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Supplementary Materials and Methods; Supplementary Figure S1. Binding kinetics of hedgehog antibodies to human and mouse hedgehog proteins using surface plasmon resonance. Supplementary Figure S2. Hedgehog ligands stimulate mGLI1 reporter activity and osteoblast differentiation in a dose-dependent manner. Supplementary Figure S3. Selective inhibition of mGLI1 or mPTC1 induction in C3H10T1/2 cells by hedgehog antibodies. Supplementary Figure S4. MEDI-5304 inhibits GLI1 expression in Colo205 xenograft stromal cells but not tumor cells. Supplementary Figure S5. Hedgehog pathway component RNA expression in primary pancreatic tumor explant model P479 and effects of MEDI-5304 on tumorspheres derived from pancreatic explant model 947. Supplementary Figure S6. Pharmacokinetics of MEDI-5304 in rats. Supplementary Figure S7. Exploratory toxicology of MEDI-5304 in rats. Supplementary Figure S8. Pharmacokinetics of MEDI-5304 from preliminary toxicology study in cynomolgus monkeys. Supplementary Figure S9. Pharmacodynamics of MEDI-5304 in cynomolgus monkeys.

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The Notch signaling pathway has been implicated in cell fate determination and differentiation in many tissues. Accumulating evidence points toward a pivotal role in blood vessel formation, and the importance of the Delta-like ligand (Dll) 4-Notch1 ligand–receptor interaction has been shown in both physiological and tumor angiogenesis. Disruption of this interaction leads to a reduction in tumor growth as a result of an increase in nonfunctional vasculature leading to poor perfusion of the tumor. MEDI0639 is an investigational human therapeutic antibody that targets Dll4 to inhibit the interaction between Dll4 and Notch1. The antibody cross-reacts to cynomolgus monkey but not mouse species orthologues. In vitro MEDI0639 inhibits the binding of Notch1 to Dll4, interacting via a novel epitope that has not been previously described. Binding to this epitope translates into MEDI0639 reversing Notch1-mediated suppression of human umbilical vein endothelial cell growth in vitro. MEDI0639 administration resulted in stimulation of tubule formation in a three-dimensional (3D) endothelial cell outgrowth assay, a phenotype driven by disruption of the Dll4-Notch signaling axis. In contrast, in a two-dimensional endothelial cell–fibroblast coculture model, MEDI0639 is a potent inhibitor of tubule formation. In vivo, MEDI0639 shows activity in a human endothelial cell angiogenesis assay promoting human vessel formation and reducing the number of vessels with smooth muscle actin-positive mural cells coverage. Collectively, the data show that MEDI0639 is a potent modulator of Dll4-Notch signaling pathway. Mol Cancer Ther; 11(8); 1650–60. ©2012 AACR.

Localized angiopoietin-2 (Ang2) expression has been shown to function as a key regulator of blood vessel remodeling and tumor angiogenesis, making it an attractive candidate for antiangiogenic therapy. A fully human monoclonal antibody (3.19.3) was developed, which may have significant pharmaceutical advantages over synthetic peptide-based approaches in terms of reduced immunogenicity and increased half-life to block Ang2 function. The 3.19.3 antibody potently binds Ang2 with an equilibrium dissociation constant of 86 pmol/L, leading to inhibition of Tie2 receptor phosphorylation in cell-based assays. In preclinical models, 3.19.3 treatment blocked blood vessel formation in Matrigel plug assays and in human tumor xenografts. In vivo studies with 3.19.3 consistently showed broad antitumor activity as a single agent across a panel of diverse subcutaneous and orthotopic xenograft models. Combination studies of 3.19.3 with cytotoxic drugs or anti–vascular endothelial growth factor agents showed significant improvements in antitumor activity over single-agent treatments alone with no apparent evidence of increased toxicity. Initial pharmacokinetic profiling studies in mice and nonhuman primates suggested that 3.19.3 has a predicted human half-life of 10 to 14 days. These studies provide preclinical data for 3.19.3 as a potential new antiangiogenic therapy as a single agent or in combination with chemotherapy or vascular endothelial growth factor inhibitors for the treatment of cancer. Mol Cancer Ther; 9(1); 145–56

Supplementary Table 1, Figure Legends 1-6 from A Human Monoclonal Anti-ANG2 Antibody Leads to Broad Antitumor Activity in Combination with VEGF Inhibitors and Chemotherapy Agents in Preclinical Models

Herein we report structure–cytotoxicity relationships for analogues of N14-desacetoxytubulyisn H 1. A novel synthetic approach toward 1 enabled the discovery of compounds with a range of activity. Calculated basicity of the N-terminus of tubulysins was shown to be a good predictor of cytotoxicity. The impact of structural modifications at the C-terminus of 1 upon cytotoxicity is also described. These findings will facilitate the development of new tubulysin analogues for the treatment of cancer.

Antibodies that target cell-surface molecules on T cells can enhance anti-tumor immune responses, resulting in sustained immune-mediated control of cancer. We set out to find new cancer immunotherapy targets by phenotypic screening on human regulatory T (Treg) cells and report the discovery of novel activators of tumor necrosis factor receptor 2 (TNFR2) and a potential role for this target in immunotherapy. A diverse phage display library was screened to find antibody mimetics with preferential binding to Treg cells, the most Treg-selective of which were all, without exception, found to bind specifically to TNFR2. A subset of these TNFR2 binders were found to agonise the receptor, inducing iκ-B degradation and NF-κB pathway signalling in vitro. TNFR2 was found to be expressed by tumor-infiltrating Treg cells, and to a lesser extent Teff cells, from three lung cancer patients, and a similar pattern was also observed in mice implanted with CT26 syngeneic tumors. In such animals, TNFR2-specific agonists inhibited tumor growth, enhanced tumor infiltration by CD8+ T cells and increased CD8+ T cell IFN-γ synthesis. Together, these data indicate a novel mechanism for TNF-α-independent TNFR2 agonism in cancer immunotherapy, and demonstrate the utility of target-agnostic screening in highlighting important targets during drug discovery., GW, BM, SG, JC-U, AS, AG-M, CB, JJ, RL, AJL, SR, RS, LJ, VV-A, RM and RWW were funded by MedImmune; JP and VB were funded by AstraZeneca PLC; JW, RSA-L and JB were funded by NIHR Cambridge Biomedical Research Centre and Kidney Research UK; JS and JF were funded by Retrogenix Ltd.

