Conversion of ATP to adenosine by CD39 and CD73 in multiple myeloma can be successfully targeted together with adenosine receptor A2A blockade.

BACKGROUND: PD1/PDL1-directed therapies have been unsuccessful for multiple myeloma (MM), an incurable cancer of plasma cells in the bone marrow (BM). Therefore, other immune checkpoints such as extracellular adenosine and its immunosuppressive receptor should be considered. CD39 and CD73 convert extracellular ATP to adenosine, which inhibits T-cell effector functions via the adenosine receptor A2A (A2AR). We set out to investigate whether blocking the adenosine pathway could be a therapy for MM. METHODS: Expression of CD39 and CD73 on BM cells from patients and T-cell proliferation were determined by flow cytometry and adenosine production by Liquid chromatograpy-mass spectrometry (HPCL/MS). ENTPD1 (CD39) mRNA expression was determined on myeloma cells from patients enrolled in the publicly available CoMMpass study. Transplantable 5T33MM myeloma cells were used to determine the effect of inhibiting CD39, CD73 and A2AR in mice in vivo. RESULTS: Elevated level of adenosine was found in BM plasma of MM patients. Myeloma cells from patients expressed CD39, and high gene expression indicated reduced survival. CD73 was found on leukocytes and stromal cells in the BM. A CD39 inhibitor, POM-1, and an anti-CD73 antibody inhibited adenosine production and reduced T-cell suppression in vitro in coculture of myeloma and stromal cells. Blocking the adenosine pathway in vivo with a combination of Sodium polyoxotungstate (POM-1), anti-CD73, and the A2AR antagonist AZD4635 activated immune cells, increased interferon gamma production, and reduced the tumor load in a murine model of MM. CONCLUSIONS: Our data suggest that the adenosine pathway can be successfully targeted in MM and blocking this pathway could be an alternative to PD1/PDL1 inhibition for MM and other hematological cancers. Inhibitors of the adenosine pathway are available. Some are in clinical trials and they could thus reach MM patients fairly rapidly.

Selective activation of anti-CD73 mechanisms in control of primary tumors and metastases.

The emerging role for CD73 in driving cancer growth and metastasis has presented opportunities to develop anti-CD73 monoclonal antibodies (mAbs) in the treatment of human cancers. Blockade of CD73 by antagonistic CD73 mAbs ameliorates tumor growth and metastasis via the inhibition of enzymatic and non-enzymatic CD73 pathways. In this study, we investigated whether Fc-receptor cross-linking represented a non-redundant mechanism by which anti-CD73 mAbs exert potent suppression of solid tumors and metastases. We engineered four anti-CD73 mAbs, each different in their ability to modulate CD73 enzymatic function and bind Fc receptors. mAbs recognizing a similar epitope of CD73 (CD73-04, TY/23 and 2C5) displayed the greatest antitumor activity. Importantly, we observed that the optimal control of metastasis by anti-CD73 mAbs involved primarily Fc receptor engagement, while suppression of solid tumors required both, enzyme inhibition and activation of Fc receptors. Engagement of Fc-receptors was also essential for optimal anti-metastatic effect in combination with either A2AR inhibitor or anti-PD-1 mAb treatment. The control of experimental metastases relied on the activation of host NK cells and IFNγ, while NK cells, CD8+ T cells and IFNγ were needed for effective antitumor effect in the spontaneous metastases model. These observations advance our understanding of the enzymatic and non-enzymatic functions of anti-CD73 mAbs in solid tumors and metastases. Altogether, these findings will greatly assist in the design of anti-CD73 mAbs to be used as either single agents or in combination with other immunotherapeutic molecules or targeted therapies.

Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain.

We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies.

Targeting CD73 in the tumor microenvironment with MEDI9447.

