Monoclonal Ab Research Articles

1899. The magnitude and durability of the antibody response to mRNA-based vaccination among SARS-CoV-2 seronegative and seropositive healthcare personnel

Abstract Background Development of robust vaccination guidelines against SARS-CoV-2 requires an understanding of the longitudinal antibody (Ab) response to vaccination and interactions with natural infection. Here, we leveraged an observational cohort study of healthcare personnel (HCP) to study the impact of prior SARS-CoV-2 infection on Ab binding and neutralization after mRNA-based vaccination over a 13 month period. Methods From July 2020 to February 2022, HCP at an academic medical center provided blood samples biweekly for 12 weeks and monthly thereafter. First and second vaccine doses became available in mid-December 2021 and boosters were available starting in October 2021. Individuals were excluded if they did not provide any samples, if baseline serostatus was unknown, and if they received a monoclonal Ab treatment for COVID. ELISA measured total immunoglobulin (Ig) and IgG binding to SARS-CoV-2 RBD. Neutralization was measured by live virus Nanoluc SARS-CoV-2ic assay. Demographics, serostatus, and vaccinations for the total study population and the sub-sample of participants with pre- and post-vaccination antibody measurements. Results Of 213 participants, 192 met inclusion criteria. A majority had detectable IgG levels 8 months after a second dose. Prior to vaccination, median total Ig was higher among seropositive vs. seronegative participants (3.7 vs 1.0, p< 0.001). After a first dose, the median total Ig response was two-fold higher in seropositive compared to seronegative participants (13.8 vs. 7.0, p=0.009). A similar pattern was noted with IgG binding and neutralization. After the second dose, median IgG increased to similar levels in both seropositive and seronegative participants (22.1 vs. 21.2, p=0.8). Neutralization after the second dose was slightly higher in seropositive vs. seronegative participants (log10 3.1 vs. 2.5, p=0.075). Durability of IgG responses after second dose of mRNA-based vaccination against SARS-CoV-2 IgG P/N measurements after 5 days post-V2 for the entire study cohort (incident seropositive: yellow circles, prevalent seropositive (red circles), seronegative (open circles) are shown. The solid lines represent Loess curves for incident and prevalent seropositive participants combined (orange line) and those who were seronegative (grey line). SARS-CoV-2-specific total Ig and IgG subtype responses among healthcare personnel before and after vaccination against SARS-CoV-2 with an mRNA-based vaccine. Total Ig P/N ratios at pre-vaccine, post-V1, post-vaccine 2 (post-V2), and post-booster dose (post-boost) timepoints by serostatus (seronegative: n(-), seropositive: n(+)) are shown in the left panel. For the pre-vaccine time point, the most recent antibody level prior to vaccination (for those who were vaccinated) or most recent antibody level overall (for those who were not vaccinated) is shown. For the post-vaccine time points, the first measurement after 5 days post-vaccination is included. Individuals who were infected with SARS-CoV-2 at any time after the first vaccine dose are shown as open circles with black outlines. The black numbers next to the circles indicate the number of days between vaccination and sample collection for seropositive individuals. SARS-CoV-2 specific IgG P/N ratios respectively at pre-vaccine, post-V1, post-V2, and post-boost timepoints by serostatus (seronegative: n(-), seropositive: n(+)) are shown in the right panel. One individual tested positive for SARS-CoV-2 by PCR shortly after the second vaccine dose (V2); post-V2 results were excluded for this participant. For the pre-vaccine time point, the most recent antibody level prior to vaccination (for those who were vaccinated) or most recent antibody level overall (for those who were not vaccinated) is shown. For the post-vaccine time points, the first measurement after 5 days post-vaccination is included. The dotted line is a P/N ratio of 2.4, the cut-off associated with 99.3% specificity (SARS-CoV-2 IgG-positive above the line, IgG-negative below). Individuals who were infected with SARS-CoV-2 at any time after the first vaccine dose are shown as open circles with black outlines. The black numbers next to the circles indicate the number of days between vaccination and sample collection for seropositive individuals. SARS-CoV-2 D614G live virus neutralization among healthcare personnel by serostatus prior to vaccination. Example neutralization curves are shown in Panel A. Panel B shows the SARS-CoV-2 D614G live virus neutralization titers displayed as EC50 for seropositive (prevalent and incident) individuals and a subset of seronegative individuals. Samples for seronegative individuals were selected by matching on age and time between vaccination and sample collection to the samples from seropositive individuals. Conclusion Antibody responses after SARS-CoV-2 vaccination persist up to 1 year with wide individual variability. Though prior infection was associated with greater Ab responses after a first dose, it did not significantly modify responses after second and third doses. Still, we observed overall slightly higher Ab levels among individuals that had a prior infection before any one of the 3 doses of vaccine. These results suggest that immunity against SARS-CoV-2 prior to vaccination has a role in initial response but does not significantly modify circulating Ab titers after multiple doses of vaccination. Disclosures All Authors: No reported disclosures.

Hevin/Sparcl1 drives pathological pain through spinal cord astrocyte and NMDA receptor signaling.

