The numbers of activated B cells were increased in SSc patients, and the up-regulation of CD5, CD86, IL-6R, and IL-21R discriminated between patients with dcSSc and those with lcSSc. Peripheral B lymphocytes from SSc patients secreted both IL-6 and TGFβ, and they activated fibroblasts in vitro.

B-cell receptor (BCR) signaling and tumor–microenvironment crosstalk both drive chronic lymphocytic leukemia (CLL) pathogenesis. Within the microenvironment, tumor cells shape the T-cell compartment, which in turn supports tumor growth and survival. Targeting BCR signaling using Bruton tyrosine kinase inhibitors (BTKi) has become a highly successful treatment modality for CLL. Ibrutinib, the first-in-class BTKi, also inhibits Tec family kinases such as interleukin-2–inducible kinase (ITK), a proximal member of the T-cell receptor signaling cascade. It is increasingly recognized that ibrutinib modulates the T-cell compartment of patients with CLL. Understanding these T-cell changes is important for immunotherapy-based approaches aiming to increase the depth of response and to prevent or treat the emergence of resistant disease. Ibrutinib has been shown to improve T-cell function in CLL, resulting in the expansion of memory T cells, Th1 polarization, reduced expression of inhibitory receptors and improved immune synapse formation between T cells and CLL cells. Investigating the modulation of BTKi on the T-cell antitumoral function, and having a more complete understanding of changes in T cell behavior and function during treatment with BTKi therapy will inform the design of immunotherapy-based combination approaches and increase the efficacy of CLL therapy.

Bruton Tyrosine Kinase inhibitors (BTKis) are a preferred treatment for patients with chronic lymphocytic leukemia (CLL). Indefinite therapy with BTKis, while effective, presents clinical challenges. Combination therapy can deepen responses, shorten treatment duration, and possibly prevent or overcome drug resistance. We previously reported on a CD19/CD3 bispecific antibody (bsAb) that recruits autologous T cell cytotoxicity against CLL cells in vitro. Compared to observations with samples from treatment-naïve patients, T cells from patients being treated with ibrutinib expanded more rapidly and exerted superior cytotoxic activity in response to the bsAb. In addition to BTK, ibrutinib also inhibits IL2 inducible T cell Kinase (ITK). In contrast, acalabrutinib, does not inhibit ITK. Whether ITK inhibition contributes to the observed immune effects is unknown. To better understand how BTKis modulate T-cell function and cytotoxic activity, we cultured peripheral blood mononuclear cells (PBMCs) from BTKi-naive, and ibrutinib- or acalabrutinib-treated CLL patients with CD19/CD3 bsAb in vitro. T-cell expansion, activation, differentiation, and cytotoxicity were increased in PBMCs from patients on treatment with either BTKi compared to that observed for BKTi-naïve patients. BTKi therapy transcriptionally downregulated immunosuppressive effectors expressed by CLL cells, including CTLA-4 and CD200. CTLA-4 blockade with ipilimumab in vitro increased the cytotoxic activity of the bsAb in BTKi-naïve but not BTKi-treated PBMCS. Taken together, BTKis enhance bsAb induced cytotoxicity by relieving T cells of immunosuppressive restraints imposed by CLL cells. The benefit of combining bsAb immunotherapy with BTKis needs to be confirmed in clinical trials.

BackgroundArthrogryposis-Renal dysfunction-Cholestasis syndrome (ARC, MIM#208085) is a rare multisystem disease due to mutations in the VPS33B and VIPAR genes, both involved in maintaining apical-basolateral cell polarity. The correlation between mutations and phenotype in the ARC Syndrome is not well described. We report on a 6 year old patient who presented with severe renal Fanconi as first manifestation of ARC related to a combined de novo mutation in the VPS33B gene.Case presentationA 6 year old girl presented during the first year of life with severe renal Fanconi as the first manifestation of ARC-Syndrome. This case presents all defining features of ARC syndrome (including liver, skin and articular manifestations) with predominantly renal impairment at presentation. This novel mutation may be associated with a pronounced renal phenotype in ARC. Furthermore, we report on the successful use of LDL-Apheresis and biliodigestive derivation for treatment of cholestatic pruritus with encouraging results.ConclusionARC is a heterogeneous disorder with early mortality. This case report contributes to a better understanding of this rare disorder, describes a novel mutation in the VPS33B gene and presents an innovative rescue treatment approach.

