Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma.

BACKGROUND: The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. METHODS: We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application. RESULTS: The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells. CONCLUSIONS: We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.

The role of rituximab in the treatment of canine lymphoma: an ex vivo evaluation.

Targeting the CD20 receptor that is common to many B-cell Non-Hodgkin's Lymphoma subtypes in people, rituximab is a chimeric monoclonal antibody which has significantly improved disease-free survival rates compared with the use of cytotoxic agents alone. This study evaluated ex vivo canine B cell binding and depletion by rituximab with flow cytometric technique as possible proof of concept for treatment of canine lymphoma. Despite immunohistochemistry supporting CD20 expression, rituximab did not bind or deplete canine B cells and it is unlikely that it will be added to the armamentarium of treatment options for canine lymphoma.

Recommendations for the evaluation of specimen stability for flow cytometric testing during drug development.

The objective of this manuscript is to present an approach for evaluating specimen stability for flow cytometric methods used during drug development. While this approach specifically addresses stability assessment for assays to be used in clinical trials with centralized testing facilities, the concepts can be applied to any stability assessment for flow cytometric methods. The proposed approach is implemented during assay development and optimization, and includes suggestions for designing a stability assessment plan, data evaluation and acceptance criteria. Given that no single solution will be applicable in all scenarios, this manuscript offers the reader a roadmap for stability assessment and is intended to guide the investigator during both the method development phase and in the experimental design of the validation plan.

Rituximab-mediated depletion of cynomolgus monkey B cells in vitro in different matrices: possible inhibitory effect of IgG.

The mechanism of rituximab-mediated depletion of nonmalignant CD20+B cells remains to be clarified. In this report, we examine contributions of complement- and cell-dependent killing to the rituximab-mediated depletion of cynomolgus monkey B cells in the in vitro assay. B cell depletion was assessed in whole blood, buffer, autologus plasma (plasma), heat-inactivated plasma (H/I plasma), and cobra venom factor (CVF)-treated plasma matrices in cynomolgus monkey and human samples. Rituximab-mediated B cell depletion in buffer appeared to be greater than that in whole blood or in autologus plasma. Heat inactivation of plasma resulted in the degree of B cell depletion closer to that seen in buffer, whereas CVF treatment of plasma had no effect on B cell depletion. Addition of IgG to the buffer decreased the degree of B cell depletion. The results of these studies imply that (i) plasma components (including complement) are not the mediators of the rituximab-triggered B cell depletion in the in vitro assay, suggesting that cell-mediated mechanisms are likely to be responsible for in vitro killing of normal B cells, and that (ii) some plasma components appear to inhibit rituximab-mediated B cell depletion in the in vitro assay, with IgG identified as a possible inhibitor component.

Differential in vivo effects of rituximab on two B-cell subsets in cynomolgus monkeys.

Cynomolgus monkeys (Macaca fascicularis) are widely used animal models in biomedical research and have been used to study new therapeutics aimed at B-cell depletion. We have recently identified two different B-cell subsets in cynomolgus monkey, with the CD20lowCD40highCD21+ subset being phenotypically closer to human B cells and having a similar responsivness to anti-CD20 mAb, rituximab, in in vitro depletion assays. Here, we show that similar to in vitro findings CD20highCD40lowCD21- and CD20lowCD40highCD21+ cynomolgus monkey B cells differ significantly in their in vivo susceptibility to rituximab, as the low dose of 0.05 mg/kg of rituximab resulted in more than 70% depletion of the former B-cell subset and virtually no depletion of the latter B-cell subset. Our data suggest that for the B-cell-targeting anti-CD20 therapeutics, depletion of CD20lowCD40highCD21+ subset rather than depletion of all cynomolgus monkey B cells is more relevant to dose-efficacy projections for humans. In addition, we show that differential cell surface expression of CD80/CD86 costimulatory molecules on the two different cynomolgus monkey B-cell subsets is similar to that identified in rhesus monkeys, suggesting that our in vivo study may be relevant to other monkey models.

Blockade of CD11a by efalizumab in psoriasis patients induces a unique state of T-cell hyporesponsiveness.

