Target modulation and pharmacokinetics/pharmacodynamics translation of the BTK inhibitor poseltinib for model-informed phase II dose selection

AbstractThe selective Bruton tyrosine kinase (BTK) inhibitor poseltinib has been shown to inhibit the BCR signal transduction pathway and cytokine production in B cells (Park et al.Arthritis Res. Ther.18, 91, 10.1186/s13075-016-0988-z, 2016). This study describes the translation of nonclinical research studies to a phase I clinical trial in healthy volunteers in which pharmacokinetics (PKs) and pharmacodynamics (PDs) were evaluated for dose determination. The BTK protein kinase inhibitory effects of poseltinib in human peripheral blood mononuclear cells (PBMCs) and in rats with collagen-induced arthritis (CIA) were evaluated. High-dimensional phosphorylation analysis was conducted on human immune cells such as B cells, CD8 + memory cells, CD4 + memory cells, NK cells, neutrophils, and monocytes, to map the impact of poseltinib on BTK/PLC and AKT signaling pathways. PK and PD profiles were evaluated in a first-in-human study in healthy donors, and a PK/PD model was established based on BTK occupancy. Poseltinib bound to the BTK protein and modulated BTK phosphorylation in human PBMCs. High-dimensional phosphorylation analysis of 94 nodes showed that poseltinib had the highest impact on anti-IgM + CD40L stimulated B cells, however, lower impacts on anti-CD3/CD-28 stimulated T cells, IL-2 stimulated CD4 + T cells and NK cells, M-CSF stimulated monocytes, or LPS-induced granulocytes. In anti-IgM + CD40L stimulated B cells, poseltinib inhibited the phosphorylation of BTK, AKT, and PLCγ2. Moreover, poseltinib dose dependently improved arthritis disease severity in CIA rat model. In a clinical phase I trial for healthy volunteers, poseltinib exhibited dose-dependent and persistent BTK occupancy in PBMCs of all poseltinib-administrated patients in the study. More than 80% of BTK occupancy at 40 mg dosing was maintained for up to 48 h after the first dose. A first-in-human healthy volunteer study of poseltinib established target engagement with circulating BTK protein. Desirable PK and PD properties were observed, and a modeling approach was used for rational dose selection for subsequent trials. Poseltinib was confirmed as a potential BTK inhibitor for the treatment of autoimmune diseases.Trial registration: This article includes the results of a clinical intervention on human participants [NCT01765478].

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Monitoring of Dendritic Cell Numbers in Patients during the Treatment of Multiple Myeloma.

Blood ◽

10.1182/blood.v106.11.5096.5096 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5096-5096

Author(s):

Lucie Kovarova ◽

Roman Hajek ◽

Adam Svobodnik ◽

Miroslav Penka ◽

Jiri Mayer

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

B Cells ◽

Dendritic Cell ◽

Healthy Volunteers ◽

Nk Cells ◽

Peripheral Blood ◽

Initial Values ◽

Hla Dr ◽

Significant Difference

Abstract Objective: In this study, the proportion of dendritic cell (DC) subsets (myeloid DC1 and plasmacytoid DC2), T cells, B cells and NK cells was evaluated in peripheral blood of patients with multiple myeloma (MM) before and during treatment including autologous transplantation. Also control group of healthy volunteers was evaluated. Methods: Flow cytometric determination of relative cells number in unmanipulated peripheral blood was based on expression of the surface antigen: T cells (CD3/CD4/CD8), B cells (CD19/CD20), NK cells (CD3/CD16/CD56) and DC (CD83/HLA-DR/CD11c and CD83/HLA-DR/CD123). Results: Significant difference (p<0.01) was found in initial values of CD83+ cells between the group of healthy volunteers (n = 15; mean count of CD83+ cells 0,26 ± 0,15%; ratio DC1/DC2 = 1,54) and the group of patients before treatment (n = 15; 0,15 ± 0,03% CD83+; DC1/DC2 = 4,55). After induction treatment with VAD regimen (vincristine, adriamycin, dexamethasone) in a group of patients was the mean percentage of DC higher (0,18 ± 0,04% CD83+ cells; DC1/DC2 = 4,77) than initial values. Administration of G-CSF again increased the total DC numbers (0,34 ± 0,11%; DC1/DC2 = 2,3) and intermediate levels of DC counts were found in the apheresis products (0,22 ± 0,05%; DC1/DC2 = 1,21). After engraftment there were found the highest relative DC numbers (0,50 ± 0,21%; DC1/DC2 = 1,85) in patients. Within six months after transplantation were achieved pretreatment DC values when compared with DC values of healthy volunteers (p<0,97) (0,24 ± 0,08%; DC1/DC2 = 1,57). Total numbers of T cells did not significantly differ during treatment only the reverse CD4/CD8 ratio was found in majority of patients within six month after the transplantation. Conclusions: Untreated patients with MM have significant lower relative numbers of peripheral blood DC in comparison with healthy volunteers. The highest number of total DC was found after engraftment. The ratio DC1/DC2 showed relative majority of DC1 subtype and its the lowest value was found in the apheresis products. Normal DC values comparable with DC values of healthy volunteers were found in patients within six months after transplantation together with the reverse CD4/CD8 ratio.

