Correlation of immune fitness with response to teclistamab in relapsed/refractory multiple myeloma in the MajesTEC-1 study.

ABSTRACT: Teclistamab, an off-the-shelf B-cell maturation antigen (BCMA) × CD3 bispecific antibody that mediates T-cell activation and subsequent lysis of BCMA-expressing myeloma cells, is approved for the treatment of patients with relapsed/refractory multiple myeloma (R/RMM). As a T-cell redirection therapy, clinical outcomes with teclistamab may be influenced by patient immune fitness and tumor antigen expression. We correlated tumor characteristics and baseline immune profiles with clinical response and disease burden in patients with R/RMM from the pivotal phase 1/2 MajesTEC-1 study, focusing on patients treated with 1.5 mg/kg of teclistamab (N = 165). Peripheral blood samples were collected at screening, and bone marrow samples were collected at screening and cycle 3. Better clinical outcomes to teclistamab correlated with higher baseline total T-cell counts in the periphery. In addition, responders (partial response or better) had a lower proportion of immunosuppressive regulatory T cells (Tregs), T cells expressing coinhibitory receptors (CD38, PD-1, and PD-1/TIM-3), and soluble BCMA and a T-cell profile suggestive of a more cytolytic potential, compared with nonresponders. Neither frequency of baseline bone marrow BCMA expression nor BCMA-receptor density was associated with clinical response to teclistamab. Improved progression-free survival was observed in patients with a lower frequency of T cells expressing exhaustion markers and immunosuppressive Tregs. Overall, response to teclistamab was associated with baseline immune fitness; nonresponders had immune profiles suggestive of immune suppression and T-cell dysfunction. These findings illustrate the importance of the contribution of the immune landscape to T-cell redirection therapy response. This trial was registered at www.ClinicalTrials.gov as #NCT03145181/NCT04557098.

Activity of ibrutinib plus R-CHOP in diffuse large B-cell lymphoma: Response, pharmacodynamic, and biomarker analyses of a phase Ib study.

INTRODUCTION: This unplanned post-hoc analysis was based on data from the phase Ib DBL1002 study (NCT01569750) and evaluated the association between molecular biomarkers and clinical response to combined treatment with ibrutinib plus rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in diffuse large B-cell lymphoma (DLBCL) subtypes. METHODS: DLBCL subtyping was conducted using immunohistochemistry. Next-generation sequencing using immunoglobulin H primers assessed minimal residual disease (MRD). A quantitative assay evaluated Bruton's tyrosine kinase (BTK) occupancy by ibrutinib in peripheral blood mononuclear cells. Targeted DNA sequencing examined genetic variants by DLBCL subtype. Secreted protein expression was evaluated with a SomaLogic analyte panel. RESULTS: Among 21 patients with DLBCL (median age 53.5 years), 17 achieved a complete response (CR) and 4 a partial response (PR). Of the 11 subtyped patients, 9 had a CR (5/7 germinal center B-cell-like [GCB] and 4/4 non-GCB) and 2 had a PR (both GCB). Nine of 12 patients tested for MRD achieved early (cycle 2 day 1) MRD negativity; most had a CR. There was near-complete BTK occupancy at 4 h postdose. Mutation analysis (n = 19) revealed variants including CREBBP, KMT2D, LRP1B, BCL2, and TNFRSF14; only 1 CD79B and TP53 each; no CARD11 or MYD88. CONCLUSIONS: In this study, first-line ibrutinib plus R-CHOP benefited patients with DLBCL, with good overall response rate and early MRD negativity. With a caveat of small sample size, our results showed that a favorable genetic profile and younger patient age may be important to beneficial clinical outcome with ibrutinib plus R-CHOP in DLBCL.

Identification of potential ibrutinib combinations in hematological malignancies using a combination high-throughput screen.

Matrix high-throughput screening (HTS) methods are increasingly employed to rapidly define potential therapeutic drug combinations. We used combination HTS to identify compounds showing synergistic anti-proliferative activity with ibrutinib, an irreversible, small-molecule inhibitor of Bruton's tyrosine kinase. The goal was to identify ibrutinib combinations with maximum synergistic effects in heme malignancy lines, particularly in non-Hodgkin lymphoma including diffuse large B-cell lymphoma (DLBCL). Growth inhibition (GI) was used to measure cell viability; synergy scores characterized strength of synergistic interaction. Single-agent ibrutinib demonstrated varying degrees of activity across 30 cell lines evaluated. In DLBCL lines, TMD8 was the most sensitive to ibrutinib (GI50 = 0.001); combinations with BCL-2 inhibitor ABT-199, and PI3K inhibitors IPI-145 and GDC-0941 showed the strongest synergistic activity. Anti-proliferative synergies were also observed with BET bromodomain inhibitor (+)-JQ1, XPO1 inhibitor selinexor, and IRAK4 inhibitor, and confirmed using apoptosis assay. These findings are intended to inform and advance treatment of B-cell malignancies.

