CUNY emails

A source was kind enough to provide us with emails that were FOIA’d from CUNY. Selected excerpts of these emails were used by a handful of twitter folk to undermine Dr. Hoau Wang and spread FuD around the investigations currently underway (at CUNY).

Part 1 – Finances

Stock holdings (June 2021)

Dr. Wang signed a disclosure where he indicated his stock holdings as of 06/29/2021 had a value of about $125,000.

From 06/14/21 – 06/29/21, Cassava’s share price traded from 75.xx to 85.xx and we can thus estimate he held ~1600 shares. This isn’t very much in terms of motive to destroy a substantial and meaningful reputation as a scientist and professor spanning decades. Additionally this underscores that all of Dr. Wang dealings with Cassava have been under appropriate service contracts.

Email request from FINRA (October 2020) 

A FINRA complaint was lodged against Cassava Sciences with regard to trading activity following the phase 2b redo. Considering the appreciable share price increase following those results, a routine request for information was sent over to Dr. Wang.  His responses leave no room for ambiguity. Below is a summary:

  • CUNY did not know the results of the p2b redo and did not communicate with Cassava Sciences on this topic prior to the public readout
  • CUNY was never unblinded
  • CUNY had no access to clinical data or demographic information of the patients

We previously discussed how the blinding protocol for a clinical trial has lots of checks and balances in place. Cassava’s p2b was quadruple-blinded. In reviewing the Citizens Petition, the FDA is likely to have looked through the logs of the electronic data capture (EDC) system while also reviewing the end-of-phase 2 meeting notes before proceeding to dismiss the petition

Part 2 – Bios Partners

Bios Partners is the lead venture capital firm behind Cognition Therapeutics (CGTX – more on them a bit later). In the email excerpt below, Tim Van Treuren, a senior analyst with Bios Partners, emails AAIC directly (instead of Dr. Wang) with questions on the SavaDx poster.

We previously saw Travis Whitfill, another senior partner with Bios Partners, take the lead to tag team Elizabeth Bik on Dr. Wang’s papers right after the CP (Citizen Petition) came out.

There seems to be more than cursory interest from Bios Partners regarding the development of PTI-125 especially considering the phase 2 trials for their lead candidate CT1812 showed robust biomarker improvements.

PTI-125 earlier showed a whole panel of biomarkers correlating and improving over 28 days. Many twitter scientists (with unimpressive credentials) frequently bring this up as results that are impossible over such short periods of time. But here there are 2 biotechs showing similar results.  Also this excerpt from a patent filed by the CGTX team is telling in that it reads just like the MoA of PTI-125.  Is it a coicidence that the lead venture firm behind CGTX (Bios Partners) is busy trying to undermine Cassava while at the same time CGTX is taking a page out of Cassava proposed mode of action? We think not.

Part 3 – Night king of shorting

Adam Feuerstein is infamous for handing out public (and private) invitations to short biotechs.  We saw this recently when he attacked Anavex Life Sciences over a delayed regulatory update, while waiting weeks to time his FuD right when the AVATAR study results dropped.

Adam has also been on record attacking Cassava Sciences with misogynistic statements and insinuations of fraud while maintaining ties with the authors of the Citizen Petition. Below is an excerpt of an email from him to Dr. Wang.

For a self proclaimed biotech expert, Adam pretends to not know what a blinded trial is. He also pretends to not know about other blinded trials where analysis is done in-house. For instance, the trailblazer-alz-2 trial which he’s mentioned a few times (without much criticism) had all its biomarker analysis done unblinded as seen below.

This study prompted donanemab to receive Breakthrough therapy designation.

Conclusion

There is an active short/distort campaign against Cassava Sciences. The CUNY emails were selectively released to paint the company in a bad light. Short investors have targeted the firm since Oct 2020 starting with FINRA complaint(s). Jordan Thomas has used insider knowledge from his time with the SEC, to file a Citizen Petition as a whistleblower representing many yet anonymous clients. He either knew or should have known that the Citizen’s Petition procedure was never meant to apply to drugs in phase 3 trials. CUNY emails including confidential information and data from previous experiments were provided via FOIA to Summit Point Capital, a hedge fund in NY. There appears to be on-going collaboration between them and some of the twitter scientists in sharing CUNY emails – this is further supported by the recent procurement of Cassava’s Phase 3 Investigator Brochure.

