Translatability of findings from cynomolgus monkey to human suggests a mechanistic role for IL-21 in promoting immunogenicity to an anti-PD-1/IL-21 mutein fusion protein.

AMG 256 is a bi-specific, heteroimmunoglobulin molecule with an anti-PD-1 antibody domain and a single IL-21 mutein domain on the C-terminus. Nonclinical studies in cynomolgus monkeys revealed that AMG 256 administration led to the development of immunogenicity-mediated responses and indicated that the IL-21 mutein domain of AMG 256 could enhance the anti-drug antibody response directed toward the monoclonal antibody domain. Anti-AMG 256 IgE were also observed in cynomolgus monkeys. A first-in-human (FIH) study in patients with advanced solid tumors was designed with these risks in mind. AMG 256 elicited ADA in 28 of 33 subjects (84.8%). However, ADA responses were only robust and exposure-impacting at the 2 lowest doses. At mid to high doses, ADA responses remained low magnitude and all subjects maintained exposure, despite most subjects developing ADA. Limited drug-specific IgE were also observed during the FIH study. ADA responses were not associated with any type of adverse event. The AMG 256 program represents a unique case where nonclinical studies informed on the risk of immunogenicity in humans, due to the IL-21-driven nature of the response.

Discovery of PF-06873600, a CDK2/4/6 Inhibitor for the Treatment of Cancer.

Control of the cell cycle through selective pharmacological inhibition of CDK4/6 has proven beneficial in the treatment of breast cancer. Extending this level of control to additional cell cycle CDK isoforms represents an opportunity to expand to additional tumor types and potentially provide benefits to patients that develop tumors resistant to selective CDK4/6 inhibitors. However, broad-spectrum CDK inhibitors have a long history of failure due to safety concerns. In this approach, we describe the use of structure-based drug design and Free-Wilson analysis to optimize a series of CDK2/4/6 inhibitors. Further, we detail the use of molecular dynamics simulations to provide insights into the basis for selectivity against CDK9. Based on overall potency, selectivity, and ADME profile, PF-06873600 (22) was identified as a candidate for the treatment of cancer and advanced to phase 1 clinical trials.

A quantitative method for detection of circulating fms related tyrosine kinase 3 (FLT-3) in acute myeloid leukemia (AML) patients.

FMS related tyrosine kinase 3 (FLT-3) is a tyrosine kinase expressed in early hematopoietic precursor cells and has roles in survival, proliferation, and differentiation. Bone marrow expression and mutagenic analysis of FLT-3 in Acute Myeloid Leukemia (AML) patients is well-characterized. However, the levels of circulating FLT-3 in serum have not been previously described. In this study we describe a quantitative electrochemiluminescent immunoassay that detects FLT-3 in human serum. Using this method we find that AML patients have elevated levels of circulating FLT-3 and these levels correlated to the percent blast counts in the bone marrow (BM).

LAG-3 Confers a Competitive Disadvantage upon Antiviral CD8+ T Cell Responses.

Ongoing clinical trials are evaluating the benefits of systemic blockade of lymphocyte activation gene-3 (LAG-3) signals to improve immunity to tumors. Those studies are founded on the well-established inhibitory role of LAG-3 in regulating CD8(+) T cells during chronic virus infection and antitumor responses. However, the T cell response in LAG-3-deficient mice is similar in size and function to that in wild type animals, suggesting LAG-3 has nuanced immune-regulatory functions. We performed a series of adoptive transfer experiments in mice to better understand the T cell-intrinsic functions of LAG-3 in the regulation of CD8(+) T cell responses. Our results indicate that LAG-3 expression by CD8(+) T cells inhibits their competitive fitness and results in a slightly reduced rate of cell division in comparison with LAG-3-deficient cells. This cell-intrinsic effect of LAG-3 was consistent across both acute and chronic virus infections. These data show that LAG-3 directly modulates the size of the T cell response and support the use of LAG-3 blockade regimens to enhance CD8(+) T cell responses.

Inflammasomes Coordinate Pyroptosis and Natural Killer Cell Cytotoxicity to Clear Infection by a Ubiquitous Environmental Bacterium.

