Pre-existing Antibodies Research Articles

Abstract 4138738: Clinical Evidence of Immune Response to Transplanted Allogeneic iPS Cell-Derived Cardiomyocytes

Background&Purpose: The clinical application of cell transplantation therapy utilizing induced pluripotent stem cells (iPS cells) for heart failure is progressing, with the primary strategy being the use of pre-prepared allogeneic iPS cells. The immune response to these transplanted cells is crucial and may affect therapeutic efficacy, necessitating the use of immunosuppressive drugs. However, detailed clinical reports on immune responses are sparse. This study aims to analyze the immune response to transplanted cells in clinical iPS cell-derived cardiomyocyte (iPSC-CM) transplants and examine the correlation between immune response and therapeutic efficacy. Methods: In the "Phase 1 multicenter clinical trial of transplantation of iPSC-CMs for ischemic cardiomyopathy" (https://jrct.niph.go.jp/en-latest-detail/jRCT2053190081), conducted as the First-in-Human trial, the immune response to transplanted cells was analyzed in four cases at our hospital. Comprehensive analysis of anti-human leukocyte antigen (HLA) antibodies in serum and single-cell RNA sequencing in peripheral blood mononuclear cells was performed to investigate their relationship with therapeutic effects on cardiac function. Results: Patients received immunosuppressive drugs (tacrolimus, mycophenolate mofetil, and steroids) for 3 months post-transplantation, followed by a 1-year follow-up. In all cases, an increase in transplant cell HLA-specific antibodies was observed after discontinuing immunosuppressive drugs (Figure 1 and 2). Single-cell analysis revealed a decrease in immunocompetent cells in peripheral blood during immunosuppressive therapy. Cardiac function analysis showed improvements in all cases except Case 2 (Figure 3), where pre-existing anti-HLA-DQ antibodies were present before transplantation. Conclusions: No immune response to the transplanted cells was observed during immunosuppressive therapy. In a case with limited therapeutic response, pre-existing antibodies to transplanted cells were detected, potentially impacting the therapeutic outcome.

Lipid Nanoparticle-Mediated Liver-Specific Gene Therapy for Hemophilia B

Background/Objectives: Hemophilia B is a hereditary bleeding disorder due to the production of liver malfunctional factor IX (FIX). Gene therapy with viral vectors offers a cure. However, applications are limited due to pre-existing antibodies, eligibility for children under 12 years of age, hepatotoxicity, and excessive costs. Lipid nanoparticles are a potential alternative owing to their biocompatibility, scalability, and non-immunogenicity. However, their therapeutic applications are still elusive due to the poor transfection efficiencies in delivering plasmid DNA into primary cells and target organs in vivo. To develop efficient liver-targeted lipid nanoparticles, we explored galactosylated lipids to target asialoglycoprotein receptors (ASGPRs) abundantly expressed on hepatocytes. Methods: We developed 12 novel liposomal formulations varying the galactose lipid Gal-LNC 5, cationic lipid MeOH16, DOPE, and cholesterol. We evaluated their physicochemical properties, toxicity profiles, and transfection efficiencies in hepatic cell lines. Among the formulations, Gal-LNC 5 could efficiently transfect the reporter plasmid eGFP in hepatic cell lines and specifically distribute into the liver in vivo. Toward developing functional factor IX, we cloned Padua mutant FIX-L in a CpG-free backbone to enhance the expression and duration. Results: We demonstrated superior expression of FIX with our galactosylated lipid nanoparticle system. Conclusions: The current research presents a specialized lipid nanoparticle system viz. Gal-LNC which is a specialized lipid nanoparticle system for liver-targeted gene therapy in hemophilia B patients that has potential for clinical use. The Gal-LNC successfully delivers a CpG-free Padua FIX gene to liver cells, producing therapeutically relevant levels of FIX protein. Among its benefits are the ideal qualities of stability, targeting the liver specifically, and maximizing efficiency of transfection. Optimization of liver-targeting lipid nanoparticle systems and function FIX plasmids will pave the way for novel lipid nanoparticle-based gene therapy products for hemophilia B and other monogenic liver disorders.

