Nanobodies counteract the toxicity of an amyloidogenic light chain by stabilizing a partially open dimeric conformation.

Light chain amyloidosis (AL) is a systemic disease where fibrillar deposition of misfolded immunoglobulin light chains (LCs) severely affects organ function and results in poor prognosis for patients, especially when heart involvement is severe. Particularly relevant in this context is the cardiotoxicity exerted by still uncharacterized soluble LC species. Here, with the final goal of identifying alternative therapeutic strategies to tackle AL amyloidosis, we produced five llama-derived nanobodies (Nbs) specific against H3, a well-characterized amyloidogenic and cardiotoxic LC from an AL patient with severe cardiac involvement. We found that Nbs are specific and potent agents capable of abolishing H3 soluble toxicity in C. elegans in vivo model. Structural characterization of H3-Nb complexes revealed that the protective effect of Nbs is related to their ability to bind to the H3 VL domain and stabilise an unexpected partially open LC dimer in which the two VL domains no longer interact with each other. Thus, while identifying potent inhibitors of LC soluble toxicity, we also describe the first non-native structure of an amyloidogenic LC that may represent a crucial step in toxicity and aggregation mechanisms.

An armed anti-immunoglobulin light chain nanobody protects mice against influenza A and B infections.

The immune system eliminates pathogen intruders such as viruses and bacteria. To recruit immune effectors to virus-infected cells, we conjugated a small molecule, the influenza neuraminidase inhibitor zanamivir, to a nanobody that recognizes the kappa light chains of mouse immunoglobulins. This adduct was designed to achieve half-life extension of zanamivir through complex formation with the much-larger immunoglobulins in the circulation. The zanamivir moiety targets the adduct to virus-infected cells, whereas the anti-kappa component simultaneously delivers polyclonal immunoglobulins of indeterminate specificity and all isotypes. Activation of antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity promoted elimination of influenza neuraminidase-positive cells. A single dose of the conjugate protected mice against influenza A or B viruses and was effective even when given several days after infection with a lethal dose of virus. In the absence of circulating immunoglobulins, we observed no in vivo protection from the adduct. The type of conjugates described here may thus find application for both anti-influenza prophylaxis and therapy.

Renal amyloidosis: a new time for a complete diagnosis.

Amyloidoses are a group of disorders in which soluble proteins aggregate and deposit extracellularly in tissues as insoluble fibrils, causing organ dysfunction. Clinical management depends on the subtype of the protein deposited and the affected organs. Systemic amyloidosis may stem from anomalous proteins, such as immunoglobulin light chains or serum amyloid proteins in chronic inflammation or may arise from hereditary disorders. Hereditary amyloidosis consists of a group of rare conditions that do not respond to chemotherapy, hence the identification of the amyloid subtype is essential for diagnosis, prognosis, and treatment. The kidney is the organ most frequently involved in systemic amyloidosis. Renal amyloidosis is characterized by acellular pathologic Congo red-positive deposition of amyloid fibrils in glomeruli, vessels, and/or interstitium. This disease manifests with heavy proteinuria, nephrotic syndrome, and progression to end-stage kidney failure. In some situations, it is not possible to identify the amyloid subtype using immunodetection methods, so the diagnosis remains indeterminate. In cases where hereditary amyloidosis is suspected or cannot be excluded, genetic testing should be considered. Of note, laser microdissection/mass spectrometry is currently the gold standard for accurate diagnosis of amyloidosis, especially in inconclusive cases. This article reviews the clinical manifestations and the current diagnostic landscape of renal amyloidosis.

Advances in the treatment of light chain amyloidosis.

