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Removing Alzheimer plaques

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Transdermal implant releases antibodies to trigger immune system to clear Alzheimer’s plaques            March 21, 2016

Test with mice over 39 weeks showed dramatic reduction of amyloid beta plaque load in the brain and reduced phosphorylation of the protein tau, two signs of Alzheimer’s
http://www.kurzweilai.net/transdermal-implant-releases-antibodies-to-trigger-immune-system-to-clear-alzheimers-plaques

 

An implant that can prevent Alzheimer’s disease. A new capsule can be implanted under the skin to release antibodies that “tag” amyloid beta, signalling the patient’s immune system to clear it before it forms Alzheimer’s plaques. (credit: École polytechnique fédérale de Lausanne

EPFL scientists have developed an implantable capsule containing genetically engineered cells that can recruit a patient’s immune system to combat Alzheimer’s disease.

Placed under the skin, the capsule releases antibody proteins that make their way to the brain and “tag” amyloid beta proteins, signalling the patient’s own immune system to attack and clear the amyloid beta proteins, which are toxic to neurons.

To be most effective, this treatment has to be given as early as possible, before the first signs of cognitive decline. Currently, this requires repeated vaccine injections, which can cause side effects. The new implant can deliver a steady, safe flow of antibodies.

Protection from immune-system rejection

Cell encapsulation device for long-term subcutaneous therapeutic antibody delivery. (B) Macroscopic view of the encapsulation device, composed of a transparent frame supporting polymer permeable membranes and reinforced with an outer polyester mesh. (C) Dense neovascularization develops around a device containing antibody-secreting C2C12 myoblasts, 8 months after implantation in the mouse subcutaneous tissue. (D and E) Representative photomicrographs showing encapsulated antibody-secreting C2C12 myoblasts surviving at high density within the flat sheet device 39 weeks after implantation. (E) Higher magnification: note that the cells produce a collagen-rich matrix stained in blue with Masson’s trichrome protocol. Asterisk: polypropylene porous membrane. Scale bars = 750 mm (B and C),100 mm (D), 50 mm (E). (credit: Aurelien Lathuiliere et al./BRAIN)

The lab of Patrick Aebischer at EPFL designed the “macroencapsulation device” (capsule) with two permeable membranes sealed together with a polypropylene frame, containing a hydrogel that facilitates cell growth. All the materials used are biocompatible and the device is reproducible for large-scale manufacturing.

The cells of choice are taken from muscle tissue, and the permeable membranes let them interact with the surrounding tissue to get all the nutrients and molecules they need. The cells have to be compatible with the patient to avoid triggering the immune system against them, like a transplant can. To do that, the capsule’s membranes shield the cells from being identified and attacked by the immune system. This protection also means that cells from a single donor can be used on multiple patients.

The researchers tested the device mice in a genetic line commonly used to simulate Alzheimer’s disease over a course of 39 weeks, showing dramatic reduction of amyloid beta plaque load in the brain. The treatment also reduced the phosphorylation of the protein tau, another sign of Alzheimer’s observed in these mice.

“The proof-of-concept work demonstrates clearly that encapsulated cell implants can be used successfully and safely to deliver antibodies to treat Alzheimer’s disease and other neurodegenerative disorders that feature defective proteins,” according to the researchers.

The work is published in the journal BRAIN. It involved a collaboration between EPFL’s Neurodegenerative Studies Laboratory (Brain Mind Institute), the Swiss Light Source (Paul Scherrer Institute), and F. Hoffmann-La Roche. It was funded by the Swiss Commission for Technology and Innovation and F. Hoffmann-La Roche Ltd.


Abstract of A subcutaneous cellular implant for passive immunization against amyloid-β reduces brain amyloid and tau pathologies

Passive immunization against misfolded toxic proteins is a promising approach to treat neurodegenerative disorders. For effective immunotherapy against Alzheimer’s disease, recent clinical data indicate that monoclonal antibodies directed against the amyloid-β peptide should be administered before the onset of symptoms associated with irreversible brain damage. It is therefore critical to develop technologies for continuous antibody delivery applicable to disease prevention. Here, we addressed this question using a bioactive cellular implant to deliver recombinant anti-amyloid-β antibodies in the subcutaneous tissue. An encapsulating device permeable to macromolecules supports the long-term survival of myogenic cells over more than 10 months in immunocompetent allogeneic recipients. The encapsulated cells are genetically engineered to secrete high levels of anti-amyloid-β antibodies. Peripheral implantation leads to continuous antibody delivery to reach plasma levels that exceed 50 µg/ml. In a proof-of-concept study, we show that the recombinant antibodies produced by this system penetrate the brain and bind amyloid plaques in two mouse models of the Alzheimer’s pathology. When encapsulated cells are implanted before the onset of amyloid plaque deposition in TauPS2APP mice, chronic exposure to anti-amyloid-β antibodies dramatically reduces amyloid-β40 and amyloid-β42 levels in the brain, decreases amyloid plaque burden, and most notably, prevents phospho-tau pathology in the hippocampus. These results support the use of encapsulated cell implants for passive immunotherapy against the misfolded proteins, which accumulate in Alzheimer’s disease and other neurodegenerative disorders.

 

A subcutaneous cellular implant for passive immunization against amyloid-β reduces brain amyloid and tau pathologies

 

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Passive immunization using monoclonal antibodies has recently emerged for the treatment of neurological diseases. In particular, monoclonal antibodies can be administered to target the misfolded proteins that progressively aggregate and propagate in the CNS and contribute to the histopathological signature of neurodegenerative diseases. Alzheimer’s disease is the most prevalent proteinopathy, characterized by the deposition of amyloid plaques and neurofibrillary tangles. According to the ‘amyloid cascade hypothesis’, which is supported by strong genetic evidence (Goate and Hardy, 2012), the primary pathogenic event in Alzheimer’s disease is the accumulation and aggregation of amyloid-β into insoluble extracellular plaques in addition to cerebral amyloid angiopathy (Hardy and Selkoe, 2002). High levels of amyloid-β may cause a cascade of deleterious events, including neurofibrillary tangle formation, neuronal dysfunction and death. Anti-amyloid-β antibodies have been developed to interfere with the amyloid-β cascade. Promising data obtained in preclinical studies have validated immunotherapy against Alzheimer’s disease, prompting a series of clinical trials (Bard et al., 2000;Bacskai et al., 2002; Oddo et al., 2004; Wilcock et al., 2004a; Bohrmann et al., 2012). Phase III trials using monoclonal antibodies directed against soluble amyloid-β (bapineuzumab and solanezumab) in patients with mild-to-moderate Alzheimer’s disease showed some effects on biomarkers that are indicative of target engagement. These trials, however, missed the primary endpoints, and it is therefore believed that anti-amyloid-β immunotherapy should be administered at the early presymptomatic stage (secondary prevention) to better potentiate therapeutic effects (Doody et al., 2014; Salloway et al., 2014). For the treatment of Alzheimer’s disease, it is likely that long-term treatment using a high dose of monoclonal antibody will be required. However, bolus administration of anti-amyloid-β antibodies may aggravate dose-dependent adverse effects such as amyloid-related imaging abnormalities (ARIA) (Sperling et al., 2012). In addition, the cost of recombinant antibody production and medical burden associated with repeated subcutaneous or intravenous bolus injections may represent significant constraints, especially in the case of preventive immunotherapy initiated years before the onset of clinical symptoms in patients predisposed to develop Alzheimer’s disease.

Therefore, alternative methods need to be developed for the continuous, long-term administration of antibodies. Here, we used an implant based on a high-capacity encapsulated cell technology (ECT) (Lathuiliere et al., 2014b). The ECT device contains myogenic cells genetically engineered for antibody production. Macromolecules can be exchanged between the implanted cells and the host tissue through a permeable polymer membrane. As the membrane shields the implanted cells from immune rejection in allogeneic conditions, it is possible to use a single donor cell source for multiple recipients. We demonstrate that anti-amyloid immunotherapy using an ECT device implanted in the subcutaneous tissue can achieve therapeutic effects inside the brain. Chronic exposure to anti-amyloid-β monoclonal antibodies produced in vivo using the ECT technology leads to a significant reduction of the amyloid brain pathology in two mouse models of Alzheimer’s disease.

 

Microglial phagocytosis study

The measurement of antibody-mediated amyloid-β phagocytosis was performed as proposed previously (Webster et al., 2001). In this study, we used either purified preparations of full mAb-11 IgG2a antibody, or a purified Fab antibody fragment. A suspension of 530 µM fluorescent fibrillar amyloid-β42 was prepared in 10 mM HEPES (pH 7.4) by stirring overnight at room temperature. The resulting suspension contained 30 µM fluorescein-conjugated amyloid-β42 and 500 µM unconjugated amyloid-β42 (Bachem). IgG-fibrillar amyloid-β42 immune complexes were obtained by preincubating fluorescent fibrillar amyloid-β42 at a concentration of 50 µM in phosphate-buffered saline (PBS) with various concentrations of purified mAb-11 IgG2a or Fab antibody fragment for 30 min at 37 °C. The immune complexes were washed twice by centrifugation for 5 min at 14 000g and resuspended in the initial volume to obtain a fluorescent fibrillar amyloid-β42 solution (total amyloid-β42 concentration: 530 µM). The day before the experiment, 8 × 104 C8-B4 cells were plated in 24-well plates. The medium was replaced with serum-free DMEM before the addition of the peptides. The cells were incubated for 30 min with fibrillar amyloid-β42 or IgG-fibrillar amyloid-β42 added to the culture medium. Next, the cells were washed twice with Hank’s Balanced Salt Solution (HBSS) and subsequently detached by trypsinization, which also eliminates surface-bound fibrillar amyloid-β42. The cells were fixed for 10 min in 4% paraformaldehyde and finally resuspended in PBS. The cell fluorescence was determined with a flow cytometer (Accuri C6; BD Biosciences), and the data were analysed using the FlowJo software (TreeStar Inc.). To determine the effect of the anti-amyloid-β antibodies on amyloid-β phagocytosis, the concentration of fluorescent fibrillar amyloid-β42 was set at 1.5 µM, which is in the linear region of the dose-response curve depicting fibrillar amyloid-β42 phagocytosis in C8-B4 cells (Fig. 2C). All experiments were performed in duplicate.