RVSV-ZEBOV vaccination in people with pre-existing immunity to Ebolavirus: an open-label safety and immunogenicity study in Guinean communities affected by Ebola virus disease (l’essai proches)

BackgroundZaire Ebolavirus disease (EVD) outbreaks can be controlled using rVSV-ZEBOV vaccination and other public health measures. People in high-risk areas may have pre-existing antibodies from asymptomatic Ebolavirus exposure that might affect response to rVSV-ZEBOV. Therefore, we assessed the impact pre-existing immunity had on post-vaccination IgG titre, virus neutralisation, and reactogenicity following vaccination.MethodsIn this prospective cohort study, 2115 consenting close contacts (“proches”) of EVD survivors were recruited. Proches were vaccinated with rVSV-ZEBOV and followed up for 28 days for safety and immunogenicity. Anti-GP IgG titre at baseline and day 28 was assessed by ELISA. Samples from a representative subset were evaluated using live virus neutralisation.ResultsTen percent were seropositive at baseline. At day 28, IgG in baseline seronegative (GMT 0.106 IU/ml, 95% CI: 0.100 to 0.113) and seropositive (GMT 0.237 IU/ml, 0.210 to 0.267) participants significantly increased from baseline (both p < 0.0001). There was strong correlation between antibody titres and virus neutralisation in day 28 samples (Spearman’s rho 0.75). Vaccinees with baseline IgG antibodies against Zaire Ebolavirus had similar safety profiles to those without detectable antibodies (63.6% vs 66.1% adults experienced any adverse event; 49.1% vs 60.9% in children), with almost all adverse events graded as mild. No serious adverse events were attributed to vaccination. No EVD survivors tested positive for Ebolavirus by RT-PCR.ConclusionsThese data add further evidence of rVSV-ZEBOV safety and immunogenicity, including in people with pre-existing antibodies from suspected natural ZEBOV infection whose state does not blunt rVSV-ZEBOV immune response. Pre-vaccination serological screening is not required.

Complex immunogenicity assessment in caplacizumab-treated patients with immune-mediated thrombotic thrombocytopenic purpura who have received plasma exchange

BackgroundISTH guidelines for immune-mediated thrombotic thrombocytopenic purpura (iTTP) treatment recommend concurrent therapeutic plasma exchange (TPE), immunosuppressive therapy (IST), and caplacizumab. TPE can complicate anti-drug antibody (ADA) measurements by transferring pre-existing antibodies (pre-Abs) into patients via donor plasma and/or diluting treatment-emergent (TE) ADAs. ObjectivesTo assess the presence of ADAs in patients with iTTP who received caplacizumab. MethodsImmunogenicity data from patients with iTTP receiving caplacizumab once daily plus TPE/IST in four clinical trials (TITAN, HERCULES, Post-HERCULES, and a trial conducted in Japanese patients) in the clinical development program were analyzed. ADA and modified ADA (mADA) assays differentiated pre-Abs from TE ADAs. A functional neutralizing antibody (NAb) assay and neutralizing epitope characterization assay (NECA) assessed the presence of ADAs with neutralizing potential. The impact of ADAs on efficacy, pharmacokinetics/pharmacodynamics, and safety was evaluated. ResultsAmong 228 patients in four studies, prevalence of pre-Abs ranged from 17.1% (TITAN) to 56.7% (HERCULES), while TE ADA prevalence ranged from 3.1% (HERCULES) to 14.3% (Japanese study). The TE NAb-positive rate ranged from 0% (Japanese study) to 12% (Post-HERCULES) using the functional NAb assay and from 2.7% (Post-Hercules) to 14.3% (Japanese study) using NECA. The presence of these antibodies did not impact treatment efficacy or safety. ConclusionsA complex immunogenicity assay strategy was required to define the pre-Ab/TE ADA status of patients with iTTP treated with caplacizumab in a clinical trial setting. In addition to the wide range of pre-Abs observed, few patients had detectable TE ADAs or NAbs, neither of which impacted on efficacy/safety.