PURPOSE OF REVIEW: After many years, the management of systemic light chain (AL) amyloidosis is entering the era of evidence-based medicine, with three recently published randomized clinical trials, a regimen (daratumumab, cyclophosphamide, bortezomib, and dexamethasone, daratumumab-CyBorD) labeled for upfront therapy, more clinical trials ongoing, and published guidelines. In this review, we discuss how current practice is changing based on this data. RECENT FINDINGS: Daratumumab-CyBorD grants unprecedentedly high rates of hematologic and organ response and became the novel standard-of-care in AL amyloidosis. The International Society of Amyloidosis and the European Hematology Association issued common guidelines for autologous stem cell transplant (ASCT) in this disease. Improved patient selection and effective induction regimens greatly reduced ASCT-related mortality. Venetoclax is emerging as a very effective option in patients harboring the common t(11;14) abnormality. Rapid and profound reduction of the amyloid free light chain can improve survival also at advanced stages. SUMMARY: Daratumumab-CyBorD is being integrated into the treatment flow-chart whereas the role of ASCT is being redefined. New approaches are being tested in clinical trials. Treatment of daratumumab-refractory patients and validation of criteria of hematologic progression to be used in clinical trials and in individual patient management are current areas of research.

Early Light Chains Removal and Albumin Levels with a Double Filter-Based Extracorporeal Treatment for Acute Myeloma Kidney.

Renal impairment in Multiple Myeloma (MM) represents one of the most important factors that influences patient survival. In fact, before the introduction of modern chemotherapy, less than 25% of patients with acute kidney injury (AKI) and MM who required dialysis recovered sufficient renal function to become independent from dialysis, with a median overall survival of less than 1 year. There are many other factors involved in determining patient survival. In this study we aimed to investigate the role of double filter-based extracorporeal treatment for removal of serum free light chains (sFLC) in acute myeloma kidney (AKI for MM) and to evaluate patient overall survival. All patients received Bortezomib-based chemotherapy and extracorporeal treatment for sFLC removal. For each session 2 dialyzers of the same kind were used. The dialytic dose was not related to the degree of renal function but to the removal of sFLC. The factors that have been found to be significantly associated with lower mortality were reduction of sFLC at day 12 and day 30, >50% reduction of sFLC at day 30, number of sessions and independence from dialysis. Among baseline characteristics, albumin level was statistically associated with the patients' outcome. Our analysis highlights the importance of the early treatment for removal of sFLC in AKI for MM. These results indicate that the early removal of sFLC can improve patient's outcome.

COVID-19 and Light Chain Amyloidosis, Adding Insult to Injury.

Light chain (AL) amyloidosis is a potentially fatal disease of monoclonal plasma cells that leads to accumulation of light chain amyloid fibrils, organ damage, and the manifestations of clinical disease. Meanwhile, coronavirus disease 2019 (COVID-19) is a disease caused by infection with the severe acute respiratory syndrome coronavirus 2 virus, with the potential to cause severe systemic illness and death. There is significant overlap in the demographics and comorbidities observed in AL amyloidosis and those associated with highest risk for severe morbidity and mortality due to COVID-19. This overlap creates unique challenges in caring for patients with AL amyloidosis, which are further compounded by the immunosuppressive nature of anti-plasma cell therapies, the need for frequent clinical assessments, and the exclusion of AL amyloidosis patients from initial COVID-19 vaccine trials. Herein, we highlight many of the relevant concerns related to COVID-19 and the treatment of AL amyloidosis, summarize a general approach for AL amyloidosis management amidst the ongoing COVID-19 pandemic, and discuss current guidance about COVID-19 vaccination of patients with AL amyloidosis.

Data mining patented antibody sequences.