 

The implantation of genetically engineered cells within a retrievable subcutaneous device leads to the continuous production of monoclonal antibodies in vivo. This technology achieves steady therapeutic monoclonal antibody levels in the plasma, offering an effective alternative to bolus injections for passive immunization against chronic diseases. Peripheral delivery of anti-amyloid-β monoclonal antibody by ECT leads to a significant reduction of amyloid burden in two mouse models of Alzheimer’s disease. The effect of the ECT treatment is more pronounced when passive immunization is preventively administered in TauPS2APP mice, most notably decreasing the phospho-tau pathology.

With the recent development of biomarkers to monitor Alzheimer’s pathology, it is recognized that a steady increase in cerebral amyloid over the course of decades precedes the appearance of the first cognitive symptoms (reviewed in Sperling et al., 2011). The current consensus therefore suggests applying anti-amyloid-β immunotherapy during this long asymptomatic phase to avoid the downstream consequences of amyloid deposition and to leverage neuroprotective effects. Several preventive clinical trials have been recently initiated for Alzheimer’s disease. The Alzheimer’s Prevention Initiative (API) and the Dominantly Inherited Alzheimer Network (DIAN) will test antibody candidates in presymptomatic dominant mutation carriers, while the Anti-Amyloid treatment in the Asymptomatic Alzheimer’s disease (A4) trial enrols asymptomatic subjects after risk stratification. If individuals with a high risk of developing Alzheimer’s disease can be identified using current biomarker candidates, these patients are the most likely to benefit from chronic long-term anti-amyloid-β immunotherapy. However, such a treatment may pose a challenge to healthcare systems, as the production capacity of the antibody and its related cost would become a challenging issue (Skoldunger et al., 2012). Therefore, the development of alternative technologies to chronically administer anti-amyloid-β antibody is an important aspect for therapeutic interventions at preclinical disease stages.

Here, we show that the ECT technology for the peripheral delivery of anti-amyloid-β monoclonal antibodies can significantly reduce cerebral amyloid pathology in two mouse models of Alzheimer’s disease. The subcutaneous tissue is a site of implantation easily accessible and therefore well adapted to preventive treatment. It is, however, challenging to reach therapeutic efficacy, as only a small fraction of the produced anti-amyloid-β monoclonal antibodies are expected to cross the blood–brain barrier, although they can next persist in the brain for several months (Wang et al., 2011; Bohrmann et al., 2012). Our results are consistent with previous reports, which have shown that the systemic administration of anti-amyloid-β antibodies can decrease brain amyloid burden in preclinical Alzheimer’s disease models (Bard et al., 2000, 2003; DeMattos et al., 2001; Wilcock et al., 2004a, b; Buttini et al., 2005; Adolfsson et al., 2012).

Remarkably, striking differences exist among therapeutic anti-amyloid-β antibodies in their ability to clear already existing plaques. Soluble amyloid-β species can saturate the small fraction of pan-amyloid-β antibodies entering the CNS and inhibit further target engagement (Demattos et al., 2012). Therefore, antibodies recognizing soluble amyloid-β may fail to bind and clear insoluble amyloid deposits (Das et al., 2001; Racke et al., 2005; Levites et al., 2006; Bohrmann et al., 2012). Furthermore, antibody-amyloid-β complexes are drained towards blood vessels, promoting cerebral amyloid angiopathy (CAA) and subsequent microhaemorrhages. The mAb-11 antibody used in the present study is similar to gantenerumab, which is highly specific for amyloid plaques and reduces amyloid burden in patients with Alzheimer’s disease (Bohrmann et al., 2012; Demattos et al., 2012; Ostrowitzki et al., 2012). We find that the murine IgG2a mAb-11 antibody efficiently enhances the phagocytosis of amyloid-β fibrils by microglial cells. In addition, ECT administration of the mAb-11 F(ab’)2 fragment lacking the Fc region fails to recruit microglial cells, and leads only to a trend towards clearance of the amyloid plaques. Therefore, our results suggest a pivotal role for microglial cells in the clearance of amyloid plaques following mAb-11 delivery by ECT. Importantly, we do not find any evidence that this treatment may cause microhaemorrhages in the mouse models used in this study. It remains entirely possible that direct binding to amyloid plaques of a F(ab’)2 fragment lacking effector functionality can contribute to therapeutic efficacy, as suggested by previous studies using antibody fragments (Bacskai et al., 2002;Tamura et al., 2005; Wang et al., 2010; Cattepoel et al., 2011). However, compared to IgG2a, the lower plasma levels achieved with F(ab’)2 are likely to limit the efficacy of peripheral ECT-mediated immunization. The exact role of the effector domain and its interaction with immune cells expressing Fc receptors, could be determined by comparing the therapeutic effects of a control antibody carrying a mutated Fc portion, similar to a previous study which addressed this question using deglycosylated anti-amyloid-β antibodies (Wilcock et al., 2006a; Fuller et al., 2014).

Remarkably, continuous administration of mAb-11 initiated before plaque deposition had a dramatic effect on the amyloid pathology in TauPS2APP mice, underlining the efficacy of preventive anti-amyloid-β treatments. In this mouse model, where tau hyperphosphorylation is enhanced by amyloid-β (Grueninger et al., 2010), the treatment decreases the number of AT8- and phospho-S422-positive neurons in the hippocampus. Furthermore, the number of MC1-positive hippocampal neurons is significantly reduced, which also indicates an effect of anti-amyloid-β immunotherapy on the accumulation of misfolded tau. These results highlight the effect of amyloid-β clearance on other manifestations of the Alzheimer’s pathology. In line with these findings, previous studies have shown evidence for a decrease in tau hyperphosphorylation following immunization against amyloid-β, both in animal models and in patients with Alzheimer’s disease (Oddo et al., 2004; Wilcock et al., 2009;Boche et al., 2010; Serrano-Pozo et al., 2010; Salloway et al., 2014).

Similar to the subcutaneous injection of recombinant proteins (Schellekens, 2005), ECT implants can elicit significant immune responses against the secreted recombinant antibody. An anti-drug antibody response was detected in half of the mice treated with the mAb-11-releasing devices, in the absence of any anti-CD4 treatment. The glycosylation profile of the mAb-11 synthesized in C2C12 myoblasts is comparable to standard material produced by myeloma or HEK293 cells (Lathuiliere et al., 2014a). Although we cannot exclude that local release by ECT leads to antibody aggregation and denaturation, it is unlikely that this mode of administration further contributes to compound immunogenicity. Because the Fab regions of the chimeric recombinant mAb-11 IgG2a contain human CDRs, it remains to be determined whether the ECT-mediated delivery of antibodies fully matched with the host species would trigger an anti-drug antibody response.

Further developments will be needed to scale up this delivery system to humans. The possibility of using a single allogeneic cell source for all intended recipients is a crucial advantage of the ECT technology to standardize monoclonal antibody delivery. However, the development of renewable cell sources of human origin will be essential to ECT application in the clinic. Although the ARPE-19 cell line has been successfully adapted to ECT and used in clinical trials (Dunn et al., 1996; Zhang et al., 2011), the development of human myogenic cells (Negroni et al., 2009) is an attractive alternative that is worth exploring. Based on the PK analysis of recombinant mAb-11 antibody subcutaneously injected in mice (Supplementary material), we estimate that the flat sheet devices chronically release mAb-11 at a rate of 6.8 and 11.8 µg/h, to reach a plasma level of 50 µg/ml in the implanted animals. In humans, injected IgG1 has a longer half-life (21–25 days), with a volume of distribution of ∼100 ml/kg and an estimated clearance of 0.2 ml/h/kg. These values indicate that the predicted antibody exposure in humans, based on the rate of mAb-11 secretion achieved by ECT in mice, would be only 10 to 20-fold lower than the typical regimens based on monthly bolus injection of 1 mg/kg anti-amyloid-β monoclonal antibody. Hence, it is realistic to consider ECT for therapeutic monoclonal antibody delivery in humans, as the flat sheet device could be scaled up to contain higher amounts of cells. Furthermore, recent progress to engineer antibodies for increased penetration into the brain will enable lowering dosing of biotherapeutics to achieve therapeutic efficacy (Bien-Ly et al., 2014; Niewoehner et al., 2014). For some applications, intrathecal implantation could be preferred to chronically deliver monoclonal antibodies directly inside the CNS (Aebischer et al., 1996; Marroquin Belaunzaran et al., 2011).

Overall, ECT provides a novel approach for the local and systemic delivery of recombinant monoclonal antibodies in the CNS. It will expand the possible therapeutic options for immunotherapy against neurodegenerative disorders associated with the accumulation of misfolded proteins, including Alzheimer’s and Parkinson’s diseases, dementia with Lewy bodies, frontotemporal lobar dementia and amyotrophic lateral sclerosis (Gros-Louis et al., 2010; Bae et al., 2012; Rosenmann, 2013).

Abbreviations
ECT
encapsulated cell technology
mAb
monoclonal antibody
sjwilliamspa

The most powerful part of the article is the methodology of the transdermal patch of genetically engineered cells which appear to give a constant stream of antibody therapy. This would be better for the authors to have highlighted because the protein delivery system is great work. However it is a shame that the Alzheimer’s field is still relying on these mouse models which are appearing to lead the filed down a path which leads to failed clinical trials. Lilly has just dropped their Alzheimer’s program and unless the filed shifts to a different hypothesis of disease etilology I feel the whole field will be stuck where it is.

References

 

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Antibody alternatives in specific aptamer 3-D scaffold binding

Curator: Larry H. Bernstein, MD, FCAP

 

 

New Proteomics Tools to Open Up Drug Discovery

Dr. Paul Ko Ferrigno, Chief Scientific Officer, and Dr. Jane McLeod, of Avacta Life Sciences

http://www.dddmag.com/articles/2016/01/new-proteomics-tools-open-drug-discovery

 

Size matters: Smaller molecules allow a tighter packing density on solid surfaces for improved signal-to-noise in assays, such as SPR or ELISA.

http://www.dddmag.com/sites/dddmag.com/files/AvactaAffimer%20stills_00006.jpg

Size matters: Smaller molecules allow a tighter packing density on solid surfaces for improved signal-to-noise in assays, such as SPR or ELISA.

