Interesting #AACR16 Abstracts and Themes

There were a number of AACR abstracts that I found interesting, and many more that just had provocative titles. Many of the symposiums and plenary sessions just had the speakers and titles of their talks, so I am sure there will be much more presented at AACR than what I can glean from the listed abstracts and titles. However, I decided to give my thoughts on a number of them, below. I kind of went down the rabbit hole, as I normally do, and made this blog post longer than I intended, so feel free to pick and choose the parts that seem most interesting. The AACR meeting starts April 16th, and while I unfortunately will not be able to go, I will absolutely be following the presentations and the coverage of the new research.

CARs that act like TCRs
Eureka Therapeutics: ET1402L1-CART, a T cell therapy targeting the intracellular tumor antigen AFP, demonstrates potent antitumor activity in hepatocellular carcinoma models (ADAP)
Eureka is a company that uses phage display to screen for antibodies that can bind to specific peptide-MHC complexes, so they can have the specificity of a T cell receptor (TCR), but in the modality of an antibody. This allows antibodies to "target" intracellular proteins. They are also developing an antibody against WT1 (called ESK1). This antibody was developed with the Scheinberg lab at MSKCC, where they made the original monoclonal antibody (described here), and more recently turned it into a CD3-bispecific antibody here. Not surprisingly, Eureka is also considering using these antibodies to make the scFv portion of a chimeric antigen receptor (CAR), and appears to have done so here, with their lead antibody ET1402, which apparently is able to target AFP. Interestingly, Adaptimmune also has an AFP program entering clinical trials, although they are using the more traditional engineered TCR approach to target it. I don't think it's clear whether it's better to target peptide-MHC complexes with normal TCRs, or with TCR-like antibody CARs (with costimulatory domains), so it will be interesting to see how it plays out. Eureka's pipeline (although very little is disclosed) is listed here.

CAR Design - new safety switches
UPenn: Reversible regulation of chimeric antigen receptor surface expression (NVS, BLCM, CLLS, JUNO, KITE)
Engineered T cells using CARs or TCRs have the potential for serious side effects, both on-target/on-tumor (CRS), and on target/off tumor (HER2 CAR lung tox), in addition to straight off-target (Titin cross-reactive MAGE-A3 TCR). There are numerous safety switch technologies different groups have developed to either remove the engineered T cells or, more recently, control CAR expression/function. The safety switch designs currently being used in the clinic generally rely on inducible removal of the engineered T cells, such as Bellicum's inducible caspase 9 approach, and Juno's truncated EGFR, although the latter has never been used as a safety switch in the clinic. In UPenn's abstract here, they wanted to design a CAR that could be inducibly down-regulated, to avoid irreversibly ablating the engineered T cells, which occurs with the previously mentioned strategies. UPenn's approach is to attach an inducible degradation component to the CAR construct. I believe the one they are using is this LID degradation system from Stanford published here. Basically, similar to Bellicum's approach, they use rapamycin or a rapamycin-related molecule to specifically bind to a protein, and in this case, reveal a degradation motif that targets the CAR to be degraded by the cell's machinery. From the abstract so far they have only tested it in culture, not in animal models, and they were able to achieve up to 80% reduction in the CAR levels by 24 hours, which they state was able to reduce the CAR-T cell's activity back down to near background levels.

There are a number of other approaches that try to tune CAR-T activity with destroying the engineered cells. One is Bellicum's inducible activation GoCAR-T approach, which uses their inducible dimerization construct to activate costimulatory signaling, as opposed to their CaspaCIDe construct, which activates cell death. Wendell Lim's lab previously published a system where they split the intracellular components of the CAR into two pieces that could be reconstituted into the full CAR after addition of a small molecule dimerizer (using a rapamycin-based molecule) to induce CAR activation. Cellectis also published a recent paper showing they could control that they could control the surface expression of CARs using novel extracellular architecture that could be regulated by a small molecule (also using rapamycin-based molecules).

These approaches are further distinguished from those that try to integrate multiple inputs to make smarter CARs that may be able to expand the potential pool of CAR-T surface targets. A recent example is a strategy from Wendell Lim's lab that was published in back-to-back cell papers which used a synthetic Notch receptor to control expression of a chimeric antigen receptor. Michel Sadelain's lab has previously published both systems that activate in response to the simultaneous presence of two antigens, or activate in response to cells that have one antigen but specifically lack the expression of a second antigen

There will also be a symposium at AACR specifically on next generation CAR approaches, which I expect will, at least in part, focus on the costimulatory domains, highlighted in these recent papers: 4-1BB vs CD28 (June), CD28 CAR + 4-1BBL surface expression (Sadelain).

TRK inhibitors face off (LOXO, RXDX)
Loxo and Ignyta are both presenting their early data for their TRK inhibitors in precision medicine clinical plenary session. While neither of the abstracts for their clinical data are available, both have published/presented case reports of clinical efficacy, as well as resistance (Loxo here & here; Ignyta here. Interestingly, there is a new abstract from Ignyta saying that the resistance mutations Loxo identified to Loxo-101 in the above presentation did not affect sensitivity to Ignyta's entrectinib (as well as other gatekeeper mutations in ALK or ROS1). This is a bit surprising, since one of those mutations was a G667 mutation, which was one of the mutated positions in the patient who became resistant to entrectinib in the paper above. It will definitely be interesting to see how the safety, efficacy, and durability of response shake out between Loxo and Ignyta for what appears to be a good target in the small % (although wide variety) of NTRK-rearranged tumors.

New MAGE-A3 TCR from NCI (KITE)
The NCI (in a CRADA with KITE) will be presenting data from its new MAGE-A3 TCR program in solid tumors in one of the immuno-oncology clinical trial plenary sessions. MAGE-A3 has been a difficult target for TCRs, with off-target neurological toxicity & cardiac toxicity in previous TCRs. This new TCR had some data presented previously showing a PR in 3/12 patients, and importantly no off target toxicity. MAGE-A3 is a cancer/testis antigen that are a class of antigens that appear to be good targets for engineered TCRs, with TCRs targeting another cancer/testis antigen, NY-ESO-1, already having some evidence of efficacy in the clinic in synovial sarcoma, melanoma (KITE), and multiple myeloma (ADAP). MAGE-A3 may also be expressed in a higher percentage of tumors than NY-ESO-1, and thus may have a greater addressable population.

Also, most likely just preclinical, but there will be a poster on an NCI/KITE KRAS TCR as well.

Immuno-oncology early trial updates (Genentech, KITE)
In the two plenary sessions for immuno-oncology trials, it appears there will be new clinical data for PD-1 (&CTLA-4) antibodies, but also some data from new targets in the second plenary session.

One presentation will be on an OX40 agonist from Genentech. OX40 is one of numerous additional T cell surface targets investigators are looking to target in combination with PD-1 or CTLA-4 antibodies. In contrast to those inhibitory receptors, where antibodies are used to block signaling, OX40 is a stimulatory receptor, and the therapeutic antibodies have been designed to activate its signaling

The other presentation will be on pegylated-IL10 from ARMO Biosciences. While there is no data in their abstract, I believe investigators have recently presented data from this program at the ITOC3 conference here (search pegylated), where they saw a 25% PR rate in RCC.

Kite will also be presenting updated CD19 CAR data in NHL, which I am sure will be closely watched.