High endothelial venule protein/SPARC-like 1 (hevin/Sparcl1) is an astrocyte-secreted protein that regulates synapse formation in the brain. Here we show that astrocytic hevin signaling plays a critical role in maintaining chronic pain. Compared with WT mice, hevin-null mice exhibited normal mechanical and heat sensitivity but reduced inflammatory pain. Interestingly, hevin-null mice have faster recovery than WT mice from neuropathic pain after nerve injury. Intrathecal injection of WT hevin was sufficient to induce persistent mechanical allodynia in naive mice. In hevin-null mice with nerve injury, adeno-associated-virus-mediated (AAV-mediated) re-expression of hevin in glial fibrillary acidic protein-expressing (GFAP-expressing) spinal cord astrocytes could reinstate neuropathic pain. Mechanistically, hevin is crucial for spinal cord NMDA receptor (NMDAR) signaling. Hevin-potentiated N-Methyl-D-aspartic acid (NMDA) currents are mediated by GluN2B-containing NMDARs. Furthermore, intrathecal injection of a neutralizing Ab against hevin alleviated acute and persistent inflammatory pain, postoperative pain, and neuropathic pain. Secreted hevin that was detected in mouse cerebrospinal fluid (CSF) and nerve injury significantly increased CSF hevin abundance. Finally, neurosurgery caused rapid and substantial increases in SPARCL1/HEVIN levels in human CSF. Collectively, our findings support a critical role of hevin and astrocytes in the maintenance of chronic pain. Neutralizing of secreted hevin with monoclonal Ab may provide a new therapeutic strategy for treating acute and chronic pain and NMDAR-medicated neurodegeneration.

Preliminary Dose-Escalation Results from a Phase 1/2 Study of GEN3014 (HexaBody®-CD38) in Patients (pts) with Relapsed or Refractory Multiple Myeloma (RRMM)

Background: CD38-targeted antibodies (Abs) are foundational treatments (Tx) for pts with newly diagnosed or RRMM. GEN3014 (HexaBody®-CD38) is a novel human IgG1 anti-CD38 monoclonal Ab (mAb) with an E430G hexamerization-enhancing mutation that facilitates highly efficient complement-dependent cytotoxicity (CDC). GEN3014 showed preclinical tumor cell killing through highly potent CDC (more efficient than daratumumab) and Ab-dependent cellular cytotoxicity and phagocytosis. We present preliminary data from dose escalation in the first-in-human, phase 1/2 trial of GEN3014 in pts with RRMM (NCT04824794). Methods: Adults with RRMM were enrolled if they had ≥3 prior lines of Tx including a proteasome inhibitor (PI) and an immunomodulatory imide drug (IMiD), were double refractory to a PI and IMiD, or had ≥2 prior lines of Tx if 1 combined a PI and IMiD. GEN3014 was tested at 6 dose levels ranging from 0.2 to 24 mg/kg and administered intravenously in 28-d cycles (first dose split equally between D1 and D2 of cycle 1, then full doses QW through cycle 2; Q2W, cycles 3-6; Q4W, cycles ≥7). Primary objectives were to determine the recommended phase 2 dose (RP2D) and maximum tolerated dose. Other endpoints included pharmacokinetic (PK) and pharmacodynamic (PD) characterization. PD biomarker analysis included immunophenotyping and quantification of complement factors and cytokines in peripheral blood. Results: As of July 10, 2022, 24 pts with RRMM were treated (median age, 65 y; range, 45-84). Pts were heavily pretreated with a median of 7 prior lines of Tx (range, 3-13). Most pts (67%) had prior exposure to a daratumumab- or isatuximab-containing regimen, with 8 pts naive to anti-CD38 mAb. GEN3014 was well tolerated; the most common Tx-emergent AEs were infusion-related reactions (IRRs; 75.0%), neutropenia (62.5%), anemia (33.3%), diarrhea (33.3%), pyrexia (25.0%), and thrombocytopenia (25.0%). IRRs were mostly low grade (G; 62.5% G1-2, 12.5% G3, no G4), occurred mainly during the first infusion, and were manageable. There were no Tx-related deaths or dose-limiting toxicities observed at doses up to 16 mg/kg, the RP2D. Disease progression led to discontinuation in 80% of pts. Among 19 response-evaluable pts (5 naive to anti-CD38 mAb, 14 refractory to anti-CD38 mAb), preliminary antitumor activity was seen. In pts naive to anti-CD38 mAb, 2 pts achieved very good partial response (1 each at 4 and 24 mg/kg); 1 achieved minimal response (MR; 16 mg/kg). In pts refractory to anti-CD38 mAb, 2 pts achieved MR (1 each at 8 and 16 mg/kg). Five pts remained on Tx (4 naive and 1 refractory to anti-CD38 mAb). Updated data will be presented. Peak GEN3014 concentrations at the end of infusion increased roughly proportionally with dose. The interval area under the concentration-time curve showed a more than proportional increase with doses ≤8 mg/kg but a proportional or less than proportional increase at doses >8 mg/kg. These results suggest target-mediated drug disposition at lower doses and a high degree of target saturation at >8 mg/kg. Trough concentrations increased with weekly administration, most notably in pts responding to Tx, indicating target cell depletion over time. GEN3014 Tx was associated with a rapid, sustained decrease in peripheral blood natural killer cells at all doses in all pts (mean 92.4% ± 9.2% peak reduction from baseline [BL], n=21). T cells transiently decreased after administration of first doses ≥4 mg/kg, followed by expansion (≥100% increase from BL), particularly in pts naive to anti-CD38 mAb. GEN3014 induced transient reduction in complement component C2 (mean 58% ± 18% peak reduction from BL, n=18) and total complement lytic activity (CH50; mean 48% ± 24% peak reduction from BL for ≥8 mg/kg, n=11) at all evaluable doses, suggesting CDC. Complement parameters rapidly returned to BL, indicating that Tx does not exhaust complement. Plasma cytokine (IL-2, IL-6, IL-8, IL-10, IFNγ, and TNFα) levels generally remained low after Tx. Conclusions: Dose-escalation results show GEN3014 had a tolerable safety profile and clinical activity in pts with RRMM, including pts with prior anti-CD38 mAb. The PK profile is characterized by target-mediated drug disposition at lower doses and signs of saturation at doses >8 mg/kg. Biomarker analyses confirm biological activity in pts with RRMM at all evaluated doses. The expansion part of the trial evaluating the 16 mg/kg RP2D is ongoing.