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Alterations of circulating lymphocyte subsets in patients with colorectal carcinoma

Cancer Immunology Immunotherapy ◽

10.1007/s00262-021-03127-8 ◽

2021 ◽

Author(s):

Johanna Waidhauser ◽

Pia Nerlinger ◽

Tim Tobias Arndt ◽

Stefan Schiele ◽

Florian Sommer ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Colorectal Carcinoma ◽

Healthy Volunteers ◽

Nk Cells ◽

Colon Carcinoma ◽

Lymphocyte Subsets ◽

Cd4 Cells ◽

Cd8 Cells ◽

Circulating Lymphocytes

Abstract Introduction Cellular immune response to cancer is known to be of great importance for tumor control. Moreover, solid tumors influence circulating lymphocytes, which has been shown for several types of cancer. In our prospective study we elucidate changes in lymphocyte subsets in patients with colorectal carcinoma compared to healthy volunteers. Methods Flow cytometry was performed at diagnosis of colon carcinoma to analyze B cells, T cells and NK cells including various subtypes of each group. Univariate and multivariate analyses including age, gender, tumor stage, sidedness and microsatellite instability status (MSI) were performed. Results Forty-seven patients and 50 healthy volunteers were included. Median age was 65 years in patients and 43 years in the control group. Univariate analysis revealed lower total lymphocyte counts, lower CD4 + cells, CD8 + cells, B cells and NKs including various of their subsets in patients. In multivariate analysis patients had inferior values of B cells, CD4 + cells and NK cells and various subsets, regardless of age and gender. Naïve, central memory and HLADR + CD8 + cells showed an increase in patients whereas all other altered subsets declined. MSI status had no influence on circulating lymphocytes except for higher effector memory CD8 + cells in MSI-high patients. Localization in the left hemicolon led to higher values of total cytotoxic T cells and various T cell subsets. Conclusion We found significant changes in circulating lymphocyte subsets in colon carcinoma patients, independent of physiological alterations due to gender or age. For some lymphocyte subsets significant differences according to tumor localization or MSI-status could be seen.

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Lymphoid and myeloid differentiation of fetal liver CD34+lineage- cells in human thymic organ culture.

Journal of Experimental Medicine ◽

10.1084/jem.180.1.123 ◽

1994 ◽

Vol 180 (1) ◽

pp. 123-132 ◽

Cited By ~ 53

Author(s):