The patent literature should reflect the past 30 years of engineering efforts directed toward developing monoclonal antibody therapeutics. Such information is potentially valuable for rational antibody design. Patents, however, are designed not to convey scientific knowledge, but to provide legal protection. It is not obvious whether antibody information from patent documents, such as antibody sequences, is useful in conveying engineering know-how, rather than as a legal reference only. To assess the utility of patent data for therapeutic antibody engineering, we quantified the amount of antibody sequences in patents destined for medicinal purposes and how well they reflect the primary sequences of therapeutic antibodies in clinical use. We identified 16,526 patent families covering major jurisdictions (e.g., US Patent and Trademark Office (USPTO) and World Intellectual Property Organization) that contained antibody sequences. These families held 245,109 unique antibody chains (135,397 heavy chains and 109,712 light chains) that we compiled in our Patented Antibody Database (PAD, http://naturalantibody.com/pad). We find that antibodies make up a non-trivial proportion of all patent amino acid sequence depositions (e.g., 11% of USPTO Full Text database). Our analysis of the 16,526 families demonstrates that the volume of patent documents with antibody sequences is growing, with the majority of documents classified as containing antibodies for medicinal purposes. We further studied the 245,109 antibody chains from patent literature to reveal that they very well reflect the primary sequences of antibody therapeutics in clinical use. This suggests that the patent literature could serve as a reference for previous engineering efforts to improve rational antibody design.

Antibody light chain variable domains and their biophysically improved versions for human immunotherapy.

We set out to gain deeper insight into the potential of antibody light chain variable domains (VLs) as immunotherapeutics. To this end, we generated a naïve human VL phage display library and, by using a method previously shown to select for non-aggregating antibody heavy chain variable domains (VHs), we isolated a diversity of VL domains by panning the library against B cell super-antigen protein L. Eight domains representing different germline origins were shown to be non-aggregating at concentrations as high as 450 µM, indicating VL repertoires are a rich source of non-aggregating domains. In addition, the VLs demonstrated high expression yields in E. coli, protein L binding and high reversibility of thermal unfolding. A side-by-side comparison with a set of non-aggregating human VHs revealed that the VLs had similar overall profiles with respect to melting temperature (T(m)), reversibility of thermal unfolding and resistance to gastrointestinal proteases. Successful engineering of a non-canonical disulfide linkage in the core of VLs did not compromise the non-aggregation state or protein L binding properties. Furthermore, the introduced disulfide bond significantly increased their T(m)s, by 5.5-17.5 ° C, and pepsin resistance, although it somewhat reduced expression yields and subtly changed the structure of VLs. Human VLs and engineered versions may make suitable therapeutics due to their desirable biophysical features. The disulfide linkage-engineered VLs may be the preferred therapeutic format because of their higher stability, especially for oral therapy applications that necessitate high resistance to the stomach's acidic pH and pepsin.

Immunoglobulin light chain, Blimp-1 and cytochrome P4501B1 peptides as potential vaccines for AL amyloidosis.

Amyloid light chain (AL) amyloidosis is a lethal disorder characterized by the pathologic deposition of clonal plasma cell-derived, fibrillogenic immunoglobulin light chains in vital organs. Current chemotherapeutic regimens are problematic in patients with compromised organ function and are not effective for all patients. Here, a platform of computer-based prediction and preclinical mouse modeling was used to begin development of a complementary, immunotherapeutic approach for AL amyloidosis. Three peptide/MHC I-binding algorithms identified immunogenic peptides from three AL plasma cell-associated proteins: (1) amyloidogenic λ6 light chains, (2) CYP1B1, a universal tumor antigen hyper-expressed in AL plasma cells and (3) B lymphocyte-induced maturation protein 1 (Blimp-1), a transcription factor required for plasma cell differentiation. The algorithms correctly predicted HLA-A(*)0201-binding native and heteroclitic peptides. In HLA-A2 transgenic mice, these peptides, given individually or in combination, induced potent CTL which kill peptide-loaded human lymphoma cells and/or lymphoma cells producing target protein. Blimp-1 peptide-immunized mice exhibited a reduced percentage of splenic, lymph node and bone marrow plasma cells and a decrease in the absolute number of splenic plasma cells demonstrating (1) presentation of target peptide by endogenous plasma cells and (2) appropriate CTL homing to lymphoid organs followed by killing of target plasma cells. These studies suggest that AL amyloidosis, with its relatively low tumor cell burden, may be an attractive target for peptide-based multivalent vaccines.

Effects of free immunoglobulin light chains on viral myocarditis.