Antibodies can undergo a variety of covalent and non-covalent degradation reactions that have adverse effects on efficacy, safety, manufacture and storage. We had identified an antibody to Angiopoietin 2 (Ang2 mAb) that neutralizes Ang2 binding to its receptor in vitro and inhibits tumor growth in vivo. Despite favorable pharmacological activity, the Ang2 mAb preparations were heterogeneous, aggregated rapidly and were poorly expressed. Here, we report the engineering of the antibody variable and constant domains to generate an antibody with reduced propensity to aggregate, enhanced homogeneity, 11°C elevated T(m), 26-fold improved level of expression and retained activity. The engineered molecule, MEDI-3617, is now compatible with the large scale material supply required for clinical trials and is currently being evaluated in Phase 1 in cancer patients. This is the first report to describe the stability engineering of a therapeutic antibody addressing non canonical cysteine residues and the design strategy reported here is generally applicable to other therapeutic antibodies and proteins.

Herein we report structure-cytotoxicity relationships for analogues of N

The c-Met proto-oncogene is a multifunctional receptor tyrosine kinase that is stimulated by its ligand, hepatocyte growth factor (HGF), to induce cell growth, motility and morphogenesis. Dysregulation of c-Met function, through mutational activation or overexpression, has been observed in many types of cancer and is thought to contribute to tumor growth and metastasis by affecting mitogenesis, invasion, and angiogenesis. We identified human monoclonal antibodies that bind to the extracellular domain of c-Met and inhibit tumor growth by interfering with ligand-dependent c-Met activation. We identified antibodies representing four independent epitope classes that inhibited both ligand binding and ligand-dependent activation of c-Met in A549 cells. In cells, the antibodies antagonized c-Met function by blocking receptor activation and by subsequently inducing downregulation of the receptor, translating to phenotypic effects in soft agar growth and tubular morphogenesis assays. Further characterization of the antibodies in vivo revealed significant inhibition of c-Met activity (≥ 80% lasting for 72-96 h) in excised tumors corresponded to tumor growth inhibition in multiple xenograft tumor models. Several of the antibodies identified inhibited the growth of tumors engineered to overexpress human HGF and human c-Met (S114 NIH 3T3) when grown subcutaneously in athymic mice. Furthermore, lead candidate antibody CE-355621 inhibited the growth of U87MG human glioblastoma and GTL-16 gastric xenografts by up to 98%. The findings support published pre-clinical and clinical data indicating that targeting c-Met with human monoclonal antibodies is a promising therapeutic approach for the treatment of cancer.

Localized angiopoietin-2 (Ang2) expression has been shown to function as a key regulator of blood vessel remodeling and tumor angiogenesis, making it an attractive candidate for antiangiogenic therapy. A fully human monoclonal antibody (3.19.3) was developed, which may have significant pharmaceutical advantages over synthetic peptide-based approaches in terms of reduced immunogenicity and increased half-life to block Ang2 function. The 3.19.3 antibody potently binds Ang2 with an equilibrium dissociation constant of 86 pmol/L, leading to inhibition of Tie2 receptor phosphorylation in cell-based assays. In preclinical models, 3.19.3 treatment blocked blood vessel formation in Matrigel plug assays and in human tumor xenografts. In vivo studies with 3.19.3 consistently showed broad antitumor activity as a single agent across a panel of diverse subcutaneous and orthotopic xenograft models. Combination studies of 3.19.3 with cytotoxic drugs or anti–vascular endothelial growth factor agents showed significant improvements in antitumor activity over single-agent treatments alone with no apparent evidence of increased toxicity. Initial pharmacokinetic profiling studies in mice and nonhuman primates suggested that 3.19.3 has a predicted human half-life of 10 to 14 days. These studies provide preclinical data for 3.19.3 as a potential new antiangiogenic therapy as a single agent or in combination with chemotherapy or vascular endothelial growth factor inhibitors for the treatment of cancer. Mol Cancer Ther; 9(1); 145–56

Radioligand binding assays for total and Ig classes of insulin antibodies (IAB) were developed and validated. For each assay, insulin-extracted serum samples were incubated with radiolabeled insulin in the presence and absence of high levels of unlabeled insulin to determine nonspecific binding and total binding, respectively. To measure total IAB, antibody-bound insulin was precipitated with a polyethylene glycol solution, washed, and counted in a gamma-counter. To measure IgG IAB, samples were treated with protein G-Sepharose beads, centrifuged, washed, and counted. For the measurement of IgA, IgE, and IgM IAB, IgG was removed from the samples and treated with anti-IgA, -IgE, or -IgM conjugated to Sepharose beads, centrifuged, washed, and counted. The acid/charcoal extraction of bound and unbound insulin from serum samples was optimized. Specificity and binding capacity of the protein G and antibody-bound beads were evaluated and optimized. The linear region of the total and IgG IAB assays was determined using serum samples containing high levels of insulin antibodies. The limit of quantitation, limit of detection, and precision for all the assays were also determined.

The objective was to develop and validate a radioligand binding assay for insulin antibodies (IABs) of the IgG1, IgG2, IgG3, and IgG4 subclasses in human serum. The validation studies focused on determining specificity, capacity, linearity, sensitivity, and precision of each assay. It was seen that our assay for IAB IgG subclasses is specific and has sufficient capacity to measure each of the subclasses in human serum. Moreover, the linear region and limits of detection and quantitation for each assay are clearly determined.

