Neuro-PASC is characterized by enhanced CD4+ and diminished CD8+ T cell responses to SARS-CoV-2 Nucleocapsid protein

IntroductionMany people with long COVID symptoms suffer from debilitating neurologic post-acute sequelae of SARS-CoV-2 infection (Neuro-PASC). Although symptoms of Neuro-PASC are widely documented, it is still unclear whether PASC symptoms impact virus-specific immune responses. Therefore, we examined T cell and antibody responses to SARS-CoV-2 Nucleocapsid protein to identify activation signatures distinguishing Neuro-PASC patients from healthy COVID convalescents.ResultsWe report that Neuro-PASC patients exhibit distinct immunological signatures composed of elevated CD4+ T cell responses and diminished CD8+ memory T cell activation toward the C-terminal region of SARS-CoV-2 Nucleocapsid protein when examined both functionally and using TCR sequencing. CD8+ T cell production of IL-6 correlated with increased plasma IL-6 levels as well as heightened severity of neurologic symptoms, including pain. Elevated plasma immunoregulatory and reduced pro-inflammatory and antiviral response signatures were evident in Neuro-PASC patients compared with COVID convalescent controls without lasting symptoms, correlating with worse neurocognitive dysfunction.DiscussionWe conclude that these data provide new insight into the impact of virus-specific cellular immunity on the pathogenesis of long COVID and pave the way for the rational design of predictive biomarkers and therapeutic interventions

A randomised, open-label, parallel group phase 2 study of antisense oligonucleotide therapy in acromegaly

Objective: ATL1103 is a second-generation antisense oligomer targeting the human growth hormone (GH) receptor. This phase 2 randomised, open-label, parallel-group study assessed the potential of ATL1103 as a treatment for acromegaly. Design: Twenty-six patients with active acromegaly (IGF-I >130% upper limit of normal) were randomised to subcutaneous ATL1103 200mg either once or twice weekly for 13 weeks and monitored for a further 8-week washout period. Methods: The primary efficacy measures were change in IGF-I at week 14, compared to baseline and between cohorts. For secondary endpoints (IGFBP3, acid labile subunit (ALS), GH, growth hormone-binding protein (GHBP)), comparison was between baseline and week 14. Safety was assessed by reported adverse events. Results and conclusions: Baseline median IGF-I was 447 and 649 ng/mL in the once- and twice-weekly groups respectively. Compared to baseline, at week 14, twice-weekly ATL1103 resulted in a median fall in IGF-I of 27.8% (P = 0.0002). Between cohort comparison at week 14 demonstrated the median fall in IGF-I to be 25.8% (P = 0.0012) greater with twice-weekly dosing. In the twice-weekly cohort, IGF-I was still declining at week 14, and remained lower at week 21 than at baseline by a median of 18.7% (P = 0.0005). Compared to baseline, by week 14, IGFBP3 and ALS had declined by a median of 8.9% (P = 0.027) and 16.7% (P = 0.017) with twice-weekly ATL1103; GH had increased by a median of 46% at week 14 (P = 0.001). IGFBP3, ALS and GH did not change with weekly ATL1103. GHBP fell by a median of 23.6% and 48.8% in the once- and twice-weekly cohorts (P = 0.027 and P = 0.005) respectively. ATL1103 was well tolerated, although 84.6% of patients experienced mild-to-moderate injection-site reactions. This study provides proof of concept that ATL1103 is able to significantly lower IGF-I in patients with acromegaly

CD49d antisense drug ATL1102 reduces disease activity in patients with relapsing-remitting MS

Objective: This study evaluated the efficacy and safety of ATL1102, an antisense oligonucleotide that selectively targets the RNA for human CD49d, the a subunit of very late antigen 4, in patients with relapsing-remitting multiple sclerosis (RRMS). Methods: In a multicenter, double-blind, placebo-controlled randomized phase II trial, 77 patients with RRMS were treated with 200 mg of ATL1102 subcutaneously injected 3 times in the first week and twice weekly for 7 weeks or placebo and monitored for a further 8 weeks. MRI scans were taken at baseline and weeks 4, 8, 12, and 16. The primary endpoint was the cumulative number of new active lesions (either new gadolinium-enhancing T1 lesions or nonenhancing new or enlarging T2 lesions) at weeks 4, 8, and 12. Results: A total of 72 patients completed the study and 74 intention-to-treat patients were assessed. ATL1102 significantly reduced the cumulative number of new active lesions by 54.4% compared to placebo (mean 3.0 [SD 6.12] vs 6.2 [9.89], p = 0.01). The cumulative number of new gadolinium-enhancing T1 lesions was reduced by 67.9% compared to placebo (p = 0.002). Treatment-emergent adverse events included mild to moderate injection site erythema and decrease in platelet counts that returned to within the normal range after dosing. Conclusions: In patients with RRMS, ATL1102 significantly reduced disease activity after 8 weeks of treatment and was generally well-tolerated. This trial provides evidence for the first time that antisense oligonucleotides may be used as a therapeutic approach in neuroimmunologic disorders. Classification: This study provides Class I evidence that for patients with RRMS, the antisense oligonucleotide ATL1102 reduces the number of new active head MRI lesions