 

In the current genomic era of very accurate DNA analyses by in situ hybridization, DNA chip analyses, and deep sequencing, it is often assumed that antibodies have an analogous ability to identify molecular targets accurately. Nothing could be further from the truth. Proteomics as a field is still lagging behind its genomic counterpart in the level of detail we can achieve, the level of data we can collect and the overall levels of accuracy and reliability that the collected data represent.

From the estimated 20,000 human genes 100,000 different possible proteins have been predicted. What is more, the variation achieved via the post-translational modification of these proteins brings another layer of complexity to cellular signalling. All this means that while studying the genetic blueprint can offer insights into the cell, it is only through examining the functional protein units that we can comprehensively map the dynamic interactions that occur within the cell to drive an organism or disease process.

 

Why not use antibodies?

Antibodies form the basis of molecular recognition in proteomics, whether this is to identify a protein within a complex mixture or label a specific protein within a cell. Both their target specificity and high binding capacity have made these molecules fantastically useful tools within diagnostics. However, the large majority of commercially available antibodies are for use as reagents in research and development, where they are simply not as well validated and issues with their manufacture have created problems that have hindered drug development.

It has proven next to impossible to develop antibodies to certain targets. This may be because of their high homology to the host protein, so the immune system fails to recognise them as different, or due to antigen processing resulting in the loss of post-translational modifications or discontinuous epitopes. However, without the necessary antibodies to investigate these targets the corresponding research avenues have remained closed and key drug targets may have been missed.

Almost worse than lacking the necessary research tools is the problem of antibody reproducibility. Matthias Uhlen revealed that of the 5,436 antibodies tested as part of the Protein Atlas project over 50 percent failed to recognize their target in at least one of two commonly used assays. Antibodies that are not specific to their target or do not recognize their target at all are responsible for increasing the cost of biological research, with an estimated $800 million spent globally every year on bad antibodies. Many published studies have had to be retracted due to antibody-derived error and those that remain unidentified in the literature will continue to lead researchers down blind alleys. This level of misinformation in the published literature is not just hindering the progression of the field, but possibly even sending it backwards and costing more than is needed — an issue not to be taken lightly when so many research budgets are coming under the knife.

More recently there has been a call from a number of leading scientists for the use of polyclonal antibodies, considered to be the worst offenders in terms of batch-to-batch irreproducibility, to be abandoned. They suggest a move towards recombinant systems of production, which would remove the restrictions of the immune system on antibody production. Yet, they state that $1 billion dollars investment would be required to re-route antibody production down this path and suggest that a period of five to ten years may be required to bring about these changes.

Simply producing recombinant antibodies rather than animal-derived affinity reagents will still leave us with a number of problems regarding their use. Antibodies are simply too large to target many smaller, hidden epitopes and the presence of key disulphide bonds within their structure makes them all too susceptible to reduction within the cell, rendering them useless for applications such as live cell imaging of molecules within the cytoplasm. Moreover, can we afford to wait another decade for a solution to this problem before we pursue protein targets for basic understanding and drug development?

 

 

 

 

Antibody alternatives for new targets and techniques

Antibody alternatives are already available to researchers in the life sciences field. They are produced either from nucleic acid or protein molecules. Aptamers are short, single-stranded RNA or DNA molecules that fold to form 3D scaffolds, which can present a specific interaction surface to allow specific binding to its target molecule. Protein scaffolds are formed from parts of or whole proteins modified to present a peptide sequence. This peptide sequence works in a similar manner to present a specific interaction surface for specific binding to a desired target.

These antibody alternatives are produced in recombinant systems, minimizing batch-to-batch variation and allowing them to be produced to theoretically any target. Additionally, as they do not use animals in their production they are generally less expensive to produce than traditional antibodies.

 

 

 

 

While companies such as Affibody and Avacta Life Sciences are aiming to open up the drug pipeline, by offering these alternative affinity reagents to previously inaccessible targets for use in research and development, many have moved into exploiting their therapeutic potential. Noxxon produce an RNA-based scaffold, Spiegelmers, which are currently in phase 2 clinical trials for diabetic nephropathy, and Molecular Partners have reached phase 3 clinical trials with their protein scaffold DARPins, for wet age-related macular degeneration.

These new antibody alternatives are smaller than traditional antibodies. This opens up the use of new technologies, such as super resolution microscopy. While the diffraction limit remained at about 250 nmm the length of the antibody at 15 nm was of little importance, tagging your molecule as accurately as necessary. Removing this limit in super resolution microscopy has meant that antibodies are now too large to provide the required level of accuracy. Instead, using an antibody alternative of only 2nm to tag your protein of interest opens up this new technique offering greater precision to intracellular localization.

 

 

 

As these scaffolds have been engineered to be fit-for-purpose many contain no intramolecular disulphide bonds and so are not sensitive to the reducing environment of the cell. This function enables their use as intracellular reporters of molecular conformation, as well as in standard assays like IHC or western blotting, so allowing scientists to use the same reagent across both intracellular and biochemical assays, thus bridging the gap between cell biology and biochemical studies.

Offering increased reproducibility, access to an increased range of applications, and the opportunity to hit previously inaccessible targets, antibody alternatives are opening up potential new avenues of drug discovery.

 

 

About the Authors

Dr. Paul Ko Ferrigno is Chief Scientific Officer at Avacta Life Sciences and a Visiting Professor at the University of Leeds. He has been working on peptide aptamers since 2001 in Leeds and at the MRC Cancer Cell Unit in Cambridge, UK where his team developed the Affimer scaffold technology.

Dr. Jane McLeod is a Scientific Writer at Avacta Life Sciences.

 

 

 

 

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Larry H Bernstein, MD, Writer, Curator
http://pharmaceutical intelligence.com/2013/06/22/ Demythologizing sharks, cancer, and shark fins/lhbern

 

Sharks have survived some 400 million years on Earth. Could their longevity be due in part to an extraordinary resistance to cancer and other diseases? If so, humans might someday benefit from the shark’s secrets—but leading researchers caution that today’s popular shark cartilage “cancer cures” aren’t part of the solution.

 

The belief that sharks do not get cancer is not supported in fact, but it is the basis for decimating a significant part of the shark population for shark fins, and for medicinal use.  The unfortunate result is that there is no benefit.

 

A basis for this thinking is that going back to the late 1800s, sharks have been fished commercially and there have been few reports of anything out of the ordinary when removing internal organs or preparing meat for the marketplace.  In addition, pre-medical students may have dissected dogfish sharks in comparative anatomy, but you don’t see reports of cancerous tumors.

 

Carl Luer of the MOTE Marine Laboratory’s Center for Shark Research in Sarasota, Florida, has been studying sharks’ cancer resistance for some 25 years.  Systematic surveys of sharks are difficult to conduct, as capturing the animals in large numbers is time-consuming, and cancer tests would likely require the deaths of large numbers of sharks. Of the thousands of fish tumors in the collections of the Smithsonian Institution, only about 15 are from elasmobranchs, and only two of these are thought to have been malignant.

 

Scientists have been studying cancerous tumors in sharks for 150 years.

 

The first chondrichthyes’ (cartilaginous fishes, including sharks) tumor was found on a skate and recorded by Dislonghamcps in 1853. The first shark tumor was recorded in 1908. Scientists have since discovered benign and cancerous tumors in 18 of the 1,168 species of sharks. Scarcity of studies on shark physiology has perhaps allowed this myth to be accepted as fact for so many years.

 

In April 2000, John Harshbarger and Gary Ostrander countered this shark myth with a presentation on 40 benign and cancerous tumors known to be found in sharks, and soon after a blue shark was found with cancerous tumors in both its liver and testes. Several years later a cancerous gingival tumor was removed from the mouth of a captive sand tiger shark, Carcharias Taurus. Advances in shark research continue to produce studies on types of cancer found in various species of shark.  Sharks, like fish, encounter and take in large quantities of environmental pollutants, which may actually make them more susceptible to tumorous growth. Despite recorded cases of shark cancer and evidence that shark cartilage has no curative powers against cancer sharks continue to be harvested for their cartilage.

 

Sharks and their relatives, the skates and rays, have enjoyed tremendous success during their nearly 400 million years of existence on earth, according to Dr. Luer. He points out that one reason for this certainly is their uncanny ability to resist disease. Sharks do get sick, but their incidence of disease is much lower than among the other fishes. While statistics are not available on most diseases in fishes, reptiles, amphibians, and invertebrates, tumor incidence in these animals is carefully monitored by the Smithsonian Institution in Washington, D.C.

 

The Smithsonian’s enormous database, called the Registry of Tumors in Lower Animals, catalogs tissues suspected of being tumorous, including cancers, from all possible sources throughout the world. Of the thousands of tissues in the Registry, most of them are from fish but only a few are from elasmobranchs. Only 8 to 10 legitimate tumors are among all the shark and ray tissues examined, and only two of these are thought to have been malignant.

 

An observation by Gary Ostrander, a Professor at Johns Hopkins University, is that there may be fundamental differences in shark immune systems so that they aren’t as prone to cancer.  The major thrust of the Motes research focuses on the immunity of sharks and their relatives the skates and rays. While skates aren’t as interesting to the public as their shark relatives, their similar biochemical immunology and their ability to breed in captivity make them perhaps more vital to Luer’s lab work.   The result is to study the differences and similarities to the higher animals, and what might possibly be the role of the immune system in their low incidence of disease.

 

This low incidence of tumors among the sharks and their relatives has prompted biochemists and immunologists at Mote Marine Laboratory (MML) to explore the mechanisms that may explain the unusual disease resistance of these animals. To do this, they established the nurse shark and clearnose skate as laboratory animals. They designed experiments to see whether tumors could be induced in the sharks and skates by exposing them to potent carcinogenic (cancer-causing) chemicals, and then monitored pathways of metabolism or detoxification of the carcinogens in the test animals. While there were similarities and differences in the responses when compared with mammals, no changes in the target tissues or their genetic material ever resulted in cancerous tumor formation in the sharks or skates.

 

The chemical exposure studies led to investigations of the shark immune system. As with mammals, including humans, the immune system of sharks probably plays a vital role in the overall health of these animals. But there are some important differences between the immune arsenals of mammals and sharks. The immune system of mammals typically consists of two parts which utilize a variety of immune cells as well as several classes of proteins called immunoglobulins (antibodies).