A Bárcena ◽

A H Galy ◽

J Punnonen ◽

M O Muench ◽

D Schols ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Organ Culture ◽

Nk Cells ◽

Progenitor Cells ◽

Fetal Liver ◽

Myeloid Differentiation ◽

Target Cells ◽

Hematopoietic Progenitors ◽

Fetal Thymus

In this article, we report that the human fetal thymus contains CD34bright cells (< 0.01% of total thymocytes) with a phenotype that resembles that of multipotent hematopoietic progenitors in the fetal bone marrow. CD34bright thymocytes were CD33-/dull and were negative for CD38, CD2, and CD5 as well as for the lineage markers CD3, CD4, and CD8 (T cells), CD19 and CD20 (B cells), CD56 (NK cells), glycophorin (erythrocytes), and CD14 (monocytes). In addition, total CD34+ lineage negative (lin-) thymocytes contained a low number of primitive myeloid progenitor cells, thus suggesting that the different hematopoietic lineages present in the thymus may be derived from primitive hematopoietic progenitor cells seeding the thymus. To investigate whether the thymus is permissive for the development of non-T cells, human fetal organ culture (FTOC) assays were performed by microinjecting sorted CD34+lin- fetal liver cells into fragments of HLA-mismatched fetal thymus. Sequential phenotypic analysis of the FTOC-derived progeny of CD34+lin- cells indicated that the differentiation into T cells was preceded by a wave of myeloid differentiation into CD14+CD11b+CD4dull cells. Donor-derived B cells (CD19+CD20+) were also generated, which produced immunoglobulins (IgG and IgM) when cultured under appropriate conditions, as well as functional CD56+CD3- NK cells, which efficiently killed K562 target cells in cytotoxicity assays. These results demonstrate that the microinjection of fetal liver hematopoietic progenitors into fetal thymic organ fragments results in multilineage differentiation in vitro.

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Engineered immunostimulatory cells can convert PBMCs from chronic lymphocytic leukemia (CLL) patients into potent tumor killing immune cells.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.7517 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 7517-7517

Author(s):

Joshua W. Keegan ◽

Frank Borriello ◽

Stacey M. Fernandes ◽

Jennifer R. Brown ◽

James A. Lederer

Keyword(s):

T Cells ◽

B Cells ◽

Tumor Cell ◽

Nk Cells ◽

Cd8 T Cells ◽

Immune Cells ◽

Killing Activity ◽

Tumor Killing ◽

Tumor Cell Killing ◽

Potent Tumor

7517 Background: Alloplex Biotherapeutics has developed a cellular therapeutic that uses ENgineered Leukocyte ImmunoSTimulatory cell lines called ENLIST cells to activate and expand populations of tumor killing effector cells from human peripheral blood mononuclear cells (PBMCs). This process leads to a 300-fold expansion of NK cells, CD8+ T cells, NKT cells, and TCRγδ T cells that are called SUPLEXA cells, which will be cryopreserved and transferred back into patients as an autologous immune cell therapy for cancer. In this study, PBMCs from CLL patients were used to generate SUPLEXA cells as a first approach to comparatively profile SUPLEXA cells from cancer patients and normal healthy volunteers (NHVs). Methods: ENLIST cell lines were engineered by expressing curated immunomodulatory proteins in the SK-MEL-2 melanoma cell line. Two million (M) PBMCs from 10 CLL patients or 2 NHVs were incubated with 0.4 M freeze/thaw killed ENLIST cells for 5 days in XVIVO-15 medium with 2% heat-inactivated human AB serum (XAB2) and then split 1:15 in XAB2 containing IL-7 and IL-15 to expand. After 9 days, SUPLEXA cells were harvested and cryopreserved. Results: Original PBMCs and matched SUPLEXA cells from each donor were thawed and characterized by mass cytometry (CyTOF) using a 47-marker antibody panel. CyTOF staining results of PBMCs from CLL patients demonstrated approximately 95% leukemia cells and few T cells, NK cells, B cells, and monocytes. CyTOF staining of SUPLEXA cells from all 10 CLL patients showed expansion of NK cells (17%), CD8 T cells (11%), and CD4 T cells (7.5%) that were similar in phenotype to SUPLEXA cells from NHVs showing high expression of granzymes and perforin that are indicative of potent tumor cell killing activity. Cancer cells in the original CLL PBMC samples were reduced to 0.78%. However, a population of non-T/non-B cells (60% ± 9.5%) was detected in SUPLEXA cells from all CLL patients that require further characterization. Next, SUPLEXA cells from CLL and NHV patients were comparatively tested for tumor cell killing activity at 2:1, 1:1, and 1:2 effector to target cell (MEL-14 melanoma cells expressing RFP) ratios. Percent killing of tumor cells by SUPLEXA cells prepared from CLL patients (77.8% ± 2.6% at 2:1) and NHVs (81.5% ± 0.3% at 2:1) were nearly identical at all effector to target ratios. Conclusions: We demonstrate for the first time that PBMCs from CLL patients can be converted into SUPLEXA cells despite low numbers of normal immune cells at baseline and the known immunologic impairment present in CLL patients. Importantly, SUPLEXA cells derived from CLL patients acquire potent tumor killing activity that is indistinguishable from SUPLEXA cells prepared from NHVs. Taken together, these findings support the feasibility of converting PBMCs from CLL patients with low percentages of NK and T cells into an autologous cellular therapy for cancer.