A Randomized, Placebo-Controlled, Phase 1 Study in Healthy Adult Volunteers of the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of ALPN303, a Potent Dual BAFF/APRIL Antagonist for the Treatment of Autoimmune Cytopenias

Background B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) are tumor necrosis factor superfamily (TNFSF) members that bind transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI), B-cell maturation antigen, and/or BAFF receptor (BAFF-R) on B cells and together support B-cell development, survival, and differentiation into antibody-secreting cells (ASC). In preclinical studies, co-neutralization of BAFF and APRIL can directly suppress ASC and reduce circulating immunoglobulins. Autoimmune hemolytic anemia (AIHA) and immune thrombocytopenic purpura (ITP) are diseases characterized by autoantibodies directed against red blood cells and platelets, respectively. Significantly higher levels of both BAFF and APRIL have been observed in the serum of patients with AIHA and ITP compared to healthy subjects, and polymorphisms in BAFF and TACI have been associated with ITP [Abdel-Hamid, et al., Am J Med, 2011; Emmerich, et al., Br J Haematol, 2007; Peng, et al., J Thromb Haemost, 2017; Yu, et al., Blood Adv, 2021]. Currently, there are no B cell targeting agents approved to treat AIHA or ITP. Rituximab, an anti-CD20 therapeutic which depletes CD20+ B cells, and thereby only indirectly reduces autoantibody production, has demonstrated some efficacy in both ITP [Neunert, et al., Blood, 2019] and AIHA [Jäger, et al., Blood Rev, 2019], though it is not approved for use in either indication. Belimumab, an anti-BAFF neutralizing antibody (Ab), has demonstrated encouraging efficacy in the treatment of ITP associated with systemic lupus erythematosus (SLE) [De Marchi, et al., Clin Exp Rheumatol, 2017] and also in combination with rituximab in patients with ITP [Mahévas, et al., Haematologica, 2021], but only had a modest impact on hematological manifestations in SLE patients [Manzi, et al., Ann Rheum Dis, 2012]. To address the shortcomings of existing B-cell-targeting agents, we have developed ALPN-303, an Fc fusion of an engineered TACI variant TNFRSF domain, which substantially improves upon the ligand affinity liabilities of wild type (WT) TACI, resulting in highly potent dual BAFF/APRIL inhibition superior to WT TACI-Fc, or to BAFF- or APRIL-specific monoclonal Abs. In preclinical studies, ALPN-303 demonstrates enhanced pharmacokinetic (PK) and immunomodulatory properties vs. WT TACI-Fc, which may translate to lower and/or less frequent doses in humans. ALPN-303 also suppresses autoantibodies and nephritis in mouse models of lupus. Taken together, these observations suggest that a potent dual BAFF/APRIL antagonist such as ALPN-303, by lowering pathogenic autoantibody levels, may be particularly beneficial in the treatment of antibody-related hematologic diseases, such as autoimmune cytopenias. Methods In this first-in-human study (NCT05034484), healthy adult volunteers have been enrolled in single ascending dose cohorts of intravenously (IV) or subcutaneously (SC) administered ALPN-303. For each IV cohort, the first 2 sentinel subjects are randomized 1:1 to receive ALPN-303 or placebo, followed by the remaining 4 subjects randomized 3:1 to receive ALPN-303 or placebo. SC cohorts are randomized 4:2 to receive a single SC dose of ALPN-303 or placebo. All subjects are followed to assess safety, PK, and pharmacodynamic (PD) effects including impacts on circulating immunoglobulins (Ig), and peripheral B-cell populations. Results ALPN-303 has been well tolerated in all cohorts evaluated to date and exhibits dose-dependent PK and expected PD effects, including dose-dependent reductions in circulating CD38+ (CD19+CD27+IgD-) ASC (Figure 1) and in serum Ig (Figure 2). To date, there have been no serious adverse events, and no events of cytokine release syndrome. Dose escalation will be complete by the time of the meeting; the presentation will include all available safety, PK, and PD data. Conclusions In this phase 1 study, ALPN-303 demonstrates acceptable preliminary safety and tolerability and exhibits dose-dependent PK and expected PD effects on circulating Ig and B-cell populations. These findings support future clinical development of ALPN-303 in patients with antibody-related diseases, including autoimmune cytopenias. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Peripheral smear reckons the nature of IgM cold agglutinins: A case‐based study asserting the role of thermal amplitude and clonality