 

Compared to the mammalian system, which is quite specialized, the shark immune system appears primitive but remarkably effective. Sharks apparently possess immune cells with the same functions as those of mammals, but the shark cells appear to be produced and stimulated differently. Furthermore, in contrast to the variety of immunoglobulins produced in the mammalian immune system, sharks have only one class of immunoglobulin (termed IgM). This Immunoglobulin normally circulates in shark blood at very high levels and appears to be ready to attack invading substances at all times.

 

Another difference lies in the fact that sharks, skates, and rays lack a bony skeleton, and so do not have bone marrow. In mammals, immune cells are produced and mature in the bone marrow and other sites, and, after a brief lag time, these cells are mobilized to the bloodstream to fight invading substances. In sharks, the immune cells are produced in the spleen, thymus and unique tissues associated with the gonads (epigonal organ) and esophagus (Leydig organ). Some maturation of these immune cells occurs at the sites of cell production, as with mammals. But a significant number of immune cells in these animals actually mature as they circulate in the bloodstream. Like the ever-present IgM molecule, immune cells already in the shark’s blood may be available to respond without a lag period, resulting in a more efficient immune response.

 

Research was being carried out during the 1980’s at the Massachusetts Institute of Technology (MIT) and at Mote Marine Laboratory designed to understand how cartilage is naturally able to resist penetration by blood capillaries. If the basis for this inhibition could be identified, it was reasoned, it might lead to the development of a new drug therapy. Such a drug could control the spread of blood vessels feeding a cancerous tumor, or the inflammation associated with arthritis.

 

The results of the research showed only that a very small amount of an active material, with limited ability to control blood vessel growth, can be obtained from large amounts of raw cartilage. The cartilage must be subjected to several weeks of harsh chemical procedures to extract and concentrate the active ingredients. Once this is done, the resulting material is able to inhibit blood vessel growth in laboratory tests on animal models, when the concentrated extract is directly applied near the growing blood vessels.  One cannot assume that comparable material in sufficient amount and strength is released passively from cartilage when still in the animal to inhibit blood vessel growth anywhere in the body.

 

Tumors release chemicals stimulating the capillary growth so a nutrient-rich blood supply is created to feed the tumorous cells. This process is called angiogenesis. If scientists can control angiogenesis, they could limit tumor growth. Cartilage lacks capillaries running through it. Why should this be a surprise?  Cartilage cells are called chondrocytes, and they fuction to produce a acellular interstitial matrix consisting of hyaluronan (complex carbohydrate formed from hyaluronic acid and chondroitin sulfate) which is protective of interlaced collagen.   Early research into the anti-angiogenesis properties of cartilage revealed that tiny amounts of proteins could be extracted from cartilage, and, when applied in concentration to animal tumors, the formation of capillaries and the spread of tumors was inhibited.

 

Henry Brem and Judah Folkman from the Johns Hopkins School of Medicine first noted that cartilage prevented the growth of new blood vessels into tissues in the 1970s. The creation of a blood supply, called angiogenesis, is a characteristic of malignant tumors, as the rapidly dividing cells need lots of nutrients to continue growing.  It is valuable to consider that these neovascular generating cells are not of epithelial derivation, but are endothelial and mesenchymal. To support their very high metabolism, tumors secrete a hormone called ‘angiogenin’ which causes nearby blood vessels to grow new branches that surround the tumor, bringing in nutrients and carrying away waste products

 

Brem and Folkman began studying cartilage to search for anti-angiogenic compounds. They reasoned that since all cartilage lacks blood vessels, it must contain some signaling molecules or enzymes that prevent capillaries from forming. They found that inserting cartilage from baby rabbits alongside tumors in experimental animals completely prevented the tumors from growing. Further research showed calf cartilage, too, had anti-angiogenic properties.

 

 

 

A young researcher by the name of Robert Langer repeated the initial rabbit cartilage experiments, except this time using shark cartilage. Indeed, shark cartilage, like calf and rabbit cartilage, inhibited blood vessels from growing toward tumors. Research by Dr. Robert Langer of M.I.T. and other workers revealed a promising anti-tumor agent obtainable in quantity from shark cartilage. The compound antagonistic to the effects of angiogenin, called ‘angiogenin inhibitor’, inhibits the formation of new blood vessels, neovascularization, that is essential for supporting cancer growth.

 

The consequence of the”shark myth” is not surprising. An inhabitant of the open ocean, the Silky Shark is ‘hit’ hard by the shark fin and shark cartilage industries – away from the prying eyes of a mostly land bound public. As a consequence of this ‘invisibility’, mortality of Silkies is difficult to estimate or regulate.  North American populations of sharks have decreased by up to 80% in the past decade, as cartilage companies harvest up to 200,000 sharks every month in US waters to create their products. One American-owned shark cartilage plant in Costa Rica is estimated to destroy 2.8 million sharks per year. Sharks are slow growing species compared to other fish, and simply cannot reproduce fast enough to survive such sustained, intense fishing pressure. Unless fishing is dramatically decreased worldwide, a number of species of sharks will go extinct before we even notice.

 

Sources:
1.  National Geographic News: NATIONALGEOGRAPHIC.COM/NEWS

 

2. Do Sharks Hold Secret to Human Cancer Fight?
by Brian Handwerk for National Geographic News.  August 20, 2003

 

3. Busting Marine Myths: Sharks DO Get Cancer!
by Christie Wilcox   November 9th 2009

 

 

 

 

 

Sand tiger shark (Carcharias taurus) at the Ne...

Sand tiger shark (Carcharias taurus) at the Newport Aquarium. (Photo credit: Wikipedia)

 

Angiogenesis

Angiogenesis (Photo credit: Wikipedia)

 

 

 

 

 

 

 

 

 

 

 

The immune response

The immune response (Photo credit: Wikipedia)

 

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Combining Nanotube Technology and Genetically Engineered Antibodies to Detect Prostate Cancer Biomarkers[1]

Writer, Curator: Stephen J. Williams, Ph.D.

acs nanoFigure of  Carbon Nanotube Transistor design with functionalized antibodies for biomarker detection.  From paper of A.T. Johnson; used with permission from A.T. Johnson)

In a literature review of the current status of the breast cancer biomarker field[2], author Dr. Michael Duffy, from University College Dublin, pondered the clinical utility of breast cancer serum markers and suggested that due to lack of sensitivity and specificity none of available markers is of value for detection of early breast cancer however these biomarkers have been shown useful in monitoring patients with advanced disease. For instance high preoperative CA15-3 is indicative of adverse patient outcome.  According to American Society of Clinical Oncology Expert Panel, however CA 15-3 may lack the sensitivity and disease specificity for breast cancer as a prognostic marker.  For panel suggestions please click on the link below:

http://www.asco.org/sites/www.asco.org/files/breast_tm_2007_changes-final.pdf

The same panel also concurred on the lack of prognostic value of other markers (for example CEA for colon cancer) but did agree that 66-73% of patients with advanced disease, who responded to therapy, showed reduction in these serum markers.  Indeed, CA125, long associated as a biomarker for ovarian cancer, does not have the sensitivity and especially the disease specificity to be a stand-alone prognostic marker[3].  Therefore, although “omics” strategies have suggested multiple possible biomarkers  for various cancers, a major issue in translating a putative biomarker to either:

1)      a clinically validated (panel) of disease-relevant biomarkers or

2)      biomarkers useful for therapeutic monitoring

is obtaining the specificity and sensitivity for detection in bio-specimens.   As discussed below, this is being achieved with the merger of nanotechnology-based sensors and bioengineering of biomolecule.

For ASCO panel suggestions of biomarkers useful in Prostate cancer please see the link below:

http://jco.ascopubs.org/site/misc/specialarticles.xhtml#GENITOURINARY_CANCER

As a side note, since 2010, ASCO has focused on reviewing and producing new guidelines for cancer biomarkers including genome sequencing:

http://www.medscape.com/viewarticle/723349

Osteopontin (OPN) and prostate cancer

Osteopontin is a phosphorylated glycoprotein secreted by activated macrophages, leukocytes, activated T lymphocytes and is present at sites of inflammation (for a review of OPN see [4]).  Osteopontin interacts with several integrins and CD44 (a putative cancer stem cell marker).  Binding of OPN to cell integrins mediates cell-matrix and cell-cell communication, stimulating adhesion, migration (through interaction with urokinase plasminogen activator {uPA}) and cell signaling pathways such as the HGF-Met pathway.  Overexpression is found on a variety of cancers including breast, lung, colorectal, ovarian and melanoma[5].  And although OPN is detected in normal tissue, it is known that OPN over-expression can alter the malignant potential of tumor cells.

Roles of osteopontin in cancer include:

  • Binding to CD44
  • Increase in growth factor signaling (HGF/Met pathway)
  • Increase uPA activity- increase invasiveness
  • Angiogenesis thru binding with αvβ3 integrin and increased VEGF expression
  • Protection against apoptosis: OPN activates nuclear factor Κβ

Some researchers have suggested it could be a prognostic marker for breast and lung cancer while there have been conflicting reports as to whether OPN expression is correlated to malignant potential in prostate cancer[6].  Osteopontin is found on tumor infiltrating macrophages, which may contribute to OPN as a prognostic marker. Breast cancer patients (disseminated carcinomas) have 4-10 times higher serum levels of OPN than found in healthy patients, although there is no difference in pre- or post-menopausal women[7].

Piezoelectric sensors have been used by the same group at Fox Chase Cancer Center to detect serum levels of the HER2 protein in breast cancer patients, for the purpose of therapeutic monitoring after anti-HER2 antibody trastuzumab (Herceptin™) therapy.  Lina Loo, in the laboratory of Dr. Gregory Adams showed the utility of using (scFv) to trastuzumab (anti-HER2) with pizo-electric nanotubes to accurately and reproducibly determine levels of serum HER2[8].  This method improved the sensitivity of serum HER2 detection over other methods such as:

  • ELISA {enzyme-linked immunoassay}
  • Luminex platforms

Please watch the following video interview concerning genetically engineered scFV antibody fragments and their use in cancer detection and treatment (with Dr. Matt Robinson and Dr. Greg Adams, from Fox Chase Cancer Center)

PLEASE WATCH VIDEO

However the advent of nanotechnology-based detection system combined with engineered affinity-based biomolecules has increased both the sensitivity and specificity of biomarker detection from complex fluids such as plasma and urine.  The advent of multiple types of biosensors, including

has given the ability to measure, with enhanced sensitivity and specificity,  putative biomarkers of disease in minute volumes of precious bio-samples.