In upcoming articles we are preparing to post more CUNY emails that reveal insightful interactions of Dr. Wang. This includes information on a potential new indication for PTI-125 and our thoughts on SavaDx.

Dr. Douglas Baker shares his personal story about Cassava Sciences’ trial patients

In this article, we briefly cover the interview of Dr. Douglas Baker, hosted by Joe Springer on Youtube live at 3 PM EST today. Dr. Baker speaks about two patients who are currently enrolled in Cassava Sciences’ phase 2 trial to test their Alzhiemer’s drug Simufilam. Cassava’s readouts of data and overall statistics so far show that the cognition improvements of the first 50 patients in this early trial are very promising. The following anecdotal, supplementary evidence of two improving patients from Dr. Baker provides further encouragement and intends to raise more awareness of Simufilam trials. We encourage our readers to watch the interview and/or read the below snippets. Ad-science members find that Dr. Baker’s description of his acquaintances’ experience is poignant, sincere and a heartfelt portrayal of disease effects and drug’s impact on the concerned individuals.

Following is our abridged version of the interview corrected for grammar, context and brevity. Q & A session has not been transcribed yet – to be added at a later point in time.

Joe Springer (Interviewer): Hello everyone, Welcome Dr. Doug Baker who knows two people that are currently enrolled in the Simufilam open label trial of Cassava Sciences. Note that this trial is not placebo-controlled. It is 12-month long and is open label, so the patients are definitely taking the drug. There is a six-month placebo extension at the end of it. Cassava sciences is also running two large Phase 3 trials (RETHINK, REFOCUS) simultaneously at this time, which are both placebo-controlled.

Dr. Doug Baker: Introducing himself.. I’m a 64 year old ophthalmologist and I specialize in Glaucoma. Alzheimer’s disease has affected my family. My father died at the age of 92 due to Alzheimer’s. My best friend and best man in my wedding – for the last year and a half we didn’t have a good relationship at all because he thought I went down to South Carolina and raided his bank account and stole all this money. He looks at me like “how could you steal all my money” and so you know, Alzheimer’s has a special place in my heart. I feel for those that have families dealing with it and hopefully we can raise the awareness of the trials and this new drug.

Joe Springer (Interviewer):  I’m sorry to hear about that heartbreaking story. You are an ophthalmologist but you happen to personally know two people that are in the Simufilam open label trial. They are not under your care as you said. Can you tell us about them? What are their symptoms and when did they enter the trial?

Dr. Doug Baker:  I know a gentleman who is in his early to mid 80s and was extremely forgetful. In one hour he would probably repeat himself 10 to 12 times for a certain question. He was unsteady from an ambulatory perspective and he had difficulty comprehending the written word. He lost interest in reading or if something was read or if he was reading something, he couldn’t really process it. 

The other individual was a charming late 70 year old female who became extremely irritable and could not remember names. She was forgetting things but the biggest thing the family had difficulty with was she went from a super sweet lady to “get out of her way” type personality. The other behavioral issues we’ve heard about, so often they could be even worse than the cognitive decline.

One individual entered the trial in december 2020 which was a difficult time because that’s right in the middle of the pandemic and the vaccines weren’t out. The family was very brave to go across the country to the trial site just outside of Miami to enter into the stud. The other individual, I believe, entered in late january or early february 2021.

Joe Springer (Interviewer): So they have been on for about a year now. How long did it take for the changes to become noticeable?

Dr. Doug Baker:  For the elderly gentleman, it took about three months and then he’s had basically steady improvement in all the areas i’ve listed. Ambulatory, reading, repeating himself and forgetfulness. For the other individual it took about four or five months. Since then both of them had steady improvement in both cognitive and behavioral issues.

Joe Springer (Interviewer): That’s remarkable to hear. I wanted to mention earlier that we also heard from Dr. Gonzalez Rojas, way back after the 2b results. I think she presented the first anecdotal evidence that patients were getting better on Simufilam and she had a number of examples. She said very similar things that you mention here. It took a couple of months and they started seeing improvements. Have there been any adverse effects or downsides?