Peripheral smear reckons the nature of IgM cold agglutinins: A case‐based study asserting the role of thermal amplitude and clonality

Development of an Anti-Zika and Anti-Dengue IgM ELISA Assay: Evaluation of Cross Reactivity and Validation.

Zika and dengue viruses (ZIKV and DENV) have been considered major global threats to humans in the past decade. The two infections display similar epidemiological and clinical manifestations. They are transmitted by the same primary vector, accounting for the co-circulation of the two viruses in regions where they are endemic. Highly specific and sensitive serological assays that are able to detect ZIKV and DENV antibodies (Abs) during the acute and convalescent phases of infections would help to improve clinical management and disease control. We report the development and characterisation of two monoclonal Abs, the ZIKV 8-8-11 and the DENV 8G2-12-21, which recognise the Zika non-structural protein 1 (NS1) and the dengue virus type 2 envelope protein, respectively. Both mAbs were used to set up enzyme-linked immunosorbent assays (ELISAs) specific for the detection of anti-Zika immunoglobulin M (IgM) and anti-dengue IgM and whose performance was similar to commercially available kits. These kits, intended to be used with the CHORUS Instruments, are rapid and require ≤ 50 µL of human serum. These tests could represent an affordable and reliable option for the rapid diagnosis of both ZIKV and DENV infections in developing countries, where these flaviviruses are endemic.

Antibody protection from SARS-CoV-2 respiratory tract exposure and infection

The COVID-19 pandemic has underscored the need to understand the dynamics of SARS-CoV-2 respiratory infection and protection provided by the immune response. SARS-CoV-2 infections are characterized by a particularly high viral load, and further by the small number of inhaled virions sufficient to generate a high viral titer in the nasal passage a few days after exposure. SARS-CoV-2 specific antibodies (Ab), induced from vaccines, previous infection, or inhaled monoclonal Ab, have proven effective against SARS-CoV-2 infection. Our goal in this work is to model the protective mechanisms that Ab can provide and to assess the degree of protection from individual and combined mechanisms at different locations in the respiratory tract. Neutralization, in which Ab bind to virion spikes and inhibit them from binding to and infecting target cells, is one widely reported protective mechanism. A second mechanism of Ab protection is muco-trapping, in which Ab crosslink virions to domains on mucin polymers, effectively immobilizing them in the mucus layer. When muco-trapped, the continuous clearance of the mucus barrier by coordinated ciliary propulsion entrains the trapped viral load toward the esophagus to be swallowed. We model and simulate the protection provided by either and both mechanisms at different locations in the respiratory tract, parametrized by the Ab titer and binding-unbinding rates of Ab to viral spikes and mucin domains. Our results illustrate limits in the degree of protection by neutralizing Ab alone, the powerful protection afforded by muco-trapping Ab, and the potential for dual protection by muco-trapping and neutralizing Ab to arrest a SARS-CoV-2 infection. This manuscript was submitted as part of a theme issue on “Modelling COVID-19 and Preparedness for Future Pandemics”.

Reevaluation of antibody-dependent enhancement of infection in anti-SARS-CoV-2 therapeutic antibodies and mRNA-vaccine antisera using FcR- and ACE2-positive cells

Many therapeutic antibodies (Abs) and mRNA vaccines, both targeting SARS-CoV-2 spike protein (S-protein), have been developed and approved in order to combat the ongoing COVID-19 pandemic. In consideration of these developments, a common concern has been the potential for Ab-dependent enhancement (ADE) of infection caused by inoculated or induced Abs. Although the preventive and therapeutic effects of these Abs are obvious, little attention has been paid to the influence of the remaining and dwindling anti-S-protein Abs in vivo. Here, we demonstrate that certain monoclonal Abs (mAbs) approved as therapeutic neutralizing anti-S-protein mAbs for human usage have the potential to cause ADE in a narrow range of Ab concentrations. Although sera collected from mRNA-vaccinated individuals exhibited neutralizing activity, some sera gradually exhibited dominance of ADE activity in a time-dependent manner. None of the sera examined exhibited neutralizing activity against infection with the Omicron strain. Rather, some ADE of Omicron infection was observed in some sera. These results suggest the possible emergence of adverse effects caused by these Abs in addition to the therapeutic or preventive effect.

Role of HLA-I Structural Variants and the Polyreactive Antibodies They Generate in Immune Homeostasis.

