Phase 2 study of the safety and efficacy of umbralisib in patients with CLL who are intolerant to BTK or PI3Kδ inhibitor therapy.

Intolerance is the most common reason for kinase inhibitor (KI) discontinuation in chronic lymphocytic leukemia (CLL). Umbralisib, a novel highly selective phosphatidylinositol 3-kinase Î´ (PI3Kδ)/CK1ε inhibitor, is active and well tolerated in CLL patients. In this phase 2 trial (NCT02742090), umbralisib was initiated at 800 mg/d in CLL patients requiring therapy, who were intolerant to prior BTK inhibitor (BTKi) or PI3K inhibitor (PI3Ki) therapy, until progression or toxicity. Primary end point was progression-free survival (PFS). Secondary end points included time to treatment failure and safety. DNA was genotyped for CYP3A4, CYP3A5, and CYP2D6 polymorphisms. Fifty-one patients were enrolled (44 BTKi intolerant and 7 PI3Kδi intolerant); median age was 70 years (range, 48-96), with a median of 2 prior lines of therapy (range, 1-7), 24% had del17p and/or TP53 mutation, and 65% had unmutated IGHV. Most common adverse events (AEs) leading to prior KI discontinuation were rash (27%), arthralgia (18%), and atrial fibrillation (16%). Median PFS was 23.5 months (95% CI, 13.1-not estimable), with 58% of patients on umbralisib for a longer duration than prior KI. Most common (≥5%) grade ≥3 AEs on umbralisib (all causality) were neutropenia (18%), leukocytosis (14%), thrombocytopenia (12%), pneumonia (12%), and diarrhea (8%). Six patients (12%) discontinued umbralisib because of an AE. Eight patients (16%) had dose reductions and were successfully rechallenged. These are the first prospective data to confirm that switching from a BTKi or alternate PI3Ki to umbralisib in this BTKi- and PI3Ki-intolerant CLL population can result in durable well-tolerated responses.

Induced Abortion and the Risk of Rh Sensitization.

Importance: While population-level data suggest Rh immunoglobulin is unnecessary before 12 weeks' gestation, clinical evidence is limited. Thus, guidelines vary, creating confusion surrounding risks and benefits of Rh testing and treatment. As abortion care in traditional clinical settings becomes harder to access, many people are choosing to self-manage and need to know if ancillary blood type testing is necessary. Objective: To determine how frequently maternal exposure to fetal red blood cells (fRBCs) exceeds the most conservative published threshold for Rh sensitization in induced first-trimester abortion. Design, Setting, and Participants: Multicenter, observational, prospective cohort study using high-throughput flow cytometry to detect circulating fRBCs in paired maternal blood samples before and after induced first-trimester abortion (medication or procedural). Individuals undergoing induced first-trimester abortion before 12 weeks 0 days' gestation were included. Paired blood samples were available from 506 participants who underwent either medical (n = 319 [63.0%]) or procedural (n = 187 [37.0%]) abortion. Exposure: Induced first-trimester abortion. Main Outcomes and Measures: The primary outcome was the proportion of participants with fRBC counts above the sensitization threshold (125 fRBCs/5 million total RBCs) after induced first-trimester abortion. Results: Among the 506 participants, the mean (SD) age was 27.4 (5.5) years, 313 (61.9%) were Black, and 123 (24.3%) were White. Three of the 506 participants had elevated fRBC counts at baseline; 1 of these patients had an elevated fRBC count following the abortion (0.2% [95% CI, 0%-0.93%]). No other participants had elevated fRBC counts above the sensitization threshold after induced first-trimester abortion. The median change from baseline was 0 fRBCs, with upper 95th and 99th percentiles of 24 and 35.6 fRBCs, respectively. Although there was a strong association between the preabortion and postabortion fRBC counts, no other baseline characteristic was significantly associated with postabortion fRBC count. Conclusions and Relevance: Induced first-trimester abortion is not a risk factor for Rh sensitization, indicating that Rh testing and treatment are unnecessary before 12 weeks' gestation. This evidence may be used to inform international guidelines for Rh immunoglobulin administration following first-trimester induced abortion.

Increased mTORC2 pathway activation in lymph nodes of iMCD-TAFRO.