Programmed cell-death 1 ligand 1 (PD-L1) is a member of the B7/CD28 family of proteins that control T-cell activation. Many tumors can upregulate expression of PD-L1, inhibiting antitumor T-cell responses and avoiding immune surveillance and elimination. We have identified and characterized MEDI4736, a human IgG1 monoclonal antibody that binds with high affinity and specificity to PD-L1 and is uniquely engineered to prevent antibody-dependent cell-mediated cytotoxicity. In vitro assays demonstrate that MEDI4736 is a potent antagonist of PD-L1 function, blocking interaction with PD-1 and CD80 to overcome inhibition of primary human T-cell activation. In vivo MEDI4736 significantly inhibits the growth of human tumors in a novel xenograft model containing coimplanted human T cells. This activity is entirely dependent on the presence of transplanted T cells, supporting the immunological mechanism of action for MEDI4736. To further determine the utility of PD-L1 blockade, an anti-mouse PD-L1 antibody was investigated in immunocompetent mice. Here, anti-mouse PD-L1 significantly improved survival of mice implanted with CT26 colorectal cancer cells. The antitumor activity of anti–PD-L1 was enhanced by combination with oxaliplatin, which resulted in increased release of HMGB1 within CT26 tumors. Taken together, our results demonstrate that inhibition of PD-L1 function can have potent antitumor activity when used as monotherapy or in combination in preclinical models, and suggest it may be a promising therapeutic approach for the treatment of cancer. MEDI4736 is currently in several clinical trials both alone and in combination with other agents, including anti–CTLA-4, anti–PD-1, and inhibitors of IDO, MEK, BRAF, and EGFR. Cancer Immunol Res; 3(9); 1052–62. ©2015 AACR.

The hedgehog pathway has been implicated in the tumorigenesis, tumor progression, and metastasis of numerous human cancers. We generated the first fully human hedgehog antibody MEDI-5304 and characterized its antitumor activity and preclinical toxicology. MEDI-5304 bound sonic hedgehog (SHH) and Indian hedgehog (IHH) with low picomolar affinity and neutralized SHH and IHH activity in cellular mGLI1 reporter assays. The antibody inhibited transcription of hedgehog target genes and osteoblast differentiation of C3H10T1/2 cells. We evaluated the activity of MEDI-5304 in vivo in model systems that allowed us to evaluate two primary hypotheses of hedgehog function in human cancer, paracrine signaling between tumor and stromal cells and cancer stem cell (CSC) self-renewal. MEDI-5304 displayed robust pharmacodynamic effects in stromal cells that translated to antitumor efficacy as a single agent in an HT-29/MEF coimplantation model of paracrine hedgehog signaling. MEDI-5304 also improved responses to carboplatin in the HT-29/MEF model. The antibody, however, had no effect as a single agent or in combination with gemcitabine on the CSC frequency or growth of several primary pancreatic cancer explant models. These findings support the conclusion that hedgehog contributes to tumor biology via paracrine tumor-stromal signaling but not via CSC maintenance or propagation. Finally, the only safety study finding associated with MEDI-5304 was ondontodysplasia in rats. Thus, MEDI-5304 represents a potent dual hedgehog inhibitor suitable for continued development to evaluate efficacy and safety in human patients with tumors harboring elevated levels of SHH or IHH. Mol Cancer Ther; 13(2); 386–98. ©2013 AACR.

Platelet-derived growth factor receptor α (PDGFRα) is a receptor tyrosine kinase that promotes cell survival and is expressed in both the tumor and the stromal components of human cancers. We have developed a fully human monoclonal antibody, MEDI-575, that selectively binds to human PDGFRα with high affinity, with no observable affinity for murine PDGFRα. To more fully characterize the role of PDGFRα in the regulation of tumor stroma, we evaluated the in vivo antitumor effects of MEDI-575 in tumor-bearing severe combined immunodeficient (SCID) mice and in genetically altered SCID mice expressing human PDGFRα in place of murine PDGFRα. We used the Calu-6 non-small cell lung cancer model because it lacks an in vitro proliferative response to PDGFRα activation. Antitumor activity was observed when the study was performed in mice expressing the human receptor, but no activity was observed in the mice expressing the murine receptor. Immunohistologic analysis of the tumors from mice expressing human PDGFRα showed a highly significant reduction in stromal fibroblast content and only minor changes in tumor proliferative index in tumors exposed to MEDI-575 compared with the results seen in vehicle-treated tumors or in tumors from mice expressing murine PDGFRα. Additional in vitro studies indicated that exposure of primary cancer-associated fibroblasts to MEDI-575 can directly affect proliferation and key signaling pathways in these cells. These results highlight the potential for observing antitumor activity with MEDI-575 through modulation of the stromal component of tumors and confirm that the PDGFRα pathway can play a role in maintaining a tumor microenvironment conducive to tumor growth.

Mutations in the failed axon connections (fax) gene have been identified as dominant genetic enhancers of the Abl mutant phenotype. These mutations in fax all result in defective or absent protein product. In a genetic background with wild-type Abl function, the fax loss-of-function alleles are homozygous viable, demonstrating that fax is not an essential gene unless the animal is also mutant for Abl. The fax gene encodes a novel 47-kD protein expressed in a developmental pattern similar to that of Abl in the embryonic mesoderm and axons of the central nervous system. The conditional, extragenic noncomplementation between fax and another Abl modifier gene, disabled, reveal that the two proteins are likely to function together in a process downstream or parallel to the Abl protein tyrosine kinase.

αvβ6 integrin expression is upregulated on a wide range of epithelial tumours, and is thought to play a role in modulating tumour growth. Here we describe a human therapeutic antibody 264RAD, which binds and inhibits αvβ6 integrin function. 264RAD cross-reacts with human, mouse and cynomolgus monkey αvβ6, and inhibits binding to all ligands including the latency-associated peptide of TGF-β. Screening across a range of integrins revealed that 264RAD also binds and inhibits the related integrin αvβ8, but not the integrins α5β1, αvβ3, αvβ5 and α4β1. In vitro 264RAD inhibited invasion of VB6 and Detroit 562 cells in a Matrigel invasion assay and αvβ6 mediated production of matrix metalloproteinase-9 in Calu-3 cells. It inhibited TGF-β-mediated activation of dermal skin fibroblasts by preventing local activation of TGF-β by NCI-H358 tumour cells in a tumour cell−fibroblast co-culture assay. In vivo 264RAD showed dose-dependent inhibition of Detroit 562 tumour growth, regressing established tumours when dosed at 20 mg/kg once weekly. The reduction in growth associated with 264RAD was related to a dose-dependent inhibition of Ki67 and phospho-ERK and a reduction of αvβ6 expression in the tumour cells, coupled to a reduction in fibronectin and alpha smooth muscle actin expression in stromal fibroblasts. 264RAD also reduced the growth and metastasis of orthotopic 4T1 tumours. At 20 mg/kg growth of both the primary tumour and the number of metastatic deposits in lung were reduced. The data support the conclusion that 264RAD is a potent inhibitor of αvβ6 integrin, with some activity against αvβ8 integrin, that reduces both tumour growth and metastasis.