Efalizumab (anti-CD11a) interferes with LFA-1/ICAM-1 binding and inhibits several key steps in psoriasis pathogenesis. This study characterizes the effects of efalizumab on T-cell activation responses and expression of surface markers on human circulating psoriatic T cells during a therapeutic trial. Our data suggest that efalizumab may induce a unique type of T-cell hyporesponsiveness, directly induced by LFA-1 binding, which is distinct from conventional anergy described in animal models. Direct activation of T cells through different activating receptors (CD2, CD3, CD3/28) is reduced, despite T cells being fully viable. This hyporesponsiveness was spontaneously reversible after withdrawal of the drug, and by IL-2 in vitro. In contrast to the state of anergy, Ca(+2) release is intact during efalizumab binding. Furthermore, lymphocyte function-associated antigen-1 (LFA-1) blockade resulted in an unexpected downregulation of a broad range of surface molecules, including the T-cell receptor complex, co-stimulatory molecules, and integrins unrelated to LFA-1, both in the peripheral circulation and in diseased skin tissue. These observations provide evidence for the mechanism of action of efalizumab. The nature of this T-cell hyporesponsiveness suggests that T-cell responses may be reduced during efalizumab therapy, but are reversible after ceasing efalizumab treatment.

Anti-BR3 antibodies: a new class of B-cell immunotherapy combining cellular depletion and survival blockade.

Removal of pathogenic B lymphocytes by depletion of monoclonal antibodies (mAbs) or deprivation of B-cell survival factors has demonstrated clinical benefit in both oncologic and immunologic diseases. Partial clinical responses and emerging data demonstrating incomplete B-cell depletion after immunotherapy fuels the need for improved therapeutic modalities. Lessons from the first generation of therapeutics directed against B-cell-specific antigens (CD20, CD22) are being applied to develop novel antibodies with additional functional attributes. We describe the generation of a novel class of B-cell-directed therapy (anti-BR3 mAbs) that combines the depleting capacity of a therapeutic mAb and blockade of B-cell-activating factor (BAFF)-BR3 B-cell survival. In mice, treatment with antagonistic anti-BR3 antibodies results in quantitatively greater reduction in some B-cell subsets and qualitatively different effects on bone marrow plasma cells compared with BR3-Fc BAFF blockade or with anti-CD20 treatment. Comparative analysis of BR3-Fc and anti-BR3 mAb reveals a lower B-cell dependence for BAFF-mediated survival in nonhuman primates than in mice. This novel class of B-cell-targeted therapies shows species characteristics in mice and primates that will guide translation to treatment of human disease.

A soluble BAFF antagonist, BR3-Fc, decreases peripheral blood B cells and lymphoid tissue marginal zone and follicular B cells in cynomolgus monkeys.

BAFF (also known as BLyS), a member of the tumor necrosis factor superfamily, plays a critical role in the maturation and development of B cells. BAFF has three receptors on B cells, the most crucial of which is BR3. In this study, we demonstrate the biological outcome of BAFF blockade in cynomolgus monkeys using a soluble fusion protein consisting of human BR3 and human IgG1 Fc. In vitro, BR3-Fc blocked BAFF-mediated survival and proliferation of cynomolgus monkey B cells. Weekly treatment of cynomolgus monkeys with BR3-Fc for 13 to 18 weeks resulted in significant B-cell reduction in the peripheral blood and in lymphoid organs. CD21(high) B cells in lymphoid tissues, a subset analogous to human marginal zone B cells, expressed nearly twofold higher BR3 levels than did CD21(med) B cells. Lymphoid tissue flow cytometric analysis showed that BR3-Fc reduced this CD21(high) B-cell subset to a greater extent than it reduced CD21(med) B cells. Dual-label immunohistochemistry and morphometric image analysis supported these results by demonstrating that BR3-Fc reduced a significant proportion of the B cells within the splenic inner and outer marginal zones. These findings should prove very useful in guiding the desired therapeutic use of BR3-Fc for autoimmune diseases in the clinic.

Depletion of B cells by a humanized anti-CD20 antibody PRO70769 in Macaca fascicularis.

