PET imaging to non-invasively study immune activation leading to antitumor responses with a 4-1BB agonistic antibody

BACKGROUND: Molecular imaging with positron emission tomography (PET) may allow the non-invasive study of the pharmacodynamic effects of agonistic monoclonal antibodies (mAb) to 4-1BB (CD137). 4-1BB is a member of the tumor necrosis factor family expressed on activated T cells and other immune cells, and activating 4-1BB antibodies are being tested for the treatment of patients with advanced cancers. METHODS: We studied the antitumor activity of 4-1BB mAb therapy using [(18) F]-labeled fluoro-2-deoxy-2-D-glucose ([(18) F]FDG) microPET scanning in a mouse model of colon cancer. Results of microPET imaging were correlated with morphological changes in tumors, draining lymph nodes as well as cell subset uptake of the metabolic PET tracer in vitro. RESULTS: The administration of 4-1BB mAb to Balb/c mice induced reproducible CT26 tumor regressions and improved survival; complete tumor shrinkage was achieved in the majority of mice. There was markedly increased [(18) F]FDG signal at the tumor site and draining lymph nodes. In a metabolic probe in vitro uptake assay, there was an 8-fold increase in uptake of [(3)H]DDG in leukocytes extracted from tumors and draining lymph nodes of mice treated with 4-1BB mAb compared to untreated mice, supporting the in vivo PET data. CONCLUSION: Increased uptake of [(18) F]FDG by PET scans visualizes 4-1BB agonistic antibody-induced antitumor immune responses and can be used as a pharmacodynamic readout to guide the development of this class of antibodies in the clinic

Cutaneous Wound Healing Through Paradoxical Mapk Activation By Braf Inhibitors

BRAF inhibitors are highly effective therapies for the treatment of BRAF(V600)-mutated melanoma, with the main toxicity being a variety of hyperproliferative skin conditions due to paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway in BRAF wild-type cells. Most of these hyperproliferative skin changes improve when a MEK inhibitor is co-administered, as it blocks paradoxical MAPK activation. Here we show how the BRAF inhibitor vemurafenib accelerates skin wound healing by inducing the proliferation and migration of human keratinocytes through extracellular signal-regulated kinase (ERK) phosphorylation and cell cycle progression. Topical treatment with vemurafenib in two wound-healing mice models accelerates cutaneous wound healing through paradoxical MAPK activation; addition of a mitogen-activated protein kinase kinase (MEK) inhibitor reverses the benefit of vemurafenib-accelerated wound healing. The same dosing regimen of topical BRAF inhibitor does not increase the incidence of cutaneous squamous cell carcinomas in mice. Therefore, topical BRAF inhibitors may have clinical applications in accelerating the healing of skin wounds.7NIH [P01 CA168585, R35 CA197633, CA-16042, AI-28697]Ressler Family FoundationGrimaldi Family FundGarcia-Corsini Family FundJames B. Pendleton Charitable TrustUCLA CFAR [5P30 AI028697]UCLA AIDS InstituteJohnson Comprehensive Cancer Center (JCCC)David Geffen School of Medicin

Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma

BACKGROUND: Approximately 75% of objective responses to anti–programmed death 1 (PD-1) therapy in patients with melanoma are durable, lasting for years, but delayed relapses have been noted long after initial objective tumor regression despite continuous therapy. Mechanisms of immune escape in this context are unknown. METHODS: We analyzed biopsy samples from paired baseline and relapsing lesions in four patients with metastatic melanoma who had had an initial objective tumor regression in response to anti–PD-1 therapy (pembrolizumab) followed by disease progression months to years later. RESULTS: Whole-exome sequencing detected clonal selection and outgrowth of the acquired resistant tumors and, in two of the four patients, revealed resistance-associated loss-of-function mutations in the genes encoding interferon-receptor–associated Janus kinase 1 (JAK1) or Janus kinase 2 (JAK2), concurrent with deletion of the wild-type allele. A truncating mutation in the gene encoding the antigen-presenting protein beta-2-microglobulin (B2M) was identified in a third patient. JAK1 and JAK2 truncating mutations resulted in a lack of response to interferon gamma, including insensitivity to its antiproliferative effects on cancer cells. The B2M truncating mutation led to loss of surface expression of major histocompatibility complex class I. CONCLUSIONS: In this study, acquired resistance to PD-1 blockade immunotherapy in patients with melanoma was associated with defects in the pathways involved in interferon-receptor signaling and in antigen presentation. (Funded by the National Institutes of Health and others.

