Welcome to Biotechr

Biotechr is written by Dr. Robert Kruse (@RobertLKruse), who holds a PhD and is currently completing his MD. His research work focused on infectious disease and immunology. This blog is focused on analyzing the latest developments in biotechnologies being developed in academia and industry, with a particular focus on biomedical therapeutics. I hope that the posts are interesting and useful, and hope you join in the discussion with guest posts on the site!

Disclaimer: The thoughts on this blog are not intended as any investment advice regarding any companies that might be discussed, and represent my opinion and not the opinions of my employer. This site is not designed to and does not provide medical advice, professional diagnosis, opinion, treatment or services to you or to any other individual.

Friday, January 27, 2017

Jounce IPO Review

Jounce Therapeutics ($JNCE) is debuting on the public markets today after raising $102 million dollars in its initial public offering. The fully diluted market cap based on the price of the shares sold would be approximately $550 million. Jounce is entering the highly active immuno-oncology (I/O) space, focusing on monoclonal antibodies, with the hopes to identify potent combinations to activate the immune system against cancer. Jounce has a high powered team of scientific co-founders and advisors, including Jim Allison, one of the pioneers of immune checkpoint blockade. They were initially seeded by Third Rock Ventures in 2013, and have picked up additional financial backers during their development. I listened to their IPO presentation (retailroadshow) and below are some of my initial thoughts. Their S-1 prospectus can be found here.

Jounce's Broad Vision
Jounce stated a number of guiding principles about how they wanted to distinguish themselves in the crowded, but exciting, space of immuno-oncology.

One tenet is that they want to make I/O more personalized, and give the right I/O treatment to the right patients. Compared to certain targeted therapy approaches using kinase inhibitors (e.g. TRK inhibitors in TRK-fusion driven cancers), I/O has often taken a less selective approach. This is because, so far, while some biomarkers in use can be useful (see nivolumab's failure in NSCLC using less PD-L1 selection than Merck), they are not great at predicting at the individual patient level who will respond or not respond, as some with no PD-L1 respond and many with high PD-L1 levels do not. Jounce stated that they want to continue to develop better biomarkers for use in guiding the development of their therapies. To accomplish this, a core part of the company is their "translational science platform", which is focused on characterizing (at both the RNA and protein level) human tumors, and their immune components, to identify potentially useful biomarkers and novel targets.

Targeting multiple immune cell types
Most immunotherapy in cancer so far has been focused on the T cell. While Jounce's first candidate (JTX-2011), an agonistic antibody of ICOS, is intended to modulate the T cell response against cancer, they intend to expand their pipeline to target other aspects of the tumor-immune interaction.

They are also focused on tumor-associated macrophages (TAMs), cells that are generally thought to be pro-tumorigenic, but could potentially be reprogrammed back into pro-inflammatory, anti-tumorigenic, cells. There has been increased interest in modulating TAMs for I/O, and targets, such as CSF1R, are being explored by a number of companies. They stated they have identified 10 novel targets to potentially modulate macrophages, and disclosed one of these at AACR last year (abstract here). That target was TIM3, which is also a hot target for T cell biology. Interestingly, they identified a novel interaction of TIM3 with an undisclosed protein on TAMs and developed antibodies that could specifically modulate this interaction. They found that these antibodies did not affect T cell activity, but induced a more pro-inflammatory (anti-tumor) phenotype in macrophages.

Based on their pipeline, they are also interested in targeting regulatory T cells as well as B cells. Jounce is also attempting to target so called "Cold Tumors" that appear to lack a strong immune response at the site of the tumor, and is associated with lack of response to current immunotherapies. A number of targets have emerged to try to improve the immune infiltrate in these tumors, such as STING agonists, CXCR4-CXCL12 blockade, and targeting desmoplasia in tumors like pancreatic cancer.

They are also developing their own PD-1 antibody (JTX-4014), which they intend to use only for combinations.

Targeting ICOS
Jounce's lead program is JTX-2011, which is an agonistic antibody against ICOS (Inducible T-cell Costimulator). Unlike targeting T cell-inhibitory proteins, such as CTLA-4 or PD-1, to boost the activity of T cells, Jounce is hoping to activate this costimulatory molecule. For background, Jounce presented some preclinical data on their antibody at AACR (abstract here) and a good review of ICOS signaling, and its role in immunity and cancer is available here. ICOS is a costimulatory molecule, similar to CD28, that is typically found on activated CD4 T cells.

