Kathy Howell

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.

A humanized monoclonal antibody targeting the β7 integrin selectively blocks intestinal homing of T lymphocytes

BACKGROUND AND PURPOSE rhuMAb Beta7 is a humanized anti‐human β7 monoclonal antibody currently in phase I in inflammatory bowel disease. rhuMAb Beta7 binds the β7 subunit of the integrins α4β7 and αEβ7, blocking interaction with their ligands. These integrins play key roles in immune cell homing to and retention in mucosal sites, and are associated with chronic inflammatory diseases of the gastrointestinal tract. The goal of this study was to evaluate the mucosal specificity of rhuMAb Beta7.

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.

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

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.

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.

B‐cell subsets in blood and lymphoid organs in Macaca fascicularis

Cynomolgus monkeys (Macaca fascicularis) are widely used animal models in biomedical research. However, the phenotypic characteristics of cynomolgus monkey (CM) B cells in peripheral blood (PB) and lymphoid organs are poorly understood.

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

BackgroundThe 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.MethodsWe 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.ResultsThe 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.ConclusionsWe 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.

Abstract A138: Evaluation of neonatal Fc receptor (FcRn) expression and function in tumor cell lines and their potential effect on IgG disposition in solid tumors.

The human neonatal Fc receptor, hFcRn, is a trafficking receptor for human IgG expressed by various cell types such as vascular endothelial cells, antigen presenting cells, and colorectal, mammary, and pulmonary epithelial cells. Binding of FcRn by monomeric IgG occurs in a pH-restricted manner and, depending on the interacting cell type, can lead to IgG recycling or transcytosis. The recycling pathway is predominant in the vascular endothelium and it traffics IgG away from lysosomal degradation; transcytotic IgG transport, on the other hand, has been more commonly observed in intestinal, breast, and lung epithelium. We hypothesize that solid tumors originating from FcRn-expressing epithelia maintain FcRn expression and that functional expression of FcRn in tumors may affect IgG subcellular trafficking, metabolism, and overall tumor disposition. In the investigations presented herein, we provide quantitative evidence of robust FcRn expression by colorectal and breast tumor cell lines as examined by Flow-Assisted Cell Sorting (FACS) and Western Blotting. Furthermore, we show by Fluorescence Microscopy in selected cell lines that FcRn is localized in both early and recycling endosomes and that its localization in recycling endosomes supports its function in these cells, which is also shown by FACS to be predominantly that of a recycling receptor. To evaluate our hypothesis in vivo, we have developed and characterized an hFcRn mouse tumor model in which hFcRn expression is conditional on doxycycline dosing. Briefly, hFcRn-transduced HM7 colorectal tumor cells (HM7-hFcRn+) implanted into athymic nude mice were evaluated as a function of growth kinetics, vascular volume, and hFcRn expression for up to 14 days following cell inoculation. We show by immunohistochemical staining and FACS substantial hFcRn expression in HM7-FcRn+ tumors relative to parental HM7 tumors during the course of study. Tumor growth and tumor vascular volumes were similar to the parental model as shown by caliper measurements and indirect (99m)Tc red blood cell labeling, respectively, suggesting that this model is appropriate for our purpose. These results provide evidence of hFcRn expression in colorectal and breast cancer cell lines commonly used in xenograft studies and suggest that such expression may lead to IgG recycling in these cell types. Furthermore, we have developed a mouse tumor model that will allow us to determine whether the recycling function of hFcRn observed in vitro translates to an in vivo setting and its effect on IgG tumor distribution, metabolism, and ultimately, anti-tumor efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A138.