number and IgM production and reduces CDR3 diversity, which could impact many biological processes and disease states that are regulated by IgM.

Specific features of a scaffolding antibody light chain.

The antigen-binding sites in conventional antibodies are formed by hypervariable complementarity-determining regions (CDRs) from both heavy chains (HCs) and light chains (LCs). A deviation from this paradigm is found in a subset of bovine antibodies that bind antigens via an ultra-long CDR. The HCs bearing ultra-long CDRs pair with a restricted set of highly conserved LCs that convey stability to the antibody. Despite the importance of these LCs, their specific features remained unknown. Here, we show that the conserved bovine LC found in antibodies with ultra-long CDRs exhibits a distinct combination of favorable physicochemical properties such as good secretion from mammalian cells, strong dimerization, high stability, and resistance to aggregation. These physicochemical traits of the LCs arise from a combination of the specific sequences in the germline CDRs and a lambda LC framework. In addition to understanding the molecular architecture of antibodies with ultra-long CDRs, our findings reveal fundamental insights into LC characteristics that can guide the design of antibodies with improved properties.

Truncation of the constant domain drives amyloid formation by immunoglobulin light chains.

AL amyloidosis is a life-threatening disease caused by deposition of immunoglobulin light chains. While the mechanisms underlying light chains amyloidogenesis in vivo remain unclear, several studies have highlighted the role that tissue environment and structural amyloidogenicity of individual light chains have in the disease pathogenesis. AL natural deposits contain both full-length light chains and fragments encompassing the variable domain (VL) as well as different length segments of the constant region (CL), thus highlighting the relevance that proteolysis may have in the fibrillogenesis pathway. Here, we investigate the role of major truncated species of the disease-associated AL55 light chain that were previously identified in natural deposits. Specifically, we study structure, molecular dynamics, thermal stability, and capacity to form fibrils of a fragment containing both the VL and part of the CL (133-AL55), in comparison with the full-length protein and its variable domain alone, under shear stress and physiological conditions. Whereas the full-length light chain forms exclusively amorphous aggregates, both fragments generate fibrils, although, with different kinetics, aggregate structure, and interplay with the unfragmented protein. More specifically, the VL-CL 133-AL55 fragment entirely converts into amyloid fibrils microscopically and spectroscopically similar to their ex vivo counterpart and increases the amorphous aggregation of full-length AL55. Overall, our data support the idea that light chain structure and proteolysis are both relevant for amyloidogenesis in vivo and provide a novel biocompatible model of light chain fibrillogenesis suitable for future mechanistic studies.

Gene copy number, gene configuration and LC/HC mRNA ratio impact on antibody productivity and product quality in targeted integration CHO cell lines.

The augmentation of transgene copy numbers is a prevalent approach presumed to enhance transcriptional activity and product yield. CHO cell lines engineered via targeted integration (TI) offer an advantageous platform for investigating the interplay between gene copy number, mRNA abundance, product yield, and product quality. Our investigation revealed that incrementally elevating the gene copy numbers of both IgG heavy chain (HC) and light chain (LC) concurrently resulted in the attainment of plateaus in mRNA levels and product titers, notably occurring beyond four to five gene copies integrated at the same TI site. Furthermore, maintaining a fixed gene copy number while varying the position of genes within the vector influenced the LC/HC mRNA ratio, which subsequently exerted a substantial impact on product titer. Moreover, manipulation of the LC/HC gene ratio through the introduction of surplus LC gene copies led to heightened LC mRNA expression and a reduction in the levels of high molecular weight species. It is noteworthy that the effects of excess LC on product titer were dependent on the specific molecule under consideration. The strategic utilization of PCR tags enabled precise quantification of transcription from each expression slot within the vector, facilitating the identification of highly expressive and less expressive slots. Collectively, these findings significantly enhance our understanding of stable antibody production in TI CHO cell lines.

Computational evidences of a misfolding event in an aggregation-prone light chain preceding the formation of the non-native pathogenic dimer.