Activating costimulatory molecules, such as CD28, had been previously tried with a "superagonistic" antibody TGN1412, which caused severe cytokine release syndrome, as detailed here. However, there were mistakes in the clinical development of that antibody, besides the target, that led to those problems. ICOS, as the name suggests, is only upregulated on already activated CD4 T cells, including both effector and regulatory T cells. Jounce hopes that this more specific expression of ICOS, such as at the tumor site, will limit broad overstimulation of the immune system. The presence on both effector and regulatory populations could confound attempts to target it. Jounce suggested that their antibody both activates effector T cells at the tumor site, as well as depletes regulatory T cells (that also express ICOS). This is presumably through antibody-dependent cell-mediated cytotoxicity. Depletion of regulatory T cells has previously been shown to be a potentially critical mechanism of action for CTLA-4 antibodies. So my assumption is that their ICOS agonist antibody is also an IgG1 antibody, like ipilimumab, capable of causing depletion of the cells it binds to. This has the potential to also cause depletion of effector T cells, but in the case of CTLA-4 seemed to be more selective for depletion of tumor-resident regulatory T cells, potentially due to their higher levels of CTLA-4 expression. How this plays out clinically with ICOS antibodies will be important to follow, and I assume we'll be seeing clinical biomarker data on effector:regulatory T cell ratios among others.

Jounce is testing its antibody both as a monotherapy, but also in combination with PD-1 antibodies. One of the rationales for this combination is that ICOS+ cells are shown to be increased in patients or mice treated with PD-1 or CTLA-4 blockade (here and here). Notably, ICOS+ cells are increased also in peripheral blood post CTLA-4 blockade in patients, which perhaps might be a concern that this will increase both the potency, but also potential toxicity, of ICOS stimulation in these combinations. Combining ICOS stimulation (through expression of ICOS ligand constitutively, in tumor models) with CTLA-4 blockade has been shown to increase the efficacy of blockade. Jounce presented data showing that, as with many immunotherapy combinations in mouse models, checkpoint blockade efficacy was enhanced by their ICOS agonist antibody.

ICOS clinical development
Jounce stated that preclinical data suggested tumors with the highest numbers of ICOS+ cells were the ones most likely to respond to therapy. This provides additional rationale for combinations (e.g. with PD-1) that increase the number of ICOS+ cells. Additionally, Jounce stated that ICOS positivity, by IHC in tumor specimens, will be an important biomarker they use to guide development. There is variability in the amount of ICOS+ cells across tumor types (typically the more "immunogenic" tumor types), as well as variability within a given tumor type. Their initial phase I/II trial testing JTX-2011 as monotherapy, and in combination with anti-PD-1, will be an all-comers trial, whereas their fast-following phase II trial will be focused on typically ICOS high tumor types and using IHC to enroll at least half of patients who have ICOS+ tumors. GSK also has an ICOS agonist antibody in clinical trials (H/T @PDRennert).

Celgene Partnership
Jounce also has a partnership with Celgene to develop a number of their products. Jounce received $225 million upfront, a $36 Million equity investment, and there is $2.3 billion in potential milestones (biobucks). There is a 60:40 US profit share on JTX-2011, with royalties on ex-US sales, and US profit sharing on 3 other targets for Celgene as well as shared profits globally on JTX-4014 (PD-1). This partnership is starting with a four year research term.

Summary and Catalysts
Jounce is backed by some of the leaders in the immuno-oncology field. Their initial target of ICOS does make rational sense based on its known biology. However, efficacy in mouse models is always difficult to project to human efficacy, and this is especially true for immunotherapy. I agree with their biomarker approach in spirit, and hope they will continue to search for predictive biomarkers for both their ICOS antibody as well as future targets. Trying to give the right immunotherapy to the right patients is something that makes a lot of sense, as I've written about previously. Additionally, I appreciate their endeavors to look beyond T cells for attractive targets, such as macrophages or tumor stroma.

Jounce has stated that their JTX-2011 all-comers monotherapy and PD-1 combo trial will readout 1H17, and their phase II in ICOS-enriched tumors may read out in 2H17. I will be looking forward to following their progress.

Disclosure: I have no position in any of the companies mentioned

Tuesday, January 10, 2017

My Top 10 New Biotechnologies of 2016 (Part I)

There were many interesting new biotechnologies published in 2016, so it was difficult to narrow it down to just 10. I ended up picking ones that either may have exciting potential to be directly translated into the clinic, or may represent approaches that I think will be important directions where biotechnology can go in the future. Here are the first 5 of my top 10 biotechnologies published in 2016 (in no particular order). Part 2 of my top 10 can be found here.

