2013.06.26 Survey: Do you outsource early preclinical research activities?

posted Jun 25, 2013, 10:13 PM by Sirid Kellermann   [ updated Nov 14, 2013, 7:55 PM ]

Over the years, we’ve gotten to know folks working in a spectrum of biopharma organizations – from large and well-established biopharma corporations, to virtual, often asset-centric companies. Despite their differences, it turns out that all types of companies may outsource some or all of their preclinical R&D – but for very different reasons, ranging from internal capacity constraints to, well, not having a wet lab at all. 

Wanting to understand more about the different motivations behind outsourcing preclinical research, we’ve created a brief survey that we would be thrilled to have you participate in. 

Particularly if your company outsources (or plans to) relatively early research activities, such as 

  • discovery research
  • lead identification
  • identifying compound mechanism of action
  • early identification of biomarkers of efficacy and side effects
...we definitely want to learn more from you! 

{This survey is now closed. Thanks for your responses!}
You may ask, “isn’t this information already out there?” If you know where we can find it, drop us a line! In our own research on the subject, we find that the early-stage outsourcing is not well-analyzed. 

For example, a 2011 Tufts CSDD study on contract research organizations (CROs) did include an “Applied Research” category(1). However, Applied Research is still fairly broad, encompassing everything from Proteomics to pharmacokinetics to lead optimization. And for the purposes of this survey, we're not interested in many of these activities, and also not ADME, toxicity, bioanalytical stuff, etc.

Notwithstanding that we occasionally hear about early preclinical partnerships - for example, Debiopharm Group's recently announced partnership with preclinical CRO Cenix BioScience to discover biomarkers that aim to enhance personalized medicine - this sector of contract research providers is smaller than the clinical CROs, and many of them (over 80%, according to Tufts CSDD) are privately held, which means information is hard to come by.

(1) Results of the Tufts study were summarized by Getz et al. in Contract Pharma ("Resizing the Global Contract R&D Services Market: A new study revises estimates of the market." Published May 30, 2012.) See more at: http://www.contractpharma.com/issues/2012-06/view_features/resizing-the-global-contract-rd-services-market/
Got two minutes?
Why not use them to complete our survey? Once you do, you’ll have a chance to win a gift certificate to an organization near and dear to our hearts, Better World Books. Besides being a great source of new and used books, BWB has raised millions of dollars for literacy and saved millions of books from landfills. So come on over and take our survey - and start thinking about what summer reading you may be spending your prize on!

(And if you've already have taken the survey - thanks!) 

If you win the Better World Books gift certificate, you could buy yourself a nice big book like this one.

If you win the coveted Better World Books gift certificate, perhaps you will select a big beach read like this lucky lady! (Source: National Gallery)

2013.04.18 IL-1: A new paradigm for an ancient family

posted Apr 18, 2013, 9:40 PM by Sirid Kellermann   [ updated Nov 15, 2013, 10:08 AM ]

IL-1, as the name implies, was the first interleukin to be identified and characterized in the early '80s. We have known for some time that IL-1 signals by binding to a heterodimeric receptor that combines a so-called primary receptor, IL-1RI, with an accessory receptor, IL-1RAcP. 

The IL-1 family and their receptors has grown over time and, with advancing research, their biological significance is emerging (see Arend, 2008; our earlier 4A update on IL-33). The ligands and their receptors can be clustered into smaller families, each of which includes ligands (IL-1ra, IL-1F7) and/or receptors (IL-1RII, SIGIRR (TIR8)) that act as inhibitory decoys, serving to block signaling in a manner that is likely to be tightly regulated, although the exact details are still being elucidated.

How IL-1 family members interact with their receptors to trigger signaling was unclear until 2010, when Wang and colleagues published the structure of IL-1beta complexed with IL-1RII (a decoy receptor) and IL-1RAcP. The results suggested an unexpected ternary configuration in which the receptor components IL-1RII and IL-1RAcP are orthogonal to each other (right panel, below), contrary to what was believed for growth hormone receptor-like complexes, that the ligand would be central in nucleating the assembly of the two coreceptors (left panel).

Since IL-1RII is a decoy receptor, there was speculation about whether this unusual configuration might be unique to nonsignaling complexes. Further confirmation that it was, in fact, likely to be shared by activating IL-1/receptor complexes was provided in 2012, when our principal consultant, Fernando Bazan, and his colleagues Christoph Thomas and K. Christopher Garcia at Stanford reported the structure of the signaling-competent heterotrimer, IL-1beta/IL-1RI/IL-1RAcp in Nature Structural & Molecular Biology.