Comparative assessment of the transduction efficiency and safety associated with the delivery of AAV9-GFP vector via lumbar puncture route to juvenile Cynomolgus Macaques with and without anti-AAV9 pre-existing antibodies

Comparative assessment of the transduction efficiency and safety associated with the delivery of AAV9-GFP vector via lumbar puncture route to juvenile Cynomolgus Macaques with and without anti-AAV9 pre-existing antibodies

Characterization of anti-AAV2 neutralizing antibody levels in sheep prior to and following intravitreal AAV2.7m8 injection

AbstractGene augmentation therapy is a promising treatment for incurable, blinding inherited retinal diseases, and intravitreal delivery is being studied as a safe alternative to subretinal injections. Adeno-Associated Viruses (AAV) are commonly-used vectors for ocular gene augmentation therapy. Naturally occurring pre-operative exposure and infection with AAV could result in presence of neutralizing antibodies (NAB’s) in patients’ serum, and may affect the safety and efficacy of treatment. Our aim was to characterize the humoral response against AAV pre- and post-intravitreal delivery of AAV2.7m8 vectors in a naturally-occurring sheep model of CNGA3 achromatopsia. Serial serum neutralization assays were performed to screen sheep for pre-exiting anti-AAV2 NAB’s, and to assess the effect of intravitreal AAV2.7m8 injection on post-operative NAB titers and intraocular inflammation in sheep. The effect of viral dose and transgene type were also assessed. Serological screening revealed pre-operative seropositivity in 21.4% of animals, with age being a risk factor for the presence of anti-AAV2 NAB’s. NAB titers increased following intravitreal AAV administration in the majority of sheep. There was no significant difference in the degree of post-operative serum neutralization between pre-operatively seronegative sheep and those with pre-existing antibodies. However, only sheep with pre-existing antibodies presented with signs of post-operative inflammation. We conclude that pre-existing anti-AAV2 NAB’s do not affect the level of post-operative NAB titers; however, they increase the risk of post-operative ocular inflammation. Our results could have implications for the management of AAV-mediated ocular gene therapies, a technology being increasingly studied and used in patients.

Exploring the Contrasts and Similarities of Dengue and SARS-CoV-2 Infections During the COVID-19 Era.

Extensive research has been conducted on the SARS-CoV-2 virus in association with various infectious diseases to understand the pathophysiology of the infection and potential co-infections. In tropical countries, exposure to local viruses may alter the course of SARS-CoV-2 infection and coinfection. Notably, only a portion of the antibodies produced against SARS-CoV-2 proteins demonstrate neutralizing properties, and the immune response following natural infection tends to be temporary. In contrast, long-lasting IgG antibodies are common after dengue virus infections. In cases where preexisting antibodies from an initial dengue virus infection bind to a different dengue serotype during a subsequent infection, there is a potential for antibody-dependent enhancement (ADE) and the formation of immune complexes associated with disease severity. Both SARS-CoV-2 and dengue infections can result in immunodeficiency. Viral proteins of both viruses interfere with the host's IFN-I signaling. Additionally, a cytokine storm can occur after viral infection, impairing a proper response, and autoantibodies against a wide array of proteins can appear during convalescence. Most of the reported autoantibodies are typically short-lived. Vaccines against both viruses alter the immune response, affecting the course of viral infection and enhancing clearance. A comprehensive analysis of both viral infections and pathogenicity is revisited to prevent infection, severity, and mortality.

Plasma exchange and intravenous immunoglobulin prolonged the survival of a porcine kidney xenograft in a sensitized, deceased human recipient.