The basic design of a biosensor is made of three components:

  1. A recognition element (I.e. antibodies, nucleic acids, enzymes)
  2. A signal transducer (electrochemical, optical, piezoelectric)
  3. Signal processor (relays and displays)

In the journal ACS Nano Mitchel Lerner from Dr. Charlie Johnson’s laboratory at University of Pennsylvania in collaboration with Fox Chase Cancer researchers in the laboratory of Dr. Matthew Robinson, describe a piezoelectric detection system for quantifying levels of osteopontin (OPN), a putative biomarker for prostate cancer[1].  In this paper Dr. Robinson’s group at Fox Chase, genetically engineered a single chain variable fragment (scFv) protein {the binding portion of the antibody} which had high affinity for OPN.  This scFv was attached to a carbon nanotube field-effect transistor (NT-FET), designed by Dr. Johnson’s group, using a chemical process called chemical functionalization {a process using diazonium salts to covalently attach scFV to NT-FET.

functionalization

Figure. Functionalization scheme for OPN attachment to carbon nanotubes. As figure 1 legend in paper states: “First, sp8 hybridized-sites are created o the nanotube sidewall by incubation in a diazonium salt solution.  The carboxylic acid group is then activated by EDC and stabilized with NHS.ScFv antibody displaces the NHS and forms an amide bond.  OPN epitope is shown in yellow and the C and N-terminuses are in orange and green respectively.” (used by permision for A.T. Charlie Johnson)

This system was then used to determine the selectivity and sensitivity of OPN from complex solutions.

Methods: 

Nanotube (NT) design

  • Grown by catalytic vapor deposition
  • Electrical contacts patterned using photo-lithography
  • Atomic Force microscopy was used to verify structure of nanotube

Chemical Linking of scFv to nanotube

  • Diazonium treatment resulted in activation and subsequent stabilization of amino (NHS) side chain
  • Amine group on lysine of scFV displaced NHS group => covalent attachment of scFV to NT
  • Atomic Force Spectroscopy used to verify linkage of scFv to nanotube

Results showed there was

  • minimal non-specific binding of OPN to the scFv
  • system allowed for detection limit of 1 pg/ml OPN (pictogram/milliliter) or 30 fM (fentomolar) in a phosphate buffered saline solution.
  •  Only a minute volume (10 µl) of sample is needed
  • Sensor able to measure million-fold  range of OPN concentrations ( from 10-3 to 103 ng/mL OPN)

Two experiments were conducted to determine the specificity of OPN to the antibody-detection system.

1st experiment

–          scFv functionalized  sensor was incubated in a solution of high concentration of BSA (450 mg/ml) to approximate nonspecific proteins in patient samples

–           minimal signal was detected

        2nd experiment

–          Functionalized NT-FET devices with a scFv based on the HER2 therapeutic antibody trastuzumab

–          There was no binding of OPN to anti-HER2 devices

–          Therefore anti OPN (23C3) scFv-functionalized carbon nanotube sensors exhibit high levels of specificity to OPN

The authors conclude “the functionalization procedure described here is expected to be generalizable to any antibody containing an accessible amine group, and to result in biosensors appropriate for detection of corresponding complementary proteins at fM concentrations”.

I had the opportunity to speak with co-author Dr. Matthew Robinson, Assistant Professor in the Developmental Therapeutics Program at Fox Chase Cancer Center about the next steps for this work.  Dr. Robinson mentioned that “at this point we have not looked in patient samples yet but our plan is to move in that direction. We need to establish sensitivity/specificity in increasingly complex samples (e.g. spiked normal serum and retrospectively in patient serum with known levels of biomarkers).” 

Cancer patients often present a complex metabolic profile.  The paper notes that OPN has a pI (isoelectric point) of 4.2, which would result in a negative charge at physiologically normal pH of 7.6. I asked Dr. Robinson about if changes in metabolic profile could hinder OPN binding to the NT-FET system would require some preprocessing of blood samples.  Dr. Robinson  agreed “that confounding variables such as additional diseases but even things like diet (i.e. is fasting necessary) need to be addressed before this platform is ready for use in clinical setting.
It is likely that sample prep will be needed to remove albumin, lower salt concentrations, etc. This could end up being problematic for biomarkers that are unstable and would degrade over the time necessary for sample prep. It is also possible that sample prep to remove albumin and other background factors could result in loss of biomarkers. This will need to be determined on a case-by-case basis with validated testing methods.”
One useful advantage of this system is the possibility of measuring multiple biomarkers, clinically important as studies has suggested that

multiple markers result in the higher sensitivity/specificity for many infrequent cancers, such as ovarian. Dr. Robinson agrees “that panels of biomarkers are likely to be better at early detection and diagnosis. In principle the platform that we describe can be set up to allow for detection of  multiple biomarkers at a time. From the biology end of things we have built antibodies against 3 different prostate cancer biomarkers for that purpose.”

Dr. Johnson  commented on the ability of the platform allowed for the simultaneous detection of multiple biomarkers, noting that ”the platform is compatible with the measurement of multiple biomarkers through the use of multiple devices, each functionalized with their own antibody.”

 

ASCO guidelines Expert Panel on Tumor Biomarkers 2007 Update for Breast Cancer:

http://www.asco.org/sites/www.asco.org/files/breast_tm_2007_changes-final.pdf 

ASCO Guidelines for Genitourinary Cancer:

Screening for Prostate Cancer With Prostate-Specific Antigen Testing: American Society of Clinical Oncology Provisional Clinical Opinion

Published in JCO, Vol. 30, Issue 24 (August 20), 2012: 3020-3025

American Society of Clinical Oncology Clinical Practice Guideline on Uses of Serum Tumor Markers in Adult Males With Germ Cell Tumors

Published in JCO, Vol 28, Issue 20 (July 10), 2010: 3388-3404

American Society of Clinical Oncology Endorsement of the Cancer Care Ontario Practice Guideline on Nonhormonal Therapy for Men With Metastatic Hormone-Refractory (castration-resistant) Prostate Cancer

Published in JCO, Vol 25, Issue 33 (November 20), 2007: 5313-5318

Initial Hormonal Management of Androgen-Sensitive Metastatic, Recurrent, or Progressive Prostate Cancer: 2006 Update of an American Society of Clinical Oncology Practice Guideline

Published in JCO, Vol. 25, Issue 12 (April 20), 2007: 1596-1605

References:

1.            Lerner MB, D’Souza J, Pazina T, Dailey J, Goldsmith BR, Robinson MK, Johnson AT: Hybrids of a genetically engineered antibody and a carbon nanotube transistor for detection of prostate cancer biomarkers. ACS nano 2012, 6(6):5143-5149.

2.            Duffy MJ: Serum tumor markers in breast cancer: are they of clinical value? Clinical chemistry 2006, 52(3):345-351.

3.            Meyer T, Rustin GJ: Role of tumour markers in monitoring epithelial ovarian cancer. British journal of cancer 2000, 82(9):1535-1538.

4.            Rodrigues LR, Teixeira JA, Schmitt FL, Paulsson M, Lindmark-Mansson H: The role of osteopontin in tumor progression and metastasis in breast cancer. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2007, 16(6):1087-1097.

5.            Brown LF, Berse B, Van de Water L, Papadopoulos-Sergiou A, Perruzzi CA, Manseau EJ, Dvorak HF, Senger DR: Expression and distribution of osteopontin in human tissues: widespread association with luminal epithelial surfaces. Molecular biology of the cell 1992, 3(10):1169-1180.

6.            Thoms JW, Dal Pra A, Anborgh PH, Christensen E, Fleshner N, Menard C, Chadwick K, Milosevic M, Catton C, Pintilie M et al: Plasma osteopontin as a biomarker of prostate cancer aggression: relationship to risk category and treatment response. British journal of cancer 2012, 107(5):840-846.

7.            Brown LF, Papadopoulos-Sergiou A, Berse B, Manseau EJ, Tognazzi K, Perruzzi CA, Dvorak HF, Senger DR: Osteopontin expression and distribution in human carcinomas. The American journal of pathology 1994, 145(3):610-623.

8.            Loo L, Capobianco JA, Wu W, Gao X, Shih WY, Shih WH, Pourrezaei K, Robinson MK, Adams GP: Highly sensitive detection of HER2 extracellular domain in the serum of breast cancer patients by piezoelectric microcantilevers. Analytical chemistry 2011, 83(9):3392-3397.

Other posts from this site on Biomarkers, Cancer, and Nanotechnology include:

Stanniocalcin: A Cancer Biomarker.

Mesothelin: An early detection biomarker for cancer (By Jack Andraka)

Squeezing Ovarian Cancer Cells to Predict Metastatic Potential: Cell Stiffness as Possible Biomarker

PIK3CA mutation in Colorectal Cancer may serve as a Predictive Molecular Biomarker for adjuvant Aspirin therapy

Biomarker tool development for Early Diagnosis of Pancreatic Cancer: Van Andel Institute and Emory University

Early Biomarker for Pancreatic Cancer Identified

In Search of Clarity on Prostate Cancer Screening, Post-Surgical Followup, and Prediction of Long Term Remission

Prostate Cancer Molecular Diagnostic Market – the Players are: SRI Int’l, Genomic Health w/Cleveland Clinic, Myriad Genetics w/UCSF, GenomeDx and BioTheranostics

Early Detection of Prostate Cancer: American Urological Association (AUA) Guideline

A Blood Test to Identify Aggressive Prostate Cancer: a Discovery @ SRI International, Menlo Park, CA

Prostate Cancer Cells: Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition

Prostate Cancer and Nanotecnology

 

Read Full Post »


Reporter: Aviva Lev-Ari, PhD, RN

 

CANCER BIOTHERAPEUTICS

ADCs, Multi-Specifics, Combined Therapies and Immunotherapy

Inaugural

TUESDAY , 5 NOVE MBER

»»PRE-CONFERENCE PLENARY SESION

16:55 Designing Receptor Binding Proteins with Highly Potent

Biological Function

Andreas Plückthun, Ph.D., Director and Professor, Biochemistry, University

of Zurich

Non-IgG molecules, unless armed with toxins or other effector units, are

usually thought to be limited in the biological responses they can elicit.

