Intratumoral delivery of tavokinogene telseplasmid yields systemic immune responses in metastatic melanoma patients.

BACKGROUND: Interleukin 12 (IL-12) is a pivotal regulator of innate and adaptive immunity. We conducted a prospective open-label, phase II clinical trial of electroporated plasmid IL-12 in advanced melanoma patients (NCT01502293). PATIENTS AND METHODS: Patients with stage III/IV melanoma were treated intratumorally with plasmid encoding IL-12 (tavokinogene telseplasmid; tavo), 0.5 mg/ml followed by electroporation (six pulses, 1500 V/cm) on days 1, 5, and 8 every 90 days in the main study and additional patients were treated in two alternative schedule exploration cohorts. Correlative analyses for programmed death-ligand 1 (PD-L1), flow cytometry to assess changes in immune cell subsets, and analysis of immune-related gene expression were carried out on pre- and post-treatment samples from study patients, as well as from additional patients treated during exploration of additional dosing schedules beyond the pre-specified protocol dosing schedule. Response was measured by study-specific criteria to maximize detection of latent and potentially transient immune responses in patients with multiple skin lesions and toxicities were graded by the Common Terminology Criteria for Adverse Events version 4.0 (CTCAE v4.0). RESULTS: The objective overall response rate was 35.7% in the main study (29.8% in all cohorts), with a complete response rate of 17.9% (10.6% in all cohorts). The median progression-free survival in the main study was 3.7 months while the median overall survival was not reached at a median follow up of 29.7 months. A total of 46% of patients in all cohorts with uninjected lesions experienced regression of at least one of these lesions and 25% had a net regression of all untreated lesions. Transcriptomic and immunohistochemistry analysis showed that immune activation and co-stimulatory transcripts were up-regulated but there was also increased adaptive immune resistance. CONCLUSIONS: Intratumoral Tavo was well tolerated and led to systemic immune responses in advanced melanoma patients. While tumor regression and increased immune infiltration were observed in treated as well as untreated/distal lesions, adaptive immune resistance limited the response.

A Four-Biomarker Blood Signature Discriminates Systemic Inflammation Due to Viral Infection Versus Other Etiologies.

The innate immune system of humans and other mammals responds to pathogen-associated molecular patterns (PAMPs) that are conserved across broad classes of infectious agents such as bacteria and viruses. We hypothesized that a blood-based transcriptional signature could be discovered indicating a host systemic response to viral infection. Previous work identified host transcriptional signatures to individual viruses including influenza, respiratory syncytial virus and dengue, but the generality of these signatures across all viral infection types has not been established. Based on 44 publicly available datasets and two clinical studies of our own design, we discovered and validated a four-gene expression signature in whole blood, indicative of a general host systemic response to many types of viral infection. The signature's genes are: Interferon Stimulated Gene 15 (ISG15), Interleukin 16 (IL16), 2',5'-Oligoadenylate Synthetase Like (OASL), and Adhesion G Protein Coupled Receptor E5 (ADGRE5). In each of 13 validation datasets encompassing human, macaque, chimpanzee, pig, mouse, rat and all seven Baltimore virus classification groups, the signature provides statistically significant (p < 0.05) discrimination between viral and non-viral conditions. The signature may have clinical utility for differentiating host systemic inflammation (SI) due to viral versus bacterial or non-infectious causes.

Immunoscore and Immunoprofiling in cancer: an update from the melanoma and immunotherapy bridge 2015.

The fifth "Melanoma Bridge Meeting" took place in Naples, December 1-5th, 2015. The main topics discussed at this meeting were: Molecular and Immuno advances, Immunotherapies and Combination Therapies, Tumor Microenvironment and Biomarkers and Immunoscore. The natural history of cancer involves interactions between the tumor and the immune system of the host. The immune infiltration at the tumor site may be indicative of host response. Significant correlations were shown between the levels of immune cell infiltration in tumors and patient's clinical outcome. Moreover, incredible progress comes from the discovery of mutation-encoded tumor neoantigens. In fact, as tumors grow, they acquire mutations that are able to influence the response of patients to immune checkpoint inhibitors. It has been demonstrated that sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. The road ahead is still very long, but the knowledge of the mechanisms of immune escape, the study of tumor neo-antigens as well as of tumor microenvironment and the development of new immunotherapy strategies, will make cancer a more and more treatable disease.

Effector memory and central memory NY-ESO-1-specific re-directed T cells for treatment of multiple myeloma.