Targeting Neoantigens (BioNTech, ZIOP, KITE)
Neoantigens are potentially personalized medicine to its most extreme for cancer therapy - directly targeting the presence of specific mutations unique to that patient's tumor. Neoantigens are thought to be a major target source for the endogenous immune response - reviewed here. Amplifying this response specifically, with vaccines or t cells (TILs or engineered TCRs), is a hot area of focus, with a sudden swelling of startups focusing on this approach. As far as I can tell, the only neoantigen vaccine presentations that will be at AACR are from BioNTech, which will present a number of neoantigen vaccines programs in Melanoma, GBM (collaboration with Immatics), and TNBC. While neoantigen vaccines are an interesting approach (and potentially easier to manufacture than neoantigen-TCR T cells), vaccines have had difficulty in producing robust efficacy in cancer, whereas neoantigen T cells (in the form of TILs) have had sometimes impressive efficacy (reviewed here). So I am reserving judgement on the potency of neoantigen vaccines until we start to see more clinical proof of concept.

While it appears Steve Rosenberg will only be presenting the MAGE-A3 TCR data, there are also two recent papers from his group showing they could prospectively identify neoantigen TCRs in peripheral blood by using PD-1 as a marker, and in a collaboration with Ziopharm, use a transposase-based system to make neoantigen-TCR T cells. Kite is also working with Rosenberg to develop neoantigen-specific TCRs. The key will be the feasibility of identifying, cloning, and expressing the neoantigen-specific TCRs, and I will be very interested in seeing how this approach is developed by the numerous groups pursuing it.

The best T cell composition for CAR-T products (KITE, JUNO)
There is also debate in what is the best T cell (or composition of T cells) for a CAR-T product. Kite Pharma will be presenting data saying that in NHL, neither the CD4:CD8 composition nor the differentiation state clearly correlated with response, while the final CAR-T product tends to be less differentiated than the initial T cell population. Paul Rennert reviewed this area in a very nice series of blog posts here. Alexey Bersenev also had a great post on this here.
 
Novartis EGFRvIII CAR-T in GBM : Phase I study of T cells redirected to EGFRvIII with a chimeric antigen receptor in patients with EGFRvIII+ glioblastoma (NVS)
While EGFRvIII seems like a great target for a CAR as it is surface protein and the target is exclusively found on the tumor, I do not believe there have been any significant responses reported for CARs targeting it. I will be interested in seeing if there have been any better responses, as well as the expansion, persistence, and trafficking to the brain (if data is available) for the CAR-T cells.

Targeting Ras
Ras has been a difficult, often called undruggable, but much sought after target in cancer due to its role in a large percentage of human tumors. Recently, there appears to be renewed emphasis on approaches to target Ras directly or downstream, and I will be interested in following what appears to be real progress in approaches to drug Ras.
There are two sessions on targeting Ras mutated tumors: Progress in Treating KRas Mutated Tumors session, and From Chemistry to the Clinic: Inhibition of K-Ras
While most of the abstracts have not been published yet, there are a number of approaches that are interesting:
Covalent inhibitors of Ras - one is described in an abstract here.
Trapping mutant KRas G12C in its inactive state - abstract (title only), paper.
Allosteric inhibition of Ras - abstract (title only), paper.

BET-bromodomain Inhibitors in NUT-midline carcinoma
There seem to be a ton of BET-bromodomain inhibitors in development, starting from JQ1, and now there seem to be multiple beginning to generate clinical data, primarily in NUT-midline carcinoma, where tumors appear to be especially dependent due to frequent NUT-BRD4 fusion events.
In a clinical trials minisymposium at AACR there will be data from: GSK525762, a selective bromodomain (BRD) and extra terminal protein (BET) inhibitor: results from part 1 of a phase I/II open-label single-agent study in patients with NUT midline carcinoma (NMC) and other cancers 

Previously there has been clinical data presented on a compound from Tensha: Clinically efficacy of the BET bromodomain inhibitor TEN-010 in an open-label substudy with patients with documented NUT-midline carcinoma (NMC)

There was also just recently a publication on a Merck-licensed compound that also had activity in NUT-midline carcinoma - here in Cancer Discovery.

Dissecting the complex ecosystem of malignant tumors with single cell RNA-Seq
While there was no abstract for this title, this is an approach that seems like it could significantly improve the resolution with which we can look at all the different cell types simultaneously and separately in a tumor. One of my favorite papers from last year used a cytolytic gene expression signature from large genomic data sets to see what tumor properties correlated with what appeared to be an immune response. I would imagine this analysis could be even more powerful in identifying ways in which the tumor and the immune system co-evolve if we had resolution of all the different cell populations instead of looking at a single tumor signature for both the tumor and all the different stromal cells. Update: The paper for the above presentation appears to just have been published in Science, here.
  
Single-cell TIL profiling (JUNO)
Juno does not appear to be presenting much work on CARs at this AACR, but I actually found this non-CAR abstract very interesting. This approach from Juno seems like it could be very powerful for identifying what is going on in individual TILs as at the single cell level, they claim to be able to sequence the TCR, while also getting a snapshot of that T cell's transcriptome and proteome. I could imagine this level of resolution could lead to the identification of additional T cell pathways of activation or repression in different T cell populations in the tumor, such as PD-1+ TILs or neoantigen-reactive TILs.

Identifying novel immunotherapy targets from human tumor data from PD-1 & CTLA-4 antibody trials:
There are so many potential immunotherapy targets for either monotherapy or combination with checkpoint blockade that it can be overwhelming. An approach I like that may help sort out which are the critical targets is starting with data sets generated from human tumors, particularly the differences in responders versus non-responders to checkpoint blockade. In this way, we can try to let what's going on in human tumors tell us what is important to escape immune surveillance. One can see this approach in a number of abstracts, such as:
The tumor immunity continuum as a framework for rational combinations or Immunogenomics and precision cancer medicine among others.

There have also been a number of recent papers published using approaches like this:
Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints

Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma

Melanoma-intrinsic β-catenin signalling prevents anti-tumour immunity.

There are many more interesting projects and themes in oncology research that I'm sure will be presented at AACR, but I will somewhat arbitrarily stop here. Any discussion is welcome here or on twitter - @Festivus159.

Baxalta making an investment in universal CAR T cells

by Robert Kruse


Last week, Baxalta ($BXLT) made a significant partnership investment up to $1.4 billion in a small, private company, Precision BioSciences, located in North Carolina. With yet another significant deal in the news, I wanted to do a deeper dive into the goals and technology of this particular partnership, to see what the upside is from a suddenly new player in the CAR T cell space.

Precision BioSciences, founded in 2006, is formed around IP centered on designer homing endonucleases, that can be modified to bind to short stretch of DNA sequences of choice. Thus, Precision is a gene editing company, a term that has gotten a lot of buzz recently in the past couple years with the advent of CRISPR-Cas9 technology. Precision doesn't have major connections to the main immunotherapy academic players (yet), which is a difference between them and Juno, Kite, and Novartis. In many ways, Precision most resembles how Cellectis ($CLLS) entered into the immunotherapy world, which I will touch upon below.

To compare the respective DNA editing technologies, one can think of homing endonucleases as similar to the Cas9 protein, except there is no guide RNA and the DNA specificity is built into the protein. Thus, every endonuclease is novel and must be engineered from scratch. Current methods to mutate the wildtype protein for the designer sequence involve mutagenic arrays and screening. Thus, while the technology and concept has been around for a long time,

To contrast with Sangamo ($SGMO) and zinc finger technologies, the zinc finger has a more customizable format for designer DNA binding. However, the protein itself doesn't have any endonuclease activity, so another nuclease domain (FokI) must be fused to the zinc finger domain to create a chimeric protein. TALENs are very similar to zinc finger nucleases in their modular design and structure, having a more user friendly construction algorithm for the DNA binding portion.