Idiopathic multicentric Castleman disease (iMCD) is a rare and life-threatening haematologic disorder involving polyclonal lymphoproliferation and organ dysfunction due to excessive cytokine production, including interleukin-6 (IL-6). Clinical trial and real-world data demonstrate that IL-6 inhibition is effective in 34-50% of patients. mTOR, which functions through mTORC1 and mTORC2, is a recently discovered therapeutic target. The mTOR inhibitor sirolimus, which preferentially inhibits mTORC1, has led to sustained remission in a small cohort of anti-IL-6-refractory iMCD patients with thrombocytopenia, anasarca, fever, renal dysfunction and organomegaly (iMCD-TAFRO). However, sirolimus has not shown uniform effect, potentially due to its limited mTORC2 inhibition. To investigate mTORC2 activation in iMCD, we quantified the mTORC2 effector protein pNDRG1 by immunohistochemistry of lymph node tissue from six iMCD-TAFRO and eight iMCD patients who do not meet TAFRO criteria (iMCD-not-otherwise-specified; iMCD-NOS). mTORC2 activation was increased in all regions of iMCD-TAFRO lymph nodes and the interfollicular space of iMCD-NOS compared with control tissue. Immunohistochemistry also revealed increased pNDRG1 expression in iMCD-TAFRO germinal centres compared with autoimmune lymphoproliferative syndrome (ALPS), an mTOR-driven, sirolimus-responsive lymphoproliferative disorder, and comparable staining between iMCD-NOS and ALPS. These results suggest increased mTORC2 activity in iMCD and that dual mTORC1/mTORC2 inhibitors may be a rational therapeutic approach.

Tumor necrosis factor receptor-associated factor 1 influences KRN/I-Ag7 mouse arthritis autoantibody production.

PURPOSE: Recently, genomewide association analysis has revealed that the Tumor Necrosis Factor Receptor-associated factor 1-Complement 5 (TRAF1-C5) containing locus on chromosome 9 was associated with an increased risk for RA. Studies in model systems suggested that either gain- or loss-of-function TRAF1 mutations have immune effects that could plausibly lead to or exacerbate the arthritis phenotype. KRN/I-A(g7) (KxB/N) is a genetic mouse model of inflammatory arthritis. We aimed to assess the impact of TRAF1 deficiency on KRN/I-A(g7) mice. METHODS: We have bred KRN/I-A(g7) mice onto a TRAF1-deficient background and followed cohorts for the spontaneous appearance of arthritis. We have also transferred KxB/N serum to B6.I-A(g7) TRAF1KO recipients. In addition, systemic autoimmunity was induced through cGVH by injecting bm12 splenocytes into TRAF1KO recipient mice. RESULTS: TRAF1-deficient KRN/I-A(g7) mice spontaneously developed severe, progressive arthritis, comparable to that seen in TRAF1-intact KRN/I-A(g7) mice. However, the anti-GPI antibody titer was significantly lower in the former group. Interestingly, the TRAF1KO mice that had background levels of anti-GPI antibodies still showed severe arthritis, although with a brief delay compared to TRAF1 sufficient mice. In addition, TRAF1KO mice were fully susceptible to passive, serum transfer experiments. In another model of autoimmunity, TRAF1KO had no effect on cGVH autoantibodies production; nor was the response to an exogenous antigen impaired. CONCLUSION: The pathogenesis of spontaneous KRN/I-A(g7) arthritis can largely proceed by TRAF1-independent pathways. The production of anti-GPI autoantibody, but not other autoantibody or antibody responses, was markedly impaired by TRAF1 deficiency. The spontaneous arthritis model in KRN mice appears to be much less antibody dependent than previously believed.

Precision targeting of autoantigen-specific B cells in muscle-specific tyrosine kinase myasthenia gravis with chimeric autoantibody receptor T cells.

Muscle-specific tyrosine kinase myasthenia gravis (MuSK MG) is an autoimmune disease that causes life-threatening muscle weakness due to anti-MuSK autoantibodies that disrupt neuromuscular junction signaling. To avoid chronic immunosuppression from current therapies, we engineered T cells to express a MuSK chimeric autoantibody receptor with CD137-CD3ζ signaling domains (MuSK-CAART) for precision targeting of B cells expressing anti-MuSK autoantibodies. MuSK-CAART demonstrated similar efficacy as anti-CD19 chimeric antigen receptor T cells for depletion of anti-MuSK B cells and retained cytolytic activity in the presence of soluble anti-MuSK antibodies. In an experimental autoimmune MG mouse model, MuSK-CAART reduced anti-MuSK IgG without decreasing B cells or total IgG levels, reflecting MuSK-specific B cell depletion. Specific off-target interactions of MuSK-CAART were not identified in vivo, in primary human cell screens or by high-throughput human membrane proteome array. These data contributed to an investigational new drug application and phase 1 clinical study design for MuSK-CAART for the treatment of MuSK autoantibody-positive MG.

