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.

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.

Phenotypes and outcome of diffuse pulmonary non-amyloid light chain deposition disease.

BACKGROUND: Light chain deposition disease (LCDD) is a very rare entity. Clinical manifestations of LCDD vary according to the organs involved. Data on pulmonary LCDD are scarce and limited to small series or case reports. This study aimed to describe the characteristics and outcome of diffuse pulmonary non-amyloid LCDD localized to the lungs. STUDY DESIGN AND METHODS: A multicenter retrospective cohort study was conducted. Clinical characteristics were collected, and chest CTs were centrally reviewed. The diagnosis of pulmonary non-amyloid LCDD was confirmed by immunohistochemistry. RESULTS: Thirty-one cases were identified (68% female), with a median age at diagnosis of 50 years (IQR 20). Baseline FEV1/FVC was < 0.70 in 45% of patients. Mean (± SD) FEV1 and DLCO were 86% ± 26.2 and 52% ± 23.9, respectively. CT revealed peculiar patterns of thin-walled cysts (58%) and thin-walled cystic bronchiectases (27%). Increased serum kappa light chain was found in 87% of patients. Histological analysis showed kappa light chain deposits in all patients, except one with lambda chain deposits. Median annual FEV1 decline was 127 ml (IQR 178) and median DLCO decline was 4.3% (IQR 4.3). Sixteen patients received immunomodulatory treatment or chemotherapy; serum light chain levels decreased in 9 cases (75%), without significant improvement in FEV1 (p = 0.173). Overall, 48% of patients underwent bilateral lung transplantation. Transplant-free survival at 5 and 10 years were 70% and 30%, respectively. An annual FEV1 decline greater than 127 ml/year was associated with increased risk of death or transplantation (p = 0.005). CONCLUSIONS: Diffuse pulmonary LCDD is characterised by female predominance, a peculiar imaging pattern with bronchiectasis and/or cysts, progressive airway obstruction and severe DLCO impairment, and poor outcome. Lung transplantation is a treatment of choice.

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.

Novel monoclonal antibodies: A really specific therapy for light chain amyloidosis.

Light chain amyloidosis is a rare disease caused by clonal plasma cells in the bone marrow generating an excessive amount of immunoglobulin light chains. These chains misfold and produce insoluble fibrils that deposit in various organs, including the heart, kidneys, liver, nervous system, and digestive tract. Life expectancy and symptoms during the course of the disease vary depending on which and how many organs are affected. Targeted plasma cell therapy has significantly advanced the clinical management of amyloidosis, with ongoing progress. However, current clinical studies are investigating innovative targets, drug combinations and treatment strategies to improve therapeutic outcomes by minimizing adverse effects and refining patient prognosis in these challenging hematological conditions. In this paper, we review the state of the art regarding the use of anti-amyloid antibodies, as a revolutionary and innovative approach in the current scenario of amyloid treatment.

Light chain deposition disease presenting with gastrointestinal disorder as primary manifestation: report of two cases and literature review.

Light chain deposition disease (LCDD) is an under-recognized condition characterized by deposition of abnormal monoclonal light chains in tissues, leading to organ dysfunction. LCDD involving the gastrointestinal tract is very uncommon, and its diagnosis is challenging. We herein report two cases of LCDD that manifested as inflammatory bowel disease-like symptoms and protein-losing gastroenteropathy. Both patients were women in their early 60s. Tissue biopsies from the gastrointestinal mucosa demonstrated extracellular deposits, which were negative by Congo red staining but positive for κ-light chain by immunohistochemistry. The recent literature on LCDD was reviewed. When patients unexpectedly show extracellular deposits in gastrointestinal biopsy specimens, evaluation of immunoglobulin chains is recommended for diagnosis of LCDD after systemic amyloidosis has been excluded.

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.

Progression free survival of myeloma patients who become IFE-negative correlates with the detection of residual monoclonal free light chain (FLC) by mass spectrometry.

Deeper responses are associated with improved survival in patients being treated for myeloma. However, the sensitivity of the current blood-based assays is limited. Historical studies suggested that normalisation of the serum free light chain (FLC) ratio in patients who were negative by immunofixation electrophoresis (IFE) was associated with improved outcomes. However, recently this has been called into question. Mass spectrometry (MS)-based FLC assessments may offer a superior methodology for the detection of monoclonal FLC due to greater sensitivity. To test this hypothesis, all available samples from patients who were IFE negative after treatment with carfilzomib and lenalidomide-based induction and autologous stem cell transplantation (ASCT) in the Myeloma XI trial underwent FLC-MS testing. FLC-MS response assessments from post-induction, day+100 post-ASCT and six months post-maintenance randomisation were compared to serum FLC assay results. Almost 40% of patients had discordant results and 28.7% of patients with a normal FLC ratio had residual monoclonal FLC detectable by FLC-MS. FLC-MS positivity was associated with reduced progression-free survival (PFS) but an abnormal FLC ratio was not. This study demonstrates that FLC-MS provides a superior methodology for the detection of residual monoclonal FLC with FLC-MS positivity identifying IFE-negative patients who are at higher risk of early progression.