The steroid hormone ecdysone initiates metamorphosis in Drosophila melanogaster by activating a cascade of gene activity that includes primary response transcriptional regulators and secondary response structural genes. The Broad-Complex (BR-C) primary response gene is composed of several distinct genetic functions and encodes a family of related transcription factor isoforms. Our objective was to determine whether BR-C isoforms were components of the primary ecdysone response in all tissues and whether tissue-specific isoform expression is associated with tissue-specific metamorphic outcomes. We used specific antibody reagents that recognize and distinguish among the Z1, Z2 and Z3 BR-C protein isoforms to study protein expression patterns during the initial stages of metamorphosis. Western blot analyses demonstrated that BR-C isoforms are induced at the onset of metamorphosis, each with unique kinetics of induction and repression. Whole-mount immunostaining showed that the BR-C proteins accumulate in the nuclei of all larval and imaginal tissues indicating that the BR-C is induced as a primary response in many tissues. Several tissues express different levels and combinations of the BR-C isoforms suggesting that the BR-C is important in determining the tissue-specific outcome of many parallel ecdysone response cascades. For example, prepupal salivary glands (destined for histolysis during metamorphosis) express Z1 isoforms while imaginal discs (destined for cell differentiation and morphogenesis) shift from the synthesis of Z2 isoforms to the synthesis of Z1 isoforms. The prepupal central nervous system (destined for tissue remodeling) expresses all isoforms, with Z3 pre-dominating. Salivary gland chromosome immunostaining indicated that BR-C proteins interact directly with numerous loci in the polytene genome. Finally, western blot analyses showed that distinct BR-C genetic functions can be correlated with single and specific BR-C protein isoforms.

Antibody drug conjugates (ADCs) combine the specificity of antibodies with the potency of small molecule cytotoxic drugs and have the potential to provide significant efficacy as a treatment for cancer. The objective of this work was to identify potent new cytotoxic ADC payloads that can be used to target diverse tumor types. Here we report for the first time the discovery of fully synthetic tubulysin payloads which belong to a class of highly cytotoxic natural products that disrupt the cellular microtubule network leading to apoptosis of tumor cells. Our fully synthetic tubulysin payloads are comprised of: (i) a tubulysin warhead that displays pM potency, (ii) a protease cleavable amino-acid sequence and (iii) a tether bearing a reactive maleimide group. Tubulysin-based ADCs were generated via site-specific conjugation of these payloads to cysteines engineered into antibodies against cancer antigen target oncofetal protein 5T4. The resulting ADCs showed potent in vitro cell killing and in vivo efficacy in multiple solid tumor xenograft models including prostate cancer, non-small cell lung adenocarcinoma, breast cancer and gastric carcinoma. Furthermore, specific structural features of the tubulysin warhead, linker design and antibody engineering were shown to impact the overall in vitro and in vivo properties of the ADCs. Thus, these synthetic tubulysin payloads represent novel microtubule network disrupting compounds that display potent preclinical anti-tumor activity as an ADC that could be advanced to the clinic. Citation Format: Dorin Toader, Jay Harper, Chris Lloyd, Rose Marwood, David Bannister, Shenlan Mao, Cui (Tracy) Chen, Haihon (Helen) Zhong, Vahe Bedian, Fengjiang Wang, Lakshmaiah Gingipalli, Melisa Vasbinder, Pamela Thompson, Ryan Fleming, Byniam Bezabeh, Nazzareno Dimasi, Changshou Gao, Adeela Kamal. Discovery of tubulysin payloads for antibody drug conjugates with potent in vitro activity and in vivo efficacy in solid tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B170.

CD40 is a member of the TNF family of receptors that has been shown to play a crucial role in enhancing dendritic cell activity and fostering anti-tumor immune responses. In this study, we demonstrate the in vitro properties and in vivo efficacious activity of the CD40 agonist antibody, CP-870,893. CP-870,893 is a fully human, IgG2 antibody that selectively interacts with CD40 at a site distinct from its ligand-binding region with a KD of 0.4 nM. It enhances the expression of MHC class II, CD54, CD86, and CD23 on human B cells in vitro. CP-870,893 also enhances dendritic cell activity as evidenced by cytokine secretion (IL-12, IL-23, IL-8), the upregulation of CD86 and CD83, and the ability to prime T cells to secrete IFNγ. In SCID-beige mice, a single parenteral injection of CP-870,893 was therapeutically effective against several CD40(pos) human tumors (B-cell lymphoma, breast, colon, and prostate) indicating direct effects on tumor cell survival and/or growth. When mice were co-implanted with human T cells and dendritic cells, the activity of CP-870,893 against CD40(pos) tumors increased, and efficacy was also observed against CD40(neg) and CD40(low) tumors demonstrating the ability of CP-870,893 to enhance anti-tumor immune function in vivo. These studies suggest that CP-870,893 has the potential to be efficacious against a wide range of tumor types through both direct and immune-mediated effects.