RATIONALE: In recent work, we have demonstrated a crucial role of mast cells in the development of viral myocarditis. Viral infection could lead to increased synthesis of free immunoglobulin light chains (FLC) and our earlier work showed that FLC can trigger mast cell activation. OBJECTIVE: We studied the possible involvement of FLC in the pathogenesis of viral myocarditis, and therapeutic effects of FLC using an animal model of viral myocarditis. METHODS AND RESULTS: DBA/2 mice were inoculated intraperitoneally with encephalomyocarditis (EMC) virus. Serum levels and concentrations in the heart of kappa FLC on day 14 in mice inoculated with EMC virus were significantly increased compared with controls. Myocardial viral concentration was significantly inhibited, the area of myocardial lesions was smaller in mice treated with kappa or lambda FLC, and survival of mice given FLC significantly improved. In contrast, an FLC antagonist deteriorated myocarditis. kappa and lambda FLC chains inhibited EMC viral replication in human amnion cells in vitro. lambda FLC significantly increased the gene expression of interleukin-10 in the heart which was previously shown to improve viral myocarditis when given exogenously. FLC also tended to increase the gene expressions of interferon-alpha and -gamma in the heart mice. CONCLUSIONS: FLC have antiviral and antiinflammatory effects and improved viral myocarditis in mice. FLC may be promising agents for the treatment of viral myocarditis.

Cloning of apoB intrabodies: specific knockdown of apoB in HepG2 cells.

Apolipoprotein (apo) B is essential for the assembly and secretion of triglyceride-rich lipoproteins made by the liver. As the sole protein component in LDL, apoB is an important determinant of atherosclerosis susceptibility and a potential pharmaceutical target. Single-chain antibodies (sFvs) are the smallest fragment of an IgG molecule capable of maintaining the antigen binding specificity of the parental antibody. In the present study, we describe the cloning and construction of two intracellular antibodies (intrabodies) to human apoB. We targeted these intrabodies to the endoplasmic reticulum for the purpose of retaining nascent apoB within the ER, thereby preventing its secretion. Expression of the 1D1 intrabody in the apoB-secreting human hepatoma cell line HepG2 resulted in marked reduction of apoB secretion. This study demonstrates the utility of an intrabody to specifically block the secretion of a protein determinant of plasma LDL as a therapeutic strategy for the treatment of hyperlipidemia.

Catalytic features and eradication ability of antibody light-chain UA15-L against Helicobacter pylori.

We have successfully developed a catalytic antibody capable of degrading the active site of the urease of Helicobacter pylori and eradicating the bacterial infection in a mouse stomach. This monoclonal antibody UA15 was generated using a designed recombinant protein UreB, which contained the crucial region of the H. pylori urease beta-subunit active site, for immunization. The light chain of this antibody (UA15-L) by itself showed a proteolytic activity to substantially degrade both UreB and the intact urease. Oral administration of UA15-L also significantly reduced the number of H. pylori in a mouse stomach. This is the first example of a monoclonal catalytic antibody capable of functioning in vivo, and such an antibody may have a therapeutic utility in the future.

Topical application of F991, an immunoglobulin free light chain antagonist, prevents development of contact sensitivity in mice.