Serum free light chains among twin siblings: is the kappa/lambda ratio genetically determined?

BACKGROUND: Serum kappa, lambda, the K/λ light chain concentrations are used for screening, diagnosis, and monitoring of patients with multiple myeloma and other plasma cell disorders. Biological variation studies conducted on healthy subjects showed that free light chains have a low within and high between-individual variation. We determined if this variation were genetically linked. METHODS: We obtained a single serum sample from 16 pairs of identical twins, 8 neonate twins, and 19 presumed directly-related siblings children, measured Κ and λ light chains and computed the Κ/λ ratio. RESULTS: As expected, Κ/λ results from each twin neonate were near identical (reflecting maternal/placental transfer). For older children and adult twins, the Κ/λ ratio form a cluster of results that were a subset of the reference range. There was one outlier, a female with a high, different from her twin sister. She likely had a monoclonal gammopathy (no followup was possible). Excluding this pair, results from neonate twins (14.4% ±10.3%) and non-neonate twins (18.0 ± 15.3%) were not significantly different. Results between non-twin siblings were more scattered (53.2%±53.4%) and different from neonate and non-neonate twin adult and children. CONCLUSION: We suggest that the Κ/λ free light chains may be genetically linked.

[Clinical Significance of Serum Free Light Chain in Monoclonal Gammopathy].

OBJECTIVE: To investigate the expression and clinical significance of serum free light chain (sFLC) in patients with monoclonal gammopathy (MG). METHODS: The peripheral blood of 98 patients with MG and 30 healthy volunteers were collected. The level of sFLC was detected by immunoturbidimetry, and the value of sFLC in diagnosis, disease severity, and efficacy evaluation was analyzed. RESULTS: Among 98 MG patients, there were 58 males and 40 females, 45 cases of monoclonal gammopathy of renal significance (MGRS), 33 cases of monoclonal gammopathy of undetermined significance (MGUS), 20 cases of hematological malignancy (HM), 58 cases of IgG type, 26 cases of IgA type, 7 cases of IgM type, 5 cases of light chain type, 2 cases of non-secreting type, 35 cases of κ type, 63 cases of λ type, 53 cases of renal insufficiency, 45 cases of normal renal function. The expression levels of sFLC-κ and sFLC-λ in MG patients were significantly higher than those of the control group (P <0.01). The expression levels of sFLC-κ and sFLC-λ in HM patients were significantly higher than MGRS and MGUS patients, and in MGRS patients were also significantly higher than MGUS patients (P <0.05). Patients with abnormal renal function had higher expression levels of sFLC-κ and sFLC-λ than patients with normal renal function (P <0.01). sFLC-κ and sFLC-λ were positively correlated with the expression level of globulin (r =0.392, r =0.435) and ß2-MG (r =0.403, r =0.468) in MG patients, as well as serum creatinine in patients with abnormal renal function (r =0.586, r =0.631), while no significant correlation was found with age, sex, albumin, lactate dehydrogenase, and serum calcium. After treatment, the levels of sFLC-κ and sFLC-λ were significantly decreased (P <0.01). CONCLUSION: sFLC is significantly elevated in MG patients and can be quickly detected with high sensitivity, which is helpful for the diagnosis of disease type, judgment of disease severity, and evaluation of therapy.

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.

Complete responses in AL amyloidosis are unequal: the impact of free light chain mass spectrometry in AL amyloidosis.

ABSTRACT: Amyloidogenic serum free light chains (sFLCs) drive disease progression in AL amyloidosis. Matrix-assisted laser desorption/ionization time of flight mass spectrometry-based FLC assay (FLC-MS) has greater sensitivity than conventional sFLC assays allowing for the detection of serological residual disease. We report the utility of FLC-MS in a large series of patients with AL amyloidosis assessing the impact of FLC-MS negativity after treatment on overall survival (OS) and organ response rates. Serum samples were analyzed using FLC-MS at diagnosis and at 6 and 12 months after treatment. The impact of FLC-MS negativity over standard hematologic responses on survival and organ response was assessed. A total of 487 patients were included; 290 (59%) and 349 (71.5%) had cardiac and renal involvement, respectively. There was 100% concordance between the light chain (LC) fibril type and LC isotype identified by FLC-MS. At 6 and 12 months, 81 (16.6%) and 101 (20.7%) were FLC-MS negative. Of those achieving a conventional hematologic complete response (CR) at 6 and 12 months, 45 (27.7%) and 64 (39%) were FLC-MS negative. At 12 months, median OS for CR + FLC-MS negative was not reached vs 108 months in CR + FLC-MS positive (P = .024). At 12 months, 70% of patients with FLC-MS negativity (vs 50% FLC-MS positive) achieved a cardiac response (P = .015). In a multivariate analysis, FLC-MS negativity at 12 months was an independent predictor of better outcomes. FLC-MS can detect persistent monoclonal light chains in a significant proportion of patients in a conventional hematologic CR. FLC-MS assessment promises to be a new standard for response assessment in AL amyloidosis.