Defective neutrophils in patients with chronic granulomatous disease (CGD) cause susceptibility to extracellular and intracellular infections. Microbes must first be ejected from intracellular niches to expose them to neutrophil attack, so we hypothesized that inflammasomes detect certain CGD pathogens upstream of neutrophil killing. Here, we identified one such ubiquitous environmental bacterium, Chromobacterium violaceum, whose extreme virulence was fully counteracted by the NLRC4 inflammasome. Caspase-1 protected via two parallel pathways that eliminated intracellular replication niches. Pyroptosis was the primary bacterial clearance mechanism in the spleen, but both pyroptosis and interleukin-18 (IL-18)-driven natural killer (NK) cell responses were required for liver defense. NK cells cleared hepatocyte replication niches via perforin-dependent cytotoxicity, whereas interferon-γ was not required. These insights suggested a therapeutic approach: exogenous IL-18 restored perforin-dependent cytotoxicity during infection by the inflammasome-evasive bacterium Listeria monocytogenes. Therefore, inflammasomes can trigger complementary programmed cell death mechanisms, directing sterilizing immunity against intracellular bacterial pathogens.

T Follicular Helper Cell-Dependent Clearance of a Persistent Virus Infection Requires T Cell Expression of the Histone Demethylase UTX.

Epigenetic changes, including histone methylation, control T cell differentiation and memory formation, though the enzymes that mediate these processes are not clear. We show that UTX, a histone H3 lysine 27 (H3K27) demethylase, supports T follicular helper (Tfh) cell responses that are essential for B cell antibody generation and the resolution of chronic viral infections. Mice with a T cell-specific UTX deletion had fewer Tfh cells, reduced germinal center responses, lacked virus-specific immunoglobulin G (IgG), and were unable to resolve chronic lymphocytic choriomeningitis virus infections. UTX-deficient T cells showed decreased expression of interleukin-6 receptor-α and other Tfh cell-related genes that were associated with increased H3K27 methylation. Additionally, Turner Syndrome subjects, who are predisposed to chronic ear infections, had reduced UTX expression in immune cells and decreased circulating CD4(+) CXCR5(+) T cell frequency. Thus, we identify a critical link between UTX in T cells and immunity to infection.

The depletion of NK cells prevents T cell exhaustion to efficiently control disseminating virus infection.

NK cells have well-established functions in immune defense against virus infections and cancer through their cytolytic activity and production of cytokines. In this study, we examined the frequency of NK cells and their influence on T cell responses in mice given variants of lymphocytic choriomeningitis virus that cause acute or persisting infection. We found increased frequencies of circulating NK cells during disseminating infection compared with uninfected or acutely infected mice. Consistent with recent reports, we observed that the depletion of NK cells in mice with disseminated infection increased peak numbers of virus-specific cytokine producing CD8(+) T cells and resulted in the rapid resolution of disseminated infection. Additionally, we show that NK cell depletion sustained T cell responses across time and protected against T cell exhaustion. The positive effects of NK cell depletion on T cell responses only occurred when NK cells were depleted within the first 2 d of infection. We find that the improved CD8(+) T cell response correlated with an enhanced ability of APCs from NK cell-depleted mice to stimulate T cell proliferation, independently of the effects of NK cells on CD4(+) T cells. These results indicate that NK cells play an integral role in limiting the CD8 T cell response and contribute to T cell exhaustion by diminishing APC function during persisting virus infection.

Design of selective, ATP-competitive inhibitors of Akt.

This paper describes the design and synthesis of novel, ATP-competitive Akt inhibitors from an elaborated 3-aminopyrrolidine scaffold. Key findings include the discovery of an initial lead that was modestly selective and medicinal chemistry optimization of that lead to provide more selective analogues. Analysis of the data suggested that highly lipophilic analogues would likely suffer from poor overall properties. Central to the discussion is the concept of optimization of lipophilic efficiency and the ability to balance overall druglike propeties with the careful control of lipophilicity in the lead series. Discovery of the nonracemic amide series and subsequent modification produced an advanced analogue that performed well in advanced preclinical assays, including xenograft tumor growth inhibition studies, and this analogue was nominated for clinical development.