Search

The programmed cell death protein 1 receptor (PD-1) and programmed death ligand 1 (PD-L1) coinhibitory pathway suppresses T-cell–mediated immunity. We hypothesized that cotargeting of PD-1 and PD-L1 with a bispecific antibody molecule could provide an alternative therapeutic approach, with enhanced antitumor activity, compared with monospecific PD-1 and PD-L1 antibodies. Here, we describe LY3434172, a bispecific IgG1 mAb with ablated Fc immune effector function that targets both human PD-1 and PD-L1. LY3434172 fully inhibited the major inhibitory receptor–ligand interactions in the PD-1 pathway. LY3434172 enhanced functional activation of T cells in vitro compared with the parent anti–PD-1 and anti–PD-L1 antibody combination or respective monotherapies. In mouse tumor models reconstituted with human immune cells, LY3434172 therapy induced dramatic and potent antitumor activity compared with each parent antibody or their combination. Collectively, these results demonstrated the enhanced immunomodulatory (immune blockade) properties of LY3434172, which improved antitumor immune response in preclinical studies, thus supporting its evaluation as a novel bispecific cancer immunotherapy.

Supplementary Figures 1-9 and Supplementary Tables 1-3

Figure S9 shows antitumor efficacy of combination therapy in the L55 NSCLC tumor xenograft model

Supplementary Table S1 - Therapeutic antibody induced EGFR ECR mutations; Supplementary Table S2 - Mean SPR KD values for binding of wild type (WT) and cetuximab resistance variants of sEGFR to immobilized Fab fragments from cetuximab (FabC225) and necitumumab (Fab11F8) and to EGF; Supplementary Table S3 - Crystallographic data collection and refinement statistics; Supplementary Table S4 - Changes in experimental binding and DSC4.1 computed energy terms for interaction of cetuximab (C225) and necitumumab (11F8) with wild type and epitope mutated EGFR variants; Supplementary Figure S1 - Binding of necitumumab and cetuximab to LK2 and HELA cells; Supplementary Figure S2 - Electron density near R468 in sEGFRd3-S468R/Fab11F8 structure; Supplementary Figure S3 - Comparison of the paratopes of necitumumab and panitumumab; Supplementary Figure S4 - Necitumumab exhibits structural plasticity in binding to sEGFRd3-S468R; Supplementary Figure S5 - Location of the epitope substitutions in the experimental test set and evaluation of the side chain conformations modeled with DSC4.1; Supplementary Figure S6 - CDR dynamics for necitumumab and cetuximab when bound to three cetuximab resistance variants, evaluated by MD simulations

Table S2 shows human immune effector ADCC and CDC activity EC50 (nM)

Supplementary Materials and Methods

The CD137 receptor plays a key role in mediating immune response by promoting T cell proliferation, survival, and memory. Effective agonism of CD137 has the potential to reinvigorate potent antitumor immunity either alone or in combination with other immune-checkpoint therapies. In this study, we describe the discovery and characterization of a unique CD137 agonist, 7A5, a fully human IgG1 Fc effector-null monoclonal antibody. The biological properties of 7A5 were investigated through in vitro and in vivo studies. 7A5 binds CD137, and the binding epitope overlaps with the CD137L binding site based on structure. 7A5 engages CD137 receptor and activates NF-κB cell signaling independent of cross-linking or Fc effector function. In addition, T cell activation measured by cytokine IFNγ production is induced by 7A5 in peripheral blood mononuclear cell costimulation assay. Human tumor xenograft mouse models reconstituted with human immune cells were used to determine antitumor activity in vivo. Monotherapy with 7A5 inhibits tumor growth, and this activity is enhanced in combination with a PD-L1 antagonist antibody. Furthermore, the intratumoral immune gene expression signature in response to 7A5 is highly suggestive of enhanced T cell infiltration and activation. Taken together, these results demonstrate 7A5 is a differentiated CD137 agonist antibody with biological properties that warrant its further development as a cancer immunotherapy.

Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy. KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers. Here we show the identification and development of an affinity-enhanced T cell receptor (TCR) that recognizes a peptide derived from the most common KRAS mutant, KRAS

The programmed cell death protein 1 receptor (PD-1) and programmed death ligand 1 (PD-L1) coinhibitory pathway suppresses T-cell–mediated immunity. We hypothesized that cotargeting of PD-1 and PD-L1 with a bispecific antibody molecule could provide an alternative therapeutic approach, with enhanced antitumor activity, compared with monospecific PD-1 and PD-L1 antibodies. Here, we describe LY3434172, a bispecific IgG1 mAb with ablated Fc immune effector function that targets both human PD-1 and PD-L1. LY3434172 fully inhibited the major inhibitory receptor–ligand interactions in the PD-1 pathway. LY3434172 enhanced functional activation of T cells in vitro compared with the parent anti–PD-1 and anti–PD-L1 antibody combination or respective monotherapies. In mouse tumor models reconstituted with human immune cells, LY3434172 therapy induced dramatic and potent antitumor activity compared with each parent antibody or their combination. Collectively, these results demonstrated the enhanced immunomodulatory (immune blockade) properties of LY3434172, which improved antitumor immune response in preclinical studies, thus supporting its evaluation as a novel bispecific cancer immunotherapy.

Background Modulation of the PD-1/PD-L1 axis through antagonist antibodies that block either receptor or ligand has been shown to reinvigorate the function of tumor-specific T cells and unleash potent anti-tumor immunity, leading to durable objective responses in a subset of patients across multiple tumor types. Results Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ monoclonal antibody that binds to human PD-L1 with high affinity and inhibits interactions of PD-L1 with its two cognate receptors PD-1 and CD80. The functional activity of LY3300054 on primary human T cells is evaluated using a series of in vitro T cell functional assays and in vivo models using human-immune reconstituted mice. LY3300054 is shown to induce primary T cell activation in vitro, increase T cell activation in combination with anti-CTLA4 antibody, and to potently enhance anti-tumor alloreactivity in several xenograft mouse tumor models with reconstituted human immune cells. High-content molecular analysis of tumor and peripheral tissues from animals treated with LY3300054 reveals distinct adaptive immune activation signatures, and also previously not described modulation of innate immune pathways. Conclusions LY3300054 is currently being evaluated in phase I clinical trials for oncology indications.

Acquired resistance to cetuximab, an antibody that targets the EGFR, impacts clinical benefit in head and neck, and colorectal cancers. One of the mechanisms of resistance to cetuximab is the acquisition of mutations that map to the cetuximab epitope on EGFR and prevent drug binding. We find that necitumumab, another FDA-approved EGFR antibody, can bind to EGFR that harbors the most common cetuximab-resistant substitution, S468R (or S492R, depending on the amino acid numbering system). We determined an X-ray crystal structure to 2.8 Å resolution of the necitumumab Fab bound to an S468R variant of EGFR domain III. The arginine is accommodated in a large, preexisting cavity in the necitumumab paratope. We predict that this paratope shape will be permissive to other epitope substitutions, and show that necitumumab binds to most cetuximab- and panitumumab-resistant EGFR variants. We find that a simple computational approach can predict with high success which EGFR epitope substitutions abrogate antibody binding. This computational method will be valuable to determine whether necitumumab will bind to EGFR as new epitope resistance variants are identified. This method could also be useful for rapid evaluation of the effect on binding of alterations in other antibody/antigen interfaces. Together, these data suggest that necitumumab may be active in patients who are resistant to cetuximab or panitumumab through EGFR epitope mutation. Furthermore, our analysis leads us to speculate that antibodies with large paratope cavities may be less susceptible to resistance due to mutations mapping to the antigen epitope. Mol Cancer Ther; 17(2); 521–31. ©2017 AACR.