Effects of CD49d-targeted antisense-oligonucleotide on α4 integrin expression and function of acute lymphoblastic leukemia cells : results of in vitro and in vivo studies

We recently demonstrated the effectiveness of blocking CD49d with anti-functional antibodies or small molecule inhibitors as a rational targeted approach to the treatment of acute leukemia in combination with chemotherapy. Antisense oligonucleotide promises to be no less specific than antibodies and inhibitors, but more interesting for pharmacokinetics and pharmacodynamics. We addressed this using the published CD49d antisense drug ATL1102. In vitro, we incubated/nucleofected the ALL cell line Kasumi-2 with ATL1102. In vivo, immunodeficient hosts were engrafted with primary ALL cells and treated with ATL1102. Changes in expression of CD49d mRNA and CD49d protein, and of cooperating gene products, including ß1 integrin and CXCR4, as well as survival in the mouse experiments were quantified. We observed dose-dependent down-regulation of CD49d mRNA and protein levels and its partner integrin ß1 cell surface protein level and, up-regulation of CXCR4 surface expression. The suppression was more pronounced after nucleofection than after incubation, where down-regulation was significant only at the higher doses. In vivo effects of ATL1102 were not sufficient to translate into “clinical” benefit in the leukemia model. In summary, antisense oligonucleotides are successful tools for specifically modulating gene expression but sufficient delivery to down-regulate CD49d in vivo may be difficult to achieve

CD49d expression determined by Western Blot 72h post nucleofection.

<p>Kasumi-2 cells were treated with antisense control (<b>-</b>), 1μM (<b>1</b>), 3 μM (<b>3</b>), 10 μM (<b>10</b>) or 30 μM (<b>30</b>) of ATL1002 and optical density was determined (normalized to control). <b>A.</b> One representative Western Blot of at least three independent experiments is shown. <b>B</b>. Mean optical density (n = 3, normalized to control) (ANOVA one way *p<0.05). Western Blot lysates from incubated Kasumi 2 cells qualitatively confirmed the observations of flow cytometric analyses, although because of high inter-assay variability did not achieve statistical significance.</p

In vivo ATL1102 <i>target effect</i> study in mouse xenograft model of B-ALL.

<p>NOD/SCID mice were engrafted with primary B-ALL cells LAX7R, then treated with ATL1002 for two weeks (group 1: s.c., group 2: i.v. n = 5 each group) <b>(A)</b>. The mice were sacrificed 3 days after the last s.c. or i.v. injection. After sacrifice, percentage of hCD45 (<b>B</b>) and CD49d mean fluorescence intensity (<b>C, D</b>) was determined by flow cytometry in peripheral blood.</p

Nucleofection or incubation of ALL cell line <i>Kasumi-2</i> with the antisense ATL1102.

<p><b>ALL cells were treated with various concentrations of ATL1102 (1</b> μ<b>M, 3</b> μ<b>M, 10</b> μ<b>M and 30</b> μ<b>M) or 30</b>μ<b>M control as indicated by grey bars. A.</b> Viability of Kasumi-2 cells at 24h, 48h and 72h post nucleofection. <b>B-M.</b> Expression of surface proteins determined by flow cytometry 24h, 48h and 72h postnucleofection. <b><i>B-C</i></b>. CD49d. <b><i>D-E</i>.</b> CXCR4. <b><i>F-G</i></b>. CD29. <b><i>H-I</i></b>. CD19. <b><i>J-K</i>.</b> CD49e. <b><i>L-M</i></b>. CD49f. Histograms show the surface marker expressed under nucleofection with the highest dose of ATL1102 (30 μM) compared to antisense control. (Analysis performed by ANOVA one way; *p<0.05, **p<0.01, ***p<0.001).</p

Quantitative RT-PCR post nucleofection / incubation.

<p><b>A.</b> Expression of CD49d in Kasumi-2 cells nucleofected with ATL1102 after 24h, 48h and 72h. <b>B.</b> Expression of CD49d in Kasumi-2 cells incubated with ATL1102 after 48h (n = 1). For technical reasons, RT-PCR for incubated cells was only performed once but observations agreed with the concurrently generated flow cytometry data as well as with the data generated by electroporating with ATL1102.</p

Incubation of ALL cell line <i>Kasumi-2</i> with the antisense ATL1102.

<p><b>ALL cells were incubated with various concentrations of ATL1102 (1</b> μ<b>M, 3</b> μ<b>M, 10</b> μ<b>M and 30</b> μ<b>M) or 30</b>μ<b>M control as indicated by grey bars. A.</b> Viability of Kasumi-2 cells at 24h, 48h and 72h post incubation. <b>-<i>B-M</i>.</b> Expression of surface proteins determined by flow cytometry 24h, 48h and 72h post incubation. <b><i>B-C</i></b>. CD49d. <b><i>D-E</i>.</b> CXCR4. <b><i>F-G</i></b>. CD29. Expression of surface proteins determined by flow cytometry 24h, 48h and 72h post incubation. <b><i>H-I</i></b>. CD19. <b><i>J-K</i>.</b> CD49e. <b><i>L-M</i></b>. CD49f. Histograms show the surface marker expressed under incubation with the highest dose of ATL1102 (30 μM) compared to antisense control. (ANOVA one way *p<0.05, **p<0.01, ***p<0.001).</p