BACKGROUND: Exposure to reactive chemicals or environmental allergens can lead to hypersensitivity reactions in the skin of predisposed people. Most of these reactions are of atopic origin, but a subgroup of patients exhibits skin hypersensitivity reactions without features of atopy. OBJECTIVE: This study was undertaken to examine the effect of inhibiting the action of Ig-free light chains in a murine model for non-atopic skin hypersensitivity by dermal application of the free light chain antagonist F991. METHODS: To study the efficacy of F991, BALB/c mice were either passively immunized with trinitrophenyl (TNP)-specific immunoglobulin light chains (IgLC) and challenged with the hapten picryl chloride (PCl) or actively skin-sensitized and challenged with dinitrofluorobenzene (DNFB). The effect of F991 or control treatment was investigated by measuring local edema formation and the production of pro-inflammatory cytokines. RESULTS: Passive immunization with TNP-specific IgLC resulted in an increase in ear swelling 2 h after PCl challenge. F991 inhibited this enhanced ear swelling in a dose-dependent manner when applied 4 h before the sensitization with IgLC. F991 also inhibited DNFB-induced contact hypersensitivity reaction in the mouse skin 2 and 24 h after challenge when applied before challenge. Besides the prophylactic action, F991 when applied 2 h after DNFB-challenge, it was also able to attenuate symptoms of the DNFB-induced hypersensitivity reaction at 24 h after challenge. We showed that the beneficial effects of F991 are restricted to the side of application. CONCLUSION: F991 is able to effectively alleviate symptoms of contact sensitivity in mice. Our study suggests that local interference with IgLC-induced allergic symptoms may be attractive in the treatment of hypersensitivity responses.

Gene therapy using adenovirus-mediated full-length anti-HER-2 antibody for HER-2 overexpression cancers.

PURPOSE: Therapeutic monoclonal antibody is increasingly applied in many clinical applications, although complicated technologies and high cost still limit their wide applications. To obtain the sustained serum antibody concentration with one single injection and lower the cost of antibody protein therapy, an adenovirus-mediated full-length antibody gene therapy was developed. EXPERIMENTAL DESIGN: Full-length antibody light-chain and heavy-chain sequences were linked with internal ribosome entry site and constructed into adenoviral vector under the control of cytomegalovirus promoter. Antibody expression in vitro and in vivo were tested with ELISA, and its antitumor efficacy was evaluated in SKOV-3-inoculated nude mice. RESULTS: Ad5-TAb-generated anti-HER-2 antibody presented the similar binding specificity with commercial trastuzumab. A single i.v. injection of 2 x 10(9) plaque-forming units of Ad5-TAb per mouse resulted in not only a sustained over 40 microg/mL serum antibody level for at least 4 weeks but also significant tumor elimination in the ovarian cancer SKOV-3-inoculated nude mice. CONCLUSIONS: An in vivo full-length antibody gene delivery system allows continuous production of a full-length antibody at high concentration after a single administration. Bioactive antibody macromolecules can be generated via gene transfer in vivo. All the data suggest that this novel adenovirus-mediated antibody gene delivery can be used for the exploitation of antibodies, without being hampered by the sophisticated antibody manufacture techniques and high cost, and, furthermore, can shorten the duration and reduce the expense of antibody developments.

Cloning of idiotype immunoglobulin genes in B cell lymphomas by anchored PCR and production of individual recombinant idiotype vaccines in Escherichia coli.

OBJECTIVES: Individual immunoglobulins expressed by B-cell lymphomas represent tumor-specific antigens ('idiotypes'). Immunization with idiotype in follicular lymphoma patients may induce specific immune responses, sustained progression-free survival, and disappearance of minimal residual disease. Manufacturing of idiotype vaccines has mostly relied on heterohybridomas established from viable lymphoma cells. This paper describes the feasibility of production of GMP-grade idiotype vaccines as recombinant Fab fragments in Escherichia coli. METHODS: IgH and IgL transcripts were analyzed by anchored PCR from 106 lymphoma and nine control biopsies. Lymphoma-derived V segments were inserted into prokaryotic expression plasmids. Recombinant idiotype Fab fragments were expressed in E. coli in a fermentation system. RESULTS: Idiotype IgH and IgL transcripts were identified in 95% of 106 lymphoma biopsies according to stringent clonality criteria. Large-scale idiotype expression was successful in 69 of 78 cases (89%) and yielded a median of 17 mg (range: 1.2-250 mg) recombinant Fab protein. After affinity chromatography, median vaccine purity was 99% heterodimeric Fab protein (range: 72-100%). Bacterial protein contamination was detectable in one vaccine only. Fab proteins with IgL lambda chains had a tendency for inferior yield and lesser purity than kappa-type Fabs. Among other structural idiotype features (isotype, V family usage, somatic hypermutation pattern, novel glycosylation sites, CDR III net charge), no consistent influences on Fab yield or purity were detected. CONCLUSIONS: Anchored PCR cloning and subsequent protein expression in E. coli provides a reliable technological basis for clinical idiotype vaccination trials.