Screening in Multiple Myeloma and Its Precursors: Are We There Yet?

BACKGROUND: Multiple myeloma (MM) is a hematological malignancy that develops over years from the asymptomatic precursors, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma. Recent evidence shows that by initiating treatment at an asymptomatic stage, outcomes in MM can be significantly improved. However, a vast majority of MM patients are diagnosed after the development of symptomatic end-organ damage and cannot reap the benefits of early treatment. The precursors of MM are easily detected by serum protein electrophoresis and free light chain assay of the serum, raising the question of whether population-based screening could detect MM at an asymptomatic stage and significantly expand the availability of early treatment in MM. Screening is a hallmark of care in many malignancies, and there are accepted criteria for when screening is appropriate. CONTENT: Here we review the available relevant evidence for the introduction of screening and discuss whether screening for MM and its precursors fulfills these criteria. We also highlight gaps in our current knowledge, most notably a lack of data on the benefits and harms of screening and the lack of a defined target population. There are ongoing studies that may fill these critical gaps in the literature, but their results are still pending. SUMMARY: Screening could lead to a paradigm shift in the care of patients with MM, but critical scientific questions need to be answered before screening of healthy individuals can be recommended. In short, we should not screen for MM and its precursors-yet.

Urine Immunofixation Electrophoresis for Diagnosis of Monoclonal Gammopathy: Evaluation of Methods for Urine Concentration.

BACKGROUND: Examination of urine by immunofixation electrophoresis (UIFE) is one of the tests recommended for screening and monitoring of monoclonal gammopathies, especially multiple myeloma. Unlike the serum free light chain measurement, a positive result on urine immunofixation is diagnostic for monoclonal immunoglobulin light chains. Urine is usually concentrated, generally by membrane filtration, prior to electrophoresis. METHODS: Alternative methods to membrane filtration for urine concentration were examined. Residual urine specimens submitted for urine protein electrophoresis were concentrated by precipitation of the proteins by ammonium sulfate salt precipitation, precipitation with ethanol and acetonitrile, and by desiccation. The concentrated specimens were subjected to immunofixation electrophoresis using antisera to free light chains (FLC). The results were compared with those from conventional immunofixation electrophoresis using specimens concentrated by membrane filtration. RESULTS: Ammonium sulfate, ethanol, and acetonitrile precipitation results were less than satisfactory. Concentration by desiccation provided results comparable, if not better than, those by membrane filtration and conventional UIFE. The cost of desiccation is minimal compared to more than $5.00/specimen cost of concentration by membrane filtration. The differences in the results with conventional UIFE and the method described here are likely due to (a) variability in the reactivity of different antisera to free monoclonal light chains, and (b) obscuration of monoclonal free light chains by co-migration with intact immunoglobulin monoclonal proteins. CONCLUSIONS: Concentrating urine by desiccation for immunofixation electrophoresis is technically simple, inexpensive, and provides results comparable to concentrating by membrane filtration. Using FLC provides a more sensitive assay than using conventional antisera.

Mode of progression in smoldering multiple myeloma: a study of 406 patients.

The approach to patients with high-risk smoldering multiple myeloma (SMM) varies among clinicians; while some advocate early intervention, others reserve treatment at progression to multiple myeloma (MM). We aimed to describe the myeloma-defining events (MDEs) and clinical presentations leading to MM diagnosis among SMM patients seen at our institution. We included 406 patients diagnosed with SMM between 2013-2022, seen at Mayo Clinic, Rochester, MN. The 2018 Mayo 20/2/20 criteria were used for risk stratification. Median follow-up was 3.9 years. Among high-risk patients who did not receive treatment in the SMM phase (n = 71), 51 progressed by last follow-up; the MDEs included: bone lesions (37%), anemia (35%), hypercalcemia (8%), and renal failure (6%); 24% met MM criteria based on marrow plasmacytosis (≥60%) and/or free light chain ratio (>100); 45% had clinically significant MDEs (hypercalcemia, renal insufficiency, and/or bone lesions). MM diagnosis was made based on surveillance labs/imaging(45%), testing obtained due to provider suspicion for progression (14%), bone pain (20%), and hospitalization/ED presentations due to MM complications/symptoms (4%). The presentation was undocumented in 14%. A high proportion (45%) of patients with high-risk SMM on active surveillance develop end-organ damage at progression. About a quarter of patients who progress to MM are not diagnosed based on routine interval surveillance testing.