Dr. Doug Baker:  No, it’s a simple 100 milligram pill that they take twice a day. There’s no stomach upset and there’s no issues at all. I mean it’s just like taking a vitamin, easily administered, easily tolerated and with no side effects.

Joe Springer (Interviewer): We know that alzheimer’s disease is a disease of continual decline so the patient should always be getting worse and so have you seen a level off or decline or an improvement

Dr. Doug Baker: I just got off the phone with one of the family members prior to coming on the show and basically they’ve noticed from the three to four month mark, a steady improvement. It’s not rapid but every month they seem to get better. From an ambulatory standpoint,  walking briskly without falling. They are not worried about him falling anymore. He might repeat himself twice in an hour versus 10 or 12 times. When they were passing around some lab work of one of his brothers, he told his brother that he’s gonna have to stop eating bacon and eggs because his cholesterol was too high. This is the guy that had lost interest in reading because he had a hard time comprehending reading and now he’s scolding his brother for his elevated cholesterol levels.

The other lady – we saw at a Greek choir concert for christmas. She could not remember names and was extremely irritable. She was back after a year to her charming personality and called everyone around the table by their name. I’m not quite sure I could do that but that was after a year of treatment.

Joe Springer (Interviewer): Wow thank you very much (for sharing)! Alzheimer’s disease is a big disease for caretakers. Can you speak about the families and loved ones and what their reaction has been?

Dr. Doug Baker: As you know, it probably affects every single family in this entire world. I would strongly encourage them to think of entering them (Alzhiemer’s affected family members) in trials. Basically, there is a 50% chance of improvement (randomized between placebo v treatment) whereas if you don’t go into the trial you have no chance of an improvement because it’s a steady march downward.

Joe Springer (Interviewer): I’m so glad you brought that up. It’s a steady march downward and it’s a disease that’s always terminally ill. Everyone, there’s https://rethink-alz.com/ or to go to cassava science’s website and it links out from there. If you have a loved one with Alzheimer’s disease, these phase 3 trials are enrolling right now. Studies show that this drug has no side effects.

Dr. Doug Baker:  If this was three years earlier I would have had my father in that study in a heartbeat.

Joe Springer (Interviewer): You treat Glaucoma as you mentioned earlier. Can you speak more about it?

Dr. Doug Baker:  It’s interesting that in Glaucoma, the elevated eye pressure damages the optic nerve. Now, the optic nerve is the largest nerve in the body. It goes straight from the brain right to the eye and if you treat somebody with advanced Glaucoma, it’s difficult. On the other hand if you pick it up early before the optic nerve has much damage it’s significantly easier to treat. You know, it’s the same thing with Alzheimer’s. If we can pick this up early at the very mild stages, certainly you have a better chance of recovering quicker and staying cognitively better versus  if you’re on your last legs. I said it’s sort of another reason to get this awareness out there so people know about it.

Joe Springer (Interviewer): Cassava sciences has received funding from the National Institute on Aging for a blood test. Right now, it’s hard to diagnose Alzheimer’s disease. You must wait until a person already has symptoms. The blood test may be able to detect Alzheimer’s disease years ahead of time, very cheap and easily without a spinal tap or anything intrusive or painful like that. 

Thank you very much Dr Baker. Dr. Baker has been gracious enough to take questions from everyone. Q & A session follows ..

An Expression Of Concern

On 12/17, J. Neurosci followed up an erratum published 10/11 with an Expression of Concern.

The first thing to point out is that the editor of the journal has been subjected to constant harassment by the Twitterati, some of whom have influential positions. Nevertheless according to COPE guidelines, accommodations must be made and in this case the expression of concern defers the legitimacy of the blots to the City University of New York (CUNY).  We note here that the J.Nsc has also updated the original paper to include the Erratum on 12/15, 2 days before issuing the Expression of Concern

This strengthens our view that the Expression of Concern is tied to the ongoing investigation. We fully expect that CUNY will be able to adjudicate this issue with the diligence it deserves. While hard evidence may be lacking to counter all accusations, Dr. Wang’s authorship of multiple papers with many different researchers, faculty and students over many decades provides a unique window into his actions, and an opportunity to determine wrongdoing. CUNY will no doubt be able to interview some of these people and weigh their opinion before making a decision.  Below are some thoughts on the blots in question:

Figure 6A & 6B: original pubpeer report

The erratum addresses these concerns, and there has been no issues raised so far on the original blots submitted for this figure.