The primary limitation to kidney transplantation is organ shortage. Recent progress in gene editing and immunosuppressive regimens has made xenotransplantation with porcine organs a possibility. However, evidence in pig-to-human xenotransplantation remains scarce, and antibody-mediated rejection (AMR) is a major obstacle to clinical applications of xenotransplantation. We conducted a kidney xenotransplantation in a deceased human recipient using a porcine kidney with five gene edits (5GE) on March 25th, 2024 at Xijing Hospital, China. Clinical-grade immunosuppressive regimens were employed, and the observation period lasted 22days. We collected and analyzed the xenograft function, ultrasound findings, sequential protocol biopsies, and immune surveillance of the recipient during the observation. The combination of 5GE in the porcine kidney and clinical-grade immunosuppressive regimens prevented hyperacute rejection. The xenograft kidney underwent delayed graft function in the first week, but urine output increased later and the single xenograft kidney maintained electrolyte and pH homeostasis from postoperative day (POD) 12 to 19. We observed AMR at 24h post-transplantation, due to the presence of pre-existing anti-porcine antibodies and cytotoxicity before transplantation; this AMR persisted throughout the observation period. Plasma exchange and intravenous immunoglobulin treatment mitigated the AMR. We observed activation of latent porcine cytomegalovirus toward the end of the study, which might have contributed to coagulation disorder in the recipient. 5GE and clinical-grade immunosuppressive regimens were sufficient to prevent hyperacute rejection during pig-to-human kidney xenotransplantation. Pre-existing anti-porcine antibodies predisposed the xenograft to AMR. Plasma exchange and intravenous immunoglobulin were safe and effective in the treatment of AMR after kidney xenotransplantation.

Adverse Impacts of PEGylated Protein Therapeutics: A Targeted Literature Review.

The beneficial effects of polyethylene glycol (PEG)-conjugated therapeutics, such as increased half-life, solubility, stability, and decreased immunogenicity, have been well described. There have been concerns, however, about adverse outcomes with their use, but understanding of those adverse outcomes is still relatively limited. The present study aimed to characterize adverse outcomes associated with PEGylation of protein-based therapeutics on immunogenicity, pharmacologic properties, and safety. A targeted review of English language articles published from 1990 to September 29, 2023, was conducted. Of the 29 studies included in this review, 18 reported adverse safety outcomes such as hematologic complications, hepatic toxicity, injection site reactions, arthralgia, nausea, infections, grade 3 or 4 adverse events (AEs), and AE-related discontinuations and dose modifications. Fifteen studies reported immunogenicity-related outcomes, such as the prevalence of pre-existing antibodies to PEG, treatment-emergent antibody response, and hypersensitivity reactions to PEGylated drugs. Seven studies reported pharmacological outcomes such as increased clearance and reduced activity in response to PEGylated drugs. This review aims to contribute to a balanced view of PEGylated therapies by summarizing the adverse outcomes or lack of benefit associated with PEGylated therapeutics reported in the literature. We identified several studies characterizing adverse outcomes, pharmacological effects, and immunogenicity associated with the use of PEGylated therapeutics. Our findings suggest that using PEGylated therapeutics may require careful monitoring for adverse safety outcomes, including screening and monitoring for pre-existing antibodies and those induced in response to PEGylated therapy, as well as monitoring and adjusting the dosing of PEGylated therapeutics.

Prevalence of Antibodies against Adeno-Associated Viruses (AAVs) in Göttingen Minipigs and Its Implications for Gene Therapy and Xenotransplantation

Adeno-associated viruses (AAV) are widely used as delivery vectors in clinical trials for in vivo gene therapy due to their unique features. Göttingen minipigs are a well-established animal model for several diseases and can be used for the efficacy and safety testing of AAV-based gene therapy. Pre-existing antibodies against AAV may influence the results of testing and, therefore, the animals should be tested for the presence of antibodies against relevant AAV serotypes. The detection of AAVs in pigs may be also important for the virus safety of xenotransplantation. In this study, we screened Göttingen minipigs from Ellegaard Göttingen Minipigs A/S, Denmark, and Marshall BioResources, USA, for antibodies against AAV1, AAV2, AAV6, AAV9 serotypes. Of the 20 animals tested, 18 had no neutralizing antibodies for all AAVs tested, none had antibodies against AAV9, only one had antibodies against AAV6, and the titers of antibodies against AAV1 and AAV2 were less than 1:100, with two exceptions. For total binding IgG, more individuals showed positivity for all the tested serotypes but, in general, the levels were low or zero. Three animals had no antibodies at all against the AAVs tested. Therefore, Göttingen minipigs could be considered an attractive animal model for gene therapy studies. Since some animals were negative for all AAVs tested, these may be selected and used as donor animals for xenotransplantation.