MEDI9447 is a human monoclonal antibody that is specific for the ectoenzyme CD73 and currently undergoing Phase I clinical trials. Here we show that MEDI9447 is a potent inhibitor of CD73 ectonucleotidase activity, with wide ranging immune regulatory consequences. MEDI9447 results in relief from adenosine monophosphate (AMP)-mediated lymphocyte suppression in vitro and inhibition of mouse syngeneic tumor growth in vivo. In contrast with other cancer immunotherapy agents such as checkpoint inhibitors or T-cell agonists, MEDI9447 drives changes in both myeloid and lymphoid infiltrating leukocyte populations within the tumor microenvironment of mouse models. Changes include significant alterations in a number of tumor micro-environmental subpopulations including increases in CD8(+) effector cells and activated macrophages. Furthermore, these changes correlate directly with responder and non-responder subpopulations within animal studies using syngeneic tumors. Combination data showing additive activity between MEDI9447 and anti-PD-1 antibodies using human cells in vitro and mouse tumor models further demonstrate the potential value of relieving adenosine-mediated immunosuppression. Based on these data, a Phase I study to test the safety, tolerability, and clinical activity of MEDI9447 in cancer patients was initiated (NCT02503774).

Co-inhibition of CD73 and A2AR Adenosine Signaling Improves Anti-tumor Immune Responses.

Preclinical studies targeting the adenosinergic pathway have gained much attention for their clinical potential in overcoming tumor-induced immunosuppression. Here, we have identified that co-blockade of the ectonucleotidase that generates adenosine CD73 and the A2A adenosine receptor (A2AR) that mediates adenosine signaling in leuokocytes, by using compound gene-targeted mice or therapeutics that target these molecules, limits tumor initiation, growth, and metastasis. This tumor control requires effector lymphocytes and interferon-γ, while antibodies targeting CD73 promote an optimal therapeutic response in vivo when engaging activating Fc receptors. In a two-way mixed leukocyte reaction using a fully human anti-CD73, we demonstrated that Fc receptor binding augmented the production of proinflammatory cytokines.

Identification of anti-tumour biologics using primary tumour models, 3-D phenotypic screening and image-based multi-parametric profiling.

BACKGROUND: Monolayer cultures of immortalised cell lines are a popular screening tool for novel anti-cancer therapeutics, but these methods can be a poor surrogate for disease states, and there is a need for drug screening platforms which are more predictive of clinical outcome. In this study, we describe a phenotypic antibody screen using three-dimensional cultures of primary cells, and image-based multi-parametric profiling in PC-3 cells, to identify anti-cancer biologics against new therapeutic targets. METHODS: ScFv Antibodies and designed ankyrin repeat proteins (DARPins) were isolated using phage display selections against primary non-small cell lung carcinoma cells. The selected molecules were screened for anti-proliferative and pro-apoptotic activity against primary cells grown in three-dimensional culture, and in an ultra-high content screen on a 3-D cultured cell line using multi-parametric profiling to detect treatment-induced phenotypic changes. The targets of molecules of interest were identified using a cell-surface membrane protein array. An anti-CUB domain containing protein 1 (CDCP1) antibody was tested for tumour growth inhibition in a patient-derived xenograft model, generated from a stage-IV non-small cell lung carcinoma, with and without cisplatin. RESULTS: Two primary non-small cell lung carcinoma cell models were established for antibody isolation and primary screening in anti-proliferative and apoptosis assays. These assays identified multiple antibodies demonstrating activity in specific culture formats. A subset of the DARPins was profiled in an ultra-high content multi-parametric screen, where 300 morphological features were measured per sample. Machine learning was used to select features to classify treatment responses, then antibodies were characterised based on the phenotypes that they induced. This method co-classified several DARPins that targeted CDCP1 into two sets with different phenotypes. Finally, an anti-CDCP1 antibody significantly enhanced the efficacy of cisplatin in a patient-derived NSCLC xenograft model. CONCLUSIONS: Phenotypic profiling using complex 3-D cell cultures steers hit selection towards more relevant in vivo phenotypes, and may shed light on subtle mechanistic variations in drug candidates, enabling data-driven decisions for oncology target validation. CDCP1 was identified as a potential target for cisplatin combination therapy.

Development of a Trispecific Antibody Designed to Simultaneously and Efficiently Target Three Different Antigens on Tumor Cells.