BackgroundKRAS is the most frequently mutated oncogene, yet mutant KRAS has historically been a challenging target for conventional small molecule drug development. Tumour specific neoantigen peptides derived from KRAS are presented by cell surface human leucocyte antigens (HLA) and form a class of shared, tumour-specific antigens that are attractive targets for immunotherapy.MethodsA T cell clone that specifically recognizes the most common KRAS G12D mutant presented as a peptide in the context of HLA-A*11:01 was isolated from healthy donor PBMCs. The affinity of the respective T cell receptor (TCR) was enhanced by phage display and the x-ray crystal structures of the affinity-enhanced TCR bound to HLA presenting mutant KRAS G12D and wildtype (KRAS WT) peptides were solved. We used structural, biochemical, and computational approaches to investigate the molecular interactions underlying TCR selectivity for mutant KRAS G12D. Finally, the high affinity TCR was engineered into a soluble T cell engaging ImmTAC (Immune mobilizing monoclonal TCR Against Cancer) molecule, IMC-KRAS-G12D, and in vitro cell-based assays were performed to evaluate its potency and selectivity.ResultsThe affinity of the engineered TCR was enhanced by a million-fold and demonstrated remarkable ability to distinguish between KRAS G12D and KRAS WT peptide presented by HLA-A*11:01. X-ray crystal structures demonstrate that TCR binding is almost identical between KRAS G12D and KRAS WT despite a binding affinity difference of >4000 fold. The mutant residue G12D is buried into the HLA peptide binding groove and acts as a secondary anchor, making it inaccessible to the TCR. Thermodynamic analysis of TCR-HLA interaction combined with molecular dynamics simulations indicates a novel mechanism of peptide selectivity, mediated by an indirect energetic mechanism driven by an induced fit in the peptide upon TCR binding. In functional assays, this molecular differentiation translated into biological specificity with IMC-KRAS-G12D mediating T cell activation in response to cells pulsed with or expressing KRAS G12D but not KRAS WT. Furthermore, IMC-KRAS-G12D was able to redirect T cell cytotoxicity towards target KRAS G12D presenting colon cancer cells, while sparing normal colon epithelial cellsConclusionsWe developed a high affinity TCR bispecific with exquisite specificity towards a common shared neoantigen, KRAS G12D, that is a relevant therapeutic target in a wide range of cancers. These findings reveal a novel molecular mechanism for TCR selectivity for a neoantigen that differs from self-antigen by only a single amino acid, with attendant implications for therapeutic development.

Background Modulation of the PD-1/PD-L1 axis through antagonist antibodies that block either receptor or ligand has been shown to reinvigorate the function of tumor-specific T cells and unleash potent anti-tumor immunity, leading to durable objective responses in a subset of patients across multiple tumor types. Results Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ monoclonal antibody that binds to human PD-L1 with high affinity and inhibits interactions of PD-L1 with its two cognate receptors PD-1 and CD80. The functional activity of LY3300054 on primary human T cells is evaluated using a series of in vitro T cell functional assays and in vivo models using human-immune reconstituted mice. LY3300054 is shown to induce primary T cell activation in vitro, increase T cell activation in combination with anti-CTLA4 antibody, and to potently enhance anti-tumor alloreactivity in several xenograft mouse tumor models with reconstituted human immune cells. High-content molecular analysis of tumor and peripheral tissues from animals treated with LY3300054 reveals distinct adaptive immune activation signatures, and also previously not described modulation of innate immune pathways. Conclusions LY3300054 is currently being evaluated in phase I clinical trials for oncology indications. Electronic supplementary material The online version of this article (10.1186/s40425-018-0329-7) contains supplementary material, which is available to authorized users.

Conformational entropy is an important component of protein-protein interactions; however, there is no reliable method for computing this parameter. We have developed a statistical measure of residual backbone entropy in folded proteins by using the ϕ-ψ distributions of the 20 amino acids in common secondary structures. The backbone entropy patterns of amino acids within helix, sheet or coil form clusters that recapitulate the branching and hydrogen bonding properties of the side chains in the secondary structure type. The same types of residues in coil and sheet have identical backbone entropies, while helix residues have much smaller conformational entropies. We estimated the backbone entropy change for immunoglobulin complementarity-determining regions (CDRs) from the crystal structures of 34 low-affinity T-cell receptors and 40 high-affinity Fabs as a result of the formation of protein complexes. Surprisingly, we discovered that the computed backbone entropy loss of only the CDR3, but not all CDRs, correlated significantly with the kinetic and affinity constants of the 74 selected complexes. Consequently, we propose a simple algorithm to introduce proline mutations that restrict the conformational flexibility of CDRs and enhance the kinetics and affinity of immunoglobulin interactions. Combining the proline mutations with rationally designed mutants from a previous study led to 2400-fold increase in the affinity of the A6 T-cell receptor for Tax-HLAA2. However, this mutational scheme failed to induce significant binding changes in the already-high-affinity C225-Fab/huEGFR interface. Our results will serve as a roadmap to formulate more effective target functions to design immune complexes with improved biological functions.