The cancer-testis antigen NY-ESO-1 is a potential target antigen for immune therapy expressed in a subset of patients with multiple myeloma. We generated chimeric antigen receptors (CARs) recognizing the immunodominant NY-ESO-1 peptide 157-165 in the context of HLA-A*02:01 to re-direct autologous CD8(+) T cells towards NY-ESO-1(+) myeloma cells. These re-directed T cells specifically lysed NY-ESO-1(157-165)/HLA-A*02:01-positive cells and secreted IFNγ. A total of 40% of CCR7(-) re-directed T cells had an effector memory phenotype and 5% a central memory phenotype. Based on CCR7 cell sorting, effector and memory CAR-positive T cells were separated and CCR7(+) memory cells demonstrated after antigen-specific re-stimulation downregulation of CCR7 as sign of differentiation towards effector cells accompanied by an increased secretion of memory signature cytokines such as IL-2. To evaluate NY-ESO-1 as potential target antigen, we screened 78 bone marrow biopsies of multiple myeloma patients where NY-ESO-1 protein was found to be expressed by immunohistochemistry in 9.7% of samples. Adoptively transferred NY-ESO-1-specific re-directed T cells protected mice against challenge with endogenously NY-ESO-1-positive myeloma cells in a xenograft model. In conclusion, re-directed effector- and central memory T cells specifically recognized NY-ESO-1(157-165)/ HLA-A*02:01-positive cells resulting in antigen-specific functionality in vitro and in vivo.

Assessing oncologic benefit in clinical trials of immunotherapy agents.

BACKGROUND: USA Food and Drug Administration approval for cancer therapy requires demonstration of patient benefit as a marker of clinical efficacy. Prolonged survival is the gold standard for demonstration of efficacy, but other end points such as antitumor response, progression-free survival, quality of life, or surrogate end points may be used. DESIGN: This study was developed based on discussion during a roundtable meeting of experts in the field of immunotherapy. RESULTS: In most clinical trials involving cytotoxic agents, response end points use RECIST based on the premise that 'effective' therapy causes tumor destruction, target lesion shrinkage, and prevention of new lesions. However, RECIST may not be appropriate in trials of immunotherapy. Like other targeted agents, immunotherapies may mediate cytostatic rather than direct cytotoxic effects, and these may be difficult to quantify with RECIST. Furthermore, significant time may elapse before clinical effects are quantifiable because of complex response pathways. Effective immunotherapy may even mediate transient lesion growth secondary to immune cell infiltration. CONCLUSIONS: RECIST may not be an optimal indicator of clinical benefit in immunotherapy trials. This article discusses alternative clinical trial designs and end points that may be more relevant for immunotherapy trials and may offer more effective prediction of survival in pivotal phase III studies.

Manipulating the host response to autologous tumour vaccines.

From our way of thinking the problem facing vaccine strategies for cancer is not that we do not have "enough" tumour antigens. The problem is we cannot induce an immune response that is sufficient to mediate tumour regression. The normal "checks and balances" found in the body prevent the sustained expansion and subsequent persistence of immune killer cells. If vaccine strategies are going to become effective treatments for cancer patients, they will need to overcome this substantial roadblock. Recent developments in immunology have provided insights into the mechanisms that regulate the expansion and persistence of T cells. This has allowed investigators to reinterpret decades-old observations suggesting that chemotherapy administered before vaccination often led to a stronger immune response. This manuscript will review experiments that offer an explanation for these observations and present pre-clinical data from our laboratory that describes an innovative new approach to combining chemotherapy and vaccination. This approach is readily translatable to the clinic and is broadly applicable to any vaccine strategy for advanced cancer.

Evaluation of a preclinical model of bone metastasis for the study of adoptive immunotherapy.

The development of new therapeutic strategies for the treatment of bone metastases strongly depends on the availability of valid animal models. In this paper, we evaluate a preclinical model of bone metastases using a technique of tumor cell injection into the left heart ventricle of mice to study the efficacy of adoptive immunotherapy. Using flow cytometric analysis and histopathological and radiological examination, we investigated whether this experimental model of bony metastases using two murine cell lines of melanoma and breast cancer would be suitable for the study of adoptive immunotherapy for these diseases. We further report that anti-CD3-activated and IL-2-expanded tumor vaccine draining lymph node cells cause regression of tumor metastases, including bone metastases, following adoptive transfer to mice bearing 3-day metastases from the D5 melanoma cell line. These promising early results lead us to conclude the following: (1). this model of experimental bone metastases is suitable for the study of immunotherapy, and (2). further studies are warranted to extend these promising early findings of the therapeutic effects of adoptive immunotherapy in this animal model.