However, Designed Ankyrin Repeat Proteins (DARPins) are particularly

versatile, because of their favorable biophysical properties, and they can be

engineered into many formats. Using DARPins generated against members

of the EGFR family, and a combination of x-ray crystallography, signaling

studies, and in vivo experiments, it will be demonstrated how molecules

could be engineered to selectively induce apoptosis in tumors, and their

mechanism of action has been deduced. New intracellular sensors will be

described for such studies.

17:45 Immunotherapy with BiTE® Antibodies

Luis Borges, Ph.D., Scientific Director, Therapeutic Innovation Unit, Amgen, Inc.

BiTE® antibodies are potent bispecific single-chain antibodies that redirect T

cells to kill tumors. They engage a tumor target and a constant region of the

T cell receptor to recruit and activate polyclonal T cells to eliminate tumors.

They have demonstrated potent efficacy in various preclinical tumor models

and have now transitioned to clinical studies. Blinatumomab, a CD19xCD3

BiTE® antibody, is in clinical development and has shown high single-agent

response rates in patients with refractory or relapsed B-ALL and B-NHL.

18:30 End of Day

Wednesday, 6 November

07:45 Registration and Morning Coffee

08:30 Chairperson’s Opening Remarks

Jason Baum, Ph.D., Principal Scientist, Research, Merrimack Pharmaceuticals, Inc.

MULTI-SPECIFIC ANTIBODY PRODUCTS

08:35 Two-in-One Antibody Targeting EGFR and HER3 and Platform

Update

Germaine Fuh, Ph.D., Senior Scientist, Antibody Engineering, Genentech, Inc.

Mutation at the antigen binding sites of a mono-specific antibody may recruit a

second binding specificity such that each Fab arm exhibits dual binding function and

IgG with this dual action Fab (DAF) can be produced as conventional IgG. Proofof-

concept is a HER2/VEGF Two-in-One antibody; EGFR/HER3 Two-in-One DAF

antibody is in clinical phase II trial for treating epithelial cancer. The talk will cover the

generation and development of the EGFR/HER3 DAF antibody including preclinical

and clinical phase I data.

09:05 MM-141, a Bispecific Antibody Co-Targeting IGF-1R and Erbb3,

Overcomes Network Adaptation by Blocking Redundant Survival

Pathways

Jason Baum, Ph.D., Principal Scientist, Research, Merrimack Pharmaceuticals, Inc.

An integrated Network Biology approach was used to design and optimize MM-

141 to overcome limitations of first generation IGF-1R therapies by also blocking

heregulin-mediated compensation through ErbB3. MM-141 potentiates the activity

of both targeted therapies and chemotherapies through the combined inhibition

of PI3K/Akt/mTOR signaling as well as control over feedback loops triggered by

these agents.

09:35 Bispecific κλ-bodies for Selective Inhibition of CD47 in Cancer Cells

Nicolas Fischer, Ph.D., Head, Research, Novimmune SA

We have used our κλ-body platform to generate CD47-neutralizing bispecific

antibodies. These fully human antibodies are composed of a CD47-specific arm

and a targeting arm, specific to a tumor associated antigen (TAA). The preferential

neutralization of CD47 on TAA-expressing cancer cells should therefore show better

pharmacological properties and a broader therapeutic window as compared to nontargeted

anti-CD47 monoclonal antibodies. The presentation will also highlight how

light chain diversity can be exploited to create bispecific antibodies with favorable

manufacturability and stability profiles that facilitate their development path.

10:05 Sponsored Presentation (Opportunity Available)

10:35 Coffee Break in the Exhibit Hall with Poster Viewing

11:05 Targeting Tumor Microenvironmental Signals with Bispecific

Antibodies

Alessandro Angelini, Ph.D., David H. Koch Institute for Integrative Cancer Research,

Massachusetts Institute of Technology (MIT)

We have developed bispecific antibodies that locally contravene soluble signaling

factors that establish the supporting tumor microenvironment that enables tumor

survival and growth. Soluble factors such as VEGF, TGF-β, and IL-8 play a demonstrated

role in tumorigenesis, and enhanced interdiction of these signals within the tumor

should enhance the therapeutic index of cancer therapy.

11:35 Novel Multi-Targeting Antibody Mixtures: Mode of Action and

Advantages Over Other Approaches

Michael Kragh, Ph.D., Director, Antibody Pharmacology, Symphogen A/S

This talk will present the selection of antibodies against tumor-related antigens to

obtain synergistic combinations, the benefits of simultaneous targeting of multiple

receptors, and examine pan-HER (EGFR, HER2 and HER3) targeting to address

tumor heterogeneity and plasticity.

12:05 Sponsored Presentation (Opportunity Available)

12:35 Luncheon Presentations (Sponsorship Opportunities Available) or

Lunch on Your Own

ADVANCES WITH CANCER IMMUNOTHERAPY

14:00 Chairperson’s Remarks

Andrea van Elsas, CSO, BioNovion B.V.

14:05 Cancer Immunotherapy Using Immune Modulating Antibodies

Andrea van Elsas, CSO, BioNovion B.V.

Immune rejection of human cancer has been an elusive goal until recently. T cell

modulating antibodies targeting CTLA-4 and the PD-1 pathway induced clinically

meaningful responses and long-term benefit in patients with metastatic cancer.

Successful immune rejection can come with significant immune related adverse

events. Immune oncology agents do not directly tumor cells but treat the patient’s

immune cells. In this presentation, the discovery of immune modulating antibodies

and their translation into clinical success will be discussed.

14:35 Immunocytokines: A Novel Potent Class of Armed Antibody

Laura Gualandi, Ph.D., Philochem A.G.

Antibodies are effective tools that can deliver molecules with potent therapeutic

activity, such as Cytokines, to the tumor site, minimizing toxic effects. Aspects like

molecular format, valence and the chosen target antigen contribute to the efficacy of

the immunocytokines in vivo. Combinatory therapeutic strategies with other agents

have also been recently investigated. This talk will cover advanced preclinical and

clinical data on armed antibodies discovered and developed by the Philogen group.

15:05 NKTT320: A Humanized Monoclonal Antibody for Cancer

Immunotherapy

Robert Mashal, CEO, NKT Therapeutics

Activation of iNKT cells has been shown to have therapeutic effects both in

PEGSummitEurope.com 7

6-7 November 2013

preclinical models and in patients with cancer, and represents an important pathway

for the immunotherapy of cancer. iNKT cells have an invariant T cell receptor (iTCR).

NKT Therapeutics is developing NKTT320, a humanized monoclonal antibody which

specifically recognizes the iTCR present exclusively on iNKT cells, and has been

shown to activate iNKT cells both in vitro and in vivo.

15:35 Refreshment Break in the Exhibit Hall with Poster Viewing

16:15 Novel Tumor-Targeted, Engineered IL-2 Variant (IL-2v)-Based

Immunocytokines for Immunotherapy of Cancer

Ekkehard Moessner, Ph.D., Group Leader, Protein Engineering, pRED, Roche Glycart A.G.

A novel class of immunocytokines will be discussed that are based on Fc containing

and also on non-Fc containing building blocks. The IL2 component is optimized for

improved performance in tumor targeting. Enhancement of in vivo efficacy, when

combined with ADCC competent antibodies, will be discussed.

ANTIBODY-DRUG CONJUGATES AND PAYLOADS

16:45 Next-Generation ADCs: Enabling Higher Drug Loading,

Alternative Payloads, and Alternative Targeting Moieties

Timothy B. Lowinger, Ph.D., CSO, Mersana Therapeutics, Inc.

The application of polymers to antibody-drug conjugate (ADC) design can provide

numerous advantages, including significantly higher capacity for drug payload;

utilization of alternative payloads not suitable for direct conjugation; improvement of

physicochemical properties; and utilization of protein recognition scaffolds beyond

the commonly used IgGs. Examples of these benefits achieved using Mersana’s

polyacetal-based conjugation system to create next-generation ADCs

will be presented.

17:15 Problem Solving Roundtable Discussions

Table 1: Engineering of Bispecific Antibodies

Moderator: Nicolas Fischer, Ph.D., Head, Research, Novimmune SA

Table 2: Antibody-Drug Conjugates: Linkers and Payloads

Moderators: Robert Lutz, Ph.D., Vice President, Translational Research &

Development, ImmunoGen, Inc.

Timothy B. Lowinger, Ph.D., CSO, Mersana Therapeutics, Inc.

Table 3: Site-Specific Conjugation of ADCs

Moderator: Pavel Strop, Ph.D., Associate Research Fellow, Protein

Engineering, Rinat-Pfizer, Inc.

Table 4: Cancer Immunotherapy: Reaping the Benefits

Moderators: Andrea van Elsas, CSO, BioNovion B.V

Luis Borges, Ph.D., Scientific Director, Amgen, Inc.

Table 5: Cancer Biotherapeutics in the Clinic

Moderators: Jason Baum, Ph.D., Principal Scientist, Research, Merrimack

Pharmaceuticals, Inc.

Martine Piccart, M.D., Ph.D., Head, Medical Oncology, Jules Bordet

Institute; Chair, ESMO (European Society for Medical Oncology)

18:15 Networking Reception in the Exhibit Hall with Poster Viewing

19:15 End of Day One

Thursday, 7 November

07:45 Breakfast Presentation (Sponsorship Opportunity Available) or

Morning Coffee

08:30 Chairperson’s Remarks

Robert Lutz, Ph.D., Vice President, Translational Research & Development,

ImmunoGen, Inc.

08:35 A Universal Chemically Driven Approach for Constructing

Homogeneous ADCs

David Jackson, Ph.D., Principle Scientist, ADC Discovery, Igenica, Inc.

Current ADCs in clinical development are heterogeneous mixtures that differ in

both DAR (drugs/antibody) and their conjugation sites. Igenica has invented novel

site-specific linkers to enable the synthesis of homogeneous ADCs. The linkers

are compatible with a variety of drug payloads and can be applied to any antibody.

Homogeneous ADCs were synthesized using the novel linkers and compared to

heterogeneous ADCs made with conventional linkers. Analytical data and activity of

the ADCs in tumor models will be presented.

09:05 Location Matters: Site of Conjugation Modulates Stability and

Pharmacokinetics of Antibody-Drug Conjugates

Pavel Strop, Ph.D., Associate Research Fellow, Protein Engineering, Rinat- Pfizer, Inc.