The anti-T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) antibody LY3321367 is a promising immune-checkpoint inhibitor that is being clinically tested for the treatment of multiple solid tumors (NCT03099109). LY3321367 was phenotypically selected from a large set of phage library Fabs that are TIM-3 binders. Tim3 has multiple protein and non-protein binding partners; hence, we attempt in this study to deconvolute the molecular mechanism of action of LY3321367. Surprisingly, LY3321367 in ELISA assays partially blocks the TIM-3/GAL-9 complex but does not block the TIM-3/CEACAM-1 complex. We also utilized X-ray crystallography to solve the structure the LY3321367-Fab complex with the IgV domain of TIM-3 at 2.0Å resolution. After solving the unbound structure of the LY3321367-Fab, we demonstrated that its CDRs do not undergo any conformational changes upon binding human TIM-3. The structural alignment of the human TIM-3/LY3321367-Fab complex with the available murine TIM-3 structure unexpectedly suggests that the 6.0Å epitope of LY3321367 overlaps the phosphatidylserine (PS) binding site on TIM-3. Consequently, we developed a cell-based assay to demonstrate that LY3321367 completely blocks the binding of soluble human TIM-3 to PS displayed on the surface of Campothecin-treated DO11.10 cells. Our structural results suggest an important role for PS in TIM-3 biology which is blocked by LY3321367. When combined with the partial blocking of GAL-9, the structural data of LY3321367 suggests that the binding sites of PS and GAL-9 may be proximal to each other on TIM-3. Citation Format: Jaafar N. Haidar, Stephen Antonysamy, Sneha Mathew, Lan Wu, Yi Zhang, Margaret C. Kearins, Leyi Shen, J. Michael Sauder, David Schaer, Kyla E. Driscoll, Michael Kalos. The molecular basis of blocking the TIM-3 checkpoint with the LY3321367 mAb in cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2753.

Antibody (Ab) humanization is crucial to generate clinically relevant biologics from hybridoma-derived monoclonal antibodies (mAbs). In this study, we integrated antibody structural information from the Protein Data Bank with known back-to-mouse mutational data to build a universal consensus of framework positions (10 heavy and 7 light) critical for the preservation of the functional conformation of the Complimentarity Determining Region of antibodies. On the basis of FR consensus, we describe here a universal combinatorial library suitable for humanizing exogenous antibodies by CDR-grafting. The six CDRs of the murine anti-human EGFR Fab M225 were grafted onto a distinct (low FR sequence similarity to M225) human FR sequence that incorporates at the 17 FR consensus positions the permutations of the naturally observed amino acid diversities. Ten clones were selected from the combinatorial library expressing phage-displayed humanized M225 Fabs. Surprisingly, 2 of the 10 clones were found to bind EGFR with stronger affinity than M225. Cell-based assays demonstrated that the 10 selected clones retained epitope specificity by blocking EGFR phosphorylation and thus hindering cellular proliferation. Our results suggest that there is a universal and structurally rigid near-CDR set of FR positions that cooperatively support the binding conformation of CDRs.

Understanding the energetic and structural response to multiple mutations in a protein−protein interface is a key aspect of rational protein design. Here we investigate the cooperativity of combinations of point mutations of a T cell receptor (TCR) that binds in vivo to HLA-A2 MHC and a viral peptide. The mutations were obtained from two sources: a structure-based design study on the TCR α chain (nine mutations) and an in vitro selection study on the TCR β chain (four mutations). In addition to combining the highest-affinity variants from each chain, we tested other combinations of mutations within and among the chains, for a total of 23 TCR mutants that we measured for binding kinetics to the peptide and major histocompatibility complex. A wide range of binding affinities was observed, from 2- to 1000-fold binding improvement versus that of the wild type, with significant nonadditive effects observed within and between TCR chains. This included an amino acid-dependent cooperative interaction between CDR1...