Immunotherapy of melanoma: a dichotomy in the requirement for IFN-gamma in vaccine-induced antitumor immunity versus adoptive immunotherapy.

The mechanism by which tumors are rejected following the adoptive transfer of tumor-specific T cells is not well characterized. Recent work has challenged the requirement for cytotoxicity mediated by either the perforin/granzyme or Fas/Fas ligand pathway in T cell-mediated tumor regression. Many reports, including ours, suggest that tumor-specific production of IFN-gamma is critical for T cell-mediated tumor regression. However, in most of these studies the evidence to support the role for IFN-gamma is only indirect. We have directly examined the requirement for IFN-gamma using IFN-gamma knockout (GKO) mice. The results show an interesting dichotomy in the requirement for IFN-gamma: Antitumor immunity induced by active-specific immunotherapy (vaccination) required IFN-gamma, whereas adoptive immunotherapy did not. In GKO mice vaccination with the GM-CSF gene-modified B16BL6-D5 tumor (D5-G6) failed to induce protective immunity against parental D5 tumor. However, adoptive transfer of effector T cells from GKO mice cured 100% of GKO mice with established pulmonary metastases and induced long term antitumor immunity and depigmentation of skin. Furthermore, in vivo neutralization of IFN-gamma by mAb treatment or adoptive transfer into IFN-gamma receptor knockout mice failed to block the therapeutic efficacy of effector T cells generated from wild-type or perforin knockout mice. Analysis of regressing metastases revealed similar infiltrates of macrophages and granulocytes in both wild-type and GKO mice. These results indicate that in this adoptive immunotherapy model, neither a direct effect on the tumor nor an indirect effect of IFN-gamma through activation of myeloid or lymphoid cells is critical for therapeutic efficacy.

Divergent roles for CD4+ T cells in the priming and effector/memory phases of adoptive immunotherapy.

The requirement for CD4(+) Th cells in the cross-priming of antitumor CTL is well accepted in tumor immunology. Here we report that the requirement for T cell help can be replaced by local production of GM-CSF at the vaccine site. Experiments using mice in which CD4(+) T cells were eliminated, either by Ab depletion or by gene knockout of the MHC class II beta-chain (MHC II KO), revealed that priming of therapeutic CD8(+) effector T cells following vaccination with a GM-CSF-transduced B16BL6-D5 tumor cell line occurred independently of CD4(+) T cell help. The adoptive transfer of CD8(+) effector T cells, but not CD4(+) effector T cells, led to complete regression of pulmonary metastases. Regression of pulmonary metastases did not require either host T cells or NK cells. Transfer of CD8(+) effector T cells alone could cure wild-type animals of systemic tumor; the majority of tumor-bearing mice survived long term after treatment (>100 days). In contrast, adoptive transfer of CD8(+) T cells to tumor-bearing MHC II KO mice improved survival, but eventually all MHC II KO mice succumbed to metastatic disease. WT mice cured by adoptive transfer of CD8(+) T cells were resistant to tumor challenge. Resistance was mediated by CD8(+) T cells in mice at 50 days, while both CD4(+) and CD8(+) T cells were important for protection in mice challenged 150 days following adoptive transfer. Thus, in this tumor model CD4(+) Th cells are not required for the priming phase of CD8(+) effector T cells; however, they are critical for both the complete elimination of tumor and the maintenance of a long term protective antitumor memory response in vivo.

Crystal structure of rhodopsin: A G protein-coupled receptor.

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) respond to a variety of different external stimuli and activate G proteins. GPCRs share many structural features, including a bundle of seven transmembrane alpha helices connected by six loops of varying lengths. We determined the structure of rhodopsin from diffraction data extending to 2.8 angstroms resolution. The highly organized structure in the extracellular region, including a conserved disulfide bridge, forms a basis for the arrangement of the seven-helix transmembrane motif. The ground-state chromophore, 11-cis-retinal, holds the transmembrane region of the protein in the inactive conformation. Interactions of the chromophore with a cluster of key residues determine the wavelength of the maximum absorption. Changes in these interactions among rhodopsins facilitate color discrimination. Identification of a set of residues that mediate interactions between the transmembrane helices and the cytoplasmic surface, where G-protein activation occurs, also suggests a possible structural change upon photoactivation.