1. Hematopoietic Stem Cell Transplants Without Myeloablation

There were three papers that came out this year on new ways to deplete hematopoietic stem cells (HSCs) to allow transplantation without the toxic myeloablative conditioning regimens that are currently used. One of the problems with conditioning regimens is they severely compromise the immune system for prolonged periods, making patients susceptible to life-threatening infections. These approaches were all tested in mouse models of transplantation and potentially reduce compromising the immune system. These could immediately be applicable to autologous stem cell transplants and gene therapy approaches of ex-vivo modified autologous cells. In fact, in bluebird bio's gene therapy trials, their main toxicities, so far, have been due to the conditioning regimens. How well these approaches would work in conditioning for allogeneic transplants is less certain, as depletion of the host immune system plays a role in allowing the engraftment of the donor HSCs. There may still be benefits (no genotoxicity & organ damage) if these approaches are combined with immune-depleting approaches (as done in the second paper) to allow allogeneic transplant. Below are the three approaches, with a brief description of each.

CD45 Immunotoxin (link)
This study used an antibody-toxin conjugate (CD45-saponin) to deplete hematopoietic cells. They found this molecule was able strongly deplete HSCs in mice, and allowed faster immune reconstitution with only transient depletion of B and T cells compared to irradiation. This approach allowed high levels of chimerism (>60%) after transplant in immunocompetent hosts. Magenta therapeutics has recently been founded to bring this approach, along with a number of other transplant-improving technologies, to the clinic.

CD47 + cKit antibodies for non-chemo conditioning (link)
In this paper from Stanford, continuing with their evaluation of the effects of CD47 antibodies (previously tested in cancer & atherosclerosis), they used a CD47 mAb to potentiate the effects of c-Kit antibodies to deplete HSCs. c-Kit is present on HSCs and their downstream progenitors. They were able to achieve high levels of chimerism (~60% HSCs) after transplant in immunocompetent hosts. Unlike the above paper, they also tested the ability of this approach in a more relevant model of allogeneic transplantation - transplanting between two different mouse backgrounds. For this they combined their approach with CD4/8 antibodies to deplete T cells to allow further suppression of the immune system and allow allogeneic engraftment, and were able to achieve ~20% HSC chimerism. A concern with this approach, which was brought up in the first paper above, was that c-Kit is also present on cardiac progenitors, gastrointestinal cells, neuronal cells, and cells of the reproductive system. Additionally, in the paper above, they found that attempting to potentiate the c-Kit antibody by conjugating it to saponin did not improve its efficacy, although this could have been for a number of reasons.

Depleting dietary valine permits nonmyeloablative mouse hematopoietic stem cell transplantation (link)
Finally, in this most recent paper, the authors found that depletion of the amino acid valine prevented HSC proliferation both in vitro and in vivo. Valine-free feed in mice also caused partial depletion of immune cells. Valine depletion in feed for three weeks allowed transplantation in immunodeficient and immunocompetent mice, but at much poorer levels of chimerism, ~10-30% vs. 60+%, compared to the above two approaches. Additionally, if valine was reintroduced into diets immediately, it would cause "refeeding syndrome" which was lethal in 10 of 27 mice, but was able to be avoided if valine was reintroduced slowly. This approach seems the furthest away from translation to the clinic of the three.

2. Directed Evolution of Bt Toxin Insecticides to Overcome Insect Resistance Mechanisms (link

I thought this paper was really interesting because it addressed a significant problem, resistance to the major insecticide, Bt toxins, by using directed evolution, an approach that is both tremendously powerful and versatile. Powerful because it uses rapid evolution to let nature figure out solutions to difficult problems without much a priori knowledge, and versatile because the general theme of directed evolution can be applied in innumerable ways depending on how you set up the experiment. For Bt toxins to work, they need to interact with proteins on the surface of insect midgut cells. Insects have started developing resistance through mutation or loss of expression of these surface proteins. The Liu lab decided to evolve Bt toxins to bind to a different midgut surface protein allowing continued insecticidal activity.