Targeting Eph (erythropoietin producing hepatoma) receptors with monoclonal antibodies is being explored as therapy for several types of cancer. To test whether simultaneous targeting of EphA2, EphA4, and EphB4 would be an effective approach to cancer therapy, we generated a recombinant trispecific antibody using the variable domain genes of anti-EphA2, anti-EphA4, and anti-EphB4 monoclonal antibodies. A multidisciplinary approach combining biochemical, biophysical, and cellular-based assays was used to characterize the trispecific antibody in vitro and in vivo. Here we demonstrate that the trispecific antibody is expressed at high levels by mammalian cells, monodispersed in solution, thermostable, capable of simultaneously binding the three receptors, and able to activate the three targets effectively as evidenced by receptor internalization and degradation both in vitro and in vivo. Furthermore, pharmacokinetic analysis using tumor-bearing nude mice showed that the trispecific antibody remains in the circulation similarly to its respective parental antibodies. These results indicate that simultaneous blockade of EphA2, EphA4, and EphB4 could be an attractive approach to cancer therapy.

Combining phenotypic and proteomic approaches to identify membrane targets in a 'triple negative' breast cancer cell type.

BACKGROUND: The continued discovery of therapeutic antibodies, which address unmet medical needs, requires the continued discovery of tractable antibody targets. Multiple protein-level target discovery approaches are available and these can be used in combination to extensively survey relevant cell membranomes. In this study, the MDA-MB-231 cell line was selected for membranome survey as it is a 'triple negative' breast cancer cell line, which represents a cancer subtype that is aggressive and has few treatment options. METHODS: The MDA-MB-231 breast carcinoma cell line was used to explore three membranome target discovery approaches, which were used in parallel to cross-validate the significance of identified antigens. A proteomic approach, which used membrane protein enrichment followed by protein identification by mass spectrometry, was used alongside two phenotypic antibody screening approaches. The first phenotypic screening approach was based on hybridoma technology and the second was based on phage display technology. Antibodies isolated by the phenotypic approaches were tested for cell specificity as well as internalisation and the targets identified were compared to each other as well as those identified by the proteomic approach. An anti-CD73 antibody derived from the phage display-based phenotypic approach was tested for binding to other 'triple negative' breast cancer cell lines and tested for tumour growth inhibitory activity in a MDA-MB-231 xenograft model. RESULTS: All of the approaches identified multiple cell surface markers, including integrins, CD44, EGFR, CD71, galectin-3, CD73 and BCAM, some of which had been previously confirmed as being tractable to antibody therapy. In total, 40 cell surface markers were identified for further study. In addition to cell surface marker identification, the phenotypic antibody screening approaches provided reagent antibodies for target validation studies. This is illustrated using the anti-CD73 antibody, which bound other 'triple negative' breast cancer cell lines and produced significant tumour growth inhibitory activity in a MDA-MB-231 xenograft model. CONCLUSIONS: This study has demonstrated that multiple methods are required to successfully analyse the membranome of a desired cell type. It has also successfully demonstrated that phenotypic antibody screening provides a mechanism for rapidly discovering and evaluating antibody tractable targets, which can significantly accelerate the therapeutic discovery process.

Development of a novel ectonucleotidase assay suitable for high-throughput screening.

5'-Ectonucleotidase (NT5E) catalyzes the conversion of adenosine monophosphate to adenosine and free phosphate. The role of this ectonucleotidase and its production of adenosine are linked with immune function, angiogenesis, and cancer. NT5E activity is typically assayed either by chromatographic quantification of substrates and products using high-performance liquid chromatography (HPLC) or by quantification of free phosphate using malachite green. These methods are not suitable for robust screening assays of NT5E activity. HPLC is not readily suitable for the rapid and efficient assay of multiple samples and malachite green is highly sensitive to the phosphate-containing buffers common in various media and sample buffers. Here the development and validation of a novel high-throughput ectonucleotidase screening assay are described, which makes use of a luciferase-based assay reagent, the Promega CellTiter-Glo kit, to measure the catabolism of AMP by NT5E. This multiwell plate-based assay facilitates the screening of potential ectonucleotidase antagonists and is unaffected by the presence of contaminating phosphate molecules present in screening samples.