X-Ray diffraction analysis of three-dimensional crystals of bovine rhodopsin obtained from mixed micelles.

Rhodopsin, a prototypic G protein-coupled receptor responsible for absorption of photons in retinal rod photoreceptor cells, was selectively extracted from bovine rod outer segment membranes, employing mixed micelles of nonyl beta-d-glucoside and heptanetriol. Highly purified rhodopsin was crystallized from solutions containing varying amounts of detergent and amphiphile. The crystals contained ground state rhodopsin molecules as judged by their red color and the linear dichroism originating from the 11-cis-retinal chromophore. However, when exposed to visible light, even at 4 degrees C, rhodopsin was bleached and the crystals decomposed. Reflections in the diffraction pattern were observed out to 3.5-A resolution at 100 K for the most ordered crystals. Diffraction data have been processed to 3.85-A resolution. The symmetry of the diffraction pattern and the systematic absences indicate that the crystals have tetragonal symmetry, space group P4(1)22 or P4(3)22, a = b = 96.51 A, c = 148.55 A. A value of 4.12 A(3)/Da for V(M) was obtained for one monomer in the asymmetric unit (eight molecules per unit cell). Our study is the first characterization of a three-dimensional crystal of a G protein-coupled receptor and may be valuable for future structural studies on related receptors of this important superfamily.

Tumor regression after adoptive transfer of effector T cells is independent of perforin or Fas ligand (APO-1L/CD95L).

The adoptive transfer of tumor-specific effector T cells can result in complete regression and cure mice with systemic melanoma, but the mechanisms responsible for regression are not well characterized. Perforin- and Fas ligand (APO-1/CD95 ligand)-mediated cytotoxicity have been proposed as mechanisms for T cell-mediated tumor destruction. To determine the role of perforin and Fas ligand (FasL) in T cell-mediated tumor regression in a murine melanoma model, B16BL6-D5 (D5), we generated D5-specific effector T cells from tumor vaccine-draining lymph nodes of wild type (wt), perforin knock out (PKO), or FasL mutant (gld) mice and treated established D5 metastases in mice with the same genotype. Effector T cells from wt, PKO and gld mice induced complete regression of pulmonary metastases and significantly prolonged survival of the treated animals regardless of their genotype. Complete tumor regression induced by PKO effector T cells was also observed in a sarcoma model (MCA-310). Furthermore, adoptive transfer of PKO and wt effector T cells provided long-term immunity to D5. Therapeutic T cells from wt, PKO, or gld mice exhibit a tumor-specific type 1 cytokine profile; they secrete IFN-gamma, but not IL-4. In these models, T cell-mediated tumor regression and long-term antitumor immunity are perforin and FasL independent.

Immune responses induced by gene gun or intramuscular injection of DNA vaccines that express immunogenic regions of the serine repeat antigen from Plasmodium falciparum.

The liver- and blood-stage-expressed serine repeat antigen (SERA) of Plasmodium falciparum is a candidate protein for a human malaria vaccine. We compared the immune responses induced in mice immunized with SERA-expressing plasmid DNA vaccines delivered by intramuscular (i.m.) injection or delivered intradermally by Gene Gun immunization. Mice were immunized with a pcdna3 plasmid encoding the entire 47-kDa domain of SERA (amino acids 17 to 382) or the N-terminal domain (amino acids 17 to 110) of SERA. Minimal antibody responses were detected following DNA vaccination with the N-terminal domain of SERA, suggesting that the N-terminal domain alone is not highly immunogenic by this route of vaccine delivery. Immunization of mice by Gene Gun delivery of the 47-kDa domain of SERA elicited a significantly higher serum antibody titer to the antigen than immunization of mice by i.m. injection with the same plasmid did. The predominant isotype subclass of the antibodies elicited to the SERA protein following i.m. and Gene Gun immunizations with SERA plasmid DNA was immunoglobulin G1. Coimmunization of mice with SERA plasmid DNA and a plasmid expressing the hepatitis B surface antigen (pCMV-s) by the i.m. route resulted in higher anti-SERA titers than those generated in mice immunized with the SERA DNA plasmid alone. Vaccination with DNA may provide a viable alternative or may be used in conjunction with protein-based subunit vaccines to maximize the efficacy of a human malaria vaccine that includes immunogenic regions of the SERA protein.

Improved recognition of native-like protein structures using a combination of sequence-dependent and sequence-independent features of proteins.