To accomplish this, the Liu lab has developed a directed evolution strategy called PACE (phage-assisted continuous evolution) which they used here to evolve protein-protein interactions. They have previously used similar direction evolution approaches to evolve gene editing proteins to improve specificity or to identify resistance mutations to HCV drugs. Here, they used PACE to evolve the Bt toxin (Cry1Ac) to bind the insect protein TnCAD, a protein structurally related to the toxin's normal target. The general scheme is shown below. They are using phage which need to bind to and enter bacteria to complete their life cycle. To bind bacteria they need to express the pIII protein, but the system is engineered so that the pIII protein is only produced if the phage expresses a Cry1Ac variant (evolving protein) that can bind to TnCAD (target). There is also a mutagenesis plasmid that causes continuous mutation to allow continued evolution in the reactor. The reactor has constant inflow and outflow to allow selective pressures to generate the enrichment of phages containing TnCAD-binding Cry1Ac variants.

In the end, they were able to evolve new Bt toxins that could interact with these alternative targets in insect cells. These Bt toxin mutants were found to be much more potent insecticides, especially against Bt toxin-resistant insects, and could lead to the production of new insecticides.

For more detail, Derek Lowe also wrote an excellent commentary on this paper on his blog.

3. A Replication-Defective Human Cytomegalovirus Vaccine for Prevention of Congenital Infection (link)

Human cytomegalovirus (HCMV) is a generally asymptomatic virus that can cause serious complications in immunosuppressed patients and newborns. There currently is no effective vaccine, and there are multiple reasons why vaccine approaches have been difficult to develop. One interesting obstacle is the ability of HCMV proteins to downregulate MHC-I expression and evade CD8+ T cell responses.

Previous live vaccines were attenuated in multiple ways, including mutations to genes in a pentameric protein complex that prevented viral entry into epithelial cells. For this vaccine, the authors restored this ability. Since this modification could strengthen the infectivity of the virus, they attenuated it further by making two essential HCMV proteins unstable in the absence of an artificial ligand (Shld-1). This system uses a degron tag based on FKBP, which binds, and is stabilized by, the rapamycin-analog Shld-1. To produce the vaccine, ligand is present during production, but is not present once given to the patient. They found the more active virus was able to produce a neutralizing antibody response against the pentameric complex, present in natural immunity to HCMV, but lacking with previous vaccines. It was able to produce this neutralizing antibody response (and CD4+ and CD8+ T cell responses) against HCMV in mouse, rabbit, and non-human primate models.

This vaccine was developed by Merck and a clinical trial is ongoing (link). Sometimes the publication of a product by a biotech/pharma company could suggest that they are no longer interested in it. However, for what it's worth, the authors at least state at the end that "Preliminary data from our ongoing clinical evaluation suggest that V160’s safety and immunogenicity profiles in human are consistent with those described in this preclinical study. V160 is therefore a promising vaccine candidate against congenital HCMV transmission."

Recently, there has been an interest in developing HCMV as a therapeutic vaccine vector to treat other chronic infectious diseases, such as HIV and tuberculosis. Vir Biotechnology recently raised over $150 million primarily centered around the development of HCMV vaccine vectors pioneered by Louis Picker & Klaus Frueh's group. One interesting thing to add is that Picker's group found that disruption of the pentameric complex (loss of Rhesus virus ortholog of UL128 & UL130, with mutations in the pentameric complex restored in Merck's vaccine) was necessary to allow the unusual MHC-E restricted immune responses they see in response to their CMV vaccines.

4. Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors

Cell therapies have exploded in popularity since the impressive early results of chimeric antigen receptor T cells (CAR-T) in ALL. The surprising potency of these cells, and the fact that they can persist in the body, can differentiate these therapies from other antibody-targeted therapeutics. However, one of the truly unique features of cell-based therapies is the ability of a cell to contain much more information than a small molecule or biologic could. In addition to adding a targeting domain and T cell activation domain, one could modify cells to do a whole host of other processes. Tools are now being developed that will hopefully be able to unlock the programmable potential of cell therapies.

Wendell Lim's group has published three (!) Cell papers this year on their new synthetic notch receptors (synNotch), which allow customizable programming of cell responses in adoptive cell therapies.

The first paper describes how synNotch receptors work. Normal Notch receptor signaling is very simple, the Notch receptor binds to its ligand, which stimulates the separation of an intracellular signaling domain that can go on to affect gene expression. Previously, Notch reporters have been developed that contain the same Notch receptor extracellular domain, but with a transcription factor internal domain that can activate GFP to report when Notch signaling is activated. The Lim lab modified this further by coming up with many different potential extracellular domains beyond the Notch receptor to allow this cassette to respond to different inputs, as well as different intracellular domains to allow it to react differently to these inputs. The schematic is shown below.