Cell-surface HLA-I molecules consisting of β2-microglobulin (β2m) associated heavy chains (HCs), referred to as Face-1, primarily present peptides to CD8+ T-cells. HCs consist of three α-domains, with selected amino acid sequences shared by all alleles of all six isoforms. The cell-surface HLA undergoes changes upon activation by pathological conditions with the expression of β2m-free HCs (Face-2) resulting in exposure of β2m-masked sequences shared by almost all alleles and the generation of HLA-polyreactive antibodies (Abs) against them. Face-2 may homodimerize or heterodimerize with the same (Face-3) or different alleles (Face-4) preventing exposure of shared epitopes. Non-allo immunized males naturally carry HLA-polyreactive Abs. The therapeutic intravenous immunoglobulin (IVIg) purified from plasma of thousands of donors contains HLA-polyreactive Abs, admixed with non-HLA Abs. Purified HLA-polyreactive monoclonal Abs (TFL-006/007) generated in mice after immunizing with Face-2 are documented to be immunoregulatory by suppressing or activating different human lymphocytes, much better than IVIg. Our objectives are (a) to elucidate the complexity of the HLA-I structural variants, and their Abs that bind to both shared and uncommon epitopes on different variants, and (b) to examine the roles of those Abs against HLA-variants in maintaining immune homeostasis. These may enable the development of personalized therapeutic strategies for various pathological conditions.

A tetravalent TREM2 agonistic antibody reduced amyloid pathology in a mouse model of Alzheimer's disease.

Triggering receptor expressed on myeloid cells 2 (TREM2) plays crucial roles in Alzheimer's disease (AD) by regulating microglia migration toward, and phagocytosis of oligomeric amyloid-β (oAβ) and amyloid plaques. Studies in rodent models of AD have shown that mice with increased TREM2 expression have reduced amyloid pathology. Here, we identified a TREM2 agonist monoclonal Ab (Ab18) by panning a phage-displayed single-chain variable fragment Ab library. By engineering the bivalent immunoglobulin G1 (IgG1) to tetra-variable domain immunoglobulin (TVD-Ig), we further increased the TREM2 activation by 100-fold. Stronger TREM2 activation led to enhanced microglia phagocytosis of the oAβ-lipid complex, migration toward oAβ, and improved microglia survival in vitro. Mechanistic studies showed increased TREM2 clustering on microglia by the tetravalent Ab18 TVD-Ig without altering microglial TREM2 amount. An engineered bispecific Ab targeting TREM2 and transferrin receptor (TfR; Ab18 TVD-Ig/αTfR) improved Ab brain entry by more than 10-fold with a broad brain parenchyma distribution. Weekly treatment of 5XFAD mice (a model of AD) with Ab18 TVD-Ig/αTfR showed a considerable reduction of amyloid burden with increased microglia migration to and phagocytosis of amyloid plaques, improved synaptic and neuronal marker intensity, improved cognitive functions, reduced endogenous tau hyperphosphorylation, and decreased phosphorylated neurofilament H immunostaining. This study demonstrated the feasibility of engineering multivalent TREM2 agonistic Ab coupled with TfR-mediated brain delivery to enhance microglia functions and reduce amyloid pathology in vitro and in vivo. This Ab engineering approach enables the development of effective TREM2-targeting therapies for AD.

The Construction of Immunoliposomes (Review)

Introduction. Due to the discovery of antibodies (Ab) targeting molecule with high specificity to the ligand, the "magic bullet" concept has been successfully implemented with various immunoconjugated drugs. Since 1980, Ab conjugates with liposomes, i.e., immunoliposomes (ILs), have been widely investigated to improve the specificity and efficacy of drug therapy. This review is devoted to the characteristic of the basic structural units of ILs on the basis of data analysis of original and review articles on the topic from PubMed, ResearchGate and CyberLeninck databases.Text. ILs are liposomes to which Ab, their fragments or other ligands are usually attached by a special linker. ILs are used to deliver antitumor, cardiovascular, antiviral, antiprotozoal drugs, genetic material, imaging molecules, etc. ILs can be derived from various phospholipids of both natural and synthetic origin, charged or neutral. The most widely used phospholipids in immunoliposomal construction are phosphatidylcholines. To increase the mechanical stability of the bilayer, sterols are introduced into the lipid composition. For selective liposome delivery, targeting ligands must be attached to the nanocarrier via the spacer arm of the PEG. Several types of end-group functionalized lipopolymers are used for this purpose, usually of the general formula X-PEG-LI, where X represents a fragment containing a reactive functional group − maleimide, biotin, cyanur, amine, etc. These lipid PEG-conjugates exhibit excellent amphiphilic properties and offer excellent advantages for the modification, formulation, and delivery of various drugs. The Ab used should enhance the accumulation of the liposomal drug in the target areas with minimal cross-reactivity with healthy tissues. Ready-made drugs based on monoclonal Ab, such as trastuzumab, cetuximab, panitumumumab, bevacizumab; commercial Ab intended for research purposes, and laboratory synthesized Ab and their fragments are used in the preparation of ILs. Ab can be attached to liposomes by two main methods: direct covalent conjugation and postinsertion.Conclusion. The results of this study allowed us to summarize the variety of literature data on the composition of ILs and the possibility of using auxiliary components to achieve the goal in the development of the drug.

