Patients with chronic lymphocytic leukemia (CLL) have an increased risk for severe COVID-19 disease and mortality. The goal of this study (NCT04746092) was to determine the efficacy of COVID-19 vaccine in patients with CLL. We evaluated humoral immune responses to BNT162b2 mRNA COVID-19 vaccine in patients with CLL and compared responses with those obtained in age-matched healthy controls. Patients received two vaccine doses, 21 days apart, and antibody titers were measured using Elecsys® Anti-SARS-CoV-2S assay after administration of the second dose. In a total of 167 patients with CLL the antibody response rate was 39.5%. A comparison between 52 patients with CLL and 52 sex- and aged-matched healthy controls, revealed a significantly reduced response rate among patients (52% vs 100%, respectively; adjusted odds ratio=0.010, 95% CI 0.001-0.162; p<0.001). Response rate was highest in patients who obtained clinical remission after treatment (79.2%), followed by 55.2% in treatment-naïve and 16% only in patients under treatment at the time of vaccination. In patients treated with either BTK inhibitors or venetoclax ± anti-CD20 antibody, response rates were considerably low (16.0% and 13.6%, respectively). None of the patients exposed to anti-CD20 antibodies <12 months prior to vaccination responded. In a multivariate analysis, the independent predictors of response were younger age, females, lack of currently active treatment, IgG levels ≥550 mg/dL and IgM levels ≥40mg/dL. In conclusion, antibody-mediated response to BNT162b2 mRNA COVID-19 vaccine in patients with CLL is markedly impaired and affected by disease activity and treatment.

Electrophiles for covalent inhibitors that are suitable for in vivo administration are rare. While acrylamides are prevalent in FDA-approved covalent drugs, chloroacetamides are considered too reactive for such purposes. We report sulfamate-based electrophiles that maintain chloroacetamide-like geometry with tunable reactivity. In the context of the BTK inhibitor ibrutinib, sulfamate analogues showed low reactivity with comparable potency in protein labeling, in vitro, and cellular kinase activity assays and were effective in a mouse model of CLL. In a second example, we converted a chloroacetamide Pin1 inhibitor to a potent and selective sulfamate acetamide with improved buffer stability. Finally, we show that sulfamate acetamides can be used for covalent ligand-directed release (CoLDR) chemistry, both for the generation of “turn-on” probes as well as for traceless ligand-directed site-specific labeling of proteins. Taken together, this chemistry represents a promising addition to the list of electrophiles suitable for in vivo covalent targeting.

Proteolysis targeting chimeras (PROTACs) are small molecules that form ternary complexes between their target and E3 ligase, resulting in ubiquitination and proteasomal degradation of the target protein. Using our own designed Bruton’s tyrosine kinase (BTK) PROTAC compounds, we show herein efficient BTK degradation in chronic lymphocytic leukemia (CLL) cells. The reversible non-covalent compound (NC-1) was the most potent and therefore we focused on this PROTAC to investigate its subsequent effects on the BCR pathway. NC-1 decreased baseline BTK phosphorylation as well as activation of BTK and other signaling molecules downstream of the BCR pathway, following IgM engagement. These effects were also obtained in samples from CLL patients with clinical resistance to ibrutinib and mutations at C481. NC-1 treatment further decreased baseline CD69 surface levels, completely abrogated its upregulation following IgM activation, decreased CLL cells migration toward SDF-1 and overcame stromal anti-apoptotic protection. In conclusion, our results indicate that targeting BTK using the PROTAC strategy could be a potential novel therapeutic approach for CLL.

Most chronic lymphocytic leukemia (CLL) clones express B-cell receptors (BcR) of both IgM/IgD isotypes; however, 5%–10% of CLL cases express isotype-switched immunoglobulin G (IgG). The early signaling and spatial patterning of the various BcRs at steady state and after activation are still fully unresolved. Herein, we show higher expression of the BcR signalosome elements and a more robust constitutive cell-intrinsic proximal BcR signaling in CLL with unmutated IGHV expressing IgM isotype (IgM U-CLL), compared with IGHV-mutated CLL (M-CLL) expressing either IgM or IgG isotypes. IgM in U-CLL is frequently located in the membrane plane in polarized patches, occasionally in caps, and sometimes inside the cells. Among M-CLL, IgM is scattered laterally in the membrane plane in a similar pattern as seen in normal B cells, whereas IgG is dispersed around the cell membrane in smaller clusters than in IgM U-CLL. Upon BcR engagement, both IgG and IgM expressing M-CLL showed attenuated signaling and only slight spatial reorganization dynamics of BcR microclusters and internalization, compared with the extensive reorganization and internalization of the BcR in IgM expressing U-CLL. The global gene signature of IgG M-CLL was closely related to that of IgM M-CLL rather than IgM U-CLL. Overall, we report fundamental differences in the basal composition, biochemical status, and spatial organization of the BcR in the three examined immunogenetic CLL subtypes that correlate with their clinical behavior. On the basis of our findings, IgG class-switched M-CLL likely represents the same disease as IgM M-CLL rather than a different biological and/or clinical entity.