"It's reasonable to predict that all members of the IL-1 family will follow this ternary complex paradigm," Fernando postulates, allowing us to develop the model below.

The IL-1 family of ligands and receptors. The eleven known ligands are shown in the outer circle, and bind to primary high-affinity IL-1 receptors (middle circle), which thereupon associate with secondary receptors (inner circle). The inhibitory effect of SIGIRR on IL-1RI and ST2 needs further substantiation. The ligands and coreceptors for IL1RAPL1 and IL1RAPL2  have not been identified to date. Adapted from Thomas et al. (2012).

In summary, this proposed ternary complex paradigm allows us to organize the entire IL-1 system - which also reveals holes that need to be filled in through target discovery, for example to build out the biology of IL-1RAPL1 and -2. These orphan receptors are interesting as they are primarily expressed in the brain, and mutations are associated with X-linked non-syndromic mental retardation mutations. 

"I don't think there are any new alpha receptors remaining to be found," Fernando comments, "so we need to look for CNS expression of one of the existing alpha receptor and piece together the ternary complex. There may, however, be additional unidentified ligands." This pairing exercise could be executed reasonably easily in vitro, but no one's reported any results - maybe because it's primarily of interest to neurobiologists. Another example of scientists in different disciplines not communicating and possibly missing some very interesting biological insights.

The paradigm allows researchers to quickly infer the contact sites between IL-1 family members and their receptors, which may speed drug design. If the history of the IL-1 family is any indication, we have yet to discover new biological with the potential for new therapeutic breakthroughs in the future.

2013.03.05 The buzz on therapeutic antibodies

posted Mar 5, 2013, 2:46 PM by Sirid Kellermann   [ updated Nov 15, 2013, 10:11 AM ]

4th & Aspen recently attended the Keystone Symposium on "Antibodies as Drugs" in Vancouver, BC. Here are some of the takeaways from this informative meeting (not least of which was that you can buy a hot dog with seaweed on it from a Japadog stand!).
Antibodies as drugs are well-proven and mature. 

With over 30 approved since 1986, and revenues nearing $60B in 2013, antibodies represent a validated approach to the therapy of inflammatory disorders, cancer, and other indications. Antibody-drug conjugates (ADCs) are coming of age, with the approval of Roche/Genentech's Kadcyla (T-DM1) shortly after this conference being the most recent testimonial to the successful introduction of ADCs in cancer immunotherapy. 

The next frontier: greater (tissue) specificity.

Although antibodies are, by nature, extraordinarily specific for their protein target, it's a challenge to identify targets that are solely expressed in diseased tissues and not healthy ones. Overcoming the toxicity caused when ADCs trigger collateral damage in healthy tissues is an area of intense activity. One interesting approach was described by Henry Lowman, CSO and founder of Cytomx, which creates protease-activated antibodies called Probodies™. Here, a linker moiety is attached to a therapeutic antibody, rendering it effectively nonbinding until said linker is cleaved by cell surface proteases known to be overexpressed in diseased tissue.

In addition, there's new developments in the combination of ADCs with kinase inhibitors. Paul Polakis mentioned that researchers at Genentech are leveraging the observation that kinase inhibitors trigger a transcriptional response that upregulates cell surface receptors, making them more receptive to killing by ADCs. Nothing published yet, but we're avidly looking forward to learning more about this approach.
Of mice and camels (or not).

Human antibody-transgenic mice are alive and well, and the reason is in the numbers: A significant proportion of clinically successful therapeutic antibodies are derived from such transgenic mice or from human B cells, likely reflecting the superiority of the natural affinity maturation process (summarized in this concise report by 
David Meininger, Executive Director of Molecular Discovery at Merck (Palo Alto)).
It's been n
early two decades since Abgenix's XenoMouse and Medarex's UltiMab transgenic mice hit the scene, and while both companies have since been respectively acquired 
by Amgen (2006) and
Bristol-Myers Squibb (2009)
, there's no shortage of new alternatives, including Regeneron's VelocImmune mice and Ablexis's AlivaMab mice. Most recently, Crescendo Bioscience introduced the TKO mouse, which generate fully human single domain antibody VH fragments that are much smaller than full-size antibodies and as such have certain desirable characteristics and can bind certain epitopes that  antibodies can't.