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Prognostic significance of natural killer cell-associated markers in gastric cancer: quantitative analysis using multiplex immunohistochemistry

Journal of Translational Medicine ◽

10.1186/s12967-021-03203-8 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Hee Young Na ◽

Yujun Park ◽

Soo Kyung Nam ◽

Jiwon Koh ◽

Yoonjin Kwak ◽

...

Keyword(s):

Gastric Cancer ◽

T Cells ◽

B Cells ◽

Nk Cells ◽

Natural Killer ◽

Immune Cells ◽

Nk Cell ◽

Critical Role ◽

Killer Cell ◽

Prognostic Significance

Abstract Background Natural killer (NK) cells mediate the anti-tumoral immune response as an important component of innate immunity. The aim of this study was to investigate the prognostic significance and functional implication of NK cell-associated surface receptors in gastric cancer (GC) by using multiplex immunohistochemistry (mIHC). Methods We performed an mIHC on tissue microarray slides, including 55 GC tissue samples. A total of 11 antibodies including CD57, NKG2A, CD16, HLA-E, CD3, CD20, CD45, CD68, CK, SMA, and ki-67 were used. CD45 + CD3-CD57 + cells were considered as CD57 + NK cells. Results Among CD45 + immune cells, the proportion of CD57 + NK cell was the lowest (3.8%), whereas that of CD57 + and CD57- T cells (65.5%) was the highest, followed by macrophages (25.4%), and B cells (5.3%). CD57 + NK cells constituted 20% of CD45 + CD57 + immune cells while the remaining 80% were CD57 + T cells. The expression of HLA-E in tumor cells correlated with that in tumoral T cells, B cells, and macrophages, but not CD57 + NK cells. The higher density of tumoral CD57 + NK cells and tumoral CD57 + NKG2A + NK cells was associated with inferior survival. Conclusions Although the number of CD57 + NK cells was lower than that of other immune cells, CD57 + NK cells and CD57 + NKG2A + NK cells were significantly associated with poor outcomes, suggesting that NK cell subsets play a critical role in GC progression. NK cells and their inhibitory receptor, NKG2A, may be potential targets in GC.

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Identification of Hub Genes and Immune Infiltration in Psoriasis by Bioinformatics Method

Frontiers in Genetics ◽

10.3389/fgene.2021.606065 ◽

2021 ◽

Vol 12 ◽

Author(s):

Wenxing Su ◽

Yuqian Wei ◽

Biao Huang ◽

Jiang Ji

Keyword(s):

T Cells ◽

B Cells ◽

Nk Cells ◽

Signaling Pathway ◽

Control Sample ◽

Gene Expression Omnibus ◽

Ppi Network ◽

Hub Genes ◽

Immune Infiltration ◽

Chemokine Signaling

BackgroundPsoriasis is a chronic, prolonged, and recurrent skin inflammatory disease. However, the pathogenesis of psoriasis is not completely clear, thus we aimed to explore potential molecular basis of it.MethodsTwo datasets were downloaded from the Gene Expression Omnibus database. After identifying the differentially expressed genes of psoriasis skin lesion samples and healthy controls, three kinds of analyses, namely functional annotation, protein-protein interaction (PPI) network, and immune infiltration analyses, were performed.ResultsA total of 152 up-regulated genes and 38 down-regulated genes were selected for subsequent analyses. Evaluation of the PPI network identified the most important module containing 13 hub genes. Gene ontology analysis showed that the hub genes have a significant enrichment effect on positive regulation of cell migration, defense response to the other organism and epithelial cell differentiation. KEGG signaling pathway analysis showed that the hub genes were significantly enriched in chemokine signaling, Toll-like receptor signaling pathway, and IL-17 signaling pathway. Compared with the normal control sample, naive B cells, CD8+ T cells, activated memory CD4+ T cells, follicular helper T cells, gamma delta T cells, resting NK cells, monocytes, M0 macrophages, M1 macrophages, activated dendritic cells and neutrophils infiltrated more, while memory B cells, naive CD4+ T cells, regulatory T cells (Tregs), activated NK cells, resting mast cells, and eosinophils infiltrated less.ConclusionTo conclude, the hub genes and pathways identified from psoriasis lesions and normal controls along with the immune infiltration profile may provide new insights into the study of psoriasis.