Figure 9A: original pubpeer report from Dr. Bik

The original in the erratum was then analyzed by Dr. Bik, and the following crop signature was flagged:

[Updated] What is likely going on with the darker background seen in the figure is that a small unexposed film was taped to the surface of a larger exposed film to run through the developer. This is done because you don’t need a full 8 x 11” or 5 x 7” film to expose a small WB, so they are usually cut into smaller pieces. The small film is then taped to a larger film because smaller films inevitably get caught in the rollers of the developer. It’s not fun digging out a stuck film from the developer. Anyways, when this occurs you can sometime see the image of the larger exposed film under the smaller taped film, which I think is what is happening here. There could be other explanations as well, including defects with the nitrocellulose membrane, which is usually cut from a large roll. What is interesting about this figure though is that the dark background appears to run through a band, which is completely illogical if the data was manipulated by cutting and pasting. As far as the 2 additional bands at the right, many researchers will run “extra samples” to try to gain more information. There is no rule that you have to report an entire figure in a publication, just provide the whole figure if questions arise of validity of the data. That fact that they actually provided an uncropped original data with the 2 extra lanes, which would undoubtedly raise more suspicion from the doubters, tells me that they are being completely honest with addressing the data manipulation claims.

Cassava critics and blots

As an example of how blots can be questioned, here is a look at an “original” blot from a paper we looked at. See figure 6C  

Setting contrast to 100%, the same figure looks like this: 

Notice the obvious contrast changes, crop signature, identical-looking blots, irregular spacing etc. We highlighted a few spots. Note that this paper is authored by Enea Miloris, one of the PhDs who constantly accuses Cassava of fraud..

We then looked for Western Blots run by Adrian Heilbut, another self-proclaimed expert on blots and a foul-mouthed critic of Dr. Wang and Dr. Burns. We found zero blots in his papers.

Elisabeth Bik already owned up to not having enough experience in the field in one of her tweets.

Put this in perspective. The twitter experts finding flaws don’t have much real-world experience running blots.  So why not look at a critic who has actually run blots over many decades?

Figure 1A Bidirectional Synaptic Plasticity Regulated by Phosphorylation of Stargazin-like TARPs

Figure 1A Blue arrows show vertical splices

Figure 4B Identification of PSD-95 Palmitoylating Enzymes

Yellow arrows show possible splice. Also one can see what looks like a partial fingerprint

The left-most bands in the PS and AD lanes have a rectangular box with a different background appearance as the other lanes.

The last three papers were co-authored by David Bredt, the same expert who found flaws with Dr. Wang’s western blots and filed a Citizen Petition anonymously for monetary benefits.

Source

The petition is also plagued with inaccuracies and we have addressed many of them in earlier posts.

Another look at Dr. Wang’s blots with contrast at 100%

Source: Fig 1: Reducing Amyloid-Related Alzheimer’s Disease Pathogenesis by a Small Molecule Targeting Filamin A
Source: Cassava Sciences phase 2a slide deck

Dr. Wang has run a lot of blots over the years and most have no issues. The phase 2a blots above show ptau levels dropping over the 28 day dosing period. Note that ptau is a crucial biomarker of AD and given that these blots look clean, its a remarkable achievement. An upcoming blog post will dwell into this further.

Our Expression of Concern

We understand that there are concerns around Dr. Wang’s research and scrutiny isn’t always a bad thing. However there are some well known folks who have dedicated themselves to harassing anyone associated with the company to serve their financial goals. These folks are at it all day picking and dicing every little detail, running a maximum pressure campaign aiming to influence investors and regulators. We want to remind folks that Simufilam has been used by Bordey Labs collaborating with Calderwood Labs at Yale School of Medicine. The following excerpt is from the mission statement of Bordey Labs.