Speaking of tiny antibodies, we were surprised by the near non-presence of any discussion of camel antibodies and their derivatives (e.g., Ablynx's nanobodies)monobodies, a binding scaffold based on fibronectin type III domains (FN3), were also MIA. Considering that a number of nanobodies and monobodies are currently in clinical trials in a variety of disease indications, we'd expected more of a presence at this conference.

Molecular engineering is a vibrant area of R&D.

In addition to FN3-based monobodies, there are a number of up-and-coming alternative recognition scaffolds. For example, Molecular Partners' Vice President of Technology Kaspar Binz discussed DARPins (Designed Ankyrin Repeat Proteins), pioneered by Andreas Pluckthün's laboratory at the University of Zürich. DARPins' small size and other biophysical features allow the design, for example, of multi-specific, linked DARPins that can bind several distinct epitopes within the same protein. Binz related how this was being leveraged to target the EGF receptor, a popular cancer target (a 2011 publication elaborates on the EGFR example). The commercial and clinical potential of DARPins has been appreciated by at least one pharma company, Allergan, which did a deal with Molecular Partners late last year focusing on diseases of the eye.

Other multispecific approaches include bi-specific T cell engagers, or BiTEs (technology acquired by Amgen through its 2012 purchase of Micromet and assets including blinatumomab, a BiTE that targets T cells expressing CD3 to malignant B cells bearing CD19). The bispecific approach is also particularly applicable in therapeutic strategies aiming to target heterodimeric surface proteins, with each binding site targeting one of the heterodimer subunits.

A few years ago, several Genentech scientists published an intriguing paper about a "two-in-one" antibody they'd engineered, using Herceptin as a starting point, that was able to recognize not only Her2 but also VEGF. This is notable not only for its protein engineering virtuosity, but also because in a practical sense the concept is similar to that of "dirty" (multi-specific) kinase inhibitors, that may prove to be more effective than single-kinase inhibitors because they target multiple signaling pathways simultaneously. There's been some buzz that the multispecific antibody feat has been replicated elsewhere, but unfortunately, no information was forthcoming at this conference.

Synthetic antibodies and scaffolds represent new ways to design therapeutics entirely computationally, as advocated by David Baker at the University of Washington. While this purely physics-based approach yields scads of potential solutions, it may not be sufficiently high throughput and requires a large amount of computing power. Andy Ellington at the University of Texas-Austin noted that of 100 such solutions screened, only 2-3 have the properties that could make them potential therapeutic candidates. Ellington advocates using high throughput sequencing of antibody complementarity-determining regions (CDRs) derived from a bona fide polyclonal human immune response to inform and 'educate' protein design computational algorithms in order to design better therapeutics more efficiently.

On another matter, it seems that the risk of immunogenicity that occupied a significant part of our own research back in the mid-2000's has been greatly diminished, as computational approaches can essentially 'anti-design' structures to edit out potentially immunogenic sequences.

Technology is only as effective as having the right targets. 

In the end, antibodies and other next-generation targeting strategies are tools whose efficacy is  directly related to the quality of their target(s). We noticed representatives from a number of larger biopharma companies at the meeting who weren't so much there for the antibody technology presentations as they were to scout for new targets and early proof-of-concept data to feed their preclinical pipelines. 

This reinforces our belief at 4th & Aspen that there's a very real demand in the biotherapeutics space not only for new technologies, but also for novel disease-relevant proteins and signaling pathways on which to focus these targeting efforts.

2013.02.04 Novel Protein Discovery: The IL-33 Story

posted Feb 4, 2013, 1:24 PM by Sirid Kellermann   [ updated Nov 14, 2013, 7:56 PM ]

Have a look at these two girls. Can you see any similarities? I can’t - and yet, they’re twin sisters. Just like these sisters, there are proteins whose folding or functional relationships can only be discerned through sophisticated analysis based on a deep understanding of the molecular evolution of protein structures. The cytokine IL-33 is an excellent example of a long-lost molecular sibling; this is the story of how it was found.