Precision BioSciences' homing endonucleases are actually synonymous with meganucleases, which was the founding seed technology of Cellectis. It appears that the two companies have IP around different proteins in the same family, thus giving them both freedom to operate. This is only where the parallels begin between the companies. Baxalta and Precision are partnering on off the shelf CAR T cells, from healthy individuals, which is the same strategy that Cellectis is employing. It is likely that precision will try to edit the same or similar gene's to Cellectis (ie eliminating the TCR and HLA genes) in order to make its product off the shelf.

In many ways, it amounts to a classic "me-too" deal in biotech. Baxalta, soon to be merged with Shire, is buying similar technology from Precision BioSciences to compete with Pfizer, who bought the US rights from Cellectis for their lead genome engineered CD19 targeting CAR T cell product. Similarly, bluebird bio acquired Precision Genome Engineering for up to $140 million back in 2014 to use their megaTAL nuclease platform for cell engineering. The Baxalta deal also harkens back to the agreement between Juno Therapeutics ($JUNO) and Editas Medicine ($EDIT) for genome editing in their CAR T cell products with CRISPR (albeit Juno is still pressing the personalized strategy). All of this amounts to that the field in general seems to agree that genome editing is key for CAR T cell therapy. The ability to remove the built in brakes on the body's immune system to trigger even more potent damage against tumors. Time will tell which genome editing technology is best, but what's unique about the T cell space is that all are likely to work with similar endpoint efficacy, with the only differences being the efficiency in processing (ie 40% edited loci batch versus 80% edited loci batch). All these strategies use proteins that are foreign to the body, but the expression of these proteins will be gone by the time the T cells are infused in the body, so that point is moot. The amount of different proteins one is transfecting into the cells also likely doesn't give one strategy an advantage versus the other, since the mRNA is electroporated in at very high efficiencies.

In general, I see it as a smart deal for Baxalta, if they are truly interested in CAR T cells, and not just placing a chip in the field. The risk is if the Precision team can truly master the immunotherapy side to capitalize on it. While genome editing is a nice feature for enhancing T cell function or making third party T cells, the tumor targets still need to be optimized, and appropriate distinguishing IP generated. If you are a fan of the off-the-shelf T cell platform, you probably already like Cellectis and will like this deal. If you are skeptical of the strategy, you will probably think this is a faulty investment. Indeed, there are two camps emerging in the CAR T cell field, and it will be interesting if there emerges a clear victor over one or the other, or a compromise where different indications decide the appropriate strategy.

Editas IPO Roadshow Review

The CRISPR revolution is about to hit the public stock markets with the upcoming Editas Medicine initial public offering. They are planning to price 5.9 million shares between $16-$18, which would give them a valuation of >$600M. While CRISPR is pervasive and widespread in its use and different applications in the biomedical research world right now, how to apply it to the clinical medicine is less certain. Toward that mission, though, Editas attempted to lay out its vision for capitalizing on Cas9 technology. Here are our thoughts on their roadshow presentation and their plan to make CRISPR therapeutics. Editas' S-1 can be found here.

How CRISPR works:
Cas9 nuclease is easily targeted to different sites in the genome through direction by a short guide RNA (gRNA) which basepairs to the target DNA sequence, making a double strand break. Note that there are Cas9 mutation variants that can only cut one DNA strand (nickase) or are completely defective in cutting, which are useful in different scenarios. Editas outlined the three basic paradigms of gene editing medicine.

   1. Cut & Revise - ex: engineered T cells probably for knockout
   2. Cut & Remove - ex: intron sequence from LCA10 protein
   3. Cut & Replace - ex: introduce a DNA template to undergo homologous recombination into a target locus. Editing of hemoglobin beta as an example.

Editas' plan for turning Cas9's editing capability into actual therapeutics seemed fairly general and light on specifics, instead listing out almost every cell and gene therapy modality that has become prominent over the years, ie: delivery with lipid nanoparticles, viral vector, ex vivo cells (HSCs or T cells), delivery routes via IV infusion or even inhalation. Target tissues of focus included: Eye, liver, muscle, lungs, bone marrow.

To identify which gRNAs induce cuts at the fewest off-target sites, they are using Keith Joung's GUIDE-Seq approach to identify where double strand breaks are occurring in the cells after adding Cas9 + the gRNAs. Editas believes this is the best way to identify potential off-target cuts, and to pick gRNAs that have the fewest or even no detectable off-target cutting, which was shown for a number of gRNAs they mentioned.

Pipeline Summary:
Eye diseases:
LCA10 (CEP290) is Editas' lead indication, planning to begin phase I trial in 2017, induce small deletion in an intron, use AAV delivery to the retina (covered more below). Other planned genetic & infectious eye diseases in discovery phase, and the disease mechanism and possibly efficacy remains to be seen pending further disclosures.

T cells - NHEJ (knockout) ex vivo (covered more below)

Hematologic diseases (B-Thal, SCD) - NHEJ or HDR strategies, ex vivo

Muscle (DMD) - small & large deletions (presumably meaning exon skipping - delivery will be the big issue. See recent papers using AAV and CRISPR to induce exon skipping in DMD)

Lung (Cystic Fibrosis) - NHEJ & HDR (see the Vertex & CRISPR Therapeutics have a collaboration - $105M upfront for CF and hemoglobinopathies)

Genetic & infectious diseases of the liver (alpha-1 antitrypsin deficiency) - NHEJ and HDR, likely necessitating in vivo delivery directly to the liver.

In general, Editas believes they can leverage progress made by others in gene therapy, particularly in the aspects of delivery. Viral vectors using AAV seem to be the primary focus right now.

General questions about Editas' strategy:

Editas showed a figure concerning the gross aspects of their platform. They don’t seem committed to any specific delivery modality (basically using what has been used previously - ie: AAV vs. nanoparticle vs. ex vivo cells), and said they are doing preclinical work in all of them. Given that delivery is the biggest issue facing CRISPR, one would have hoped for more details ahead of the IPO. They did not present any specific improvements in delivery they've made, except for using smaller Cas9 for AAV (potentially the S. aureus Cas9 as published previously by Feng Zhang.) to fit into the vector genome size.

From genengnews.com

Editas then showed the ability to edit genes in vivo, targeting the endogeneous Factor VII gene in mice. The exact delivery mechanism was unspecified however (most likely AAV) for the presumably liver targeting approach. The figure reported protein levels down of ~40-65% depending on guide RNA, as measured by ELISA in the serum. It's uncertain that this level of knockdown would be significant enough to modulate any disease to a great extent, although that will largely depend on the target.

Finally, the competitive advantage over Zinc fingers, TALENs, and meganucleases is not very clear for single targets in genetic medicines, where you need to take time to develop the drug anyway (see the Genome biology figure for a comparison of zinc finger and CRISPR strategies). It is faster to use CRISPR for researchers & easy to design, which explains its widespread popularity, but this advantage is actually nullified in the clinical world. It is possible that Sangamo, Cellectis, and others championing these alternative genome editing platforms might see a resurgence in interest due to CRISPR-Cas9 patent exclusivity. The ability, in vivo, to make multiple double strand cuts using a single AAV vector may be an advantage for CRISPR-Cas9 over the other technologies, useful for the "Cut & Remove" types of edits - exemplified in their lead indication of LCA10 discussed further below.