Comparative analysis of lymphocyte activation marker expression and cytokine secretion profile in stimulated human peripheral blood mononuclear cell cultures: an in vitro model to monitor cellular immune function.

Activation of lymphocytes is a complex, yet finely regulated cascade of events that results in the expression of cytokine receptors, production and secretion of cytokines and expression of several cell surface molecules that eventually lead to divergent immune responses. Assessing the qualitative and quantitative nature of lymphocyte function following immunotherapy provides valuable information about the immune responses mediated by a therapeutic agent. To facilitate evaluation of the immunomodulatory activity of therapeutic agents, we have established a platform of in vitro immunoassays with normal human peripheral blood mononuclear cells (PBMCs) treated with several polyclonal activators that are known to exhibit different modes of action. We evaluated the kinetics of cell surface marker expression and cytokine release from PBMCs stimulated in parallel with various activating agents over a time course. These stimulating agents induced early (CD69 and CD71) and late (CD25 and HLA-DR) activation markers to varying antigen densities, indicated different cytokine profiles, and showed differential inhibition with dexamethasone (DEX), an inhibitor of early signaling events. Based on the association or correlation of the kinetics of activation marker expression and secreted cytokines, the results of our study indicate the appropriate time points for the simultaneous measurement of both these activation products. This study defines the kinetics for both measures of T cell activation and provides a comprehensive review with various polyclonal activators that can serve as a reference for monitoring lymphocyte function in clinical study samples.

Stability of immunophenotypic markers in fixed peripheral blood for extended analysis using flow cytometry.

The analysis of whole blood samples by flow cytometry for pharmacodynamic and biomarker assessments in clinical studies has been limited by the necessity to test these samples within a short time frame after blood collection. In most clinical studies, blood specimens are shipped to a centralized testing facility; it is critical to demonstrate specimen stability over a period of time which will encompass the time elapsed between specimen collection and testing. A possible solution to overcome this limitation is the use of a fixative to preserve the cell surface antigen stability in whole blood. We examined the stability of markers for T lymphocytes (CD3, CD4, CD8, CD45RA, and CD45RO), B lymphocytes (CD19), NK cells (CD16+CD56), activation (CD25 and HLA-DR), chemokine receptors (CCR5 and CXCR3) and skin homing (CLA) in fixed blood over 7 days and used this information to select the markers for global clinical studies. These assays with selected markers were further validated using fit-for-purpose approach (Lee et al., 2006) and to set the sample acceptability criteria for use in clinical sample testing. Most of the markers examined were stable when collected in Cyto-Chex® BCT for one week with the exception of the activation markers on T cells.

Positive treatment effects of ustekinumab in psoriasis: analysis of lesional and systemic parameters.

Ustekinumab, a human anti-interleukin (IL)-12/IL-23p40 monoclonal antibody has demonstrated significant efficacy in patients with moderate-to-severe psoriasis. Skin lesion biopsies, cell surface markers on peripheral blood lymphocytes, and ex vivo T-helper (Th)1/Th2 cytokine responses from peripheral blood mononuclear cells (PBMC) from patients receiving ustekinumab 45 or 90 mg, or placebo were evaluated at baseline and week 12. Inflammatory serum protein levels were measured at baseline, week 2 and week 12. At week 12, median epidermal thickness decreased from 312.1 to 132.7 microm, and median levels of cellular proliferation (Ki67) and T-cell infiltration (CD3) decreased by 84.3% and 70.7%, respectively, in the combined ustekinumab group (all P < or = 0.002). Serum levels of tumor necrosis factor (TNF)-alpha, C-C motif ligand 27 (CCL27) and other inflammatory cytokines remained unchanged. Minimal variation in the percentage of T cells expressing cutaneous lymphocyte antigen (CLA) was observed following ustekinumab treatment, with no significant variation in the percentage of cells expressing CD45RA, CD45RO, CD25, human leukocyte antigen-DR (HLA-DR), and C-X-C motif receptor 3 (CXCR3). No apparent effect on the magnitude of Th1/Th2 responses to external stimuli in PBMC was observed following placebo or ustekinumab treatment. Ustekinumab improves histological psoriasis measures, with minimal impact on the systemic immune system.