A novel orf virus vector-based COVID-19 booster vaccine shows cross-neutralizing activity in the absence of anti-vector neutralizing immunity

ABSTRACT Next-generation COVID-19 vaccines are being developed to expand the breadth of coverage against existing and future variants and to extend the duration of protection. Prime-2-CoV_Beta is an orf virus (ORFV) based multi-antigen COVID-19 vaccine that co-expresses Spike (S) and Nucleocapsid (N) antigens. The safety and immunogenicity of Prime-2-CoV_Beta is investigated in a phase 1 first-in-human (FIH) dose-finding trial (ORFEUS study, ClinicalTrials.gov: NCT05367843). Participants of two age groups (18–55 and 65–85 years) who previously completed at least two doses of mRNA vaccines were enrolled and sequentially assigned to different dose groups to receive one intramuscular dose of 3 × 105, 3 × 106, 1.5 × 107, or 3 × 107 plaque-forming units (PFU) of Prime-2-CoV_Beta on day 1 and a second dose on day 29. Here, we report safety and immunogenicity data collected up to 6 months after the first study vaccination. Prime-2-CoV_Beta is safe and well tolerated and elicits immune responses at higher dose levels in participants aged 18–55. A single dose of 3 × 107 PFU boosted binding and cross-neutralizing antibody responses that are maintained through 6 months after the first booster vaccination. Polyfunctional S-specific CD4+ and CD8+ T cell responses are observed after vaccination. No pre-existing or vaccine-induced neutralizing anti-vector antibodies are detected. Our findings highlight the potential of the ORFV vector as a safe platform for future vaccine design, which provides the ability to deliver multiple antigens and allows for repeat immunization.

Identification of a novel neutralization epitope in rhesus AAVs

Adeno-associated viruses (AAVs) are popular gene therapy delivery vectors, but their application can be limited by anti-vector immunity. Both preexisting neutralizing antibodies (NAbs) and post-administration NAbs can limit transgene expression and reduce the clinical utility of AAVs. The development of novel AAVs will advance our understanding of AAV immunity and may also have practical applications. In this study, we identified five novel AAV capsids from rhesus macaques. RhAAV4282 exhibited 91.4% capsid sequence similarity with AAV7 and showed similar tissue tropism with slightly diminished overall signal. Despite this sequence homology, RhAAV4282 and AAV7 showed limited cross-neutralization. We determined a cryo-EM structure of the RhAAV4282 capsid at 2.57Å resolution and identified a small segment within the hypervariable region IV, involving seven amino acids that formed a shortened external loop in RhAAV4282 compared with AAV7. We generated RhAAV4282 and AAV7 mutants that involved swaps of this region and showed that this region partially determined neutralization phenotype. We termed thisregion the hypervariable region IV neutralizing epitope (HRNE). Our data suggests that modification of the HRNE can lead to AAVs with altered neutralization profiles.

IL-12 encoding oNDV synergizes with CAR-T cells in orthotopic models of non-small cell lung cancer

Systemic administration of oncolytic viruses (OVs) is a promising approach for targeting metastatic solid tumors, but their anti-tumor activity is limited by pre-existing neutralizing antibodies against common human viruses. Therefore, investigators have developed OVs derived from non-human host viruses. Successful implementation of this strategy requires that the viral vector selectively infects and replicates within human cancer cells. Newcastle disease virus (NDV) is an avian paramyxovirus that as NDV-based OVs (oNDVs) have demonstrated safety and activity against multiple human tumors in clinical trials. Their use as a single agent, however is insufficient to cure tumors. Similarly, CAR-T cells enable systemic targeting of cancer cells but have limited anti-tumor effects against bulky solid tumors, in part due to the immunosuppressive tumor environment. In this study, we evaluated the anti-tumor effects of combining systemic oNDV and CAR-T cell treatments. In models of non-small cell lung carcinoma (NSCLC), we found that oNDV itself and IL-12 derived from oNDV enhance HER2-directed CAR-T cell anti-tumor activity and persistence in vitro and in vivo, leading to superior control of NSCLC tumors compared with either agent alone in vivo. Our data indicate that oNDV enhances the anti-tumor effects of HER2.CAR-T cells, thus controlling the growth of orthotopic NSCLC tumors.