T cell applications:

We like the potential of the Juno Therapeutics alliance that Editas has established, and that was emphasized many times throughout the presentation. The delivery and editing targets are fairly clear, with translational application direct to improving cancer immunotherapy. For the goals of the Juno collaboration, Editas outlined the need to improve persistence of T cells & overcome inhibitory microenvironment signals. Editas showed they could edit 80-90% of T cells for one target, while only achieving 40% editing of the second undisclosed target. There were also some effects on the viability of the edited cells, with a decrease to 50-60% normal viability, which Editas still considers well tolerated. The first CRISPR T cell application seems to be the knockout, and so it would not be a surprise if they are potentially targeting PD-1 and/or CTLA-4.

We have a couple of concerns though. First, there is risk in Juno being a big winner of the CAR-T cell battle, whose own story will continue to evolve over the next years. We also wonder if the IP will make it fall through on making this license special and exclusive. Other companies are doing genome editing of CAR T cells with different modalities, and some also having CRISPR partnerships. We want to know if they have specific IP on T cell applications, or if their claim is more on the general terms of owning the Cas9 technology. For example, the UCSF paper recently (which had Jennifer Doudna has an author) discussed CRISPR editing in T cells, and UCSF filed a patent on the technology. It is our presumption Editas does not own this due to the ongoing dispute, meaning this will flow to another company. Our fear is that CRISPR will be generalizable to all immunotherapy companies soon, meaning the Juno/Editas advantage is diminished, after which the revenue stream from the deal is solely based on the success of Juno's platform, which Editas has no control over.

Leber's Congenital Amaurosis Type 10 as their First Treatment Target:

Editas will go after Leber Congenital Amaurosis 10 (LCA10) as their first indication. LCA10 is a disease that results in progressive blindness. LCA can be caused by many different mutations, but Editas will focus on the subset caused by CEP290 mutations (LCA type 10), of which there is no approved therapy. Spark therapeutics has used AAV vectors to treat LCA type 2 patients with mutations in a different gene (RPE65) by re-expressing the protein, with positive phase III results recently released for 1 year post-treatment. The mutation in the CEP290 gene is a single base pair mutation in an intron causing inappropriate splicing of the protein. This leads to truncation of protein by causing insertion of an abnormal exon that contains a premature stop codon. The theory is that if one just cuts out that cryptic splice site with 2 guide RNAs (as outlined in the figure below), removing that stretch of DNA, then normal splicing and protein expression will occur.

Concerning the data presented, Editas only has in vitro data from patient cells showing correction. By analyzing RNA in patient derived fibroblasts with LCA, they observed ~1.5 units normal CEP290 RNA, ~4 units mutant CEP290 RNA before treatment. After treatment, with either pair of gRNAs they tested, this caused a flip to ~3.5-4x normal RNA, ~1x mutant RNA (presumably no selection for cells that received Cas9 & guides). By protein, this RNA change approximately doubles the full length CEP290 protein produced in this bulk population of cells. No in vivo modeling of this approach or any phenotypic readout was shown. Indeed, Editas said they were finishing the in vivo work in 2016 in order to file the IND late this year for trials in 2017. While the genetics of the disease and logic of this therapeutic approach appear well understood, showing efficient delivery and editing in vivo will be important to further de-risk this approach.

There are a number of reasons this disease makes sense as a first target for Editas. The disease does not involve changing the coding sequence of the protein, meaning that the normal protein can be restored through deletion and an immune response against normal CEP290 will not happen (see the Editas figure above). The eye is immunopriveleged and should in theory have less or no reaction to the vector. Eye delivery efficiency using AAV is well documented in previous clinical trials (Spark has been successful (mostly) with an AAV to the eye, Avalanche and others have been less successful, but may be due to the AAV or the target), so Editas knows they can theoretically reach the target cells. The disease is in theory monogeneic and has a well defined mechanism. These points are all positives, which led to the selection of the LCA10 indiction.

As far as potential downsides for this target, one concern is how much protein will be needed for therapeutic efficacy. Is double the amount of correct protein enough for disease pathology, or will the presence of the pathologic protein in mixture in corrected cells still cause centrosome and cilia dysfunction and cell death over time?

We also have some questions about the age of treatment, since this disorder leads to cell death in the cones, and the time point of restoration might be crucial in order to rejuvenate them. Secondary injury to the retina could permanently put the cones on a path toward destruction even if the centrosome assembly is saved. We have concerns about their all-in-one vector approach for treating the disease. It's not specified when or how the Cas9 protein expression will ever be turned off, permanently marking those cells as immunogenic potentially. Furthermore, any AAV vector leaking out of the ocular cavity could prime an immune response to Cas9, which might eventually find its way to the eye, in spite of the anatomical barriers. Lastly, we wonder if a gene therapy competitor could simply deliver full length CEP290, although the very large size of the protein may make it less amenable to fitting the full coding sequence in an AAV. This also applies to many other applications of gene editing technology, that there could be competition from more traditional gene therapy approaches. In that regard, the stability of genome editing in host chromosomes is an advantage over episomal driven expression of a transgene from AAV, and while AAV is the current preferred in vivo gene therapy vector, a lentiviral approach could potentially solve this problem. As mentioned above, competition for this indication could come from other gene editing platforms that can cut the same sites that Editas is targeting, however, it may be easier to fit all the machinery to make the 2 different cuts here with Cas9 and 2 gRNAs versus multiple separate zinc fingers or TALENs.

Strengths of Editas Medicine:

The company appears to be the financial and intellectual leader in the CRISPR space so far. In particular, the financial aspect is crucial since they will have the most money to fund initial trials and future trials, as well as purchase more academic IP related to CRISPR technology.

As the Chief Financial Officer outlined in explaining how they are building Editas the business, they have focused on building an impressive list of business leadership & scientific founders/advisors (Feng Zhang, David Liu, Keith Joung, George Church). Furthermore, beyond the foundational CRISPR patent dispute (see below), Editas does have a myriad of patents for applications that they will own and will at least block anyone else from pursuing that same strategy, suggesting that cross-licensing will at least be a backup plan in the future. As they outlined in their figure to the right, currently for their patents, their IP estate is directed to both platform technology & therapeutic products, with 21 issued patents & >180 pending patents licensed. Editas itself has filed 31 patents pending.

Financials:
$43M Series A
$25M upfront from Juno
$120M Series B
$150M Cash, at least 24 months runway before IPO

What to watch on Editas Going Forward:
There is a plan to release more proof of concept (presumably additional preclinical) data each year at medical conferences for multiple programs. A potential challenge for Editas in the public markets might be investor patience, as they will not enter clinical trials until 2017. To this end, Editas emphasized these proof of concept data releases and potential further business development as important catalysts to keep investors interested in the story and maintaining more visible progress.

Lastly, there was only minor acknowledgement, in an appendix slide, of the upcoming patent interference hearings between University of California (Doudna), University of Vienna (Charpentier) versus MIT/Harvard/Broad patents. Editas has the licenses from the Broad/MIT/Harvard estate, so will be hoping for their victory. The legal case will not be covered here, but it could add significant volatility to the stock when the rulings are being made, although this will play out over a number of years.

Summary:
Editas led off the presentation referencing the 6,000 diseases with genetic mutations 95% and no approved therapy. While enticing, this statement overstates the potential of their platform in targeting all of these diseases, following the general hype in the media around CRISPR. However, there are other options like classical gene therapy and cell therapy, as well as other gene editing technologies that could go after the same indications. It is still uncertain which of these options will ultimately win. A major question for Editas' in vivo applications will be the efficiency of delivery to the target tissue, which appears to currently be piggybacking off of the routes of administration used for other gene and cell therapies. Thus, Editas will also share the same potential and difficulties as gene therapies in terms of delivering sufficient amounts of their therapy to target tissues, and target selection will be key. There is also the elephant in the room of who will really own Cas9 and genome editing? While carrying immense promise, the gene editing story will take some time to begin to unfold, and its full potential will not be known for many years. With those risks stated, the prospect of a dominant CRISPR therapeutic company on the public stock markets is very exciting, with a platform technology that could truly revolutionize medicine if properly executed.