Complete genome sequence analysis of Seneca Valley virus-001, a novel oncolytic picornavirus.

The complete genome sequence of Seneca Valley virus-001 (SVV-001), a small RNA virus, was determined and was shown to have typical picornavirus features. The 7280 nt long genome was predicted to contain a 5' untranslated region (UTR) of 666 nt, followed by a single long open reading frame consisting of 6543 nt, which encodes a 2181 aa polyprotein. This polyprotein could potentially be cleaved into 12 polypeptides in the standard picornavirus L-4-3-4 layout. A 3' UTR of 71 nt was followed by a poly(A) tail of unknown length. Comparisons with other picornaviruses showed that the P1, 2C, 3C and 3D polypeptides of SVV-001 were related most closely to those of the cardioviruses, although they were not related as closely to those of encephalomyocarditis virus and Theiler's murine encephalomyelitis virus as the latter were to each other. Most other regions of the polyprotein differed considerably from those of all other known picornaviruses. SVV-001 contains elements of an internal ribosome entry site reminiscent of that found in hepatitis C virus and a number of genetically diverse picornaviruses. SVV-001 is a novel picornavirus and it is proposed that it be classified as the prototype species in a novel genus named 'Senecavirus'.

Seneca Valley virus, a systemically deliverable oncolytic picornavirus, and the treatment of neuroendocrine cancers.

BACKGROUND: Numerous clinical trials have demonstrated that oncolytic viruses can elicit antitumor responses when they are administered directly into localized cancers. However, the treatment of metastatic disease with oncolytic viruses has been challenging due to the inactivation of viruses by components of human blood and/or to inadequate tumor selectivity. METHODS: We determined the cytolytic potential and selectivity of Seneca Valley Virus-001 (SVV-001), a newly discovered native picornavirus, in neuroendocrine and pediatric tumor cell lines and normal cells. Suitability of the virus for intravenous delivery in humans was assessed by blood inactivation assays. Safety was evaluated in vivo using an immune-competent mouse model, and efficacy was evaluated in vivo in athymic mice bearing tumors derived from human small-cell lung cancer and retinoblastoma cell lines. RESULTS: Cell lines derived from small-cell lung cancers and solid pediatric cancers were at least 10,000-fold more sensitive to the cytolytic activity of SVV-001 than were any of the adult normal human cells tested. Viral infectivity was not inhibited by human blood components. Intravenous doses up to 1 x 10(14) virus particles (vp) per kg were well tolerated, and no dose-limiting toxicity was observed in immune-competent mice. A single intravenous dose of 1 x 10(8) vp per kg into athymic mice bearing preestablished small-cell lung or retinoblastoma tumors resulted in complete, durable responses in ten of ten and five of eight mice, respectively. CONCLUSIONS: SVV-001 has potent cytolytic activity and high selectivity for tumor cell lines having neuroendocrine properties versus adult normal cells. Systemically administered SVV-001 has potential for the treatment of metastatic neuroendocrine cancers.

Systemic gene-directed enzyme prodrug therapy of hepatocellular carcinoma using a targeted adenovirus armed with carboxypeptidase G2.

Hepatocellular carcinoma is the fifth most common cancer worldwide, and there is no effective therapy for unresectable disease. We have developed a targeted systemic therapy for hepatocellular carcinoma. The gene for a foreign enzyme is selectively expressed in the tumor cells and a nontoxic prodrug is then given, which is activated to a potent cytotoxic drug by the tumor-localized enzyme. This approach is termed gene-directed enzyme prodrug therapy (GDEPT). Adenoviruses have been used to target cancer cells, have an intrinsic tropism for liver, and are efficient gene vectors. Oncolytic adenoviruses produce clinical benefits, particularly in combination with conventional anticancer agents and are well tolerated. We rationalized that such adenoviruses, if their expression were restricted to telomerase-positive cancer cells, would make excellent gene vectors for GDEPT therapy of hepatocellular carcinoma. Here we use an oncolytic adenovirus to deliver the prodrug-activating enzyme carboxypeptidase G2 (CPG2) to tumors in a single systemic administration. The adenovirus replicated and produced high levels of CPG2 in two different hepatocellular carcinoma xenografts (Hep3B and HepG2) but not other tissues. GDEPT enhanced the adenovirus-alone therapy to elicit tumor regressions in the hepatocellular carcinoma models. This is the first time that CPG2 has been targeted and expressed intracellularly to effect significant therapy, showing that the combined approach holds enormous potential as a tumor-selective therapy for the systemic treatment of hepatocellular carcinoma.