Peripheral blood lymphocytes from patients with Sezary syndrome (SzS) frequently demonstrate decreased surface expression of transforming growth factor beta receptor II (TGFbetaRII). The mechanism of this low TGFbetaRII expression remains unknown. Because mutations within the poly-A tract of the TGFbetaRII sequence (nucleotides 709-718) were shown to result in diminished TGFbetaRII expression in other types of malignant tumors, we examined the sequence of the TGFbetaRII poly-A tract in two SzS-derived cell lines and in peripheral blood SzS cells from 17 SzS patients and 4 control, healthy individuals using DNA sequencing and single-stranded conformation polymorphism (SSCP) analysis. A standard bidirectional, automated sequence analysis of the RT-PCR-generated cDNA TGFbetaRII fragment showed a heterogenous population of the normal length, 10-, with admixed, shortened, 9-base poly-A stretches. Surprisingly, this mixture was present not only in the cells from 5 SzS patients and 2 SzS cell lines, but also in cells from 2 healthy control individuals. Importantly, the proportion of the shortened, 9-base fragments was markedly reduced or practically eliminated when the procedure was modified by usage of high-fidelity DNA polymerase, labeled primers and/or cloned RT-PCR products, which indicates that the presence of the shortened, 9-base fragments represented a procedural phenomenon rather than a true deletional mutation within an allele of the TGFbetaRII gene. Accordingly, SSCP analysis of genomic DNA did not reveal any mutations within the poly-A tract-containing region. These results indicate that a mechanism different from mutations in the polyadenine tract underlies the diminished TGFbetaRII expression in SzS cells and that the results of an unmodified, direct sequence analysis of homopolymeric base streaches in RT-PCR-derived cDNA should be interpreted with caution.

Regulatory T cells (Treg cells) are observed within multiple tumor types and contribute to immune evasion. Effective and specific targeting of Treg cells by antibodies is hampered by the lack of a unique surface marker. With the aim of identifying such a marker, human Treg cells were isolated from peripheral blood, expanded in vitro and used as a source of antigen in phage display selections with a diverse library of designed ankyrin repeat proteins (DARPins). The resulting DARPins were screened for binding to multiple cell-types, and approximately thirty with preferential binding to Treg cells were identified. All of these DARPins were determined to bind to the third and/or fourth cysteine-rich extracellular domains of tumor necrosis factor receptor II (TNFRII, also known as TNFRSF1B or CD120b). Expression of TNFRII has previously been demonstrated on Treg cells, and TNFRII is shown here also to be expressed on CD8+ T cells following exposure to cognate antigen and on the surface of tumor-infiltrating T cells in the 4T1 mouse tumor model. Furthermore, TNFRII mRNA was detected at increased levels in tumor-infiltrating leukocytes isolated from cancer patients. Functional characterisation of the anti-TNFRII DARPins and mAbs revealed an ability to enhance T-cell activation in response to polyclonal and antigen specific stimuli, and in Treg cell suppression assays. These studies demonstrate the utility of DARPins for phenotypic selections to identify drug targets using primary human lymphocytes and highlight TNFRII as a potential therapeutic target enriched on Treg cells. Citation Format: Geoff Williams, Judith Anderton, Vahe Bedian, Jane Coates Ulrichsen, Andrea Gonzalez-Munoz, Sandrine Guillard, Olivia Harris, James Hair, Andrew Leishman, Arthur Lewis, Jacques Moissan, Ralph Minter, Bina Mistry, Julie Parmentier, Edmund Poon, Amy Popple, Steve Rust, Alan Sandercock, Ross Stewart, Viia Valge-Archer, Robert W. Wilkinson. Identification of tumor necrosis factor receptor II as a regulatory T cell target for cancer immunotherapy using designed ankyrin repeat protein phenotypic selections. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4613. doi:10.1158/1538-7445.AM2014-4613

The genetic code presents an important conceptual challenge within the broader context of the origin of life. Translation of genetic information captures a fundamental property of living systems, i.e. the ability of decoding proteins (e.g. aminoacyl-tRNA synthetases) to reproduce themselves from self-contained RNA/DNA descriptors. Silvano Colombano and I, as graduate students with Howard Pattee in the 1970s, focused on achieving this closure of self-description and self-reproduction in the genetic code. Simulation and analysis of competitive models that allowed alternate code assignments, exploring initial conditions, arbitrary descriptor-catalyst relationships, and degree of non-linearity, indicated that these dynamical systems undergo bifurcations, transforming initial ambiguous stable states to unstable states. New, stable, steady states, progressively closer to a code, became available as the descriptor parameters were varied. The efficiency of utilization of raw materials for the production of a coding family of catalysts is proposed as a selection criterion that drives such systems towards a coded state.

In order to identify novel proteins associated with various stages of macrophage phagocytosis, we have generated monoclonal antibodies that recognize phagosomes. Purified Fc receptor-mediated phagosomes, isolated by feeding IgG-conjugated magnetic beads to LPS-primed murine peritoneal macrophages, were used as the immunogen. An immunofluorescence screen was used to isolate and single-cell clone approximately 150 monoclonal antibodies that recognize mouse macrophage phagosomes as well as labeling other cellular components in patterns which are frequently distinct from those observed with previously characterized phagosome-associated proteins. Predominant morphological categories (in addition to phagosome labeling) include staining of one or more of the following: cytoskeletal patterns, vesicular patterns and plasma membrane localization. In this paper, we describe the antibody screen, preliminary characterization of the antibodies and our identification of the antigens for three representative monoclonal antibodies. These antibodies identify a plasma membrane associated receptor (Mac-1, a subunit of the complement receptor), an actin binding protein (coronin-2) and a vesicular protein (amphiphysin II). Some of the antibodies recognize many cell types, whereas other antibodies are apparently macrophage specific as assessed by flow cytometry and histology. Remarkably, several of the antibodies cross-react with the phagocytic slime mold, Dictyostelium discoideum, recognizing phagosomes and other cellular elements as assessed by immunofluorescence and immunoblots. These results indicate that macrophage phagocytosis has both conserved ancestral features and unique specialized aspects associated with the role of these phagocytes in immunity.