Disclosure: DM owns shares of Sangamo.

Does Cellectis have an infection problem?

by Robert Kruse

Cellectis ($CLLS) has sparked much interest among investors, scientists, and patients for the allogeneic CAR T cell therapy platform. It offers the potential for a centralized manufacturing of T cells at a single site, and then distribution of frozen vials around the world to hospitals to infuse locally to cancer patients. The manufacturing, distribution, and payment model fits well within pharmaceutical paradigms today. This contrasts with the autologous approaches being pursued by Juno ($JUNO), Kite ($KITE), and Novartis ($NVS) for their CAR T cell therapy platforms.

The Cellectis strategy consists of a few key elements: (note I will focus on their lead UCART19 product and not their other proposed strategies. more information here).

1) Knockout of the TCR genes removes the ability of allogeneic cells to bind to host MHC class I molecules and become activated, attacking host tissue (graft versus host disease). Another human's T cells will not have undergone thymic deletion to your different set of HLA molecules, which is the biological reason behind this discrepancy.

2) Knockout of CD52 in transfused allogeneic cells allows the use of an anti-CD52 antibody (Alemtuzumab) to kill host T cells, providing an ongoing selective advantage for the transfused T cells, and also to prevent host T cells from attacking those T cells.

3) General chemotherapy and lymphodepletion, like the UPenn and Novartis protocols, helps CAR T cell homeostatic expansion, but also in the case of Cellectis, helps protects their cells from other immune effectors, which would recognize the varied MHC class I molecules on the infused product surface as foreign.

4) Suicide gene in addition to CAR T cell expression for additional safety feature to eliminate the T cells

5) Cellectis has an opportunity in the market for patients whose chemotherapy has rendered their cells ineffective donors. Having healthy T cells from another human being should be a major advantage in this instance.


There are potential issues with this approach that I have yet seen discussed among press releases by the company and other scientific commentary. Prominently, I see the potential for the Cellectis strategy to make the patient highly susceptible to infections, mitigating the ability of the therapy to achieve its endpoints.

Before the start of therapy, the patient is conditioned to remove most of their current immune cells. This lymphodepletion leads to loss of T and B cells fighting infection. CAR T cell therapies right now are similar to patients undergoing bone marrow transplants. When one infuses the allogeneic CAR T cells, these cells aren't matched to the patient's HLA molecules, so can't recognize any viral peptides inside host cells. Indeed, the CAR T cells don't even have TCR molecules even if Cellectis did try to match donor CAR T cells to patients' HLA types. During the provision of cancer therapy, the patient must constantly be in an immunosuppressed state in order to avoid Cellectis' CAR T cell destruction by the host immune system, which alternatively makes the patient highly susceptible to infections. The T cell population in the patient is mostly incapable of the regular job of surveying peptides inside cells, presenting major potential issue in the clinical management of these patients, who could perhaps require hospitalization during the duration of their CAR T cell treatment while immunosupressed, causing the costs of the procedure to skyrocket, in spite of the initial allogeneic manufacturing costs being much cheaper. I am making an assumption here that the UCART19 clearance of cancer will take 20-30 days, similar to the autologous trials from UPenn. In the UPenn trials, the most monitored time is just the first few days for cytokine release syndrome, but otherwise the patients can leave the hospital after emerging from this window and return as necessary.

Contrast this with the approach of Juno, Kite, and Novartis. The patient is lymphodepleted, but the infused autologous CAR T cells are polyclonal in their TCRs and should have the capacity to fight off any infections, in essence, re-constituting the immunity to pathogens in addition to their tumor fighting capabilities.

A solution that Cellectis or any other company could pursue is a compromise of the two models. The answer is possibly found with Bellicum's ($BLCM) strategy of infusion allogeneic haploidentical T cells in order to provide infection protection in bone marrow patients, and some measure of graft versus host disease. It could easily be imagined that these haploidentical cells retaining TCR genes could be manufactured with CAR T cells. A bank of CAR T cells could be created with various HLA types, and then infused into patients with some partial HLA matches in order to provide a measure of limited infection protection. Of course, these partially HLA-matched T cells would likely cause some measure of GVHD in some patients meaning that a safety switch would be needed (Cellectis has their own safety switch for this purpose). As an aside, this proposal is different from allogeneic (but not off-the shelf) CAR-T cells that are made from donor T cells after an allogeneic transplant, with some data recently presented at ASH here. Furthermore, lymphodepletion would still be necessary in the protocol to protect the proposed allogeneic T cells from being recognized as foreign. Nevertheless, this strategy of using partially HLA-matched allogeneic T cells as the CAR T cell effector would solve the infection problem and permit a safer and more judicious use of allogeneic products for cancer immunotherapy.

Final Thoughts on Bluebird Prior to ASH

Here's what I'll be looking for in the ASH data bluebird bio will present over the next week. I thought it was worth a short post since there has been some new and relevant data relating to the BCMA CAR-T program partnered with Celgene, and also there was some confusion before/during their Piper Jaffray presentation over the path forward with LentiGlobin. I previously wrote a more detailed post on the data in the ASH abstracts for bluebird's LentiGlobin gene therapy product here.

LentiGlobin in B-Thalassemia
My main focus for B-Thalassemia will be how the new data influences the development path forward. Bluebird presented at the Piper Jaffray healthcare conference where they tried to clear up some confusion over whether the development path for LentiGlobin had changed. The confusion came from an investor meeting prior to the conference (link). Bluebird reiterated that its development path had not changed. They added that they are constantly trying to improve their products, and they look at minor changes that can be incorporated into the current program, as well as other changes that might be for "next-generation" products that would require a new IND.

Just as a refresher, in May, bluebird gave an update on their regulatory path forward in June after discussions with both EU & US health agencies (link).

For the EU, they believe they could apply for conditional approval based on their current HGB-204 & 205 studies, which are expected to complete enrollment soon. Importantly, the proposed primary endpoint would be transfusion reduction. For the US, they believe they could apply for accelerated approval based on their pivotal studies HGB-207 and 208, which have yet to be initiated (15 B-Thal patients per study), with accelerated converting to full approval based on longer term follow-up data. For the US, the proposed primary endpoint is transfusion independence. While this was the current thinking in May, the plans were subject to change with further data and discussions with regulators.

Considering the consistent ability, so far, of LentiGlobin to induce transfusion independence in non-B0/B0 B-Thalassemia patients, but not B0/B0 patients, bluebird has mentioned the possibility of developing the product differently for these 2 populations. However, bluebird emphasized it was still too early to be making decisions yet. To completely speculate with this in mind, and the above regulatory outlook, I could see bluebird developing LentiGlobin, as is, for the EU since transfusion reduction is the proposed primary endpoint, and LentiGlobin should cause reduced transfusions in B0/B0 patients as well. For the US, I could see them splitting off the B0/B0 patients, and focusing on the non-B0/B0 patients, which they say are 2/3rds of the addressable population. If, later, they can improve the amounts of T87Q hemoglobin LentiGlobin treated patients produce to consistently 8+ g/dl with either future production tweaks or follow-on products, then I could see them going after the B0/B0 genotype again, with the goal of consistent transfusion independence.

So with this in mind, what I'll be looking for in the B-Thalassemia data from their ASH data is - are 100% of the non-B0/B0 patients transfusion independent (or trending) by 1 year post-treatment, and what is the level of transfusion reduction in B0/B0 patients?