In this first paper they use synNotch receptors in a variety of ways in additional cell types other than T cells. Additionally, more than one synNotch circuit can be used even within the same cell.

In their second paper, published back-to-back in Cell, they used a synNotch circuit to increase the specificity of CAR-T cells to require the presence of two antigens. Normally CAR-T cells are directed against a single antigen (CD19 etc.) however, not many cancers express a surface antigen that is specific only to the tumor and not essential normal tissue. To increase specificity, a number of groups have developed approaches to require the simultaneous presence of two antigens on the target, which will hopefully single out tumor cells and reduce activation against normal cells possessing only one of the two antigens. The Sadelain lab has also published approaches requiring target cells to possess two antigens simultaneously or specifically one antigen but lacking a second. The Lim lab used their synNotch circuit to direct expression of the CAR only when the T cell binds a first antigen, with the CAR recognizing cells containing a second antigen. The schematic is shown below.

They make T cells that should react only against cells with two (artificial) antigens, and find that the cells do in fact have specificity for cells expressing both. In vivo, when cells expressing one antigen were injected in one side of a mouse and cells expressing both were injected on the other, the engineered cells primarily localized and prevented the growth of the cells expressing both antigens. The ability to maintain specificity to both antigens will be critical for safety, as there is the potential for the CAR to be expressed after interacting with antigen A, and then be able to move around the body and react with a normal cell that only expresses antigen B. However, the ability of a T cell to make decisions from two inputs instead of one could potentially drastically increase the tumor types and targets amenable to CAR-T targeting.

Lastly, the Lim lab more recently published a paper using synNotch circuits to use T cells to deliver different payloads, such as immunomodulatory antibodies, bispecific antibodies, cytokines, or cytotoxic agents localized to target cells expressing a specific antigen. Some of the possibilities are shown below:

This is one of numerous ways to use T cells to cause local changes, which could be critical for enabling higher local concentrations or decreasing systemic concentrations of molecules that could have undesired systemic effects. The synNotch platform is impressive due to its modular nature and ability to be customized, which perhaps will allow it to be applied to multiple therapeutic applications.

One potential concern with all of these approaches is the potential immunogenicity of these additional proteins being produced by the cell. Although how much, if any, this will limit cell persistence/activity is unknown.

This work is being commercialized by Cell Design Labs, as a spinout from the Lim lab.

5. Targeting of Cancer Neoantigens with Donor-derived T Cell Receptor Repertoires (link)

There has been an increasing realization of the importance of the endogenous immune response against neoantigens, immunogenic mutations present only in a patient's cancer. Clinical trials of tumor infiltrating lymphocytes (TILs) and checkpoint inhibitors, like anti-PD-1 and anti-CTLA-4, have demonstrated that the reaction against neoantigens may be critical for the efficacy of these therapies, and boosting a response against neoantigens is an attractive therapeutic approach. Directly targeting neoantigens, with vaccines or T cell receptors (TCRs) is complicated by identifying which ones to target. Steve Rosenberg's group has done a lot of work with TILs and has demonstrated that T cells present in the patient's own tumor can react against specific neoantigens and induce long term tumor regressions (examples here and here). One questions is whether or not the patient's own immune system has produced a robust response against all the potential immunogenic antigens to allow all potential reactive T cells to be detected. This is what Ton Schumacher's group set out to investigate in this paper, where they attempted to identify if T cells existed in healthy donors that could react against patient-specific neoantigens.

The authors used autologous dendritic cells (from donors) loaded with tandem minigenes expressing different predicted neoantigens present in a patient's tumor, and cocultured them with donor T cells to determine if the T cells could react against any of the neoantigens. Across three patients, they could identify T cells reactive against 11 of the 57 predicted neoantigens. Only 1 of the 11 neoantigen-reactive T cells was detectable in TILs from a patient, indicating this is a way to identify a broader repertoire of TCRs reactive to patient-specific mutations. Additionally, this study attempted to expand our understanding of what makes an immunogenic neoantigen. They found that higher stability of the peptide-MHC complex for a neoantigen was statistically significantly associated with being able to identify T cells reactive against it.

This approach provides a way to prospect for neoantigen-reactive TCRs and expand the pool of potential TCRs beyond what is found in a patient, with potentially significant implications for neoantigen-targeting therapies.