Neonatal SARS-CoV-2 mRNA Vaccination Efficacy Is Influenced by Maternal Antibodies.

Vaccination in pregnancy guards against infection. Maternal antibodies, however, can inhibit antibody production in neonates. We sought to determine the effects of maternal vaccination on neonatal immune response to a SARS-CoV-2 mRNA vaccine. We hypothesized that mRNA-lipid nanoparticles (LNP) vaccination allows for a de novo neonatal antibody response even in the presence of vertically transmitted maternal antibodies. Female mice were vaccinated with SARS-CoV-2 spike receptor binding domain (RBD) mRNA-LNPs. Mice were then bred, and 21-day-old pups were inoculated with the same mRNA-LNPs. Spike-specific IgG ELISAs were performed using mouse serum. A SARS-CoV-2 spike protein peptide library to perform peptide ELISAs characterized high affinity binding domains within the spike protein. Results were analyzed with one-way ANOVAs with Tukey's multiple comparisons tests. Compared to pups of unvaccinated dams, there were high levels of spike-specific IgG detected in the pups of vaccinated dams at 3 weeks of life (p < 0.0001). After neonatal vaccination, pups of unvaccinated dams had higher spike-specific serum IgG than pups of vaccinated dams at 12 weeks of life (p < 0.001). Antibody specificity to peptide moieties within spike RBD were similar when comparing a vaccinated dam to her pup at Week 3 of life, with different binding affinities observed in the pups by Week 15 of life. Pre-existing maternal antibodies may partially blunt the initial neonatal antibody response to mRNA-LNPs vaccination. This vaccine strategy, however, does not prohibit the subsequent development of a broad range of RBD antibody specificities that may be protective.

Reviewing The Latest Treatments for Haemophilia

Abstract: Advancements in haemophilia treatment have been significant in the first 20 years of the 21st century. However, the progress started with the fractionation of plasma in 1946. The first concentrates were developed after discovering FVIII in frozen plasma cryoprecipitate and FIX in the supernatant in the early 1960s, leading to initial attempts at replacement therapy. Unfortunately, due to the lack of screening methods for viral pathogens, people with haemophilia (PWH) received contaminated concentrates, leading to infections with the hepatitis A virus, hepatitis C virus, and human immunodeficiency virus. Thankfully, by 1985, viral screening methods and virucidal techniques were developed, making concentrates safe. The introduction of chromatography steps with monoclonal antibodies in the production process led to increasingly pure products. However, the problem of immunogenicity of administered concentrates persists. The development of alloantibodies against FVIII in a significant percentage of PWH is a serious adverse effect of replacement therapy. The next major advancement came with cloning the F8 and F9 genes, which allowed the production of factor concentrates using recombinant DNA technology. The injected FVIII and FIX molecules have a relatively short circulating half-life in the plasma of people with haemophilia A and B, approximately 12 and 18 hours, respectively. Methods such as conjugating the factor molecule with fragment crystallizable of IgG1, albumin, or adding polyethylene glycol have been applied to prolong the plasma half-life and extend the interval between injections, especially for risk. The next frontier in haemophilia therapy is the development of durable and potentially curative treatments, such as gene addition therapy. Experiments in haemophilia B have shown promising long-lasting responses. However, the results of gene therapy for haemophilia A have not been as remarkable, and the durability of the treatment is yet to be demonstrated. The long-term safety, predictability, durability, and efficacy of gene therapy for haemophilia A and B remain open questions. Currently, only healthy adult PWH has been enrolled in gene therapy clinical trials, and further studies are needed before gene therapy can be widely applied to children and those with pre-existing antibodies against the delivery vector.

Determining the Degree of Sulfo-tag Conjugation to AAV5 Vectors by LC-HRMS and Evaluating the Effects on Antibody Binding Affinity and Bridging Assay Sensitivity.