LentiGlobin in Sickle Cell Patients
There has not been as much recent discussion about LentiGlobin for sickle cell disease (SCD), but I'll be looking for longer follow-up on the 1st treated patient on clinical & blood endpoints, and their current T87Q level. I would not expect much of an increase in T87Q at this point, as I've mentioned in previous posts, however, the ~50% T87Q would seem likely to be sufficient for significant clinical benefit.

There will also be a poster presentation with data on at least 2 new patients treated with LentiGlobin. These patients were treated with a lower cell dose than the first patient, and also one patient had a lower vector copy number (VCN) in the drug product. So I will be looking for the levels of T87Q these patients have been able to produce, both in terms of the total amount of T87Q and the relative amount of T87Q to sickle hemoglobin (HbS).

Bluebird has also suggested that they have been performing assays to determine the percent of modified cells, in addition to just giving the average VCN across all cells. This is another way to measure how well they are able to transduce the CD34+ cells with the LentiGlobin vector in their drug product, and potentially track the levels in the patient. I could see this being a useful number to know, particularly for SCD, where in hematopoietic stem cell transplants (HSCT), mixed chimerism can occur, with data suggesting that you only need 10-30% of HSCs to be corrected to functionally cure a patient. I previously wrote about mixed chimerism and other considerations for LentiGlobin in SCD in more detail with Zack, @BioTerp, which can be found here. How much a single copy of the LentiGlobin vector can protect a red blood cell from sickling is unknown, but is likely to be at least somewhat beneficial based on levels of fetal hemoglobin of 30% being known to be protective.

BCMA CAR in Multiple Myeloma
The importance of ASH data for Bluebird's BCMA CAR program has increased, as an additional late-breaking abstract will have 1st in human clinical data of a different BCMA CAR in a phase I trial at the NIH - to be presented on Tuesday December 8th.

This study comes from James Kochenderfer, whom bluebird has a cooperative research agreement with. I believe bluebird has licensed the BCMA patents from the NIH as well. The BCMA CAR used in this program does have some differences from the one bluebird will use, but considering the relative consistency of the data across different CD19 CARs (CD28 or 4-1BB), there is significant read-through to bluebird's program (comparison of different BCMA programs). In the NIH study, the CAR has a CD28 costimulatory domain, versus the 4-1BB domain bluebird is using. Importantly, in two patients at the highest cell dose given, both had responses, with one patient achieving a complete response. It will be important to see how durable these responses are, and if any additional patients have been treated at this dose, although not much time may have elapsed from when this abstract was submitted. The cytokine release syndrome seen in both patients who received this cell dose is still significant, but was considered similar to those seen in CD19 CAR treated patients. This is probably the most crucial safety parameter I'll be keeping an eye on. I could see this presentation getting substantial attention at ASH, as the expansion of CAR-T therapies beyond CD19 has been a big open question.


Bluebird has mentioned the study in recent webcasts, and I expect them to try to use it to draw excitement to their program. Bluebird itself has 3 poster presentations at ASH on their BCMA program (1, 2, 3), however, I would say none of them really adds that much new data to what they've already presented. On using CD28 versus 4-1BB as a costimulatory domain, it is still too early to tell the differences in safety or efficacy yet for CD19 targeted CARs. A recent preclinical paper suggests that on a per cell basis, CD28 costimulatory cells have greater tumoricidal activity whereas 4-1BB costimulatory cells have better persistence. In the clinic, results using either costimulatory domain have been impressive, but it's still too early to tell if either has an advantage - reviewed here. Despite the potential differences in the CAR construct, my confidence and interest in bluebird's BCMA program has increased after the release of this new abstract, and I will be very interested in any updates from the NIH trial.


Competing Approaches in B-Thalassemia and SCD
Lastly, a big thing I will be keeping an eye on at ASH are data from other approaches in B-Thalassemia and SCD. Specifically, I am interested in clinical data from Bellicum on their approach to make allogeneic HSCTs safer (including in B-Thalassemia and SCD), clinical data from Global Blood Therapeutics on their small molecule drug for SCD, and preclinical data on a different gene therapy approach for both diseases by Sangamo. I discussed them a bit more in my previous post on ASH abstracts. Additionally, Timothy Sullivan had a very good post on Bellicum's approach here. One interesting way bluebird could improve the safety of their approach compared to even the presumably improved-safety allogeneic approach of Bellicum would be to change their conditioning regimen, such as depleting only CD34+ cells, instead of myeloablation with busulfan. This might improve some of the negative effects of the conditioning regimen, such as immune recovery. Bluebird has mentioned this as a possibility, so it will be interesting to follow any potential updates on that, however, this would be a significant change and would certainly be a follow-on product requiring separate clinical development.

Edit: @BioTerp also reminded me about additional similar approaches using viral transduction of HSCs with a hemoglobin (or modified hemoglobin) expressing vector - one being developed by Donald Kohn, whose lab has published many preclinical papers here, as well as another from MSKCC with economic partner Errant Gene Therapeutics. Although I do not believe there will be new data from either group presented at ASH.

Conclusion
There will be a lot of new information coming out of ASH to consider for bluebird - new LentiGlobin data, 1st in human BCMA CAR data, as well as early data from other competing approaches. I'll be looking for more clarity on the development pathway for LentiGlobin, and the consistency of the results given the current treatment protocol. I think the BCMA CAR data from the NIH program might generate additional focus on bluebird's program, and it will be interesting to see how bluebird tries to frame that data in the context of their program.






Disclosure: I own shares of BLUE, BLCM, SGMO

Bluebird Bio's Lentiglobin - Is the Sky Falling? (ASH 2015 Abstract Review)

While I don't think the sky is falling for bluebird's LentiGlobin, sentiment is certainly changing, as the story "seems" to have shifted from "it can cure every patient" to "the therapy wears off over time and it doesn't work for everybody". However, I feel this is more a shift in sentiment than their data getting significantly weaker. I think people were way too optimistic if they were expecting cures for every patient, but the pendulum may have swung a bit too far to the negative side. So here I'll take a look at bluebird bio's most recently released data in their 2015 ASH abstracts and how the whole picture is coming together for the efficacy of LentiGlobin.

I've recently written a number of other posts on bluebird, mostly on LentiGlobin in sickle cell disease (SCD) and B-Thalassemia and their results presented at the EHA meeting in June. Check them out if you want additional context for this post:
Posts 1, 2, 3, 4.

The abstract that appears to be the main focus of people's attention is on further follow-up data on patients in the Northstar (HGB-204) study, which is enrolling patients with B-Thalassemia.

HGB-204 Abstract

So they've treated 10 patients with B-Thalassemia major by July 31st with LentiGlobin, 5 with the B0/B0 genotype, where no functional HbA hemoglobin is made, and 5 who are heterozygous for B0 and have one functional or hypomorphic copy of beta globin (BE etc.). The headline is that none of the 3 patients evaluable (>6 months follow up) who have B0/B0 became transfusion independent. However, the 4 evaluable patients with non-B0/B0 all have been transfusion free for >90 days. Up until now all patients treated (only 4 - 2 in the Northstar study, 2 in HGB-205) had achieved transfusion independence for at least 90 days of follow up. While perhaps some had expectations that every patient would achieve transfusion independence, that was always unlikely to be the case based on their previous data. Total hemoglobin needs to be at least >8 g/dl (probably better if 9-10 g/dl) to avoid transfusions, and looking back at their data from last ASH shows this might be right at the edge of what they're able to achieve with LentiGlobin for B0/B0 patients. Here's the data on their first 4 patients they presented last year:

3 patients had B0/BE (patients 1201, 1202 and 1102) and reached transfusion independence. You can see those patients are still getting a contribution in their total hemoglobin from HbE (from BE allele) and HbF (fetal hemoglobin). The B0/B0 patient, 1106, whom they also called transfusion free, only has HbF to add to their HbA-T87Q hemoglobin. While they were off to a good start with already 6.8 g/dl T87Q being made, it would probably need to get ~8 g/dl to achieve stable transfusion independence, assuming they continue to get some contribution from HbF.