Today, IL-33 and its receptor, ST2, are the focus of intense research in both academia and industry. But as recently as 2005, ST2 was an intriguing but orphan IL-1 family receptor whose function remained elusive since its cloning 16 years earlier by Shin-Ichi Tominaga’s group. Investigators at several biotechnology companies (notably Millennium - now Takeda) deduced that dysregulation of ST2 was likely tied to chronic autoimmune and inflammatory diseases, and sought to block its activity with antibodies. 

4th & Aspen's principal consultant, Fernando Bazan, recalls: “Although research discerned the expression pattern of ST2, and gleaned that it was involved in certain Th2-like immune responsesthe field could not move forward without identifying the physiological ligand and understanding the triggering of ST2 signaling inside cells.” Yet, despite many efforts, the ST2 ligand stubbornly eluded identification.

Fernando and his team at the DNAX Research Institute finally cracked the case, based on the logic that since ST2 is a distant member of the IL-1 receptor family, it should engage a β-trefoil fold cytokine from the IL-1 family. Using structural insights and the power of expanding genomic databases, Fernando iteratively refined his computational searches until a candidate molecule was fished out of a genomic screen of canine trauma-induced genes. This protein, and its deduced human homologue, lacked a signal peptide, but instead possessed a hallmark pro-domain similar to non-classically secreted IL-1 cytokines, as well as a predicted β-trefoil domain.

This discovery was the newest member of the IL-1 family, christened IL-33. Fernando and his colleagues rapidly cloned and expressed the globular domain of IL-33 and showed that the cytokine binds and signals through ST2. “Finally finding the ligand for ST2 permitted us and others to show that inflammation is exacerbated when you stimulate ST2-bearing cells with recombinant IL-33,” Fernando recalls.

The team’s seminal publication in Immunity has been cited over 850 times, and sparked research that attests to IL-33’s diverse roles in a range of disorders including infection, cardiovascular disease, and kidney disease. As for its role in inflammation, “we now know that the Th2 connection is likely due to the stimulation by IL-33 of a very specialized class of IL-33 responsive immune cells called innate lymphoid cells or ILCs, which then secrete Th2-like cytokines,” says Fernando. “That means that the field now has new cellular targets for therapeutic efforts above and beyond the discrete IL-33 signaling complex, benefiting  people suffering from a wide range of debilitating diseases.”

Fernando continued to characterize IL-33 with colleagues at Genentech and Stanford, culminating in publications in Structure and Nature Structural & Molecular BiologyThis work has led to a comprehensive picture of IL-1 family receptor complexes.

So, what can the IL-33 story teach us? Just like those twin girls in the picture look nothing like each other, in the protein world as well, it's important to focus on deeper evolutionary links to gain clues about the relationships between molecules, complexes and signaling networks. 

And armed with that kind of information, therapeutics R&D is a lot less guesswork.

2012.12.13 How Frizzled domains really work

posted Feb 4, 2013, 1:21 PM by Sirid Kellermann   [ updated Nov 14, 2013, 10:43 AM ]

Fernando Bazan shares the fascinating story of how he and his colleagues at Genentech and Stanford University elucidated the true nature of ligand binding to Frizzled (Fz) and Smoothened (Smo). These findings have had significant and continuing impact on the design of next-generation oncology therapeutics. 

Wnt and Hedgehog (Hh) are families of secreted, lipid-tagged proteins that play critical roles in cell development, tissue patterning, and organ development. The evolutionarily conserved intracellular signaling pathways triggered by these morphogens have captured significant research interest, because (1)malfunctions in these pathways are implicated in many types of cancers; (2) emerging regulatory ties to immune cell activation; and (3) mechanistic links to sensory cilia trafficking and signaling.

Wnts impact cell signaling directly by binding a family of Frizzled (Fz) GPCRs. In contrast, Hhs exert their effects indirectly, by engaging a cell surface transporter called Patched and triggering the release of unknown, possibly sterol-like molecules that act as activating ligands for a distant Fz-like GPCR, Smoothened (Smo). Smo in particular has been the target of  vigorous drug development efforts, although with the more recent appreciation of the role of Wnt signaling in cancer, Fz's are increasingly garnering attention as therapeutic targets for intervention.