Bruton tyrosine kinase (BTK) is essential for B-cell receptor (BCR) signaling, a driver of chronic lymphocytic leukemia (CLL). Covalent inhibitors bind C481 in the active site of BTK and have become a preferred CLL therapy. Disease progression on covalent BTK inhibitors is commonly associated with C481 mutations. Here, we investigated a targeted protein degrader, NRX-0492, that links a non-covalent BTK binding domain to cereblon, an adaptor protein of the E3 ubiquitin ligase complex. NRX-0492 selectively catalyzes ubiquitylation and proteasomal degradation of BTK. In primary CLL cells, NRX-0492 induced rapid and sustained degradation of both wild-type and C481 mutant BTK at half maximal degradation concentration (DC50) of ≤0.2 nM and DC90 of ≤0.5 nM, respectively. Sustained degrader activity was maintained for at least 24 hours after washout and was equally observed in high-risk (deletion 17p) and standard-risk (deletion 13q only) CLL subtypes. In in vitro testing against treatment-naïve CLL samples, NRX-0492 was as effective as ibrutinib at inhibiting BCR mediated signaling, transcriptional programs, and chemokine secretion. In patient-derived xenografts, orally administered NRX-0492 induced BTK degradation and inhibited activation and proliferation of CLL cells in blood and spleen and remained efficacious against primary C481S mutant CLL cells collected from a patient progressing on ibrutinib. Oral bioavailability, >90% degradation of BTK at sub-nanomolar concentrations and sustained pharmacodynamic effects after drug clearance make this class of targeted protein degraders uniquely suitable for clinical translation, in particular as a strategy to overcome BTK inhibitor resistance. Clinical studies testing this approach have been initiated (NCT04830137, NCT05131022).

Chronic lymphocytic leukemia (CLL) is an immunosuppressive disease characterized by increased infectious morbidity and inferior anti-tumor activity of immunotherapies. Targeted therapy with Bruton's tyrosine kinase inhibitors (BTKis), or the Bcl-2 inhibitor venetoclax, have profoundly improved treatment outcomes in CLL. To overcome or prevent drug resistance and extend duration of response after time-limited therapy, combination regimens are tested. Anti-CD20 antibodies that recruit cell and complement mediated effector functions are commonly used. Epcoritamab (GEN3013), an anti-CD3xCD20 bispecific antibody (bsAb) that recruits T cell effector functions, has demonstrated potent clinical activity in patients with relapsed CD20+ B-cell non-Hodgkin's lymphoma. Development in CLL is ongoing. To characterize epcoritamab mediated cytotoxicity against primary CLL cells, peripheral blood mononuclear cells (PBMCs) from treatment-naïve and BTKi-treated patients, including patients progressing on therapy were cultured with epcoritamab alone or in combination with venetoclax. Ongoing treatment with a BTKi and high effector:target ratios were associated with superior in vitro cytotoxicity. Cytotoxic activity was independent of CD20 expression on CLL cells, and observed in samples from patients progressing on a BTKi. Epcoritamab induced significant T-cell expansion, activation, and differentiation into Th1 and effector memory cells, in all patient samples. In patient-derived xenografts, epcoritamab reduced blood and spleen disease burden compared to mice receiving a non-targeting control. In vitro, the combination of venetoclax with epcoritamab induced superior killing of CLL cells than either agent alone. These data support the investigation of epcoritamab in combination with BTKis or venetoclax to consolidate responses and target emergent drug resistant subclones.