The chemokine receptor CXCR4 is a seven-transmembrane G-protein coupled receptor that mediates chemotaxis and cell migration upon stimulation via its ligand, stromal-derived factor 1 (SDF-1), also called CXCL12. CXCR4 is normally expressed on bone marrow stem and progenitor cells, various circulating lymphocytes, endothelial precursor cells, tissue macrophages and fibroblasts but the aberrant overexpression of CXCR4 is linked to various hematological malignancies, solid tumors and metastatic neoplasms. Moreover, CXCR4 overexpression is correlated with poor prognosis in many types of cancer, including breast, ovarian, colon, pancreatic, AML and glioblastomas. CXCR4 inhibition using siRNA, small-molecule and peptide inhibitors has demonstrated that it can inhibit tumor growth by blocking tumor cell survival/proliferation, metastasis, angiogenesis and tumor immune infiltrates. Here we describe a novel, fully human, antagonistic antibody to CXCR4, MEDI3185, which blocks SDF-1 binding to CXCR4. MEDI3185 has picomolar binding affinity to human CXCR4 and exhibits no significant binding to other chemokine receptors such as CCR4 or CXCR3. In vitro studies demonstrated that MEDI3185 inhibited tumor cell migration, blocked SDF-1 induced tumor cell signaling and induced apoptosis of tumor cells. In preclinical human tumor xenograft models in mouse, MEDI3185 showed single-agent tumor growth inhibition in multiple myeloma and B-cell Burkitt's lymphoma models and had combination activity in ovarian models. In addition, MEDI3185 extended survival as combination therapy in mouse models of CLL and also blocked lung tumor burden in a disseminated ovarian model. Combined, these data suggest that MEDI3185 is a potent CXCR4 antibody for the treatment of both hematological and solid tumors because it has pleiotropic effects on tumor biology that may enhance the efficacy of the current standard of care. Citation Format: Adeela Kamal, Youzhen Wang, Philipp Steiner, Anne-Marie Mazzola, Leslie Wetzel, Melissa Passino, Brenda McDermott, Keven Huang, Vahe Bedian, Norman Greenberg. MEDI3185, a potent anti-CXCR4 antibody, inhibits tumor cell migration, signaling and tumor growth in preclinical models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5462. doi:10.1158/1538-7445.AM2013-5462

OBJECTIVE: Oncofetal fibronectin reactive with antibody FDC-6 has been associated with trophoblastic implantation and chorion structural stability. Abnormal release of this fibronectin into cervical and vaginal secretions has identified patients at risk for preterm labor and delivery. The aim of this study was to determine whether trophoblast-oncofetal fibronectin contains other novel epitopes distinct from the FDC-6 binding site. STUDY DESIGN: Antitrophoblast fibronectin hybridomas were generated and screened by comparative immunoassays. One specific monoclonal antibody, X18A4, was identified and compared with antibody FDC-6 by immunocytochemical and immunoblot analyses. Both antibodies were also evaluated in "sandwich"-type double monoclonal immunosorbent assays. RESULTS: X18A4 and FDC-6 bind avidly and noncompetitively to distinct epitopes within oncofetal fibronectin. They exhibit similar immunohistochemical staining of the extracellular matrix within placental tissue, ovarian epithelial tumors, and cultured trophoblasts. However, in contrast to FDC-6, X18A4 has no detectable binding activity to human plasma fibronectin, and its binding to oncofetal fibronectin was unaffected by enzymatic deglycosylation. Immunoblot analyses of oncofetal fibronectin proteolytic digests suggest that X18A4 binds near or within the alternatively spliced type III connecting segment domain. CONCLUSIONS: X18A4 identifies and binds with high affinity to a new epitope within oncofetal fibronectin, distinct from the FDC-6 binding site. Because X184A displays no detectable binding to plasma fibronectin, it could be used as an important adjunctive antibody for enhancing the specificity of clinically based oncofetal fibronectin diagnostic assays.

Cancerous cells emerge within the body following accumulation of deleterious genetic mutations. These mutations alter the phenotype of a cancer cell marking it as distinct from the surrounding host; an immunological state termed “altered self”. These cells, like other non-self entities such as viruses and bacteria, are recognised by the immune system and marked for destruction, a process known as “immune surveillance”. B7-H1 expression by tumour cells is believed to aid tumours in evading detection and elimination by the immune system. B7-H1 functions in this respect via several alternative mechanisms including driving exhaustion and anergy of tumour infiltrating T lymphocytes, stimulating secretion of immune repressive cytokines into the tumour micro-environment, stimulating repressive regulatory T cell function and protecting B7-H1 expressing tumour cells from lysis by tumour cell specific cytotoxic T cells. Using hybridoma technology and high throughput screening MedImmune has identified a series of fully human antibodies specific for human B7-H1. Further characterisation of these antibodies led to the identification of a single high affinity antibody, MEDI 4736, with the ability to relieve B7-H1 mediated suppression of T cell activation in vitro and to enhance sub-optimal T cell activation in a mixed lymphocyte reaction. In vitro testing shows that MEDI 4736 does not trigger non-specific cytokine release in whole blood, and is only able to activate T cells in the context of an active T cell receptor signal. A surrogate anti-mouse B7-H1 antibody shows significant anti-tumour activity in a syngeneic model when dosed in combination with chemotherapy. Similarly MEDI 4736 is able to inhibit tumour growth in a novel in vivo xenograft model, via a mechanism that is dependent on the presence of tumour specific human T cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-158. doi:10.1158/1538-7445.AM2011-LB-158