To understand the role of conjugation site, we developed an enzymatic method for

site-specific antibody-drug conjugation. This allowed us to attach diverse compounds

at multiple positions and investigate how the site influences stability, toxicity, and

efficacy. We show that the conjugation site has significant impact on ADC stability

and pharmacokinetics in a species-dependent manner. With this method, it is

possible to produce homogeneous ADCs and tune their properties to maximize the

therapeutic window.

09:35 Development of Second Generation Duocarmycin ADCs with

Superior Therapeutic Window

Marion Blomenröhr, Ph.D., Program Manager Biopharmaceuticals, Synthon

Biopharmaceuticals

The first generation ADCs have successfully exploited the mAb-driven tumor cell

targeting for optimization of efficacy, but have failed to reduce off-target toxicities.

This presentation will highlight Synthon’s second generation Linker-Drug technology

and its complementarity with novel proprietary duocarmycin payloads yielding highly

stable and potent ADCs, with an improved in vivo therapeutic window.

10:05 Producing Better Antibody-Drug Conjugates Sponsored by

(ADCs) Using ThioBridge™ Conjugation

Antony Godwin, Ph.D., Director, Science & Technology, PolyTherics Ltd

Next-generation antibody-drug conjugates will be required to be less heterogeneous

and have better stability. PolyTherics has developed ThioBridge™ for improved

conjugation of a cytotoxic payload at the disulfides bonds of antibodies, antibody

fragments and other targeting proteins. With ThioBridge™, the resulting ADC

has the benefit of reduced heterogeneity, as the drug to antibody ratio is limited

to a maximum of 4 with little DAR 0 species. Stability is also enhanced, as unlike

single thiol conjugation approaches at disulfides, ThioBridge™ is not prone to

drug deconjugation reactions in serum. In vitro and in vivo data for mAb and Fab

conjugates with an established payload confirms specific binding and activity.

10:35 Coffee Break in the Exhibit Hall with Poster Viewing

»»PLENARY SESION

11:05 Medical Treatment of HER2 Positive Breast Cancer: Two

Decades of a Fascinating History and More to Come

Martine Piccart, M.D., Ph.D., Head, Medical Oncology, Jules Bordet

Institute; Chair, ESMO (European Society for Medical Oncology)

The talk will cover multiple aspects of anti-HER2 treatment in breast cancer.

It will present a summary of the clinical results obtained with trastuzumab

and several other anti-HER2 drugs in breast cancer (lapatinib, TDM1,

pertuzumab). Issues like the treatment duration, biomarkers of resistance

to treatment will be debated. Finally it will discuss future promising

research strategies: neoadjuvant trials, comparison between anti-HER2

agents, combinations of these drugs and functional imaging.

11:50 Antibody-Drug Conjugates: From Bench to Bedside and Back

Robert Lutz, Ph.D., Vice President, Translational Research & Development,

ImmunoGen, Inc.

Antibody-drug conjugates are emerging as an exciting approach to the

development of antibody-based therapeutics. The growing preclinical and

clinical experience with maytansinoid conjugates such as Kadcyla (T-DM1) is

leading to an enhanced understanding regarding critical attributes for target

antigens, antibodies, payloads and linkers. The translational knowledge

is being incorporated into research and development efforts for the next

generation of ADC candidates.

12:35 End of Cancer Biotherapeutics

http://www.pegsummiteurope.com/PEGS_Europe_Content.aspx?id=123176&libID=123124

 

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CD47: Target Therapy for Cancer

Author/Curator: Tilda Barliya

“A research team from Stanford University’s School of Medicine is now one step closer to uncovering a cancer treatment that could be applicable across the board in killing every kind of cancer tumor” (1). It appeared that their antibody-drug against the CD47 protein, enabled the shrinking of all tumor cells. After completing their animal studies the researchers now move into a human phase clinical trials. CD47 has been previously studied and evaluated for its role in multiple cells, some of this data however, is somewhat controversy. So where do we stand?

CD47

CD47 (originally named integrin-associated protein (IAP)) is a cell surface protein of the immunoglobulin (Ig) superfamily, which is heavily glycosylated and expressed by virtually all cells in the body and overexpressed in many types of cancer  including breast, ovarian, colon, prostate and others (3). CD47 was first recognized as a 50 kDa protein associated and copurified with the  Alpha-v-Beta-3 integrin in placenta and neutrophil granulocytes and later shown to have the capacity to regulate integrin function and the responsiveness of leukocytes to RGD-containing extracellular matrix proteins. CD47 has also been shown to be identical to the OA-3/OVTL3 antigen highly expressed on most ovarian carcinomas (4,5).

CD47 consists of an extracellular IgV domain, a five times transmembrane-spanning domain, and a short alternatively spliced cytoplasmic tail. In both humans and mice, the cytoplasmic tail can be found as four different splice isoforms ranging from 4 to 36 amino acids, showing different tissue expression patterns (3).

CD47 interactions (3, 6):

  • Thrombospondin-1 (TSP-1) – a secreted glycoprotein that plays a role in vascular development and angiogenesis. Binding of TSP-1 to CD47 influences several fundamental cellular functions including cell migration and adhesion, cell proliferation or apoptosis, and plays a role in the regulation of angiogenesis and inflammation.
  • Signal-regulatory protein-alpha (SIRPα) – an inhibitory transmembrane receptor present on myeloid cells. The CD47/SIRPα interaction leads to bidirectional signaling, resulting in different cell-to-cell responses including inhibition of phagocytosis, stimulation of cell-cell fusion, and T-cell activation.
  • Integrins – several membrane integrins, most commonly integrin avb3. These interactions result in CD47/integrin complexes that effect a range of cell functions including adhesion, spreading and migration

These interactions with multiple proteins and cells types create several important functions, which include:

  • Cell proliferation – cell proliferation is heavily dependent on cell type as both activation and loss of CD47 can result in enhanced proliferation. For example, activation of CD47 with TSP-1 in wild-type cells inhibits proliferation and reduces expression of stem cell transcription factors. In cancer cells however, activation of CD47 with TSP-1 increases proliferation of human U87 and U373 astrocytoma. it is likely that CD47 promotes proliferation via the PI3K/Akt pathway in cancerous cells but not normal cells (7).  Loss of CD47 allows sustained proliferation of primary murine endothelial cells and enables these cells to spontaneously reprogram to form multipotent embryoid body-like clusters (8).
  • Apoptosis – Ligation of CD47 by anti-CD47 mAbs was found to induce apoptosis in a number of different cell types (3). For example: Of the two SIRP-family members known to bind the CD47 IgV domain (SIRPα and SIRPγ), SIRPα as a soluble Fc-fusion protein does not induce CD47-dependent apoptosis, hile SIRPα or SIRPγ bound onto the surface of beads induces apoptosis through CD47 in Jurkat T cells and the myelomonocytic cell line U937.
  • Migration – CD47  role on cell migration was first demonstrated in neutrophils, these effects were shown to be dependent on avb3 integrins, which interact with and are activated by CD47 at the plasma membrane. In cancer, Blocking CD47 function has been shown to inhibit migration and metastasis in a variety of tumor models. Blockade of CD47 by neutralizing antibodies reduced migration and chemotaxis in response to collagen IV in melanomaprostate cancer and ovarian cancer-derived cells (9).
  • Angiogenesis – The mechanism of the anti-angiogenic activity of CD47 is not fully understood, but introduction of CD47 antibodies and TSP-1 have been shown to inhibit nitric oxide (NO)-stimulated responses in both endothelial and vascular smooth muscle cells (10). More so, CD47 signaling influences the SDF-1 chemokine pathway, which plays a role in angiogenesis (11). (12)
  • Inflammatory response – Interactions between endothelial cell CD47 and leukocyte SIRPγ regulate T cell transendothelial migration (TEM) at sites of inflammation. CD47 also functions as a marker of self on murine red blood cells which allows RBC to avoid phagocytosis. Tumor cells can also evade macrophage phagocytosis through the expression of CD47 (2, 13).

It appears that CD47 ligation induce different responses, depending on cell type and partner for ligation.

Therapeutic and clinical aspect of CD47 in human cancer:

CD47 is overexpressed in many types of human cancers  and its known function as a “don’t eat me” signal, suggests the potential for targeting the CD47-SIRPα pathway as a common therapy for human malignancies (2,13). Upregulation of CD47 expression in human cancers also appears to influence tumor growth and dissemination. First, increased expression of CD47 in several hematologic malignancies was found to be associated with a worse clinical prognosis, and in ALL to predict refractoriness to standard chemotherapies (13, 14-16). Second, CD47 was demonstrated to regulate tumor metastasis and dissemination in both MM and NHL (13, 17).

Efforts have been made to develop therapies inhibiting the CD47-SIRPα pathway, principally through blocking monoclonal antibodies directed against CD47, but also possibly with a recombinant SIRPα protein that can also bind and block CD47.

Figure 2

Chao MP et al. 2012 Combination strategies targeting CD47 in cancer

While monotherapies targeting CD47 were efficacious in several pre-clinical tumor models, combination strategies involving inhibition of the CD47-SIRPα pathway offer even greater therapeutic potential. Specifically, antibodies targeting CD47-SIRPα can be included in combination therapies with other therapeutic antibodies, macrophage-enhancing agents, chemo-radiation therapy, or as an adjuvant therapy to inhibit metastasis (13).

For example, anti-SIRPα antibody was found to potentiate  antibody-dependent cellular cytotoxicity (ADCC) mediated by the anti-Her2/Neu antibody trastuzumab against breast cancer cells (18).  CD47–SIRPα interactions and SIRPα signaling negatively regulate trastuzumab-mediated ADCC in vitro and antibody-dependent elimination of tumor cells in vivo

More so, chemo-radiation therapy-mediated upregulation of cell surface calreticulin may potentially augment the activity of anti-CD47 antibody. However, this approach may also lead to increased toxicity as cell surface calreticulin is expressed on non-cancerous cells undergoing apoptosis, a principle effect of chemo-radiation therapy (19).