KRN/I-Ag7 mouse arthritis is independent of complement C3.

BACKGROUND: KRN/I-A(g7) (KxB/N) is a mouse model of inflammatory arthritis, which resembles human rheumatoid arthritis. Arthritis in these animals is caused by autoreactivity to a ubiquitously expressed autoantigen, glucose-6 phosphate isomerase. Tolerance is broken at both the T cell and B cell level. The sera from KRN/I-A(g7) mice can induce mouse arthritis in healthy mice. Complement components of the alternative complement pathway, including C3, have been shown to be required in induction of mouse arthritis by serum transfer. METHODS: We have bred KRN/I-A(g7) mice onto a C3-deficient background and followed cohorts for the spontaneous appearance of arthritis. We have also transferred KxB/N serum to B6.I-A ( g7 ) recipients. RESULTS: C3-deficient KRN/I-A(g7) mice spontaneously developed severe, destructive arthritis, comparable to that seen in C3-intact KRN/I-A(g7) mice. However, serum transfer experiments confirmed the strong requirement for C3 in the passive model. CONCLUSION: The pathogenesis of spontaneous KRN/I-A(g7) arthritis can largely proceed by complement-independent pathways and must have pathology effector mechanisms in addition to those seen in the passive serum transfer model.

Effects of systemic inflammation on relapse in early breast cancer.

Chronic inflammation has been a proposed mechanism of resistance to aromatase inhibitors in breast cancer. Stratifying by HER2 status, a matched case-control study from the Wellness After Breast Cancer-II cohort was performed to assess whether or not elevated serum inflammatory biomarkers (C-Reactive protein [CRP], interleukin-6 [IL-6], and serum amyloid A [SAA]) and/or the presence of a high-risk IL-6 promoter genotype were associated with recurrence of hormone receptor positive (HR+) early breast cancer. Estrogen levels were also measured and correlated with biomarkers and disease outcomes. CRP and SAA were significantly associated with an increased risk of recurrence in the HR+/HER2- group, but not the HR+/HER2+ group. Mean serum estrogen levels were non-significantly elevated in patients who relapsed vs. non-relapsed patients. Surprisingly, high-risk IL-6 promoter polymorphisms were strongly associated with HER2+ breast cancer relapse, which has potential therapeutic implications, as elevated intracellular IL-6 has been associated with trastuzumab resistance in pre-clinical models.

Disrupted Mer receptor tyrosine kinase expression leads to enhanced MZ B-cell responses.

Control of lymphocyte homeostasis is essential to ensure efficient immune responses and to prevent autoimmunity. Splenic marginal zone B cells are important producers of autoantibodies, and are subject to stringent tolerance mechanisms to prevent autoimmunity. In this paper, we explore the role of the Mer tyrosine kinase (Mertk) in regulating autoreactive B cells. This receptor tyrosine kinase serves to bind apoptotic cells, to mediate their phagocytosis, and to regulate subsequent cytokine production. Mice lacking Mertk suffer from impaired apoptotic cell clearance and develop a lupus-like autoimmune syndrome. Here we show that such Mertk-KO mice have expanded numbers of splenic marginal zone B cells. Mertk-KO mice bearing a DNA-specific immunoglobulin heavy-chain transgene (3H9) produced anti-DNA antibodies that appeared to be secreted largely by marginal zone B cells. Finally, Mertk-KO mice developed greater antibody responses after NP-Ficoll immunization than their B6 counterparts. Taken together, our data show that Mertk has a major effect on the development of the marginal zone B-cell compartment. Mertk is also important in establishing DNA-specific B-cell tolerance in 3H9 anti-DNA transgenic mice.

T cell-independent spontaneous loss of tolerance by anti-double-stranded DNA B cells in C57BL/6 mice.