Deregulated Wnt signaling has been implicated in a wide range of cancer types, both through truncating mutations in the tumor suppressor protein Adenomatous Polyposis Coli (APC), most prevalent in colorectal cancer, as well as overexpression of Wnt ligands and receptors. Canonical Wnt signaling involves the regulated degradation of the beta catenin protein. In the absence of Wnt signaling, cytosolic beta catenin levels are maintained at low levels, as a result of its phosphorylation-dependent ubiquitination and subsequent proteasomal degradation. This degradation is mediated by the ‘destruction complex’, whose constituents include Glycogen Synthase Kinase 3 (GSK3β), Casein Kinase 1 (CK1) and the scaffolding proteins Axin and APC. The efficient assembly of this complex depends upon the steady state levels of its components, such as Axin, which when overexpressed has been shown to decrease beta catenin levels and inhibit Wnt signaling. Recent reports suggest that the poly-ADP-ribosylating Tankyrase enzymes can bind to and regulate Axin levels, hence providing promising targets for treating Wnt-dependent tumors. We have identified potent, low nM small molecule inhibitors of the Wnt pathway, which inhibit Tankyrases 1 and 2, stabilize Axin, deplete beta catenin protein levels and modulate a set of Wnt-regulated genes in colorectal cell lines in a similar manner as seen when using siRNA to beta catenin. Furthermore, using siRNAs to Tankyrase 1 and 2 we have shown that co-depletion of both enzymes is required for Axin stabilization and Wnt pathway inhibition. We have also successfully demonstrated that our lead molecules cause prolonged Axin stabilization and Wnt pathway inhibition in vivo following oral BID dosing in colon xenografts carrying APC mutations. However, in contrast to previous reports, using molecular tools and our lead compounds (e.g. inducible Axin cell lines, compound rescue experiments using siRNAs to Axin 1/2), we have shown that Axin stabilization has no effect on the viability/proliferation of APC mutant colorectal cell lines, under conditions where siRNA to beta catenin was able to cause significant growth inhibition. Some potential mechanistic rationales for this lack of phenotypic effect will be presented. Understanding the consequences of Tankyrase inhibition in vivo in relevant disease models will be important to establish the clinical utility of these molecules in colorectal cancer and beyond. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5196. doi:10.1158/1538-7445.AM2011-5196

The eyes of the marine snail Bulla gouldiana act as circadian pacemakers. The eyes exhibit a circadian variation in spontaneous optic nerve compound action potential frequency in constant darkness, and are involved in controlling circadian rhythms in behavioral activity expressed by the animal. To initiate an investigation of the molecular aspects of circadian rhythmicity in the Bulla eye and to identify specific molecular markers in the nervous system, we raised monoclonal antibodies (MAb) to the eye and screened them for specific patterns of staining in the eye and brain. Several MAb recognize antigens specific to groups of neurons in the brain, whereas others stain antigens found only in the eye. In addition, some antigens are shared by the eye and the brain. The antigens described here include molecules that mark the lens, retina, neural pathways between the eye and the brain, specific groups of neurons within the central ganglia, and an antigen that is shared by basal retinal neurons (putative ocular circadian pacemaker cells) and glia. These molecular markers may have utility in identifying functionally related groups of neurons, elucidating molecular specializations of the retina, and highlighting pathways used in transmission of information between the retina and the brain.

The Drosophila developmental antigen recognized by the monoclonal antibody F7D6 is expressed in dividing embryonic and imaginal cells but is lost from all differentiating fissues except electrogenic cells of the nervous system and spontaneously contracting muscles. The 63 kDa antigen is associated with the inner surface of plasma membranes and is expressed in several classes of fumorous mutants of Drosophila. The monoclonal antibody was used for immunoprecip-itating the antigen for biochemical characterization and for screening expression vector cDNA libraries. Here we report that this oncodevelopmental antigen is a phosphoprotein and a serine-threonine specific protein kinase. A 1.6 kb cDNA isolated by immunological screening of an ovarian library hybridized to a single band on polytene chromosomes, localizing the gene to 72F on the left arm of the third chromosome. Immunofluorescence assays of deficiency stocks in the region confirmed the location of the gene and identity of the cDNA clone, and mapped the gene between the left breakpoints of Df(3L) st1100.62 and Df(3L) sti7, i.e., between 72F3–7 and 73A1–2. The biochemical and genetic properties indicate that this is a novel growth-related kinase of Drosophila.

2539 Background: CD40 is expressed on B-cells, monocytes, dendritic cells, other normal tissues and tumors. Previous studies showed that CD40 stimulation enhances antigen presentation, breaks tolerance, bypasses T-cell help, and induces apoptosis in CD40pos tumor cells. We report the in vitro activity and primate pharmacokinetics of a human anti-CD40 agonist antibody, CP-870,893, currently in clinical trials for the treatment of cancer. Methods: CP-870,893 was identified as a CD40 agonist antibody by screening lead molecules generated through the Abgenix Xenomouse® platform. Agonist activity was determined using upregulation of B-cell and monocytes derived dendritic cell surface markers, as well as dendritic cell IL-12 induction. BIAcore and equilibrium binding were utilized to determine affinity, and competition studies with CD40L were conducted on BIAcore. CP-870,893 was administered to cynomolgus monkeys i.v. at various doses, serum antibody levels were evaluated over time in an ELISA assay, and B-cell markers were monitored by FACS. Results: CP-870,893 (IgG2, kappa) binds CD40 with sub-nanomolar affinity, and does not block binding of CD40L. When human whole blood is incubated with CP-870,893, upregulation of key surface molecules involved in antigen presentation (MHC Class II, CD80, CD86, CD23 and ICAM-1) is observed with an EC50 of 5–50 ng/ml. Human monocytes derived dendritic cells, when stimulated with CP-870,893, upregulate activation markers (MHC Class II, CD80 and CD83) with an EC50 of 100–300 ng/ml, and secrete high levels of IL-12p40. In the presence of a second stimulus, such as LPS, human dendritic cells also secreted bioactive IL12-p70 when stimulated with CP-870,893 (EC50 ∼ 150 ng/ml). In addition, a CD40 positive human B-cell tumor line, when stimulated with CP-870,893, becomes susceptible to killing by human CTLs. In cynomolgus monkey studies, the clearance of CP-870,893 decreased with increasing dose. Circulating B-cell numbers decreased, and surface molecules were upregulated on B-cells. Conclusions: These data support the potential utility of CP-870,893 as an immune enhancing agent in cancer immunotherapy, by activating antigen presenting cells, and by enhancing the immunogenicity of CD40 positive tumor cells. [Table: see text]