It appears that this patient was one of the two B0/B0 HGB-204 patients that required a single transfusion, based on the new ASH abstract. I haven't heard many discussing this B0/B0 patient, and it would have potentially made for an even worse headline of having a previously "cured" patient no longer being cured. This was basically the headline a couple weeks ago when a patient treated with an older version of the T87Q expressing vector required a transfusion after 7 years of being transfusion free. I'll get back to my thoughts on that patient later.

Getting back to the more recent B0/B0 patient, I had hoped that her T87Q hemoglobin would increase enough to remain transfusion independent. You can see they still have some HbA (blue) from previous blood transfusions in their total hemoglobin, and this would need to be made up for by the patient's own hemoglobin after the transfused cells have completely gone. So it was probably premature to announce this patient as "transfusion free" (ASH 2014 PR), and it's probably best to wait until all the transfused HbA goes away. In any case, having a single transfusion over the course of about a year is still a significant reduction in transfusions considering the entry criteria for the study requires at baseline at least 8 transfusions per year or >100 mL/kg/year of transfusions. It's also possible the patient will become transfusion independent if their T87Q production goes up a bit more, which it has in some of their previously treated patients. So, I will be interested if their production of T87Q has increased, and by how much, since last year. Importantly, this patient had a relatively high vector copy number (VCN) in their CD34+ LentiGlobin product (1.5), so if they wanted to more consistently achieve >8 g/dl T87Q in B0/B0 patients they might need to achieve ~2 VCN, like in patient 1202, who has now been holding steady at around 9.7 g/dl T87Q for almost a year.

How tight a correlation between VCN, CD34+ cell dose, and the quantity of T87Q patients produce will become clearer as they continue to treat more patients. My general feeling is VCN will be more important for determining peak levels of T87Q. However, I could imagine low CD34+ cell doses might cause reduced chimerism - where a lower percentage of old hematopoietic stem cells get replaced by transplanted ones - and thus cause lower levels of T87Q production. So far, though, it seems like the VCN they see in the LentiGlobin product they administer is similar to the amount they see in patients' peripheral blood, so they are likely getting relatively good reconstitution:

Another thing I noted from the abstract was that a patient had relatively modest T87Q production (just 1.9 g/dl), as well as one patient having very low VCN in the CD34+ cell product they received (just 0.3 VCN). It will be interesting to see if the patient given the 0.3 VCN product is also the one producing the very low levels of T87Q, and if this is the B0/B0 patient who is still considered transfusion dependent. It's surprising a patient was treated with that low VCN, as I thought the original release criteria for LentiGlobin on this study was 0.5 VCN - page 43 here.

Turning, briefly, to the other 2 abstracts, which had less significant data updates, the abstract for the HGB-205 study includes the first sickle cell patient treated (previously reported data at this year's EHA meeting), and follow up data on patients 1201 & 1202, whom I mentioned above in the 2014 ASH data.

HGB-205 Abstract

All this data looks consistent with previous releases, both B-Thalassemia patients have maintained their T87Q production steady for over a year. The SCD patient's T87Q levels continued to increase now at 9 months compared to 6 month data at the EHA meeting. In SCD, the ratio of sickling hemoglobin (HbS) to anti-sickling hemoglobin (T87Q + HbF) is more important than the absolute quantity (g/dl) of T87Q produced. The levels bluebird believes will be sufficient to significantly reduce symptoms is 30% anti-sickling hemoglobin, which they have already achieved. I am a bit more conservative, as I had outlined in previous posts, about what percentage one might need to achieve, thinking it could be slightly higher than 30% depending on how evenly the T87Q hemoglobin is distributed throughout the patient's red blood cells. However, the ~50% anti-sickling hemoglobin achieved in this patient is impressive, and I would expect to be therapeutically beneficial. So far the patient does not have any reported no hospitalizations or blood transfusions. It is not surprising that the T87Q levels are leveling off somewhat. They're actually slightly higher than I would have expected at 48% now from 40% at 6 months. A major reason for the large increases in the %T87Q in the patient's blood from months 3-6 came from having their previously transfused blood cells (carrying HbA) die off, raising the relative amount of T87Q. So I am not expecting a significant increase in T87Q% when they present updated data at ASH. I will be looking to see the maintenance of T87Q levels, and if the other efficacy measures in this patient continue to look promising.

Lastly, the abstract for the HGB-206 trial contains only information on the LentiGlobin drug product that was infused into 2 patients with SCD.

HGB-206 Abstract

It's dangerous to mobilize hematopoietic stem & progenitor cells in SCD patients to enter the blood stream, which is how CD34+ cells were collected for the previously treated B-Thalassemia patients. So SCD patients need to have CD34+ cells harvested directly from bone marrow. This typically leads to lower amounts of CD34+ cells, as you can see the cell dose in these two patients is <3 x10^6 cells/kg, compared to 5.6 x10^6 cells/kg for the previous SCD patient, and normally 8 x10^6 cells/kg or higher in their previously treated B-Thalassemia patients. The first patient listed in this abstract also has a fairly low VCN of 0.5/0.6. It will be important to see how this low of a CD34+ cell dose affects the kinetics and total production of T87Q in these patients. For the patient with the 0.5/0.6 VCN dose (compared to 1.2/1.0 VCN for the SCD patient we have 9 month data on) they might produce significantly less T87Q. This patient may not make the 30% threshold bluebird has been using, however, an increase to 10-20% of anti-sickling hemoglobin could still have some therapeutic benefit.

Transfusions needed patient treated with original HPV569 vector
Another recent event was the disclosure that Subject 3, a B0/BE patient who had achieved transfusion independence after being treated with a previous version of bluebird's vector, required 2 transfusions after almost 7 years of transfusion independence. Since people were hoping for sustained transfusion independence, this news certainly sounded disappointing with potential implications for the duration of the efficacy for this approach. However, if we look at the levels of T87Q and total hemoglobin this patient had, they were always on the borderline of needing a transfusion, making only 2.7 g/dl T87Q and having total hemoglobin levels fluctuating between 8 and 9 g/dl over the past 5 years.

Importantly, bluebird noted in their press release that the levels of T87Q and VCN levels in peripheral blood have remained "largely unchanged". Unfortunately, the data have not been released to see exactly what "largely unchanged" means, but I doubt there was a sudden drop in T87Q based on the modest but relatively stable levels of T87Q the patient was producing. It does warrant keeping an eye on to see if T87Q levels do drop over long time periods. If there had been silencing of the viral construct, or perhaps an immune reaction against cells containing the transgene, one would have imagined there would have been a preferential drop in T87Q compared to total hemoglobin levels, which does not seem to be the case here. It's unclear how long viral gene expression can last in cells, but there is some data for transduced T cells that it can continue to last over 10 years, and potentially longer. 1st generation retrovirally transduced CAR-T cells still can be found with CAR expression more than a decade later. Additionally, there is another presentation at ASH where the authors retrovirally transduced a Thymidine Kinase safety switch into transplanted T cells, and they also saw that expression can be maintained for over 10 years. However, it will take further follow up in more patients before we get a better sense of the duration of efficacy for bluebird's LentiGlobin.