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Characterization of the DNAM-1, TIGIT and TACTILE Axis on Circulating NK, NKT-Like and T Cell Subsets in Patients with Acute Myeloid Leukemia

Cancers ◽

10.3390/cancers12082171 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2171

Author(s):

Isabel Valhondo ◽

Fakhri Hassouneh ◽

Nelson Lopez-Sejas ◽

Alejandra Pera ◽

Beatriz Sanchez-Correa ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

T Cell ◽

Healthy Volunteers ◽

Nk Cells ◽

Myeloid Leukemia ◽

Cell Activation ◽

Nk Cell ◽

T Cell Subsets ◽

Acute Myeloid

Background: Acute myeloid leukemia (AML) remains a major clinical challenge due to poor overall survival, which is even more dramatic in elderly patients. TIGIT, an inhibitory receptor that interacts with CD155 and CD112 molecules, is considered as a checkpoint in T and NK cell activation. This receptor shares ligands with the co-stimulatory receptor DNAM-1 and with TACTILE. The aim of this work was to analyze the expression of DNAM-1, TIGIT and TACTILE in NK cells and T cell subsets in AML patients. Methods: We have studied 36 patients at the time of diagnosis of AML and 20 healthy volunteers. The expression of DNAM-1, TIGIT and TACTILE in NK cells and T cells, according to the expression of CD3 and CD56, was performed by flow cytometry. Results: NK cells, CD56− T cells and CD56+ T (NKT-like) cells from AML patients presented a reduced expression of DNAM-1 compared with healthy volunteers. An increased expression of TIGIT was observed in mainstream CD56− T cells. No differences were observed in the expression of TACTILE. Simplified presentation of incredibly complex evaluations (SPICE) analysis of the co-expression of DNAM-1, TIGIT and TACTILE showed an increase in NK and T cells lacking DNAM-1 and co-expressing TIGIT and TACTILE. Low percentages of DNAM-1−TIGIT+TACTILE+ NK cells and DNAM-1− TIGIT+TACTILE+ CD56− T cells were associated with a better survival of AML patients. Conclusions: The expression of DNAM-1 is reduced in NK cells and in CD4+ and CD8+ T cells from AML patients compared with those from healthy volunteers. An increased percentage of NK and T cells lacking DNAM-1 and co-expressing TIGIT and TACTILE is associated with patient survival, supporting the role of TIGIT as a novel candidate for checkpoint blockade.

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Expression of the G-protein--coupled receptor BLR1 defines mature, recirculating B cells and a subset of T-helper memory cells

Blood ◽

10.1182/blood.v84.3.830.830 ◽

1994 ◽

Vol 84 (3) ◽

pp. 830-840 ◽

Cited By ~ 21

Author(s):

R Forster ◽

T Emrich ◽

E Kremmer ◽

M Lipp

Keyword(s):