Source

Calderwood labs have done research on FLNA and how it can have cryptic binding sites. Below is a screenshot of a slide deck presented by Dr. Bordey in the recent past. 

Dr. Bordey’s lab observed Simufilam/NT-125’s treatment effects in preventing seizures associated with TSC, and in preventing cell enlargement that leads to TSC. They also have reason to believe this may be a similar path for Alzheimer’s treatment.

We question why Dr. Wang would need to fake so much science for a drug that seems to have some effect on TSC. We also question all the antagonism towards Cassava when their drug, if not better, is as promising as the multitude of other drugs undergoing clinical trials for AD – see figure below.

In our view, the short and distort crowd are willing to go to any length including preventing an effective treatment for AD from reaching the market. We ask the FDA, NIH, CUNY and other prominent players to step in and ensure completion of these trials. 

Of shorts and blots

This article looks at specific accusations in the citizens petition supplement attachment 1

Example #1: “Manipulated WB”. The CP claims that different WBs were “spliced together” to make a figure in a 2005 paper. They point out a “bowtie” effect of several bands as supporting evidence. However, anyone with WB experience has seen this bowtie effect. In theory, it can be caused by proteins sticking to the sides of the wells or a small amount of extract entering the space between the wells and the plates that support the gel on each side. This effect is often seen when the top of the gel is exposed to air too long prior to loading.

Example #2: “Falsified WB”. This claim states that the same beta-actin control WB was used in 2 different papers published 5 years apart. The CP takes a beta-actin WB, which is low resolution, stretches it vertically, then blows it up and compares it to a WB published 5 years earlier. Although they claim the blots are identical, to me they look completely different. For example: 1) the left side of the band in lane 1 of the 2005 publication has a notch that is not in the 2010 WB; 2) band images between lanes 2 and 3 are clearly different; 3) the right sides of the bands in lane 4 are also different. I also have difficulty taking this claim seriously. The labs likely ran many beta-actin WBs between the 5-year timeframe the 2 papers were published, but it is claimed they somehow elected to risk their scientific careers to include 4 control lanes from a previous publication? A new beta-actin WB could be run in half a day. This accusation suggests that the scientists who contributed to the CP are not very experienced in molecular biology.

Example #3: “Reused WB”. This claim takes 2 WB panels of a figure and compares them side-by-side, suggesting they are identical. This claim could have some credibility, though it is more likely a mistake in constructing the figure for publication than scientific misconduct. Although I can’t say for sure that the 2 WBs in question are indeed the same, they do look very similar, though one is a slightly darker exposure. When WBs are exposed to film, we generally get several exposures from light to dark. If the films aren’t labelled properly, things can get mixed up when the graduate student/post-doc generates the figure for publication, especially if two of the WBs look similar. If the 2 blots are indeed the same, the PI, other authors, or reviewers probably should have asked if this was indeed an error. I’m inclined to think this was an error in making the figure and not intentional since there is nothing covert going on here- the 2 WBs are basically side-by-side in the same figure.

Example #4: “Band insertion into WBs”. The concerns raised here include: 1) irregular spacing between lanes; 2) FLNA bands not looking correct since it is a large (290 kDa) protein; 3) bands looking identical between lanes; and 4) white halos around bands. In my opinion, this claim is completely baseless and shows a lack of experience of the scientists that generated the CP. Irregular spacing between bands is routinely seen with self-poured gels. It is explained by the teeth of the comb used to cast the gel not being completely straight when the gel is polymerized. The claim that FLNA shouldn’t run very far in a gel because of its large size is inaccurate because proteins will run as far through a gel as a current is applied. Longer gels could have been used. Also, the researchers could have used gradient gels that allow for larger proteins to be better resolved in the gel. As far as the control beta-actin bands looking identical, they are basing this assumption on a common “tadpole-like” appearance of the bands. However, this tadpole effect is commonly seen if the gel is polymerized next to an air current (e.g. in a fume hood). The air current can cause the sides of the wells to lose moisture during polymerization resulting in a tadpole-like effect on the same sides of the wells of each lane, which is seen here. As far as halos around bands, most films used to expose WBs have a threshold level of exposure that is designed to limit background. You often see this halo effect directly on the exposed film, especially with stronger bands. The white halo could also be a compression effect, caused when the figure levels were adjusted for publication.