Modulating the PD-L1/PD-1 axis in man through function blocking antibodies can release potent anti-tumor immunity, leading to durable objective responses across multiple tumor types. Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ antibody capable of binding human PD-L1 with high affinity and inhibiting binding to its two cognate receptors, PD-1 and CD80. LY3300054 is an antagonist monoclonal antibody recognizing human PD-L1 with high affinity (KD 0.08 nM), selected and derived from a scFv phage library. LY3300054 was engineered and expressed as an IgG1-Fc null monoclonal antibody to ablate immune effector function. Multiple in vitro assays, including mixed leukocyte reaction (MLR) and tetanus toxoid recall assay were utilized to demonstrate LY3300054 potent functional activity in enhancing the activation of primary human T cells in culture. The biological activity of LY3300054 on T cells was also shown to be enhanced by co-administration of anti-CTLA4 mAb (ipilimumab) in MLR. Further, we evaluated LY3300054 activity in vivo using xenograft mouse tumor models reconstituted with human immune cells. LY3300054 demonstrated anti-tumor activity in both NCI-H292 xenografts co-implanted with human PBMCs and HCC827 xenograft model upon infusion of human T cells. LY3300054 in vivo activity was also tested in HCC827- and Ov79-bearing immunodeficient NSG/NOG mice reconstituted with human hematopoietic stem cells. In this setting, LY3300054 displayed robust anti-tumor and immunomodulatory effects exemplified by T cell inflamed phenotype in the tumor and peripheral tissues. High-content molecular profiling identified distinct gene expression changes indicative of T cell activation in all models tested. A mutational strategy based on integrating the PD-1/PD-L1 structure data with the orthologous sequence data of PD-L1 has identified a residue on PD-L1, which is part of the PD-L1/PD-1 interface, that is critical for the species specificity of LY3300054. This study demonstrates that LY3300054 is novel anti-PD-L1 monoclonal antibody, capable of potently enhancing human T cell function both in vitro and in vivo, and provides previously not described insights into the effects of PD-L1 blockade on the intra- and extra-tumoral immune response. LY3300054 is currently under clinical evaluation in monotherapy and combination with other therapeutic modalities in multiple tumor types (NCT02791334; NCT03099109; NCT02791334; NCT02791334) Citation Format: Carmine Carpenito, Yiwen Li, George X. Wang, Maria S. Malabunga, Jaafar N. Haidar, Amelie Forest, Mary Y. Murphy, Gerald E. Hall, Cindy Wang, Leyi Shen, Andreas Sonyi, Darin Chin, Anthony L. Pennello, Ivan V. Inigo, David Surguladze, Yung-mae Yao, Douglas Burtrum, Ruslan D. Novosiadly, Kris Persaud, Dale L. Ludwig, Michael D. Kalos. Preclinical characterization of the anti-PD-L1 monoclonal antibody LY3300054 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2730.

Pace et al. (1995) [1] recommended an equation used to predict extinction coefficient of a protein. However, no antibody data was included in the development of this equation. The main objective of this study was to therefore investigate how the predicted value of the extinction coefficient is comparable to the experimentally determined extinction coefficient of antibodies measured by the Edelhoch method. We have measured the extinction coefficients (ɛ) of 13 IgG1 monoclonal antibodies (mAbs) in phosphate buffer at pH 7.2. The maximum variability in the experimentally measured extinction coefficient of a given mAb molecule was found to be about 2%. Experimentally determined extinction coefficients of all mAbs were found to be lower than the predicted value, with the maximum difference found to being 4.7%. The highest and lowest values of experimental extinction coefficient among the thirteen IgG1 monoclonal antibodies obtained were 230525.9M(-1)cm(-1) (i.e. 1.55(mg/ml)(-1)cm(-1)) and 191,411.6M(-1)cm(-1) (i.e. 1.29(mg/ml)(-1)cm(-1)). A difference of