T Cells ◽

B Cells ◽

Cord Blood ◽

G Protein ◽

Blood Cells ◽

Memory Cells ◽

T Helper ◽

G Protein Coupled Receptor ◽

Color Flow ◽

G Protein Coupled

Abstract The G-protein-coupled receptor BLR1 related to receptors for chemokines and neuropeptides has been identified as the first lymphocyte-specific member of the gene family characterized by seven transmembrane-spanning regions. Using a high-affinity anti-BLR1 monoclonal antibody (MoAb) and three-color flow cytometry it is shown that BLR1 expression on peripheral blood cells is limited to B cells and to a subset of CD4+ (14%) and CD8+ (2%) lymphocytes. T cells expressing BLR1 were positive for CD45R0, were negative for interleukin-2 receptors, show high levels of CD44, and show low levels of L-selectin. The majority of CD4+ cells originating from secondary lymphatic tissue, but none of cord blood- derived T cells, express BLR1. These observations suggest that BLR1 is a marker for memory T cells. Furthermore, BLR1 expression was detected on all CD19+ peripheral or tonsillar B lymphocytes, but only on a fraction of cord blood cells and bone marrow cells expressing CD19, sIgM, or sIgD. Interestingly, activation of both mature B and T cells by CD40 MoAb and CD3 MoAb, respectively, led to complete downregulation of BLR1. These data suggest that the G-protein-coupled receptor BLR1 is involved in functional control of mature recirculating B cells and T- helper memory cells participating in cell migration and cell activation.

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The Complexity of Microglial Interactions With Innate and Adaptive Immune Cells in Alzheimer’s Disease

Frontiers in Aging Neuroscience ◽

10.3389/fnagi.2020.592359 ◽

2020 ◽

Vol 12 ◽

Author(s):

Season K. Wyatt-Johnson ◽

Randy R. Brutkiewicz

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

T Cells ◽

B Cells ◽

Nk Cells ◽

Immune Cells ◽

Reactive Microglia ◽

Adaptive Immune Cells ◽

Affected Relative ◽

Peripheral Immune Cells

In the naïve mouse brain, microglia and astrocytes are the most abundant immune cells; however, there is a complexity of other immune cells present including monocytes, neutrophils, and lymphocytic cells, such as natural killer (NK) cells, T cells, and B cells. In Alzheimer’s disease (AD), there is high inflammation, reactive microglia, and astrocytes, leaky blood–brain barrier, the buildup of amyloid-beta (Aβ) plaques, and neurofibrillary tangles which attract infiltrating peripheral immune cells that are interacting with the resident microglia. Limited studies have analyzed how these infiltrating immune cells contribute to the neuropathology of AD and even fewer have analyzed their interactions with the resident microglia. Understanding the complexity and dynamics of how these immune cells interact in AD will be important for identifying new and novel therapeutic targets. Thus, this review will focus on discussing our current understanding of how macrophages, neutrophils, NK cells, T cells, and B cells, alongside astrocytes, are altered in AD and what this means for the disorder, as well as how these cells are affected relative to the resident microglia.

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Maturation of Lymphocyte Immunophenotypes and Memory T Helper Cell Differentiation During Development in Mice

Developmental Immunology ◽

10.1155/2000/56106 ◽

2000 ◽

Vol 8 (1) ◽

pp. 47-60 ◽

Cited By ~ 5

Author(s):

Omar R. Fagoaga ◽

Steven. M. Yellon ◽

Sandra. L. Nehlsen-Cannarella

Keyword(s):

Immune Response ◽

T Cells ◽

Cell Differentiation ◽

B Cells ◽

Cell Subset ◽

T Helper Cell ◽

Natural Killers ◽

Memory Cells ◽

Spleen Lymphocyte ◽

T Helper

The goal of this study was to systematically investigate the ontogeny of lymphoid populations throughout postnatal development. In CD-1 mice, peak lymphocyte numbers occurred in blood on postnatal day 10 (dl0) including those for natural killers (NK1.1), B cells (CD19), T helper (CD3CD4), naïve T helper (CD4CD62LposCD44low), memory T helper (CD4CD62LnegCD44high), and T cytotoxic (CD3CD8) cells. As percent of total lymphocytes, peaks were achieved by d10 for all T helper subtypes but not B cells which declined to a nadir. In spleen, lymphocyte numbers increased exponentially after d10. Proportionately, NK and T cells peaked on d10, declined by d20, and increased 2–3-fold by d45. Naive T cells constituted the majority of lymphocytes during development while memory cells gained to 2.2% (blood) and 12 % (spleen) by d20. C57BL/6 mice had similar profiles except that the B cell nadir and T cell subset peaks were at d5. Peripheralization of critical numbers of lymphocytes by d10, and importantly, development of a repertoire of memory cells by d20, may define immune response capabilities that close the period of immaturity for the neonate.

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