Pre-existing anti-AAV antibodies can be detected using ligand binding-based assay formats. One such format is the MSD-based bridging assay, which uses sulfo-tag-labeled AAV vectors as detection reagents. However, no method has been developed to accurately measure the degree of sulfo-tag labeling on AAV vectors. To fill this gap, we developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method to assess the degree of labeling (DoL) of sulfo-tag on AAV5 vectors, enabling the measurement of the DoL on AAV5 at six increasing levels of sulfo-tag challenge ratio. In addition, a Biacore-based assay was used to evaluate the binding affinity between an anti-AAV5 monoclonal antibody and various sulfo-tag labeled AAV5 vectors. The results indicated that increased DoL of sulfo-tag labeling on AAV5 did not compromise the binding affinity.Our study further employed the MSD-bridging assay to detect the binding Signal/Noise (S/N) ratios of four anti-AAV5 monoclonal antibodies (mAbs) to various sulfo-tag-labeled AAV5 vectors. The findings revealed a strong correlation between the degree of sulfo-tag labeling and both the S/N ratios and the sensitivity of MSD bridging assays. This result underscores the importance of optimizing the labeling of detection reagents to enhance assay sensitivity for detecting anti-AAV5 antibodies.

Rocuronium-specific antibodies drive perioperative anaphylaxis but can also function as reversal agents in preclinical models.

Neuromuscular blocking agents (NMBAs) relax skeletal muscles to facilitate surgeries and ease intubation but can lead to adverse reactions, including complications because of postoperative residual neuromuscular blockade (rNMB) and, in rare cases, anaphylaxis. Both adverse reactions vary between types of NMBAs, with rocuronium, a widely used nondepolarizing NMBA, inducing one of the longest rNMB durations and highest anaphylaxis incidences. rNMB induced by rocuronium can be reversed by the synthetic γ-cyclodextrin sugammadex. However, in rare cases, sugammadex can provoke anaphylaxis. Thus, additional therapeutic options are needed. Rocuronium-induced anaphylaxis is proposed to rely on preexisting rocuronium-binding antibodies. To understand the pathogenesis of rocuronium-induced anaphylaxis and to identify potential therapeutics, we investigated the memory B cell antibody repertoire of patients with suspected hypersensitivity to rocuronium. We identified polyclonal antibody repertoires with a high diversity among V(D)J genes without evidence of clonal groups. When recombinantly expressed, these antibodies demonstrated specificity and low affinity for rocuronium without cross-reactivity for other NMBAs. Moreover, when these antibodies were expressed as human immunoglobulin E (IgE), they triggered human mast cell activation and passive systemic anaphylaxis in transgenic mice, although their affinities were insufficient to serve as reversal agents. Rocuronium-specific, high-affinity antibodies were thus isolated from rocuronium-immunized mice. The highest-affinity antibody was able to reverse rocuronium-induced neuromuscular blockade in nonhuman primates with kinetics comparable to that of sugammadex. Together, these data support the hypothesis that antibodies cause anaphylactic reactions to rocuronium and pave the way for improved diagnostics and neuromuscular blockade reversal agents.

Positive rates and distribution characteristics of pre-existing anti-human-leukocyte-antigen antibodies in patients with different hematological diseases