Concern 3.2 “Evidence of data manipulation from human tissue”. It is claimed that Figure 12 of a 2017 paper shows a WB with 12 lanes for the control protein NR1 and 13 lanes for PCLgamma1. It is also suggested that WBs from different experiments were spliced together to make the figure. The 12/13 lane issue is an obvious error that likely occurred when making the figure for publication, but in no way rises to the level of scientific misconduct. As far as splicing together 2 different WBs, please refer to Response to General Concern #1 above about manipulating low-resolution figures and spliced WBs. I honestly don’t see much evidence of splicing for the top WB. Also, on the bottom WB, the change in pixels appears to wrap around the top of the band on the left side of the proposed splice site. It would be difficult to crop the figure this way. Another thing to point out is that the 3 lanes on the right of both WBs are running lower in the gel than the band directly to the left, which is slightly higher than the other bands to its left. However, the proposed splice sites are 4 lanes from the right on the top WB and 3 lanes from the right on the bottom WB. These factors support that the figure was not generated by splicing 2 WBs spliced together.

Appendix items/Additional areas of concern:

Claim #4: It is claimed that the top WBs cannot be FLNA based on the banding pattern. Please see Response to Example #4 above. Also, note that Albumin was used as the control protein here, which is a comparatively large (66 kDa) for a control protein and suitable for analysis of large proteins, such as FLNA.

Claim #6: It is claimed that several papers between 2010-2017 don’t show control WBs for co-immunoprecipitation experiments that evaluate the interaction of beta-amyloid with alpha7-nicotinic acetylcholine receptors. This claim is true, but there is a very good reason for it. Beta-amyloid are small (10 kDa) peptides of 40-43 amino acids in length. Their small size makes them very difficult to analyze by WB, other than running specialized gels that would likely preclude the analysis of larger interacting proteins, such as alpha7nAChR (60+ kDa). Most companies pre-validate their antibodies for use in certain applications, such as immunoprecipitation experiments.

E.2 Additional suspicious WBs (1) and probable band duplication (2):

1) Suspicious WBs. The CP shows several examples (pages 30-31) of control bands looking the same between WBs suggesting that the researchers must have duplicated these images. To me, this is a ridiculous claim. I see clear differences between the bands in question and I think most others do as well.

2A) Probable band duplication. Regarding the white halo around bands on WBs, see Response to Example #4 above.

2B) FLNA bands look identical (page 33). The CP highlights 3 FLNA bands that they claim are duplicated, but I see clear differences. For example, differences are seen with the bottom of the right and middle bands and top of the right bands.

2C) Five IRbeta bands look identical (page 34). There are clear differences in the sizes of the notch at the bottom left of each band, as well as the slopes of the bands on the top right.

2D) Clipping multiple blots together (page 35). The clip effect that is seen when the proteins run through an air bubble in the gel, which is common with self-poured gels.

2E) Tampering of WBs (pages 37-39). It is claimed that bands are inserted into various WBs in a 2006 Nature Medicine publication. This claim is borderline ridiculous and is likely why it appears at the end of the CP. Dr. Wang is first author on this publication, so the implication being made is that he started manipulating data early in his career and continues to do so today as head of an academic laboratory. Firstly, Nature Medicine is an outstanding journal, so the data was thoroughly vetted by experts in the field prior to publication. See Response to General Concern #1 regarding manipulation of low-resolution WB images and drawing conclusions. Also, several of the examples shown as evidence of data manipulation support the opposite. For example, on page 38 the background pixels around the bands are irregularly shaped. It is hard to believe that Dr. Wang would have cropped each of the bands for insertion as irregular shapes (which would be difficult in 2006) instead of just using a box shape. Also, if differences in background pixels are evidence of data manipulation, why are there also irregular shaped pixels around the text in the figures? Were they copied from other figures as well? It is obvious that the differences in background pixels observed are the direct result of the scientists who constructed the CP altering the contrast and brightness of already low-resolution figures.

Notes from a Molecular Biologist

The author of this post received a Ph.D. degree in Molecular Biology and has been an academic researcher since 2003. His laboratory studies cancer and other human diseases and routinely run western blots (~1,000/year) for their studies.