We describe the development of a scoring function based on the decomposition P(structure/sequence) proportional to P(sequence/structure) *P(structure), which outperforms previous scoring functions in correctly identifying native-like protein structures in large ensembles of compact decoys. The first term captures sequence-dependent features of protein structures, such as the burial of hydrophobic residues in the core, the second term, universal sequence-independent features, such as the assembly of beta-strands into beta-sheets. The efficacies of a wide variety of sequence-dependent and sequence-independent features of protein structures for recognizing native-like structures were systematically evaluated using ensembles of approximately 30,000 compact conformations with fixed secondary structure for each of 17 small protein domains. The best results were obtained using a core scoring function with P(sequence/structure) parameterized similarly to our previous work (Simons et al., J Mol Biol 1997;268:209-225] and P(structure) focused on secondary structure packing preferences; while several additional features had some discriminatory power on their own, they did not provide any additional discriminatory power when combined with the core scoring function. Our results, on both the training set and the independent decoy set of Park and Levitt (J Mol Biol 1996;258:367-392), suggest that this scoring function should contribute to the prediction of tertiary structure from knowledge of sequence and secondary structure.

Examining the immune response in sentinel lymph nodes of mice and men.

Recently, it was recognized that an immune response develops along one of two major pathways. One leads to a destructive immune response (type 1), while the alternative leads to a nondestructive immune response (type 2). Our studies in animal models suggest that therapeutic vaccines induce a tumor-specific type 1 immune response while ineffective vaccines induce a type 2 response. These results have led us to examine the immune response in sentinel lymph nodes draining tumor vaccines of patients entered onto clinical trials for melanoma, breast and renal cell cancer.

Stable transformation of Toxoplasma gondii based on a pyrimethamine resistant trifunctional dihydrofolate reductase-cytosine deaminase-thymidylate synthase gene that confers sensitivity to 5-fluorocytosine.

To improve genetic models available for the analysis of apicomplexan protozoan parasites, bacterial sequences encoding the 427 amino acid cytosine deaminase (CD) gene were fused, in-frame, to an engineered linker domain of the high level pyrimethamine resistant form of the parasite bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. Toxoplasma gondii was transformed with the plasmid containing the fused pyrimethamine resistant dihydrofolate reductase-cytosine deaminase-thymidylate synthase (DHFRm2m3-CD-TS) gene and parasites were selected in a high level of pyrimethamine. Transfected parasites that acquired resistance to pyrimethamine were cloned and evaluated for expression of the CD genetic marker. CD transgenic parasites acquired a high sensitivity to 5-fluorocytosine due to the intraparasitic conversion of this non-toxic prodrug to the cytotoxic compound 5-fluorouracil. Exogenously supplied cytosine or uracil rescued the growth of CD transgenic T. gondii parasites that were cultured in the presence of cytotoxic concentrations of 5-fluorouracil or 5-fluorocytosine. Bacterial CD fused to the pyrimethamine resistant DHFR-TS marker provides a novel genetic tool for new positive and negative genetic selection strategies in several protozoan parasites. An advantage of the CD genetic marker is that it is derived from a bacterial gene and can therefore be used in nearly any parasite genetic background for negative selection. This novel system should facilitate new approaches for the development of improved model genetic systems for the biological investigation of apicomplexan parasites.

Identification of the calcium binding site and a novel ytterbium site in blood coagulation factor XIII by x-ray crystallography.

The presence or absence of calcium determines the activation, activity, oligomerization, and stability of blood coagulation factor XIII. To explore these observed effects, we have determined the x-ray crystal structure of recombinant factor XIII A2 in the presence of calcium, strontium, and ytterbium. The main calcium binding site within each monomer involves the main chain oxygen atom of Ala-457, and also the side chains from residues Asn-436, Asp-438, Glu-485, and Glu-490. Calcium and strontium bind in the same location, while ytterbium binds several angstroms removed. A novel ytterbium binding site is also found at the dimer two-fold axis, near residues Asp-270 and Glu-272, and this site may be related to the reported inhibition by lanthanide metals (Achyuthan, K. E., Mary, A., and Greenberg, C. S. (1989) Biochem. J. 257, 331-338). The overall structure of ion-bound factor XIII is very similar to the previously determined crystal structures of factor XIII zymogen, likely due to the constraints of this monoclinic crystal form. We have merged the three independent sets of water molecules in the structures to determine which water molecules are conserved and possibly structurally significant.