Naturally Occurring Anti-Idiotypic Antibodies Portray a Largely Private Repertoire in Immune-Mediated Thrombotic Thrombocytopenic Purpura.

Rare immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a life-threatening disease resulting from a severe autoantibody-mediated ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motifs, member 13) deficiency. Acute iTTP episodes are medical emergencies, but when treated appropriately >95% of patients survive. However, at least half of survivors will eventually experience a relapse. How remission of an initial episode is achieved and factors contributing to reemergence of anti-ADAMTS13 Abs and a relapsing course are poorly understood. In acquired hemophilia and systemic lupus erythematosus, anti-idiotypic Abs counteracting and neutralizing pathogenic autoantibodies contribute to remission. We selected and amplified the splenic anti-idiotypic IgG<sub>1</sub> Fab κ/λ repertoire of two relapsing iTTP patients on previously generated monoclonal inhibitory anti-ADAMTS13 Fabs by phage display to explore whether anti-idiotypic Abs have a role in iTTP. We obtained 27 single anti-idiotypic Fab clones, half of which had unique sequences, although both patients shared four H chain V region genes (V<sub>H</sub>1-69*01, V<sub>H</sub>3-15*01, V<sub>H</sub>3-23*01, and V<sub>H</sub>3-49*03). Anti-idiotypic Fab pools of both patients fully neutralized the inhibitor capacity of the monoclonal anti-ADAMTS13 Abs used for their selection. Preincubation of plasma samples of 22 unrelated iTTP patients stratified according to functional ADAMTS13 inhibitor titers (>2 Bethesda units/ml, or 1-2 Bethesda units/ml), with anti-idiotypic Fab pools neutralized functional ADAMTS13 inhibitors and restored ADAMTS13 activity in 18-45% of those cases. Taken together, we present evidence for the presence of an anti-idiotypic immune response in iTTP patients. The interindividual generalizability of this response is limited despite relatively uniform pathogenic anti-ADAMTS13 Abs recognizing a dominant epitope in the ADAMTS13 spacer domain.

TIP: A phase I/II study of MGTA-117, an anti-CD117 antibody-drug conjugate, in patients with adult acute myeloid leukemia (AML) and myelodysplasia with excess blasts (MDS-EB).

TPS3156 Background: Hematopoietic stem cell transplantation (HSCT) is used with curative intent for AML and MDS-EB. MGTA-117 is a novel Ab-drug conjugate (ADC) in development for conditioning prior to HSCT. MGTA-117 selectively targets CD117 (c-Kit) with a human monoclonal Ab to CD117 conjugated to an amanitin payload that depletes CD117-expressing cells by inhibiting RNA polymerase II. Human hematopoietic stem cells and AML tumor cells express high levels of CD117, and MGTA-117 potently depletes these target cells, with an IC50 of <10pM in vitro. MGTA-117 has demonstrated in vitro and in vivo stability, confirming its characterization as a highly potent and selective agent. In a primate GLP toxicology study, MGTA-117 maximally depleted bone marrow stem cells at a dose not associated with evidence of toxicity in other tissues. Dose-dependent reduction of peripheral reticulocytes, produced from CD117+ erythroid precursors in the bone marrow, was an early and time sensitive biomarker of bone marrow CD117+ cell depletion. Higher doses were associated with the elevation of transaminases and histopathology that were asymptomatic and transient. Highest Non- Severely Toxic Dose (HNSTD) was used to establish the starting dose in this First-in-Human study. Based upon dose exposure and allometric scaling, it is expected that the clinical exposures after a 0.02 mg/kg dose in the first human cohort will provide an optimal > 100-fold safety margin over exposures observed after the 0.3 mg/kg dose that was the HNSTD in the primate GLP toxicology study. Methods: This phase I/II, multicenter, open-label, dose-escalation study will investigate the safety, tolerability, PK profile, PD activity, and blast depletion activity of MGTA 117 given intravenously as a single dose in adults with R/R AML or MDS-EB. Patients must be 18-75 yrs, have a WHO-defined diagnosis of CD117+ R/R AML or MDS-EB with ≥5% marrow myeloblasts. Patients must have ECOG PS ≤2, and adequate hepatic, renal, and cardiac function. The primary objective is to establish a minimum safe and biologically effective (MSBE) dose of MGTA-117 in R/R AML and MDS-EB patients based on safety and CD117 receptor occupancy (RO) in circulating leukemic blasts after dosing. The observation period for dose limiting toxicities is 21 days. Patients will be followed for changes in reticulocyte, neutrophil, and platelet counts in PB and percent change from baseline in leukemic blasts or stem/progenitor cells in PB and/or BM. CD117 receptor occupancy by MGTA-117 will be measured and MSBE dose will be based on safety and receptor occupancy. The study is designed with the possibility that subjects would proceed to HSCT >28 days after MGTA-117 administration, if eligible per the local transplant practices. Clinical trial information: NCT05223699.

Induced antibodies directed to the angiotensin receptor type 1 provoke skin and lung inflammation, dermal fibrosis and act species overarching

ObjectiveTo determine contributions and functions of autoantibodies (Abs) directed to the angiotensin receptor type 1 (AT1R), which are suggested to be involved in the pathogenesis of AT1R Abs-related diseases such...

