Cris Kamperschroer

Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity

Abstract4-1BB (CD137, TNFRSF9) is a costimulatory receptor expressed on several subsets of activated immune cells. Numerous studies of mouse and human T cells indicate that 4-1BB promotes cellular proliferation, survival, and cytokine production. 4-1BB agonist mAbs have demonstrated efficacy in prophylactic and therapeutic settings in both monotherapy and combination therapy tumor models and have established durable anti-tumor protective T-cell memory responses. PF-05082566 is a fully human IgG2 that binds to the extracellular domain of human 4-1BB with high affinity and specificity. In preclinical studies, this agonist antibody demonstrated its ability to activate NF-κB and induce downstream cytokine production, promote leukocyte proliferation, and inhibit tumor growth in a human PBMC xenograft tumor model. The mechanism of action and robust anti-tumor efficacy of PF-05082566 support its clinical development for the treatment of a broad spectrum of human malignancies.

Human lymphocyte activation assay: an in vitro method for predictive immunotoxicity testing.

Preclinical immunotoxicity assessments may be performed during pharmaceutical drug development in order to identify potential cause for concern prior to use in the clinic. The in vivo T-dependent antibody response (TDAR) is widely used in this regard, given its sensitivity to known immunosuppressive compounds, but may be impractical early in drug development where quantities of test article are limited. The goal of the current work is to develop an in vitro human cell-based assay that is sensitive to immunosuppression, uses relatively small quantities of test article, and is simple to perform with moderate to high throughput. Ideally, this assay would require the cooperation of multiple cellular compartments to produce a response, similar to the TDAR. Although the Mishell–Dutton assay (in vitro mouse splenic sheep red blood cell response) has been used for this purpose, it shows considerable inter-laboratory variability, and rodent cells are used which leads to potential difficulty in translation of findings to humans. We have developed an assay that measures an influenza antigen-specific response using frozen-stored human peripheral blood mononuclear cells, which we have termed the human lymphocyte activation (HuLA) assay. The HuLA assay is sensitive to cyclosporine, dexamethasone, rapamycin, mycophenolic acid, and methotrexate at concentrations within their respective therapeutic ranges. Although proliferation is the primary endpoint, we demonstrate that flow cytometry approaches may be used to characterize the proliferating lymphocyte subsets. Flu antigen-specific proliferation in the HuLA assay primarily involves both CD4+ and CD8+ T-lymphocytes and B-lymphocytes, although other lymphocyte subsets also proliferate. In addition, flu-specific antibody-secreting cells can be measured in this assay by ELISPOT, a response that is also sensitive to known immunosuppressive compounds. The HuLA assay represents a relatively straightforward assay with the capability of detecting immune suppression in human cells and can be applied to compound ranking and immunotoxicity assessment.

A summary of meeting proceedings for ‘Measuring immune responses in non-human primates for drug development—Opportunities and challenges for predicting human efficacy and immunotoxicity’

Non-human primates (NHP) are important pre-clinical toxicology models, particularly in cases where therapeutics such as monoclonal antibodies (mAbs) only crossreact with NHP. Therapeutics can be toxic to the immune system because of unexpected effects or because of exaggerated pharmacology of those intended to enhance or suppress immune responses in order to treat immune-related diseases. To adequately assess the risk of potentially immunotoxic drugs in NHP, we need more advanced tools to measure their effects on the immune system. In March 2010, on the 25 Anniversary of the Immunotoxicology Specialty Section of the Society of Toxicology (SOT), several methods for measuring immune responses in NHP were presented and discussed at the annual SOT meeting in Salt Lake City. The session was co-chaired by Cris Kamperschroer and Herve Lebrec, who also gave presentations. JoAnne Flynn and Amitinder Kaur also gave presentations on methods they use for measuring NHP immune responses to important human pathogens. The presentations covered issues with more established Jo ur na l o f Im m un ot ox ic ol og y D ow nl oa de d fr om in fo rm ah ea lth ca re .c om b y T ec hn is ch e U ni ve rs ite it E in dh ov en o n 11 /2 1/ 14

Developmental toxicity assessment of tanezumab, an anti-nerve growth factor monoclonal antibody, in cynomolgus monkeys (Macaca fascicularis).