Highlights:

  • Phagocytic cells, macrophages, regulate tumor growth through phagocytic clearance
  • CD47 binds SIRPα on phagocytes which delivers an inhibitory signal for phagocytosis
  • A blocking anti-CD47 antibody enabled phagocytic clearance of many human cancers
  • Phagocytosis depends on a balance of anti-(CD47) and pro-(calreticulin) signals
  • Anti-CD47 antibody synergized with an FcR-engaging antibody, such as rituximab

Summary

Evasion of immune recognition is a major mechanism by which cancers establish and propagate disease. Recent data has demonstrated that the innate immune system plays a key role in modulating tumor phagocytosis through the CD47-SIRPα pathway. Careful development of reagents that can block the CD47/SIRPα interaction may indeed be useful to treat many forms of cancer without having too much of a negative side effect in terms of inducing clearance of host cells. Therapeutic approaches inhibiting this pathway have demonstrated significant efficacy, leading to the reduction and elimination of multiple tumor types.

Dr. Weissman says: “We are now hopeful that the first human clinical trials of anti-CD47 antibody will take place at Stanford in mid-2014, if all goes wellClinical trials may also be done in the United Kingdom”. These clinical trials must be designed so that the data they generate will produce a valid scientific result!!!

REFERENCES

1. By Sara Gates:  Cancer Drug That Shrinks All Tumors Set To Begin Human Clinical Trials. http://www.huffingtonpost.com/2013/03/28/cancer-drug-shrinks-tumors_n_2972708.html

2. Willingham SB, Volkmer JP, Gentles AJ, Sahoo D, Dalerba P, Mitra SS, Wang J, Contreras-Trujillo H, Martin R, Cohen JD, Lovelace P, Scheeren FA, Chao MP, Weiskopf K, Tang C, Volkmer AK, Naik TJ, Storm TA, Mosley AR, Edris B, Schmid SM, Sun CK, Chua MS, Murillo O, Rajendran P, Cha AC, Chin RK, Kim D, Adorno M, Raveh T, Tseng D, Jaiswal S, Enger PØ, Steinberg GK, Li G, So SK, Majeti R, Harsh GR, van de Rijn M, Teng NN, Sunwoo JB, Alizadeh AA, Clarke MF, Weissman IL. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proc Natl Acad Sci U S A. 2012 Apr 24;109(17):6662-6667. http://www.pnas.org/content/early/2012/03/20/1121623109

3. Oldenborg PL. CD47: A Cell Surface Glycoprotein Which Regulates Multiple Functions of Hematopoietic Cells in Health and Disease. ISRN Hematology Volume 2013 (2013), Article ID 614619, 19 pages.  http://www.hindawi.com/isrn/hematology/2013/614619/

4. G. Campbell, P. S. Freemont, W. Foulkes, and J. Trowsdale, “An ovarian tumor marker with homology to vaccinia virus contains an IgV- like region and multiple transmembrane domains,”Cancer Research, vol. 52, no. 19, pp. 5416–5420, 1992. http://cancerres.aacrjournals.org/content/52/19/5416.long

5. L. G. Poels, D. Peters, Y. van Megen et al., “Monoclonal antibody against human ovarian tumor-associated antigens,” Journal of the National Cancer Institute, vol. 76, no. 5, pp. 781–791, 1986. http://www.ncbi.nlm.nih.gov/pubmed/3517452

6. CD47. Wikipedia. http://en.wikipedia.org/wiki/CD47

7. Sick E, Boukhari A, Deramaudt T, Rondé P, Bucher B, André P, Gies JP, Takeda K (February 2011). “Activation of CD47 receptors causes proliferation of human astrocytoma but not normal astrocytes via an Akt-dependent pathway”. Glia 59 (2): 308–319. http://www.ncbi.nlm.nih.gov/pubmed/21125662

8. Kaur S, Soto-Pantoja DR, Stein EV, Liu C, Elkahloun AG, Pendrak ML, Nicolae A, Singh SP, Nie Z, Levens D, Isenberg JS, Roberts DD.  “Thrombospondin-1 Signaling through CD47 Inhibits Self-renewal by Regulating c-Myc and Other Stem Cell Transcription Factors”Sci Rep 2013: 3: 1673. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3628113/

9. Shahan TA, Fawzi A, Bellon G, Monboisse JC, Kefalides NA. “Regulation of tumor cell chemotaxis by type IV collagen is mediated by a Ca(2+)-dependent mechanism requiring CD47 and the integrin alpha(V)beta(3)”. J. Biol. Chem 2000. 275 (7): 4796–4802. http://www.jbc.org/content/275/7/4796

10. Isenberg JS, Ridnour LA, Dimitry J, Frazier WA, Wink DA, Roberts DD. “CD47 is necessary for inhibition of nitric oxide-stimulated vascular cell responses by thrombospondin-1”. J. Biol. Chem  2006. 281 (36): 26069–26080.  http://www.jbc.org/content/281/36/26069

11. Smadja DM, d’Audigier C, Bièche I, Evrard S, Mauge L, Dias JV, Labreuche J, Laurendeau I, Marsac B, Dizier B, Wagner-Ballon O, Boisson-Vidal C, Morandi V, Duong-Van-Huyen JP, Bruneval P, Dignat-George F, Emmerich J, Gaussem P. “Thrombospondin-1 is a plasmatic marker of peripheral arterial disease that modulates endothelial progenitor cell angiogenic properties”. Arterioscler. Thromb. Vasc. Biol  2011. 31 (3): 551–559. http://atvb.ahajournals.org/content/31/3/551

12. G. D. Grossfeld, D. A. Ginsberg, J. P. Stein et al., “Thrombospondin-1 expression in bladder cancer: association with p53 alterations, tumor angiogenesis, and tumor progression,” Journal of the National Cancer Institute 1997 vol. 89, no. 3, pp. 219–227. http://www.scopus.com/record/display.url?eid=2-s2.0-18744423089&origin=inward&txGid=9C86356DDB0B6816ACCBF90F9CA44E92.WlW7NKKC52nnQNxjqAQrlA%3a2

13. Chao MP, Weissman IL, Majeti R. “The CD47-SIRPα pathway in cancer immune evasion and potential therapeutic implications”Curr. Opin. Immunol 2012. 24 (2): 225–32. http://www.sciencedirect.com/science/article/pii/S095279151200012Xhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3319521/

14. Majeti R, Chao MP, Alizadeh AA, Pang WW, Jaiswal S, Gibbs KD, Jr, van Rooijen N, Weissman IL. Cd47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell. 2009;138(2):286–299. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726837/

15. Chao MP, Alizadeh AA, Tang C, Jan M, Weissman-Tsukamoto R, Zhao F, Park CY, Weissman IL, Majeti R. Therapeutic antibody targeting of cd47 eliminates human acute lymphoblastic leukemia.Cancer Res. 2011;71 (4):1374–1384. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3041855/

16. Chao MP, Alizadeh AA, Tang C, Myklebust JH, Varghese B, Gill S, Jan M, Cha AC, Chan CK, Tan BT, Park CY, et al. Anti-cd47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-hodgkin lymphoma. Cell. 2010;142(5):699–713. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943345/

17. Chao MP, Tang C, Pachynski RK, Chin R, Majeti R, Weissman IL. Extranodal dissemination of non-hodgkin lymphoma requires cd47 and is inhibited by anti-cd47 antibody therapy. Blood.2011;118(18):4890–4901. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3208297/

18. Zhao XW, van Beek EM, Schornagel K, Van der Maaden H, Van Houdt M, Otten MA, Finetti P, Van Egmond M, Matozaki T, Kraal G, Birnbaum D, et al. Cd47-signal regulatory protein-alpha (sirpalpha) interactions form a barrier for antibody-mediated tumor cell destruction. Proc Natl Acad Sci U S A.2011;108(45):18342–18347. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3215076/

19. Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, Castedo M, Mignot G, Panaretakis T, Casares N, Metivier D, et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med. 2007;13(1):54–61. http://www.ncbi.nlm.nih.gov/pubmed/17187072

Other related articles on this Open Access Online Scientific Journal include the following:

I. By: Larry Bernstein MD. Treatment for Metastatic HER2 Breast Cancer https://pharmaceuticalintelligence.com/2013/03/03/treatment-for-metastatic-her2-breast-cancer/

II. By: Tilda Barliya PhD. Colon Cancer.  https://pharmaceuticalintelligence.com/2013/04/30/colon-cancer/

III. By: Ritu Saxena PhD. In focus: Triple Negative Breast Cancer. https://pharmaceuticalintelligence.com/2013/01/29/in-focus-triple-negative-breast-cancer/

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Author and Curator: Chloe Thomas, Manager, Scientific Sessions and Education at Heart Rhythm Society

 

One step further towards an HIV vaccine

Statistics show that approximately 34 million people are infected with the Human Immunodeficiency Virus. Within the last years, important steps have been taken in finding treatments and medications against HIV. The study introduced in this article is a helpful contribution to the development of an HIV vaccine.

Cloning antibodies

Researchers working in the California Institute of Technology have focused more closely on the binding mechanism of the virus to the human cell. Leading a study which was published in the Science Magazine in 2011, they departed from the fact that a passive transfer of HIV neutralizing antibodies can prevent an infection and might therefore even be valuable for the creation of an HIV vaccine. As the number of naturally occurring antibodies is relatively low, the researchers intended to discover whether these antibodies belong to a larger group of molecules which might turn out useful studies of the infection. By cloning more than 500 HIV antibodies taken from four different infected individuals, they discovered that all of them produced a large number of potent HIV antibodies which mimic the binding to CD4. By isolating the potent anti-HIV antibodies of infected people, the scientists have begun to develop ways in order to neutralize subtypes of the infection. The researchers have found a strong version of an anti-HIV antibody, which is named NIH45-46. These antibodies that target the binding site of the host receptor (namely CD4) interact with the protein gp120. This protein sits on the viruses and helps the virus enter the cell, and thus mainly contributes to the infection process. The interaction between antibody and the protein leads to neutralizing the virus and thus may avoid infection. Knowing this, the scientists were able to develop an even stronger type, named NIH45-46G54W, which employs the described mechanism more effectively. The next step the researchers are advocating is a clinical testing period for the newly-created effective antibody. Through that, they hope to gain further information on understanding the neutralization of the virus which might even help in developing a vaccine against HIV.

Scientific research: a long process

Despite the success of the study, it is important to note that an analysis in the laboratories and a clinical testing phase has to be conducted over a long period of time in order to bring about representative results. Methods have to be considered, antibodies suppliers like here have to be contacted, and data have to be evaluated. For that reason, the development of an HIV vaccine cannot happen overnight, but should be furthered patiently.

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