Impact of IgG Isotype on the Induction of Antibody-Dependent Cellular Phagocytosis of HIV by Human Milk Leukocytes.

Approximately 100,000 mother-to-child transmission (MTCT) events of HIV via human milk feeding occur each year. However, only about 15% of infants milk-fed by untreated HIV+ mothers become infected, suggesting a protective effect of the milk itself. Infants ingest 105-108 maternal leukocytes daily via milk, which remain functional beyond ingestion. Such function may be elicited by maternal milk antibody (Ab). Though IgA is dominant in milk, most HIV-specific milk Abs are of the IgG subclass, highlighting the importance of investigating the function of each IgG isotype in the milk context. Though Ab effector function mediated by the constant (Fc) domain via interaction with Fc Receptors (FcRs), such as Ab-dependent cellular phagocytosis (ADCP), are critical in protecting against HIV infection, ADCP is largely unexplored as it relates to mitigation of MTCT. Presently we report the ADCP activity of milk leukocytes against HIV particles and immune complexes (ICs), using 57 unique samples from 34 women, elicited by IgG1/2/3/4 of monoclonal (m)Ab 246-D. Granulocyte ADCP of HIV was most potent compared to other phagocytes when elicited by IgG1/3/4. IgG1/3 activated granulocytes similarly, exhibiting 1.6x-4.4x greater activity compared to IgG2/4, and a preference for virus compared to ICs. Notably, CD16- monocyte ADCP of a given target were unaffected by isotype, and CD16+ monocytes were poorly stimulated by IgG1. IgG2/4 elicited potent IC ADCP, and in terms of total leukocyte IC ADCP, IgG4 and IgG3 exhibited similar function, with IgG4 eliciting 1.6x-2.1x greater activity compared to IgG1/IgG2, and CD16+ monocytes most stimulated by IgG2. These data contribute to a more comprehensive understanding of Fc-mediated functionality of milk leukocytes, which is critical in order to develop therapeutic approaches to eliminating this route of MTCT, including mucosal administration of mAbs and/or a maternal vaccination aimed to elicit a potent milk Ab response.

Characterization of Apparently Paradoxical Thyrotropin Binding Inhibitory Immunoglobulins With Neutral Bioactivity.

ContextThe thyrotropin (TSH) receptor (TSH-R) autoantibody activity is clinically measured by inhibition of labeled ligand (TSH or M22) binding to the TSH-R (TSH-binding inhibitory immunoglobulin [TBII]) or by stimulation (TSH-R stimulating antibody [TSAb]) or inhibition (TSH-R blocking antibody [TSBAb]) of 3′,5′-cyclic adenosine 5′-monophosphate (cAMP) production in isolated cells.ObjectiveWe experienced a patient with hypothyroid Graves disease (GD) having strong positive TBII but with almost neutral bioactivities on the TSH-R. The aim of this study is the characterization of this apparently paradoxical TBII (serum sample S).MethodsWe first compared the TBII, TSAb, and TSBAb activities of serum sample S with mixtures of stimulating (S-mAb) and blocking monoclonal Ab (B-mAb). Next, we serially measured cAMPs stimulated by various serum samples in the presence or absence of TSH.ResultsMixtures of S-mAb and B-mAb did not reproduce the characteristics of serum sample S. Instead, serum sample S had a unique feature that blocked the TSH-stimulated cAMP initially but disappeared the blocking activity thereafter to reach the control level.ConclusionWe present here the TBIIs with neutral bioactivities found in the patient with autoimmune thyroid disease, which strongly inhibit TSH binding to the TSH-R but exerts neither TSAb nor TSBAb activity. Differences in the methods of detecting TRAb between TBII in vitro and bioassay may cause the discrepancy. Although serum sample S may be an extreme example, a variety of TRAb that not only stimulates or blocks but also interferes with TSH-R binding for only a short time may exist in the serum samples of GD patients.

Monoclonal antibodies from B cells of patients with anti-MDA5 antibody-positive dermatomyositis directly stimulate interferon gamma production

Anti-melanoma differentiation-associated gene 5 (MDA5) antibody (Ab) positive dermatomyositis (anti-MDA5 DM) is a rare entity associated with poor prognosis and multiple immunologic abnormalities. These include the presence of autoAbs and deleterious interferon (IFN)-gamma production in the severe form of the disease. Here, we show that the autoAbs profile differs between patients, depending on disease severity, and that autoAbs from B cells of patients directly stimulate IFN-gamma production by peripheral blood cells. Serum of 29 anti-MDA5 DM patients were analyzed by indirect immunofluorescence (IIF) on Hep-2 cells, to identify patterns associated with poor outcome. Seventeen (59%) serum gave a specific cytoplasmic MDA5 pattern on Hep-2 cells, while 12 (41%) gave an unspecific pattern. Specific MDA5 pattern was associated with a higher risk to develop interstitial lung disease (p = 0.003). Monoclonal autoAbs were generated from B cell clones of two patients with extreme clinical presentation, one who developed a lethal form of the disease, and the other with a favorable outcome. Supernatants of the autoreactive B cell clones that gave an IIF cytoplasmic pattern were tested for their abilities to stimulate IFN-gamma production by peripheral blood cells. Out of 120,000 B cell clones analyzed, 12 produced monoclonal Abs that triggered direct IFN-gamma secretion by peripheral blood cells, by a monocyte-dependent mechanism. None of them was directed against the MDA5 antigen. Altogether, these findings demonstrated that autoAbs other than the highly specific anti-MDA5 Ab are direct contributors of the IFN-gamma upregulation that is linked to the severity of the disease.