Sangamo also brought up in their 3rd quarter conference call concerns about the lentiviral integrations in this patient, and clonal dominance. Previously, cells containing a viral integration near HMGA2 grew in prevalence, a potential concern suggesting preferential growth of this clone. However, that clone ended up decreasing over time:

Other clones have increased in prevalence among the vector-modified cells recently, but bluebird suggests this is potentially due to random increases and decreases in prevalence over time, which could be exacerbated by the fact that so few cells were efficiently transduced such that, inherently, any given clone could grow or shrink considerably. There were only approximately 100 integration sites for the original patient treated, whereas there were many more in the more recently treated patients. An integration site analysis of their more recent patients suggests they are currently maintaining a very polyclonal population of engrafted cells:

 

From https://twitter.com/BioTerp/status/610431838151143425

Additionally, other self-inactivating (SIN) lentiviral vectors similar to bluebird's are also showing improved safety compared to earlier gamma-retroviruses so far, as well as signs of efficacy. A number of them are being presented at the same ASH session as bluebird:
- Safety and Clinical Benefit of Lentiviral Hematopoietic Stem Cell Gene Therapy for Wiskott-Aldrich Syndrome
- Gene Therapy Using a Self-Inactivating Lentiviral Vector Improves Clinical and Laboratory Manifestations of Wiskott-Aldrich Syndrome

- Lentiviral Hematopoietic Stem Cell Gene Therapy for Older Patients with X-Linked Severe Combined Immunodeficiency

Safety & efficacy data in Wiskott-Aldrich Syndrome was also published here with integration site analysis.

Continued safety, in particular the risk of insertional oncogenesis, is still a significant consideration, but I think the data so far suggests these SIN-lentiviral vectors are likely considerably safer than earlier gamma retroviral ones.

Conclusions:
In conclusion, I don't think the data presented here is out of line with bluebird's previously released data, but expectations were probably too high. LentiGlobin, with the average VCN and CD34+ cell dose given so far seems to be able to consistently induce transfusion independence in B-Thalassemia patients with B0/BE. In B0/B0 patients, the average VCN and cell dose do not seem to consistently be able to produce sufficient levels of T87Q to achieve transfusion independence, but will still likely be therapeutically beneficial and cause transfusion reductions. If bluebird can more consistently treat B0/B0 patients with VCNs on the higher side of what they've been able to achieve so far, it's possible they could achieve transfusion independence. It's also possible that currently treated patients will have T87Q levels increase over time to those sufficient for transfusion independence, but I'm not expecting large increases 6 months post-treatment. I think we will continue to learn considerably more about the relationship between VCN, CD34+ cell dose, and the kinetics of T87Q production from their upcoming presentations at ASH and in subsequent data updates. Bluebird has stated that only about 1/3rd of B-Thalassemia major patients are of the B0/B0 genotype, and their therapy will still probably be beneficial to those patients by reducing transfusion frequency, if not transfusion independence. Their data in the first SCD patient looks very promising, but the follow-up is still early, and subsequent SCD patients may have been treated with less efficacious doses. In total, I still think bluebird's approach appears to be of significant therapeutic benefit if the results they've seen so far continue.

None of my blog posts are intended as investment advice, and I intentionally don't typically go into valuation, I normally focus only on my interpretation of the data. There are, however, additional approaches for treatments in B-Thalassemia and SCD that are being presented at ASH that look promising as well. So I'll briefly look at a couple below:

Additional approaches in B-Thalassemia and SCD being presented at ASH
There are a number of other companies with interesting therapeutic approaches to B-Thalassemia or SCD that will be presenting pre-clinical and clinical data at ASH as well, they include:

Bellicum - Allogeneic HSCT is curative in patients with B-Thalassemia and SCD, in addition to other monogenic blood diseases and also is potentially curative in the treatment of hematologic malignancies. Bellicum is trying to make allogeneic transplants safer by adding back T cells containing their inducible caspase safety switch to T cell-depleted allogeneic transplants. Adding back T cells potentially can improve early immune reconstitution and hopefully reduce potentially lethal infections. Their safety switch is added to improve the safety of having donor T cells in the transplant, so T cells can be depleted if they begin to cause GVHD. Bellicum will be presenting at ASH on this approach, including in patients with B-Thalassemia. They have stated already that 4 patients with B0/B0 Thalassemia have been treated without complications and are transfusion independent. It's not surprising the transplant is curative, but the main things I will be looking for are levels of infection (and maybe measures of virus-specific T cells), as well as instances of GVHD and how well it's handled by activation of the suicide switch. In a number of patients from earlier studies, the approach has worked quite well in resolving GVHD rapidly, while surprisingly maintaining antiviral immunity post-depletion, published here.

For me, I have not researched enough to know for how many patients this would shift the risk:reward balance in favor of doing HSCTs, but I think how Bellicum's approach might effect this balance will become clearer as their data matures.

They have 3 ASH abstracts on this approach:
- BPX-501 Cells (donor T cells transduced with iC9 suicide gene) Are Able to Clear Life-Threatening Viral Infections in Children with Primary Immune Deficiencies Given Alpha/Beta T-Cell Depleted HLA-Haploidentical Hematopoietic Stem Cell Transplantation (haplo-HSCT)
- Clinical Outcome after Adoptive Infusion of BPX-501 Cells (donor T cells transduced with iC9 suicide gene) in Children Given Alpha/Beta T-Cell Depleted HLA-Haploidentical Hematopoietic Stem Cell Transplantation (haplo-HSCT): Preliminary Results of a Phase I-II Trial
- Immune Reconstitution after Adoptive Infusion of BPX501 Cells (donor T cells transduced with iC9 suicide gene) in Children Given Alpha/Beta T-Cell Depleted HLA-Haploidentical Hematopoietic Stem Cell Transplantation (haplo-HSCT): Preliminary Phenotypic and Functional Results of a Phase I-II Trial

Sangamo - Sangamo is targeting an erythroid-specific enhancer that controls BCL11A expression. BCL11A restricts fetal hemoglobin levels, and mutations that affect BCL11A, or an erythroid-specific enhancer, have been shown to cause higher levels of fetal hemoglobin in patients. Sangamo is using their zinc finger approach to try to disrupt one or both copies of the enhancer that controls BCL11A erythroid expression. BCL11A is not just expressed in erythroid cells, so targeting the erythroid-specific enhancer is probably a better therapeutic approach. This approach looks promising as an alternative way to induce therapeutically beneficial levels of fetal hemoglobin in gene-modified autologous transplants. They will be presenting a number of preclinical studies on the approach in oral presentations at ASH:
- Clinical-Scale Genome Editing of the Human BCL11A Erythroid Enhancer for Treatment of the Hemoglobinopathies
- Genome Editing of the Bcl11A Erythroid Specific Enhancer in Bone Marrow Derived Hematopoietic Stem and Progenitor Cells for the Treatment of Sickle Cell Disease

Some background papers on BCL11A and fetal hemoglobin:
2008: Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A.
2011: A functional element necessary for fetal hemoglobin silencing. 
2011: Correction of sickle cell disease in adult mice by interference with fetal hemoglobin silencing.
2013: An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level.

Global Blood Therapeutics - will also be presenting data at ASH on their oral small molecule drug for SCD.
GBT440, a Potent Anti-Sickling Hemoglobin Modifier Reduces Hemolysis, Improves Anemia and Nearly Eliminates Sickle Cells in Peripheral Blood of Patients with Sickle Cell Disease



Disclosure: I own shares of Bluebird and Bellicum, no position currently in Sangamo or Global Blood Therapeutics.