Introduction: The prognostic significance of immunoglobulin heavy-chain variable region gene (IGHV) mutational status in chronic lymphocytic leukemia (CLL) is well established. Previous studies have shown that CLL patients with mutated IGHV (M-IGHV) have a better prognosis, manifested in a longer time-to first treatment (TTFT) and overall survival (OS). Here we present an analysis on the impact of IGHV mutational status in an Israeli cohort of patients with CLL. Methods: A total of 254 patients with CLL (diagnosed from 1991 to 2020), followed at the Tel-Aviv Sourasky Medical Center were included. The IGHV mutational status has been determined by next-generation sequencing (NGS) or cDNA Sanger. The sequences with a germline homology 98% or higher were considered unmutated, and those with a homology less than 98% as mutated CLL. IGHV subsets were analyzed using the ARResT/AssignSubsets website. All data were statistically analyzed by IBM SPSS Statistics 27 (IBM Corporation, Armonk, NY, USA), P-values were two-sided, and the significance level was determined at a<0.05 were considered significant. Results: Out of 254 patients, 132 (52.0%) had an unmutated IGHV gene (UM-IGHV), and 122 (48.0%) were mutated (M-IGHV). At diagnosis, most patients (Table 1) were ≤65 years of age (n=157, 61.8%), males (n=160, 63.0%) and had an absolute lymphocytic count equal to or less than (≤)15.0 x10 9/L (n=115, 52.5%). Advanced Binet stage was more commonly associated with UM-IGHV (n=44, 57.9%) (P=0.033). Among 240 patients (94.1%), the most frequently used VH gene segments (Figure 1) included; VH1-69 (n=35, 14.6%), VH4-34 (n=24, 10.0%), VH1-2 (n=17, 7.1%), and VH3-23 (n=16, 6.7%). Six major B-cell receptors (BCR) subsets (Figure 2) were identified in 21/180 of patients (11.7%), which most commonly included: CLL#1 (n=6, 28.6%), CLL#4 (n=5, 23.8%), and CLL#2(n=3, 14.3%). Patients with M-IGHV had a longer time to first treatment (TTFT) (P<0.001, hazard ratio (HR)=2.5, 95% confidence interval [1.8-3.5], Figure 3A) and a better overall survival (OS) (P=0.002, HR=2.7, [1.4-5.1]) compared to those with UM-IGHV gene (Figure 3B). In multivariate analyses of the entire cohort; for TTFT (Table 2A), males (P=0.002, HR=1.9, [1.3-2.9]), >65 years of age at diagnosis (P=0.008, HR=1.8, [1.2-2.7]), advanced Binet stage (P<0.001, HR=2.4, [1.5-3.8]) and UM-IGHV mutational status (P=<0.001, HR=2.0, [1.3-2.9]) were found to be significant predictors for shorter TTFT, while in a multivariate analysis for OS (Table 2B), only >65 years of age at diagnosis (P=0.010, HR=2.6, [1.3-5.5]) and UM-IGHV mutational status (P=0.004, HR=2.9, [1.4-5.9]) were found to be significant factors for shorter OS. In multivariate analyses performed separately for each IGHV mutational status group; males (P=0.021, HR=2.1, [1.1-4.0]), age >65 years (P=0.002, HR=3.2, [1.5-6.4]) and advanced Binet stage (P<0.001, HR=4.1, [1.9-8.7]) were found as independent predictors for a shorter TTFT in M-IGHV patients, while only males retained a statistically significance in UM-IGHV patients (P=0.017 HR=2.0, [1.1-3.5]). Furthermore, in a multivariate analysis for OS; age>65 years at diagnosis (P=0.039, HR=4.3, [1.1-17.0]) was the only independent predictor in M-IGHV patients, while no variable maintained its statistical significance in UM-IGHV cases. Conclusion: As previously studied, our cohort demonstrates that M-IGHV CLL patients have better TTFT and OS. However, the rate of BCR subsets in Israel appears to be lower than expected. A separated multivariate analysis for M-IGHV and UM-IGHV patients revealed different independent predictors for TTFT and OS to each of the IGHV mutational status groups. Further studies with larger cohorts are required to deeply study these differences and provide further clinical insight into the pathophysiology of CLL. Figure 1 Figure 1. Disclosures Joffe: AstraZeneca: Consultancy; Epizyme: Consultancy. Herishanu: AbbVie: Honoraria, Research Funding; Janssen: Honoraria; Roche: Honoraria; Medison: Honoraria.