Targeting B-cell receptor signaling with ibrutinib, the first-in-class irreversible inhibitor of Bruton tyrosine kinase, has become a highly successful treatment modality for Chronic Lymphocytic Leukemia (CLL) patients. However, there remains a need for adjunct treatments to deepen responses and prevent or treat resistant disease. Ibrutinib also inhibits inducible T-cell kinase (ITK) which is hypothesized to improve antitumor T-cell immunity. We developed a CD19/CD3 bispecific antibody (bsAb) in a 100 kDa single chain-Fv-Fc format (19/3-scFv-Fc). We previously reported increased T-cell activation and cytotoxic activity by the bsAb in blood samples from ibrutinib-treated patients compared to treatment-naïve patients. To better understand how ibrutinib affects T-cell function and increases cytotoxic activity we assessed T-cell responses to the bsAb in samples from ibrutinib-treated patients (12 ± 2 months on ibrutinib) and treatment-naïve (TN) patients. We cultured peripheral blood mononuclear cells (PBMCs) from both patient groups with bsAb and measured CLL cell death, T-cell expansion, activation, and differentiation by flow cytometry. After 5 days of exposure, the CD19/CD3 bsAb induced superior killing of ibrutinib-treated CLL cells, with a median specific-killing of 97% for ibrutinib-treated compared to 77 % for treatment-naïve patient samples (n = 15; p= 0.008). In vitro treatment with CD19/CD3 bsAb induced ≥ 5-fold expansion of autologous CD8 and CD4 T cells and an increase of CD45RO+CCR7- effector memory and CD45RO+CCR7+ central memory CD4 T-cells in response to bsAb (Table 1). We also observed a shift towards Th1 (CCR6-CXCR3+) polarization, and higher T-cell granzyme B expression in response to CD19/CD3 bsAb. While T cells from both patient groups responded to the bsAb, all these effects were more pronounced in ibrutinib-treated samples compared to treatment-naïve samples (Table 1). There was a 13-fold increase of the Th1/Th2 ratio in PBMCs from ibrutinib-treated patients compared to a 7-fold increase in the treatment-naïve group. Moreover, the bsAb induced a significant increase of activation markers HLADR (p=0.0013; p=0.0006) and CD27 (p< 0.001; p=0.0114) on CD4 and CD8 T cells in the ibrutinib-treated patient samples, while there was no significant change in T-cell activation in the treatment-naïve group. Granzyme B expression in CD8 T cells was high even in untreated samples and its upregulation by the bsAb was comparable in both patient groups. However, we observed a 25-fold increase in granzyme B positive CD4 T cells in ibrutinib-treated samples, 5-fold higher than in the treatment-naïve samples. In summary, the enhanced cytotoxic activity of T cells in response to the bsAb in samples from ibrutinib-treated patients correlated with increased memory cell differentiation, higher T-cell activation, and a strong shift towards a Th1 phenotype. As ibrutinib enhances anti-CLL activity of autologous T cells, bsAb immunotherapy may work well with concurrent ibrutinib treatment. These data support the investigation of bsAbs in combination with ibrutinib for immunotherapy of CLL. The mechanisms underlying the enhanced T-cell responses remain to be fully elucidated. In particular, it will be important to define whether inhibition of ITK by ibrutinib plays a role in enhancing T-cell dependent cytotoxicity. Supported by the intramural program of the NHLBI/NIH. Disclosures Rader: NIH: Patents & Royalties: ROR1 mAb 2A2. Wiestner:Acerta: Research Funding; Merck: Research Funding; Nurix: Research Funding; Pharmayclics: Research Funding.

Chronic Lymphocytic Leukemia (CLL) is a heterogeneous B cell neoplasm ranging from indolent to rapidly progressive disease. Leukemic cell subsets with regulatory properties evade immune clearance; however, the contribution of such subsets during CLL progression is not completely elucidated. Here, we report that CLL B cells crosstalk with their immune counterparts, notably by promoting the regulatory T (Treg) cell compartment and shaping several helper T (Th) subsets. Among various constitutively- and BCR/CD40-mediated factors secreted, tumour subsets co-express two important immunoregulatory cytokines, IL10 and TGFβ1, both associated with a memory B cell phenotype. Neutralizing secreted IL10 or inhibiting the TGFβ signalling pathway demonstrated that these cytokines are mainly involved in Th- and Treg differentiation/maintenance. In line with the regulatory subsets, we also demonstrated that a CLL B cell population expresses FOXP3, a marker of regulatory T cells. Analysis of IL10, TGFβ1 and FOXP3 positive subpopulations frequencies in CLL samples discriminated 2 clusters of untreated CLL patients that were significantly different in Tregs frequency and time-to-treatment. Since this distinction was pertinent to disease progression, the regulatory profiling provides a new rationale for patient stratification and sheds light on immune dysfunction in CLL.