Systemic lupus erythematosus is characterized by loss of tolerance to DNA and other nuclear Ags. To understand the role of T cells in the breaking of tolerance, an anti-DNA site-specific transgenic model of spontaneous lupus, B6x56R, was studied. T cells were eliminated by crossing B6x56R with CD4(-/)(-) or TCRbeta(-/-)delta(-/-) mice, and the effects on anti-dsDNA serum levels, numbers of anti-dsDNA Ab-secreting cells, and isotypes of anti-dsDNA were analyzed. In addition, the development and activation of B cells in these mice were examined. Surprisingly, the presence of T cells made little difference in the development and character of the serum anti-dsDNA Ab in B6x56R mice. At 1 mo of age, anti-dsDNA Abs were somewhat lower in mice deficient in alphabeta and gammadelta T cells. Levels of Abs later were not affected by T cells, nor was autoantibody class switching. B cell activation was somewhat diminished in T cell-deficient mice. Thus, in the B6 background, the presence of an anti-dsDNA transgene led the production of autoantibodies with a specificity and isotype characteristic of murine systemic lupus erythematosus with little influence from T cells. TLR9 also did not appear to play a role. Although we do not yet understand the mechanism of this failure of immunoregulation, these results suggest that similar processes may influence autoimmunity associated with clinical disease.

The concentration of fetal red blood cells in first-trimester pregnant women undergoing uterine aspiration is below the calculated threshold for Rh sensitization.

OBJECTIVES: To calculate the minimum fetal red blood cell concentration required to cause maternal Rh sensitization; validate the use of a flow cytometry protocol below that concentration; preliminarily assess the concentrations of fetal red blood cells in pregnant women before and after uterine aspiration. STUDY DESIGN: Using pre-existing literature, we calculated the lowest concentration of fetal red blood cells found to cause sensitization within adult female circulation. We validated a two-color flow cytometry protocol using fluorescently labeled antibodies to Hemoglobin F (expressed by fetal red blood cells and adult F cells) and Carbonic Anhydrase (expressed in red blood cells during the third trimester and postnatally) by titrating second trimester cord blood into non-pregnant adult blood. We applied this flow cytometry protocol in a prospective cohort study of 42 pregnant women at 5-12 weeks gestational age undergoing uterine aspiration for induced or spontaneous abortion. RESULTS: The calculated threshold for causing Rh sensitization was 250 fetal red blood cells per 10 million total red blood cells. We showed a linear relationship between observed and expected fetal red blood cell fractions in titrated samples. Fetal red blood cell counts were more reliable when samples acquired by flow cytometry contained at least 1 million red blood cells. All 37 subjects with evaluable paired samples demonstrated fetal red blood cell concentrations below the calculated threshold for Rh sensitization both pre- and post-procedure. The fetal RBC concentrations increased from a mean of 4.5 (median 0, range 0-57) fetal RBCS pre- to a mean of 8.6 (median 2, range 0-32) fetal RBCs post- per 10 million total RBCs (p < 0.001). CONCLUSIONS: Flow cytometry was capable of separately quantifying fetal red blood cells and maternal F cells to very dilute concentrations. Fetal red blood cell exposure in the first trimester was well below the calculated threshold for maternal Rh sensitization in our cohort. Larger studies are warranted to confirm our pilot study findings, fill this evidence gap and inform universal guidelines for administering Rh immunoglobulin after first trimester uterine aspiration. IMPLICATIONS: Fetal red blood cell exposure following first trimester uterine aspiration is well below the calculated threshold for maternal Rh sensitization in our cohort.

Combination immunotherapy after ASCT for multiple myeloma using MAGE-A3/Poly-ICLC immunizations followed by adoptive transfer of vaccine-primed and costimulated autologous T cells.