Biology and Clinical Relevance of HCMV-Associated Adaptive NK Cells.

Natural killer (NK) cells are an important component of the innate immune system due to their strong ability to kill virally infected or transformed cells without prior exposure to the antigen (Ag). However, the biology of human NK (hNK) cells has largely remained elusive. Recent advances have characterized several novel hNK subsets. Among them, adaptive NK cells demonstrate an intriguing specialized antibody (Ab)-dependent response and several adaptive immune features. Most adaptive NK cells express a higher level of NKG2C but lack an intracellular signaling adaptor, FcϵRIγ (hereafter abbreviated as FcRγ). The specific expression pattern of these genes, with other signature genes, is the result of a specific epigenetic modification. The expansion of adaptive NK cells in vivo has been documented in various viral infections, while the frequency of adaptive NK cells among peripheral blood mononuclear cells correlates with improved prognosis of monoclonal Ab treatment against leukemia. This review summarizes the discovery and signature phenotype of adaptive NK cells. We also discuss the reported association between adaptive NK cells and pathological conditions. Finally, we briefly highlight the application of adaptive NK cells in adoptive cell therapy against cancer.

Therapeutic and prophylactic treatment with a virus-specific antibody is highly effective in rodent models of Chikungunya infection and disease

Chikungunya virus (CHIKV) has re-emerged as a significant human pathogen in the 21st century, causing periodic, and sometimes widespread, outbreaks over the past 15 years. Although mortality is very rare, a debilitating arthralgia is very common and may persist for months or years. There are no antivirals that are approved for the treatment of CHIKV infection, and current treatment options consist of supportive care only. Herein, we demonstrate the efficacy of a CHIKV-specific antibody in the prophylactic and therapeutic treatment of CHIKV in mouse models of disease. The fully human anti-CHIKV monoclonal Ab SVIR023 demonstrated broad in vitro activity against representative strains from the three major CHIKV clades. Therapeutic treatment with SVIR023 administered 1- or 3-days post-infection resulted in reduced virus in various tissues in a dose- and time-dependent manner. Prophylactic treatment up to 4 weeks prior to virus challenge was also effective in preventing disease in mice. Mice treated with SVIR023 and infected with CHIKV were resistant to secondary challenge and no evidence of antibody enhancement of disease was observed. Treatment with SVIR023 was effective in mouse models of CHIKV infection and disease and further evaluation towards clinical development is warranted.

The Diagnostic Value of Pan-Trk Expression to Detect Neurotrophic Tyrosine Receptor Kinase (NTRK) Gene Fusion in CNS Tumours: A Study Using Next-Generation Sequencing Platform.

Background: Neurotrophic tyrosine receptor kinase (NTRK) fusion has been detected in rare types of CNS tumours, which can promote tumorigenesis. The efficacy of Trk inhibitor became a significant therapeutic interest. Our aim was to investigate whether Pan-Trk immunohistochemistry (IHC) is a reliable and efficient marker for detecting NTRK-fusion in different brain tumours. Methods: This study included 23 patients diagnosed with different types of CNS tumours. Testing for Pan-Trk IHC with monoclonal Ab (EPR17341) has been performed on all FFPE tissues. Parallelly, NTRK-rearrangements were tested using both DNA and RNA-based next-generation sequencing (NGS) assay using TruSight Onco500 platform. Results: The cohort included eight pilocytic astrocytomas, one oligodendroglioma, six IDHwildtype glioblastomas, four IDHmutant grade four astrocytomas, and one sample of each (astroblastoma, central neurocytoma, medulloblastoma, and liponeurocytoma). The mean age was 35 years; seven cases were in the paediatric age group, and 16 were adult. Pan-Trk expression was detected in 11 (47.8%) tumours, and 12 (52.1%) tumours showed no Pan-Trk expression. Nine Cases (82%) with different Pan-Trk expressions did not reveal NTRK-rearrangement. The other two positively expressed cases (liponeurocytoma and glioblastoma) were found to have NTRK2-fusions (SLC O 5A1-NTRK2, AGBL4-NTRK2, BEND5-NTRK2). All the 12 cases (100%) with no Pan-Trk expression have shown no NTRK-fusions. There was no statistically significant association between Pan-Trk expression and NTRK-fusion (p = 0.217). The detection of NTRK- fusions using NGS had high specificity over NTRK-fusion detection by using Pan-Trk IHC. Conclusion: Pan-Trk IHC is not a suitable tissue-efficient biomarker to screen for NTRK-fusions in CNS tumours, however RNA-based NGS sequencing should be used as an alternative method.