Clinical data of 1 494 patients with hematological diseases who were scheduled to receive allogeneic hematopoietic stem cell transplantation and received the anti-human-leukocyte-antigen (HLA) antibody test for the first time at the First Affiliated Hospital of Soochow University from 2016 to 2018 was collected to analyze the positive rates and distribution characteristics of different types of pre-existing anti-HLA antibodies in patients with different hematological diseases. Among 1 494 patients with hematological diseases, there were 849 males and 645 females, aged [31 (17, 45)] years, and included 577 cases of acute myeloid leukemia (AML), 373 cases of acute lymphocytic leukemia (ALL), 234 cases of aplastic anemia (AA), 175 cases of myelodysplastic syndrome (MDS), and 135 cases of other diseases. The total positive rate of pre-existing anti-HLA antibodies was 25.1% (375/1 494), among which the positive rates of anti-HLA class Ⅰ, anti-HLA class Ⅱ, and anti-HLA class Ⅰ+Ⅱ antibodies were 11.2% (168/1 494), 4.9% (73/1 494), and 9.0% (134/1 494), respectively.The total positive rates of pre-existing anti-HLA antibodies in patients with MDS、AA、AML、ALL and other diseases were 40.6% (71/175), 30.8% (72/234), 26.2% (151/577), 12.3% (46/373), and 25.9% (35/135), respectively, with statistically significant difference (P<0.001). The positive rates of anti-HLA class Ⅰ, anti-HLA class Ⅱ, and anti-HLA class Ⅰ+Ⅱ antibodies in patients with different hematological diseases showed statistically significant differences (all P<0.001). Given the varying positive rates and distribution characteristics of pre-existing anti-HLA antibodies among patients with different hematological diseases, anti-HLA antibody test should be performed before receiving hematopoietic stem cell transplantation.

Endocrine Adverse Events Related To Immune Checkpoint Inhibitor Treatment: Relationship Between Antibodies and Severity of Thyroid Dysfunction.

The purpose of this study was to identify predictive and risk factors for the development of immune-related endocrinopathies and to analyze the incidence and characteristics of immune-related endocrinopathies in our population. A retrospective, single-centre cohort carried out at Gregorio Marañón Hospital between January 2018 -December 2019. A total of 163 patients were enrolled. In January 2018 and December 2019, we treated patients who underwent ICI treatment in the Medical Oncology Department of General University Hospital Gregorio Marañón, a tertiary care public hospital in Madrid, as part of an observational, retrospective, single-center cohort study. Endocrinopathies were diagnosed in 19.5% of the patients (n = 32). The tumours with the highest incidence of endocrinopathies were non-small cell lung cancer (25,9%), kidney cell cancer (25%) and hepatocarcinoma (20%). Among the 32 patients who developed endocrinopathy, 18,8%, 19,13%, and 21,28% received anti-CTLA-4, anti-PD-1 and anti-PDL-1, respectively. Thyroid dysfunction was the most frequent endocrinopathy (12,8%). A higher percentage of patients with negative antiTPO and antiTG antibodies developed G1 hypothyroidism compared to patients with positive antibodies who developed a higher proportion of G2 hypothyroidism. The presence of an initial phase of thyrotoxicity was not related to greater severity. We observed longer progression-free survival in patients who developed thyroid dysfunction. Pre-existing antibodies were independently associated with endocrinopathies. Moreover, our study let us conclude that the presence of thyroid autoantibodies may be related to its severity. It is important to determine anti-thyroid antibodies prior to the start of immunotherapy as a risk factor for thyroid dysfunction, which in turn is a prognostic marker.

Advances in immunotoxin engineering: precision therapeutic strategies in modern oncology.

Immunotoxins (ITs) are specialized therapeutic agents designed for targeted treatment, particularly in cancer therapy. They consist of a monoclonal antibody or antibody fragment linked to a potent cytotoxic agent, such as bacterial- or plant-derived toxins like diphtheria toxin, ricin, or pseudomonas exotoxin. The monoclonal antibody component specifically binds to antigens expressed on the surface of target cells, facilitating the internalization of the IT. Once inside the cell, the cytotoxic agent is released, disrupting essential cellular processes and leading to cell death. This targeted approach minimizes damage to healthy tissues while effectively eliminating diseased cells. The production of ITs involves two primary methods: recombinant fusion and chemical conjugation. In recombinant fusion, genetic engineering is used to create a fusion protein that combines the antibody and toxin, ensuring precise control over their ratio and functionality. In chemical conjugation, pre-existing antibodies are chemically linked to toxins, allowing for greater flexibility in combining different antibodies and cytotoxic agents. Each method has its advantages and challenges, influencing the specificity, production complexity, and therapeutic potential of the resulting ITs. As research advances, ITs continue to show promise not only in oncology but also in treating other diseases, including inflammatory conditions and atherosclerosis. The precise targeting and potent effects of ITs make them a valuable tool in the development of new therapeutic strategies.