Two intravenous studies with tanezumab, an anti-nerve growth factor monoclonal antibody, were conducted in pregnant cynomolgus monkeys to assess potential effects on pregnancy and pre- and postnatal development. Study 1 evaluated infants up to 12 months of age following weekly maternal dosing (0, 0.5, 4 or 30 mg/kg; 18 per group) from gestation day (GD) 20 through parturition. Study 2 evaluated infants 2 months postnatally following weekly maternal dosing (0, 0.5 or 30 mg/kg; 20-21 per group) from GD 20 through 48. In the absence of maternal toxicity, tanezumab increased stillbirth and post-birth infant mortality/morbidity, decreased infant growth and resulted in microscopic changes in the peripheral sympathetic and sensory nervous system of the infants at all doses. Decreased primary antibody responses and increased incidences in skin changes in infants were also observed. The no-observed-adverse-effect-level for maternal toxicity was 30 mg/kg and <0.5 mg/kg for developmental toxicity.

Summary of roundtable discussion meeting: Non-human primates to assess risk for EBV-related lymphomas in humans

Epstein-Barr virus (EBV)-associated lymphomas are a known risk for immunosuppressed individuals. Non-clinical methods to determine the potential of new immunomodulatory compounds to produce EBV-associated lymphomas (hazard identification) have not been developed. Since lymphocryptovirus (LCV) in non-human primates (NHP) has similar characteristics to EBV in humans, a Roundtable meeting was held in October 2010 to explore how the potential for EBV-related lymphomas in humans can be assessed by using surrogate biomarkers for lymphoma risk in NHP toxicity studies. Stakeholders from regulatory agencies, academia, and industry came together to determine the research gaps and potential benefits and considerations of such an approach given the current state-of-the-science. Key conclusions from the discussion included considerations raised about the potential usefulness of LCV-related biomarkers from NHP studies since there is significant controversy over the reliability of using EBV viral load or EBV-specific T-lymphocytes to predict for lymphoproliferative disorders in transplant patients. In addition, there are technical challenges that need to be further addressed in order to develop methods to measure LCV viral load and LCV-specific T-lymphocytes from cynomolgus monkeys.

Measuring T-cell responses against LCV and CMV in cynomolgus macaques using ELISPOT: Potential application to non-clinical testing of immunomodulatory therapeutics

Abstract A number of immunomodulatory therapeutics increase the risk of disease associated with latent herpesviruses such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV), a member of the lymphocryptovirus (LCV) family that infects humans. The diseases associated with loss of immunity to these viruses can have major impacts on patients as well as on the commercial viability of the immunomodulatory therapeutics. In an effort to develop non-clinical methods for measuring effects on anti-viral immunity, we have developed an interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISPOT) assay to quantify the number of CMV or LCV-reactive T-cells in peripheral blood of cynomolgus macaques. After optimization of various parameters, the IFN-γ ELISPOT assay was characterized for specificity, intra-assay, monkey-to-monkey, and longitudinal variability and sensitivity to immunosuppression. The results show that nearly all animals have detectable responses against both CMV and LCV and responses were derived from T-cells specific to the virus of interest. Analyses of variability show assay reproducibility (≤23% CV), and that variability over time in anti-viral responses in individual animals (larger for LCV than for CMV) was ∼2-fold in most animals over a 3-month time period, which is predicted to allow for detection of drug-induced changes when using group sizes typical of non-clinical studies. In addition, the IFN-γ ELISPOT assay was capable of detecting decreases in the numbers of CMV and LCV reactive T-cells induced by immunosuppressive drugs in vitro. This assay may allow for non-clinical assessment of the effects of immunomodulatory therapeutics on anti-viral T-cell immunity in monkeys, and may help determine if therapeutics increase the risk of reactivating latent viral infections.

The genomic sequence of lymphocryptovirus from cynomolgus macaque.