Modulation of CLA, IL-12R, CD40L, and IL-2Ralpha expression and inhibition of IL-12- and IL-23-induced cytokine secretion by CNTO 1275.

Cytokines interleukin (IL)-12 and IL-23 are implicated in the pathogenesis of psoriasis. IL-12 causes differentiation of CD4+ T cells to interferon-gamma (IFN-gamma)-producing T helper 1 (Th1) cells, while IL-23 induces differentiation to IL-17-producing pathogenic Th17 cells. The effects of the monoclonal antibody to IL-12/23 p40 subunit (CNTO 1275) on IL-12 receptor (IL-12R) expression, markers associated with skin homing, activation, and cytokine secretion were investigated in vitro using human peripheral blood mononuclear cells (PBMCs) from healthy donors. PBMCs were activated in the presence or absence of recombinant human (rh) IL-12 or rhIL-23, with or without CNTO 1275. CNTO 1275 inhibited upregulation of CLA, IL-12R, IL-2Ralpha and CD40L expression and also inhibited IL-12- and IL-23-induced IFN-gamma, IL-17A, tumor necrosis factor (TNF)-alpha, IL-2, and IL-10 secretion. Thus, the therapeutic effect of CNTO 1275 may be attributed to the IL-12/23 neutralization, resulting in decreased expression of skin homing and activation markers, and IL-12- and IL-23-induced cytokine secretion.

Co-expression of IL-12 receptors along with CXCR3 and CD25 on activated peripheral blood T lymphocytes.

IL-12 receptors (IL-12R) play a critical role in maintaining IL-12 regulation of T helper-1 (Th1) type immune responses. We studied the expression of two IL-12R, beta1 and beta2 on peripheral blood mononuclear cells (PBMCs) from normal donors, stimulated with polyclonal activators in the presence or absence of exogenous rhIL-12. Unstimulated peripheral blood T lymphocytes (PBTs) expressed moderate levels of IL-12Rbeta1 and very low to undetectable levels of IL-12Rbeta2. Superantigens and anti-CD3+anti-CD28 induced higher expression of both IL-12R on PBTs than PHA-P stimulation. Exogenous rhIL-12 further enhanced the PHA-P or anti-CD3+anti-CD28 induced IL-12Rbeta2 expression. Only a fraction of mitogen activated IL-12Rbeta1+ or beta2+ T lymphocytes co-expressed CD25 (with further enhancement by exogenous rhIL-12), while a higher percentage of these cells were CXCR3+. The majority of superantigen or anti-CD3+anti-CD28-induced IL-12R+ PBTs were positive for both CD25 and CXCR3 markers. Our results indicated differential induction of IL-12R expression that correlated with up regulation of CD25 and CXCR3 expression on activated PBTs and provide a useful insight for monitoring these markers during treatment of Th1 type inflammatory diseases.

Cutaneous lymphocyte antigen expression on activated lymphocytes and its association with IL-12R (beta1 and beta2), IL-2Ralpha, and CXCR3.

The majority of T lymphocytes that infiltrate psoriatic lesions express cutaneous lymphocyte antigen (CLA), a skin homing receptor involved in the influx of memory T cells to cutaneous sites. We investigated CLA expression on normal human peripheral blood mononuclear cells (PBMCs) and evaluated its association with IL-12 receptors, chemokine receptor, CXCR3, and IL-2Ralpha. PBMCs were stimulated in vitro with or without polyclonal activators (mitogen, or superantigens, or anti-CD3+anti-CD28) in the presence or absence of exogenous rhIL-12. The percentage of CLA+ T lymphocytes increased significantly after superantigen stimulation compared to anti-CD3+anti-CD28 or mitogen activation. The majority of activation induced CLA+ T lymphocytes co-expressed IL-12Rbeta1, IL-12Rbeta2, CXCR3, and CD25 in the presence of rhIL-12. Our results indicate that CLA expression on activated T lymphocytes is IL-12 and activation dependent and correlates with the expression of IL-12 receptors, IL-2Ralpha, and CXCR3. Monitoring the levels of Th1 differentiation markers such as CXCR3 and IL-12Rbeta2 along with activation marker, CD25 on skin homing CLA+ T lymphocytes may provide insight into the mechanism of action of immunotherapies directed against Th1 type skin inflammatory diseases.