PRO70769 is a humanized IgG1 monoclonal antibody against the CD20 molecule that is present on normal and malignant B cells. PRO70769 is being evaluated for treatment of B-cell-mediated diseases and is in a phase 1 trial for rheumatoid arthritis. As part of the preclinical toxicology evaluation, B-cell depletion profiles and safety of PRO70769 were assessed in cynomolgus monkeys. Animals were administered drug (IV) on days 1 and 15 with 10, 50, or 100 mg/kg PRO70769 and killed 2 weeks after the second dose and after a 3-month recovery period. In a parallel study, animals were not necropsied but instead were retreated with a second cycle of PRO70769 administered under an identical regimen. PRO70769 suppressed B cells in the blood to undetectable levels and significantly reduced B cells in lymphoid tissues. Splenic B cells were depleted to a greater extent compared with lymph node B cells. A second cycle of treatment resulted in a greater extent of depletion in lymphoid tissues compared with the depletion observed after one cycle of treatment; however, residual B cells in lymphoid tissues were still detectable, even at the highest dose. The rate of B-cell recovery in peripheral blood appeared similar between one and two cycles of treatment. Upon depletion there was a change in the profile of lymph node B-cell subsets. After recovery, B-cell subsets were reconstituted to normal levels. Depletion of CD20-expressing cells and lymphoid follicular atrophy were the only treatment-related effects.

Effect of anti-CD20 monoclonal antibody, Rituxan, on cynomolgus monkey and human B cells in a whole blood matrix.

BACKGROUND: Cynomolgus monkeys are widely used animal models in biomedical research. The differences between cynomolgus monkey and human B cells are not completely understood. However, these differences are of crucial importance for interpretation of data from studies on new therapeutic agents aimed at B-cell depletion, such as anti-CD20 monoclonal antibodies. METHODS: Multicolor fluorescence-activated cell sorting analysis of peripheral blood B cells was performed on samples treated ex vivo with the anti-CD20 therapeutic monoclonal antibody, Rituxan, in a whole blood matrix. RESULTS: In contrast to humans, cynomolgus monkeys had two distinct B-cell subsets, CD20highCD40lowCD21- and CD20lowCD40highCD21+. These B-cell subsets had a 2.5-fold difference in the EC50 for Rituxan binding and differed significantly in their in vitro susceptibility to Rituxan depletion. Human B cells were similar to the CD20lowCD40highCD21+ cynomolgus monkey B cells with regard to their EC50 for Rituxan and response to Rituxan in a whole blood matrix assay. CD21 was upregulated, whereas CD40 was downregulated at incubation with Rituxan in the CD20lowCD40highCD21+ monkey and human B cells in a concentration-dependent manner. CONCLUSIONS: These findings have direct implications for in vivo studies of therapeutic agents that target B cells in cynomolgus monkeys and for extrapolation of the results to humans. In addition, our data are consistent with the model in which CD20, CD21, and CD40 exist in a supramolecular complex that is affected by anti-CD20 monoclonal antibodies.

Efalizumab (anti-CD11a)-induced increase in peripheral blood leukocytes in psoriasis patients is preferentially mediated by altered trafficking of memory CD8+ T cells into lesional skin.

Therapeutic administration of efalizumab, a humanized antibody to CD11a, induces a marked but reversible increase of peripheral lymphocytes in psoriasis patients. In this study, 13 patients were treated with 12 weekly subcutaneous doses (2 mg/kg/week) of efalizumab, and all 13 patients had increases in leukocyte counts. This increased white blood cell count was mainly due to a 3- to 4-fold increase in the number of circulating CD3(+) lymphocytes during active treatment. Both naive and memory populations of CD4(+) and CD8(+) lymphocytes in the peripheral blood increased, with the largest increase observed in memory CD8(+) T cells. This CD8(+) memory T cell subset is a prominent T cell population found in psoriatic skin. An increase in Type 1 (IFN-gamma producing) T cells was also observed during treatment. Both components of LFA-1, CD11a and CD18, were downregulated during treatment, and surprisingly the integrins CD11b and beta 7 were similarly reduced. We conclude that efalizumab most likely blocks cutaneous entry of memory CD8(+) T cells, a highly disease-relevant cell population. The relatively smaller increase in naive peripheral blood T cells could be attributed to reduced trafficking of naive T cells.