Targeting of interleukin 2 to human ovarian carcinoma by fusion with a single-chain Fv of antifolate receptor antibody.

To provide a new tool for the immunotherapy of human ovarian carcinoma, we constructed a fusion protein between interleukin-2 (IL-2) and the single-chain Fv (scFv) of MOV19, a monoclonal antibody directed against alpha-folate receptor (alpha-FR), known to be overexpressed on human nonmucinous ovarian carcinoma. This was accomplished by fusing the coding sequences in a single open reading frame and expressing the IL-2/MOV19 scFv chimera under the control of the murine immunoglobulin K promoter in J558L plasmacytoma cells. The design allowed the construction of a small molecule combining the specificity of MOV19 with the immunostimulatory activity of IL-2. This might improve the tissue penetration and distribution of the fusion protein within the tumor, reduce its immunogenicity, and avoid the toxicity related to the systemic administration of IL-2. The IL-2/MOV19 fusion protein was stable on purification from the cell supernatant and was biologically active. Importantly, this construct was able to target IL-2 onto the surface of alpha-FR-overexpressing tumor cells and stimulated the proliferation of the IL-2-dependent CTLL-2 cell line as well as that of human resting peripheral blood lymphocytes. In a syngeneic mouse model, IL-2/MOV19 scFv specifically targeted a-FR gene-transduced metastatic tumor cells without accumulating in normal tissues, due to its fast clearance from the body. Prolonged release of IL-2/MOV19 scFv by in vivo transplanted J558-EF6.1 producer cells protected 60% of mice from the development of lung metastases caused by an i.v. injection of a-FR gene-transduced tumor cells. Moreover, treatment with IL-2/MOV19 scFv, but not with recombinant IL-2, significantly reduced the volume of s.c. tumors. The pharmacokinetics and biological characteristics of IL-2/NMOV19 scFv might allow us to combine the systemic administration of this molecule with the adoptive transfer of in vitro retargeted T lymphocytes for the treatment of ovarian cancer, thereby providing local delivery of IL-2 without toxicity.

Peptide-antibody conjugates for tumour therapy: a MHC-class-II-restricted tetanus toxin peptide coupled to an anti-Ig light chain antibody can induce cytotoxic lysis of a human B-cell lymphoma by specific CD4 T cells.

Anti-idiotype antibody therapy of B-cell lymphomas, despite numerous promising experimental and clinical studies, has so far met with limited success. Tailor-made monoclonal anti-idiotype antibodies have been injected into a large series of lymphoma patients, with a few impressive complete tumour remissions but a large majority of negative responses. The results presented here suggest that, by coupling to antilymphoma idiotype antibodies a few molecules of the tetanus toxin universal epitope peptide P2 (830-843), one could markedly increase the efficiency of this therapy. We show that after 2-hr incubation with conjugates consisting of the tetanus toxin peptide P2 coupled by an S-S bridge to monoclonal antibodies directed to the lambda light chain of human immunoglobulin, human B-lymphoma cells can be specifically lysed by a CD4 T-lymphocyte clone specific for the P2 peptide. Antibody without peptide did not induce B-cell killing by the CD4 T-lymphocyte clone. The free cysteine-peptide was also able to induce lysis of the B-lymphoma target by the T-lymphocyte clone, but at a molar concentration 500 to 1000 times higher than that of the coupled peptide. Proliferation assays confirmed that the antibody-peptide conjugate was antigenically active at a much lower concentration than the free peptide. They also showed that antibody-peptide conjugates required an intact processing function of the B cell for peptide presentation, which could be selectively inhibited by leupeptin and chloroquine.(ABSTRACT TRUNCATED AT 250 WORDS)