Antitumor efficacy and tumor-selective replication with a single intravenous injection of OAS403, an oncolytic adenovirus dependent on two prevalent alterations in human cancer.

A potentially promising treatment of metastatic cancer is the systemic delivery of oncolytic adenoviruses. This requires engineering viruses which selectively replicate in tumors. We have constructed such an oncolytic adenovirus, OAS403, in which two early region genes are under the control of tumor-selective promoters that play a role in two key pathways involved in tumorigenesis. The early region E1A is controlled by the promoter for the E2F-1 gene, a transcription factor that primarily upregulates genes for cell growth. The E4 region is under control of the promoter for human telomerase reverse transcriptase, a gene upregulated in most cancer cells. OAS403 was evaluated in vitro on a panel of human cells and found to elicit tumor-selective cell killing. Also, OAS403 was less toxic in human hepatocyte cultures, as well as in vivo when compared to an oncolytic virus that lacked selective E4 control. A single intravenous injection of 3 x 10(12) vp/kg in a Hep3B xenograft mouse tumor model led to significant antitumor efficacy. Additionally, systemic administration in a pre-established LNCaP prostate tumor model resulted in over 80% complete tumor regressions at a tolerable dose. Vector genome copy number was measured in tumors and livers at various times following tail vein injection and showed a selective time-dependent increase in tumors but not livers over 29 days. Furthermore, efficacy was significantly improved when OAS403 treatment was combined with doxorubicin. This virus holds promise for the treatment of a broad range of human cancers including metastatic disease.

An oncolytic adenovirus selective for retinoblastoma tumor suppressor protein pathway-defective tumors: dependence on E1A, the E2F-1 promoter, and viral replication for selectivity and efficacy.

The use of oncolytic adenoviruses as a cancer therapeutic is dependent on the lytic properties of the viral life cycle, and the molecular differences between tumor cells and nontumor cells. One strategy for achieving safe and efficacious adenoviral therapies is to control expression of viral early gene(s) required for replication with tumor-selective promoter(s), particularly those active in a broad range of cancer cells. The retinoblastoma tumor suppressor protein (Rb) pathway is dysregulated in a majority of human cancers. The human E2F-1 promoter has been shown to be selectively activated/derepressed in tumor cells with a defect in the Rb pathway. Ar6pAE2fE3F and Ar6pAE2fF are oncolytic adenoviral vectors (with and without the viral E3 region, respectively) that use the tumor-selective E2F-1 promoter to limit expression of the viral E1A transcription unit, and, thus, replication, to tumor cells. We demonstrate that the antitumor activity of Ar6pAE2fF in vitro and in vivo is dependent on the E2F-1 promoter driving E1A expression in Rb pathway-defective cells, and furthermore, that its oncolytic activity is enhanced by viral replication. Selective oncolysis by Ar6pAE2fF was dependent on the presence of functional E2F binding sites in the E2F-1 promoter, thus linking antitumor viral activity to the Rb pathway. Potent antitumor efficacy was demonstrated with Ar6pAE2fF and Ar6pAE2fE3F in a xenograft model following intratumoral administration. Ar6pAE2fF and Ar6pAE2fE3F were compared with Addl1520, which is reported to be molecularly identical to an E1B-55K deleted vector currently in clinical trials. These vectors were compared in in vitro cytotoxicity and virus production assays, after systemic delivery in an in vivo E1A-related hepatotoxicity model, and in a mouse xenograft tumor model after intratumoral administration. Our results support the use of oncolytic adenoviruses using tumor-selective promoter(s) that are activated or derepressed in tumor cells by virtue of a particular defective pathway, such as the Rb pathway.