I thought it might be helpful to post an expert’s review of the concerns raised in the Labaton Sucharow Citizen Petition (CP) regarding western blot (WB) data for Cassava Science’s Alzheimer’s drug. Simufilam has demonstrated remarkable improvement in cognition in Alzheimer’s patients at 12 months of treatment in an open label study and the drug is entering two pivotal phase III studies.

Western blotting is a research tool that we use to analyze proteins, including their levels, modifications, and association with other proteins. Protein extracts are prepared from cells or tissue, denatured using a detergent and heating, and then loaded onto a gel, which is a matrix of polyacrylamide, and then a current applied. This allows for proteins to be separated based on their size (i.e. molecular weight (MW)). After the gel is “run”, the proteins are transferred (i.e. blotted) onto a membrane and then probed with an antibody specific for whatever protein you want to analyze. The blot is then probed again, this time with a secondary antibody that is enzyme-linked and binds to the first antibody, thus allowing for detection using a chemiluminescent reagent that produces light where the first antibody binds the membrane (thus producing what is known as a “band”). All WBs include analysis of a control protein (e.g. actin, GAPDH) to confirm that equal amounts of protein extract were loaded in each lane.

The first thing to understand about WBs is that they are semi-quantitative. They can basically tell you if there are differences in levels (increases, decreases) or presence of a modification or binding protein. Secondly, the western blotting technique is somewhat of an artform. It takes practice knowing what procedures are best to analyze a specific protein. Thirdly, there are a lot of variables. Researchers use different first and second antibodies, gel conditions, membrane supports, washing and exposing conditions. There is no standardized protocol. For example, many academic labs that are on a budget will pour their own gels as opposed to buying more expensive pre-made gels. With the former, you have more control over your analysis, but the WBs can often look a little dirtier, lanes slightly unevenly spaced, or bands looking not so sharp. Though the results, in terms of levels etc., should be the same no matter what variations are used.

My overall impression of the CP is that it does exactly what it set out to do- confuse the average investor and seed doubt so that they sell their shares. The average investor has no idea what a WB is let alone how to objectively analyze them. The majority of concerns raised are easily explained and a small number of others are obvious errors that likely occurred during generating figures for publication. There is no obvious evidence of systematic data manipulation or scientific misconduct. Someone with knowledge of molecular biology likely helped put together the CP, but many of the concerns raised show a shallow understanding of WB technique and data analysis. Below, I provide responses to general concerns and then a point-by-point analysis of each concern raised in the CP.

General concerns raised in the petition:

1.    It is stated that “the western blot data presented by Wang/Burns are almost always overexposed and highly processed, which has been repeatedly seen in previously reported examples of image manipulation.” Several of the studies scrutinized in the CP were published between 2005-2012. It is important to realize that during this time WBs were visualized by exposing the membrane to film. Control proteins generally give very strong signals due to their high level of expression in cells and use of validated antibodies that give the best (i.e. strongest) signal. Also, analysis of low-expressed proteins requires more extract to be run per lane to see the signal. These factors can often lead to control bands that are strong (i.e. overexposed). Today, most researchers use digital imaging systems to expose WBs, which avoids issues with overexposure. As far as the claim of “highly processed” images, know that most research papers published pre-2010 used images of 300-600 dpi or lower. Images retrieved from online papers, as was done for the CP, are likely even lower resolution. In my opinion, it is dangerous to take a low-resolution WB and adjust the contrast and brightness levels to attempt to uncover evidence of data manipulation. For example, the CP repeatedly shows changes in background pixels as evidence of data manipulation, but there are many factors that could give this effect. Over the years, I’ve seen things as simple as streaks on a film caused by the processor or how the membrane is wrapped with layers of plastic wrap to prevent drying from influencing the background of the figure.