Advances in PET Detection of the Antitumor T Cell Response

Positron emission tomography (PET) is a powerful noninvasive imaging technique able to measure distinct biological processes in vivo by administration of a radiolabeled probe. Whole-body measurements track the probe accumulation providing a means to measure biological changes such as metabolism, cell location, or tumor burden. PET can also be applied to both preclinical and clinical studies providing three-dimensional information. For immunotherapies (in particular understanding T cell responses), PET can be utilized for spatial and longitudinal tracking of T lymphocytes. Although PET has been utilized clinically for over 30 years, the recent development of additional PET radiotracers have dramatically expanded the use of PET to detect endogenous or adoptively transferred T cells in vivo. Novel probes have identified changes in T cell quantity, location, and function. This has enabled investigators to track T cells outside of the circulation and in hematopoietic organs such as spleen, lymph nodes, and bone marrow, or within tumors. In this review, we cover advances in PET detection of the antitumor T cell response and areas of focus for future studies

Tools for probing 2A sequence space

Foot-and-mouth disease virus (FMDV) 2A is an oligopeptide composed of only 18 amino acids that can mediate a co-translational cleavage at its own C- terminus. It has been observed that 2A sequences do not show cleavage activity within bacterial organisms. Why 2A lacks activity in a prokaryotic organism such as E.coli is unclear. A series of plasmids designed to provide a phenotypic screen for 2A-mediated cleavage (in prokaryotes) were developed. Even though no active 2A sequences were found in bacteria, this system can easily be adapted to eukaryotic cells and will also be very useful in mutagenic studies on 2A sequences. Furthermore, 2A[subscript(FMDV)] has been used in the construction of a reporter of stress in the cell. This may allow us to open a new approach in the use of 2A oligopeptide, which had already been widely used to co-express genes of interest with reporter proteins, in biotechnology and gene therapy. Theiler’s murine encephalomyelitis cardiovirus (TMEV) 2A has the same role as in FMDV but is 150 aa in length instead of the 18 aa in FMDV. It also presents the same C-terminal motif but what is the function of the remaining ~85% of the cardiovirus 2A sequence remains a mystery. To this end we have produced antibodies against TMEV-2A, to study the role of 2A[subscript(TMEV)] within the cell. Database searches probing for 2A’s C-terminal conserved motif (- DxExNPGP-) has identified many 2A-like sequences, not only within picornaviruses but also in trypanosomes, insect and cellular genes. These remarkable findings indicate that the control of protein synthesis by 2A is not solely confined to the Picornaviridae. Bioinformatics analyses of all the known 2A-like sequences, comparing all the different upstream sequences, show a clear pattern on the organization of residues in the upstream region. The discovery of this 2A oligopeptide has led to a breakthrough in protein co- expression technology. It has been used as a highly effective new tool for the co- expression of multiple proteins from a single ORF in plant biotechnology and also gene therapy applications. Although we have gained substantial insights into the general features and biological significance of this process, a great deal still needs to be uncovered about the structural and mechanistic details of this unique mechanism of action

Tools for probing 2A sequence space

Foot-and-mouth disease virus (FMDV) 2A is an oligopeptide composed of only 18 amino acids that can mediate a co-translational cleavage at its own C- terminus. It has been observed that 2A sequences do not show cleavage activity within bacterial organisms. Why 2A lacks activity in a prokaryotic organism such as E.coli is unclear. A series of plasmids designed to provide a phenotypic screen for 2A-mediated cleavage (in prokaryotes) were developed. Even though no active 2A sequences were found in bacteria, this system can easily be adapted to eukaryotic cells and will also be very useful in mutagenic studies on 2A sequences. Furthermore, 2A[subscript(FMDV)] has been used in the construction of a reporter of stress in the cell. This may allow us to open a new approach in the use of 2A oligopeptide, which had already been widely used to co-express genes of interest with reporter proteins, in biotechnology and gene therapy. Theiler’s murine encephalomyelitis cardiovirus (TMEV) 2A has the same role as in FMDV but is 150 aa in length instead of the 18 aa in FMDV. It also presents the same C-terminal motif but what is the function of the remaining ~85% of the cardiovirus 2A sequence remains a mystery. To this end we have produced antibodies against TMEV-2A, to study the role of 2A[subscript(TMEV)] within the cell. Database searches probing for 2A’s C-terminal conserved motif (- DxExNPGP-) has identified many 2A-like sequences, not only within picornaviruses but also in trypanosomes, insect and cellular genes. These remarkable findings indicate that the control of protein synthesis by 2A is not solely confined to the Picornaviridae. Bioinformatics analyses of all the known 2A-like sequences, comparing all the different upstream sequences, show a clear pattern on the organization of residues in the upstream region. The discovery of this 2A oligopeptide has led to a breakthrough in protein co- expression technology. It has been used as a highly effective new tool for the co- expression of multiple proteins from a single ORF in plant biotechnology and also gene therapy applications. Although we have gained substantial insights into the general features and biological significance of this process, a great deal still needs to be uncovered about the structural and mechanistic details of this unique mechanism of action.EThOS - Electronic Theses Online ServiceGBUnited Kingdo