An improved method of high-resolution, two-dimensional gel electrophoresis has been used to study the patterns of protein synthesis during embryonic development of Drosophila melanogaster . Small groups of synchronized embryos were radiolabeled directly by microinjection with [ 35 S]methionine, and the patterns of synthesis of [ 35 S]methionine-labeled proteins from 15 developmental stages between 1.5 and 18.5 hr post-fertilization evaluated. Over 1200 proteins were resolved, and changes in the temporal pattern of synthesis of many of these have been analyzed in the present study. Major qualitative and quantitative differences in the relative rates of synthesis of large numbers of proteins were found in the stages examined. These have been grouped into several different classes. In particular, one group consisted of proteins displaying a dramatic fall in levels of synthesis from preblastoderm through cellular blastoderm. Within this group, some proteins disappeared completely by gastrulation, whereas others underwent a phase of elevated synthesis at a later stage of development. A second group consisted of proteins that were found at all stages examined. All polypeptides in this group underwent a phase of elevated rate of synthesis post cellular blastoderm. A third group consisted of stage-specific proteins that were synthesized during a specific embryonic stage. Temporal changes in the relative rates of synthesis of many proteins were found to be clustered in a few limited periods of rapid change that occurred every 4 to 5 hr. In addition, subsets of proteins increasing during one such period were observed to decrease during a later period, indicating the possibility of some form of coordinate regulation. The time interval between a period of increase and decrease in rates of synthesis of many proteins was observed to occur every 9–10 hr.

The inhibition of protein kinase activity by the isoquinoline sulfonamide, H-8, lengthens the period of the Bulla ocular circadian rhythm in a dose-dependent manner. In addition, the phosphorylation of 5 proteins is markedly affected by H-8. The observed correlation between H-8's period lengthening effects, and its effects on the phosphorylation of specific proteins, suggests that: (1) these proteins are candidate components regulating the period of the circadian rhythm; and (2) the dailychanges in membrane potential underlying the circadian rhythm are mediated by similar mechanisms that serve to change neural function in other systems; modulation of protein kinases.

The problem of code origin is presented in the context of increasingly complex events in the origin of life. The likely sequence of events appears to progress from abiotic synthesis of biological monomers to polymers to formation of protocells, which would be capable of competition and further evolution. We propose that rate of polymer formation was a critical controlling parameter of rate of protocell propagation. This would lead to selection of autocatalytic and mutually catalytic reactions of polymer formation. Primitive proteins would catalyze polynucleotide formation, and polynucleotides could be used as anvils of noncoded polypeptide synthesis. Proteins that could catalyze this latter reaction (assignment catalysts) would play an important role in subsequent evolution of a genetic code. Competing populations of assignment catalysts would possess very nonlinear dynamics of production of the catalysts themselves. An analysis of this dynamics shows that it has a rich family of bifurications which would provide a pathway for gradual approach to a genetic code. The selection criterion in this process would be efficiency of utilization of monomers and energy for the production of assignment catalysts.

We have identified early embryo proteins related to the segmentation gene Krueppel by (35S)methionine pulse labelling and two-dimensional gel electrophoresis. Protein synthesis differences shared by homozygous embryos of two Krueppel alleles when compared to heterozygous and wild-type embryos are reported. The study was extended to syncytial blastoderm stages by pulse labelling and gel analysis of single embryos, using Krueppel-specific proteins from gastrula stages as molecular markers for identifying homozygous Krueppel embryos. Localized expression of interesting proteins was examined in embryo fragments. The earliest differences detected at nuclear migration stages showed unregulated synthesis in mutant embryos of two proteins that have stage specific synthesis in normal embryos. At the cellular blastoderm stage one protein was not synthesized and two proteins showed apparent shifts in isoelectric point in mutant embryos. Differences observed in older embryos included additional proteins with shifted isoelectric points and a number of qualitative and quantitative changes in protein synthesis. Five of the proteins with altered rates of synthesis in mutant embryos showed localized synthesis in normal embryos. The early effects observed are consistent with the hypothesis that the Krueppel product can be a negative or positive regulator of expression of other loci, while blastoderm and gastrula stage shiftsmore » in isoelectric point indicate that a secondary effect of Krueppel function may involve post-translational modification of proteins.« less

The 63-kDa antigen recognized by the monoclonal antibody F7D6 is present in all Drosophila embryonic cells and disappears from most tissues as each one reaches its final, differentiated state. Larval tissues lose the antigen around the time of hatching, imaginal tissues lose it during metamorphosis, and germ cells lose it during gametogenesis (Bedian et al: Devel Biol 115:105-118, 1986). The nervous system and spontaneously contracting musculature of the gut and gonads are exceptions and remain antigen positive at all stages. The F7D6 antigen appears to be associated with dividing, undifferentiated cells and electrogenic cells. This prompted us to test tumors for antigen presence. We tested four different recessive mutants that give rise to four different types of tumorous transformation: the embryonic tumor Notch, several larval melanotic tumors, the imaginal disc tumor 1(2)gl, and three alleles of the ovarian tumor otu. In all cases, tumorous tissues in homozygotes contained the F7D6 antigen. The electrophoretic mobility of the antigen appeared to be unaltered in tumorous tissues compared to normal cells, but the antigen is expressed at higher levels. The antigen is found on the cytoplasmic surface of plasma membranes and appears to be a marker of undifferentiated normal and tumorous cells. Similarities and differences between the F7D6 antigen and Drosophila c-src protein are discussed.

A model is presented for the emergence of a primitive genetic code through the selection of a family of proteins capable of executing the code and catalyzing their own formation from polynucleotide templates. These proteins are assignment catalysts capable of modulating the rate of incorporation of different amino acids at the position of different codons. The starting point of the model is a polynucleotide based polypeptide construction process which maintains colinearity between template and product, but may not maintain a coded relationship between amino acids and codons. Among the primitive proteins made are assumed to be assignment catalysts characterized by structural and functional parameters which are used to formulate the production kinetics of these catalysts from available templates. Application of the model to the simple case of two letter codon and amino acid alphabets has been analyzed in detail. As the structural, functional, and kinetic parameters are varied, the dynamics undergoes many bifurcations, allowing an initially ambiguous system of catalysts to evolve to a coded, self-reproductive system. The proposed selective pressure of this evolution is the efficiency of utilization of monomers and energy. The model also simulates the qualitative features of suppression, in which a deleterious mutation is partly corrected by the introduction of translational error.