Activin is a member of the TGFβ family of cytokines involved in the control of cell proliferation. We have previously shown that the majority of clinically nonfunctioning pituitary tumors do not respond to activin-induced growth suppression. Human pituitary tumors specifically express alternatively spliced activin type I receptor Alk4 mRNAs, producing C-terminus truncated isoforms designated Alk4-2, 4-3, and 4-4. However, it is not known whether these truncated activin receptors suppress activin effects on cell proliferation in human pituitary cells. Therefore, we investigated activin signaling in a human pituitary tumor cell line, HP75, derived from a clinically nonfunctioning pituitary tumor. HP75 cells express activin A mRNA and secrete activin A, as measured by ELISA and a functional bioassay. TGFβ administration decreases the proliferation of HP75 cells, suggesting that the signaling pathway shared by TGFβ and activin is functional in this cell line. However, activin neither inhibits cell proliferation nor stimulates reporter gene expression in HP75 cells, indicating that activin signaling is specifically blocked at the receptor level. HP75 cells express all truncated activin type I receptor Alk4 isoforms, as determined by RT-PCR. Because truncated Alk4 receptor isoforms inhibit activin signaling by competing with the wild-type receptor for binding to activin type II receptors, we hypothesized that overexpression of wild-type activin type I receptor will restore activin signaling. In HP75 cells, cotransfection of the wild-type activin type I receptor Alk4-1 expression vector increases activin-responsive reporter activity. Furthermore, transfection with wild-type activin receptor type I results in activin-mediated suppression of cell proliferation. These data indicate that truncated Alk4 isoforms interfere with activin signaling pathways and thereby may contribute to uncontrolled cell growth. Overexpression of the wild-type Alk4-1 receptor restores responsiveness to activin in human pituitary tumor-derived cells.

Somatostatin (SST) acts through a family of seven transmembrane domain G protein-coupled receptors to inhibit hormone secretion and cell proliferation in a variety of neuroendocrine tissues. In normal and neoplastic human pituitary somatotroph cells, SST-specific receptor types (SSTR) 1, 2, 3, and 5 are prevalently expressed, and SST and its analogs have been shown to inhibit GH secretion. However, in somatotroph adenomas, little is known regarding: 1) effects of SST and its analogs on pituitary tumor proliferation; 2) the relationship between the effects of SST analogs on GH secretion and tumor cell proliferation; and 3) whether SSTR expression predicts the antiproliferative effects of SST analogs in human somatotroph tumors. We investigated the effects of SST-14, lanreotide, and SSTR 2 (BIM-23190) and SSTR 5 (BIM-23268) specific analogs in 18 somatotroph pituitary adenomas in primary culture. Our results showed that cell proliferation was significantly inhibited by SST-14, lanreotide, BIM-23190, and BIM...

T-cell receptors (TCRs) are proteins that recognize peptides from foreign proteins bound to the major histocompatibility complex (MHC) on the surface of an antigen-presenting cell. This interaction enables the T cells to initiate a cell-mediated immune response to terminate cells displaying the foreign peptide on their MHC. Naturally occurring TCRs have high specificity but low affinity toward the peptide-MHC (pepMHC) complex. This prevents the usage of solubilized TCRs for diagnosis and treatment of viral infections or cancers. Efforts to enhance the binding affinity of several TCRs have been reported in recent years, through randomized libraries and in vitro selection. However, there have been no reported efforts to enhance the affinity via structure-based design, which allows more control and understanding of the mechanism of improvement. Here, we have applied structure-based design to a human TCR to improve its pepMHC binding. Our design method evolved based on iterative steps of prediction, testing, and generating more predictions based on the new data. The final design function, named ZAFFI, has a correlation of 0.77 and average error of 0.35 kcal/mol with the binding free energies of 26 point mutations for this system that we measured by surface plasmon resonance (SPR). Applying the filter that we developed to remove nonbinding predictions, this correlation increases to 0.85, and the average error decreases to 0.3 kcal/mol. Using this algorithm, we predicted and tested several point mutations that improved binding, with one giving over sixfold binding improvement. Four of the point mutations that improved binding were then combined to give a mutant TCR that binds the pepMHC 99 times more strongly than the wild-type TCR. Proteins 2009. © 2008 Wiley-Liss, Inc.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view about chromatin structure has emerged, including its interrelationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded novel mechanistic and evolutionary insights about the functional landscape of the human genome. Together, these studies are defining a path forward to pursue a more-comprehensive characterisation of human genome function.