As Fernando recalls, “I started looking at these receptors soon after arriving at Genentech. The company had generated small molecule antagonists of Smo that showed efficacy against basal cell carcinoma - in fact, Erivedge was approved by the FDA for treatment of BCC earlier this year. I was part of a team that sought to find a structural explanation for how these drugs worked. I pursued the notion that Smo, besides possessing an obvious binding site in its core GPCR cavity, may have additional binding site(s). Identifying these alternative pockets would help us better explain the enigmatic mechanisms of action of diverse Smo-active drugs in the literature (which ran the gamut from full agonists to full antagonists) and allow us to more intelligently design effective compounds.”

Fz's and Smo both possess cysteine-rich Fz ectodomains. Fernando’s work led to the surprising proposal that the Fz module is endowed with a natural binding cavity (or groove) within a cage-like array of  disulfide-linked helices. Moreover, this unique fold is found in several other protein families, including the NPC2 cholesterol transporter as well as folate receptors. As Fernando describes in the accompanying video, this connection between apparently unrelate proteins is not immediately obvious by visual inspection or comparison using standard computational approaches.

These structural and evolutionary links are intriguing, considering that NPC2 and folate receptors are known to shuttle cholesterol and folate-like molecules, respectively, while the proposed ligands for Fz and Smo are lipidated Wnt proteins and oxysterols, respectively. Taking the analysis further, Fz domains appear to be related to pheromone- and lipid-binding secreted proteins found in insects.

This work led to the prediction that the lipid moiety of Wnts is a critical component of their interactions with Fz's. After Fernando and his colleague, Fred de Sauvage, published this insight in 2009 paper in Cell, it provided a tantalizing clue to solving the structure of Wnts, which have historically been intractable to crystallography since no one could find a way to make significant amounts of pure, stable, and soluble Wnt protein. K. Christopher Garcia’s group at Stanford leveraged Fernando’s prediction by cleverly coexpressing the Fz ectodomain with Wnt, thereby stabilizing the protein and creating sufficient amounts to be crystallized.

“In our Cell paper, we hypothesized that all Fz ectodomains would be found to bind lipids, sterols or folates. This is supported by observations that at least some agonists and antagonists of Smo do not appear to bind in the GPCR’s cavity. Might they be acting allosterically, by interacting with Smo’s Fz domain?”
- Fernando Bazan


The Garcia team published the structure of Wnt in 2012 in Science, revealing how Wnt grips the globular Fz ectodomain at two sites (see video at 2:10), somewhat like a ball being pinched between a thumb and a forefinger – where the tip of the Wnt "thumb" has a lipid moiety that docks in Fz's hydrophobic groove, in accord with Fernando's proposal that this was a lipid-sequestering site. 

Listen to Dr. Garcia tell the story in this Science podcast.

Moreover, the structure of Wnt showed that it possesses a remarkably complex, novel fold. “I realized that this odd Wnt structure was actually a molecular tapestry of several modular folds, each conferring a unique functional attribute to the morphogen,” says Fernando. “There are two key folds, as we outlined in a subsequent 2012 paper, coauthored by Dr. Garcia and his group in Developmental Cell: an N-terminal saposin-like module designed to interact with lipid membranes, fused to a C-terminal domain reminiscent of a degenerate cystine-knot cytokine fold. The former module contains the lipid attachment site, while the latter cytokine-like domain is charged with making a second, protein-protein contact with Fz. That second domain also likely engages the LRP family member that completes the ternary Wnt/Fz/LRP complex. Quite likely, Wnts evolved from the ancient fusion of two genes encoding these smaller protein domains, creating a molecule with composite functions.”

New possibilities in drug development
The prediction that Smo, Fz, and other proteins expressing Fz ectodomains interact with their ligands through lipid binding, in addition to insights about the structure of the Fz ligand, Wnt, open new possibilities for the design of small molecule compounds and biotherapeutics (video, 4:09). The Fz ectodomain’s lipid-binding groove is a natural hotspot for drug binding. In the case of biotherapeutics, deconstructing Wnt into its component parts through protein engineering can help create superbinders that interact extremely tightly with receptors that bear Fz ectodomains, while antibodies directed against the lipid binding groove may be effective in blocking the binding of Wnts.

"The (field) has been ignoring that there's this readily druggable domain that will be an excellent antagonist binding site," Fernando concludes (video, 7:08).

And there’s a third therapeutic strategy, as Fernando points out: “We may be able to specifically target Wnt’s interaction with LRP5/6, the second signaling receptor of the Fz supercomplex, through the interaction site found in Wnt’s cytokine-like subdomain. That’s an area of pretty intensive research activity right now.”

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