PURPOSE: Myeloma-directed cellular immune responses after autologous stem cell transplantation (ASCT) may reduce relapse rates. We studied whether coinjecting the TLR-3 agonist and vaccine adjuvant Poly-ICLC with a MAGE-A3 peptide vaccine was safe and would elicit a high frequency of vaccine-directed immune responses when combined with vaccine-primed and costimulated autologous T cells. EXPERIMENTAL DESIGN: In a phase II clinical trial (NCT01245673), we evaluated the safety and activity of ex vivo expanded autologous T cells primed in vivo using a MAGE-A3 multipeptide vaccine (compound GL-0817) combined with Poly-ICLC (Hiltonol), granulocyte macrophage colony-stimulating factor (GM-CSF) ± montanide. Twenty-seven patients with active and/or high-risk myeloma received autografts followed by anti-CD3/anti-CD28-costimulated autologous T cells, accompanied by MAGE-A3 peptide immunizations before T-cell collection and five times after ASCT. Immune responses to the vaccine were evaluated by cytokine production (all patients), dextramer binding to CD8(+) T cells, and ELISA performed serially after transplant. RESULTS: T-cell infusions were well tolerated, whereas vaccine injection site reactions occurred in >90% of patients. Two of nine patients who received montanide developed sterile abscesses; however, this did not occur in the 18 patients who did not receive montanide. Dextramer staining demonstrated MAGE-A3-specific CD8 T cells in 7 of 8 evaluable HLA-A2(+) patients (88%), whereas vaccine-specific cytokine-producing T cells were generated in 19 of 25 patients (76%). Antibody responses developed in 7 of 9 patients (78%) who received montanide and only weakly in 2 of 18 patients (11%) who did not. The 2-year overall survival was 74% [95% confidence interval (CI), 54%-100%] and 2-year event-free survival was 56% (95% CI, 37%-85%). CONCLUSIONS: A high frequency of vaccine-specific T-cell responses were generated after transplant by combining costimulated autologous T cells with a Poly-ICLC/GM-CSF-primed MAGE-A3 vaccine.

Silencing of microRNA-21 in vivo ameliorates autoimmune splenomegaly in lupus mice.

MicroRNAs (miRNAs) have been implicated in B cell lineage commitment, regulation of T cell differentiation, TCR signalling, regulation of IFN signalling, and numerous other immunological processes. However, their function in autoimmunity, and specifically in systemic lupus erythematosus (SLE), remains poorly understood. B6.Sle123 is a spontaneous genetic mouse model of SLE characterized by autoantibody production, lymphosplenomegaly, and glomerulonephritis. We identified several differentially regulated miRNAs in B and T lymphocytes of B6.Sle123 mice. We found that miR-21 expression in lupus B and T cells is up-regulated and that in vivo silencing of miR-21 using a tiny seed-targeting LNA reversed splenomegaly, one of the cardinal manifestations of autoimmunity in B6.Sle123 mice, and de-repressed PDCD4 expression in vivo and in vitro. In addition, treatment with anti-miR-21 altered CD4/CD8 T cell ratios and reduced Fas receptor-expressing lymphocyte populations. Our study shows that tiny LNAs can be used to efficiently antagonize endogenous miRNAs in peripheral lymphocytes in vivo and in primary lymphocytes cultured ex vivo and can alter the course of a spontaneous genetic disease in mice.

In vivo, multimodal imaging of B cell distribution and response to antibody immunotherapy in mice.

BACKGROUND: B cell depletion immunotherapy has been successfully employed to treat non-Hodgkin's lymphoma. In recent years, increasing attention has been directed towards also using B-cell depletion therapy as a treatment option in autoimmune disorders. However, it appears that the further development of these approaches will depend on a methodology to determine the relation of B-cell depletion to clinical response and how individual patients should be dosed. Thus far, patients have generally been followed by quantification of peripheral blood B cells, but it is not apparent that this measurement accurately reflects systemic B cell dynamics. METHODOLOGY/PRINCIPAL FINDINGS: Cellular imaging of the targeted population in vivo may provide significant insight towards effective therapy and a greater understanding of underlying disease mechanics. Superparamagnetic iron oxide (SPIO) nanoparticles in concert with near infrared (NIR) fluorescent dyes were used to label and track primary C57BL/6 B cells. Following antibody mediated B cell depletion (anti-CD79), NIR-only labeled cells were expeditiously cleared from the circulation and spleen. Interestingly, B cells labeled with both SPIO and NIR were not depleted in the spleen. CONCLUSIONS/SIGNIFICANCE: Whole body fluorescent tracking of B cells enabled noninvasive, longitudinal imaging of both the distribution and subsequent depletion of B lymphocytes in the spleen. Quantification of depletion revealed a greater than 40% decrease in splenic fluorescent signal-to-background ratio in antibody treated versus control mice. These data suggest that in vivo imaging can be used to follow B cell dynamics, but that the labeling method will need to be carefully chosen. SPIO labeling for tracking purposes, generally thought to be benign, appears to interfere with B cell functions and requires further examination.