Lymphocryptoviruses such as Epstein-Barr virus (EBV) cause persistent infections in human and non-human primates, and suppression of the immune system can increase the risk of lymphocryptovirus (LCV)-associated tumor development in both human and non-human primates. To enable LCV infection as a non-clinical model to study effects of therapeutics on EBV immunity, we determined the genomic DNA sequence of the LCV from cynomolgus macaque, a species commonly used for non-clinical testing. Comparison to rhesus macaque LCV and human EBV sequences indicates that LCV from the cynomolgus macaque has the same genomic arrangement and a high degree of similarity in most genes, especially with rhesus macaque LCV. Genes showing lower similarity were those encoding proteins involved in latency and/or tumor promotion or immune evasion. The genomic sequence of LCV from cynomolgus macaque should aid the development of non-clinical tools for identifying therapeutics that impact LCV immunity and carry potential lymphoma risk.

Quantitative PCR assays reveal high prevalence of lymphocryptovirus as well as lytic phase gene expression in peripheral blood cells of cynomolgus macaques.

Lymphocryptoviruses such as Epstein-Barr virus (EBV) are important pathogens in both human and non-human primates, particularly during immunosuppression. Immunomodulatory molecules that may suppress antiviral immunity are commonly tested in the cynomolgus macaque. To enable the study of lymphocryptovirus (LCV) in this non-clinical model, PCR-based assays were developed to measure LCV viral load, as well as transcripts for the lytic phase LCV gene, BALF-2. Results from studies employing these assays showed that LCV genome was detected in the oropharyngeal epithelium of all cynomolgus monkeys tested, and the majority had viral genome in peripheral blood mononuclear cells (PBMCs). The results also revealed LCV lytic phase gene expression not only in the oropharynx of most monkeys, but also in PBMCs of approximately one half of monkeys tested. This unexpected finding suggests that initiation of the lytic gene expression cascade occurs often in the peripheral blood cells of healthy monkeys.

Abstract 2667: In vitro properties and pre-clinical activity of PF-06801591, a high-affinity engineered anti-human PD-1

Monoclonal-antibody-based therapies targeting the immune checkpoint receptors have become the new standard of care in many cancers. Antibodies that specifically target programmed death receptor-1 (PD-1) or its cognate ligand, programmed death receptor ligand-1 (PD-L1), alone or in combination, have yielded clinical benefits and durable responses in patient subsets with various cancers (including metastatic melanoma, NSCLC, RCC, urothelial cancer, cHL and others). Here we report on the biophysical characteristics and non-clinical antagonistic activities of PF-06801591. PF-06801591 is a humanized anti-PD-1 antibody of human IgG4 isotype, that binds selectively and with similar potency to human and cynomolgus monkey PD-1 receptor (EC50 ~50 pM) and blocks its interaction with its cognate ligands PD-L1 and PD-L2 (IC50 Citation Format: Sawsan Youssef, Yasmina Abdiche, HoangKim Nguyen, Joyce Chou, Sherman Michael Chin, Cris Kamperschroer, Patricia A. Schneider, Eugenia Kraynov, Heike I. Krupka, Arvind Rajpal, John Lin. In vitro properties and pre-clinical activity of PF-06801591, a high-affinity engineered anti-human PD-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2667. doi:10.1158/1538-7445.AM2017-2667

Abstract 4384: Targeting of 4-1BB by monoclonal antibody, PF-05082566, enhances T cell function and promotes antitumor activity

4-1BB (CD137, TNFRSF9) is a costimulatory receptor expressed in an activation induced manner on several subsets of immune cells. Numerous studies of mouse and human T cells indicate that 4-1BB promotes enhanced cellular proliferation, survival, and cytokine production. 4-1BB agonist mAbs have demonstrated efficacy in prophylactic and therapeutic settings in both monotherapy and combination therapy tumor models and have established durable anti-tumor protective T cell memory responses. PF-05082566 is a fully human IgG2 which binds to the extracellular domain of human 4-1BB with high affinity and specificity. In preclinical studies this agonist antibody demonstrated its ability to activate NF-κB and induce downstream cytokine production, promote leukocyte proliferation, and inhibit tumor growth in a human PBMC xenograft tumor model. The mechanism of action and robust anti-tumor efficacy of PF-05082566 support its clinical development for the treatment of a broad spectrum of human malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4384. doi:1538-7445.AM2012-4384