Antibody light chain amyloidosis is a disorder in which protein aggregates, mainly composed of immunoglobulin light chains, deposit in diverse tissues impairing the correct functioning of organs. Interestingly, due to the high susceptibility of antibodies to mutations, AL amyloidosis appears to be strongly patient-specific. Indeed, every patient will display their own mutations that will make the proteins involved prone to aggregation thus hindering the study of this disease on a wide scale. In this framework, determining the molecular mechanisms that drive the aggregation could pave the way to the development of patient-specific therapeutics. Here, we focus on a particular patient-derived light chain, which has been experimentally characterized. We investigated the early phases of the aggregation pathway through extensive full-atom molecular dynamics simulations, highlighting a structural rearrangement and the exposure of two hydrophobic regions in the aggregation-prone species. Next, we moved to consider the pathological dimerization process through docking and molecular dynamics simulations, proposing a dimeric structure as a candidate pathological first assembly. Overall, our results shed light on the first phases of the aggregation pathway for a light chain at an atomic level detail, offering new structural insights into the corresponding aggregation process.

Biophysical characterization of human-cell-expressed, full-length κI O18/O8, AL-09, λ6a, and Wil immunoglobulin light chains.

Immunoglobulin light chain (AL) amyloidosis involves the deposition of insoluble monoclonal AL protein fibrils in the extracellular space of different organs leading to dysfunction and death. Development of methods to efficiently express and purify AL proteins with acceptable standards of homogeneity and structural integrity has become critical to understand the in vitro and in vivo aspects of AL protein aggregation, and thus the disease progression. In this study, we report the biophysical characterization of His-tagged and untagged versions of AL full-length (FL) κI and λ6 subgroup proteins and their mutants expressed from the Expi293F human cell line. We used an array of biophysical and biochemical methods to analyze the structure and stability of the monomers, oligomerization states, and thermodynamic characteristics of the purified FL proteins and how they compare with the bacterially expressed FL proteins. Our results demonstrate that the tagged and untagged versions of FL proteins have comparable stability to proteins expressed in bacterial cells but exhibit multiple unfolding transitions and reversibility. Non-reducing SDS-PAGE and analytical ultracentrifugation analysis showed presence of monomers and dimers, with an insignificant amount of higher-order oligomers, in the purified fraction of all proteins. Overall, the FL proteins were expressed with sufficient yields for biophysical studies and can replace bacterial expression systems.

Facile One-Step Generation of Camelid VHH and Avian scFv Libraries for Phage Display by Golden Gate Cloning.

Since its development in the 1980s, the Nobel Prize-awarded phage display technology has been one of the most commonly used in vitro selection technologies for the discovery of therapeutic and diagnostic antibodies. Besides the importance of selection strategy, one key component of the successful isolation of highly specific recombinant antibodies is the construction of high-quality phage display libraries. However, previous cloning protocols relied on a tedious multistep process with subsequent cloning steps for the introduction of first heavy and then light chain variable genetic antibody fragments (VH and VL). This resulted in reduced cloning efficiency, higher frequency of missing VH or VL sequences, as well as truncated antibody fragments. With the emergence of Golden Gate Cloning (GGC) for the generation of antibody libraries, the possibility of more facile library cloning has arisen. Here, we describe a streamlined one-step GGC strategy for the generation of camelid heavy chain only variable phage display libraries as well as the simultaneous introduction of heavy chain and light chain variable regions from the chicken into a scFv phage display vector.

Amyloidogenic light chains impair plasma cell survival.

Systemic light chain amyloidosis (AL) is a clonal plasma cell disorder characterized by the deposition of misfolded immunoglobulin light chains (LC) as insoluble fibrils in organs. The lack of suitable models has hindered the investigation of the disease mechanisms. Our aim was to establish AL LC-producing plasma cell lines and use them to investigate the biology of the amyloidogenic clone. We used lentiviral vectors to generate cell lines expressing LC from patients suffering from AL amyloidosis. The AL LC-producing cell lines showed a significant decrease in proliferation, cell cycle arrest, and an increase in apoptosis and autophagy as compared with the multiple myeloma LC-producing cells. According to the results of RNA sequencing the AL LC-producing lines showed higher mitochondrial oxidative stress, and decreased activity of the Myc and cholesterol pathways. The neoplastic behavior of plasma cells is altered by the constitutive expression of amyloidogenic LC causing intracellular toxicity. This observation may explain the disparity in the malignant behavior of the amyloid clone compared to the myeloma clone. These findings should enable future in vitro studies and help delineate the unique cellular pathways of AL, thus expediting the development of specific treatments for patients with this disorder.