2.    It is repeatedly said in the CP that the authors should produce the original WB data in order to satisfy their concerns. I’m pretty sure the scientists behind the CP know that this request is unreasonable. Most researchers will store data after publication in case questions do arise. In a perfect world, films and electronic WB data would be stored forever, but students leave the lab, labs downsize or move to new institutes, and thumb drives get lost. In addition, journals generally don’t require researchers to keep data for longer than a year or so. The fact that Drs. Wang/Burns have not “answered” the WB concerns of the CP is likely not due to them hiding something but rather that it is impossible to retrieve data used in a publication 10-15 years ago. Journals rely on a thorough analysis of the data by the reviewers, which are generally 3 experts in the field. If a reviewer had a problem with any of the WBs in question, they could have asked for them to be repeated. If they thought the data were manipulated in any way, the reviewers would have instantly rejected the paper and likely notified the journal.

3.    The CP repeatedly says that their analysis indicates fraud or scientific misconduct. Scientific misconduct is a very serious claim. As scientists, we welcome critiques of our data presented in research papers or presentations. That is part of being a scientist. But know that scientists are trained from early in their careers and throughout about what constitutes misconduct. We take refresher courses and sign statements when we submit research papers regarding the validity of our work. Every researcher knows that being accused of misconduct can instantly ruin a career. In my years as an academic researcher, I have never known of anyone who was even accused of scientific misconduct. I’m not saying that it doesn’t happen, but there is a big difference between a paper mill in China that is duplicating figures in different publications and an established researcher whose lab has consistently published quality papers and is supported by NIH funding. It is improbable to think that Drs. Wang/Burns have systematically produced fraudulent research and manipulated data for research publications and grant applications without a colleague, collaborator, or reviewer detecting it for 15+ years.

In the next article, I will follow up on specific allegations made in the petition.

Citizen Petition Supplement

The petition against Cassava Sciences was supplemented on Sep 8. We shall examine some questions arising from this supplement and provide our comments in this post.

Concern #1: Authenticity and Availability of Phase 2a Data—Western Blots

The petition claims the original western blot images should be available at Cassava’s premises
Can someone point us to a regulation that requires this?

The petition claims corresponding authors in a paper must store, maintain, and validate data.
The role of the corresponding author according to International Committee of Medical Journal Editors (ICMJE) is shown below

Source :http://www.icmje.org/recommendations/browse/roles-and-responsibilities/defining-the-role-of-authors-and-contributors.html

Concern #2: Phase 2b Biomarker Redo Was Not Done by an “Outside Lab”
Concern #3: Which Outside Lab Did the First Phase 2b Biomarker Study?

These concerns were already presented in the original petition and are just repeated in the supplement. Here is the relevant paragraph from Cassava Sciences’ Form 10-K where they mention that blinded conditions were maintained.

Source: https://www.cassavasciences.com/static-files/0e33310f-8cec-4c7b-8444-8ae4bc25116f

We also tried to find other study protocol designs where names of the specific lab(s) were listed and mostly came away empty-handed. Most big and small pharmas do not list the names of the labs they use; many do not even upload study protocols or results onto clinicaltrials.gov. This list includes drugs that were granted SPA. When we did find a trial granted SPA with a study protocol, the shipping address for the samples in that study was blacked out. But by carefully studying the protocol we found that the shipping recipient was the sponsoring pharma. Our point here is simple – the independence of labs is only truly guaranteed by blinding requirements and not affiliation. If this were not the case, it would be impossible to trust trial sites and labs used in developing countries, as many big pharmas do.

On this front we plan to watch out for the study design of Cassava Sciences’ phase 3 studies for Simufilam where we may learn how they plan to leverage their new CRO.

Concern #4 Likely Lab Errors or Manipulation in the Phase 2b Redo Study

The petition claims that CSF/plasma albumin ratio baseline does not match clinically observed ratios

In the methods section of the phase 2b paper, it is mentioned that optical densitometry is used in determining CSF albumin levels. It is therefore fairly clear that the CSF/Plasma albumin baseline ratio also used the same measure. Our team member highlighted this fact on twitter first. 

Albumin constitutes a high portion of plasma protein (~60%) and can easily hit the upper end of the optical density range due to saturation. This usually causes an under measurement of plasma albumin and can bump up the CSF/plasma albumin ratio significantly as seen in the baseline provided by Cassava. The clinical ratio Qalb, which is usually measured by a more precise ELISA assay, cannot therefore be directly compared to this technique.

In Cassavas’ defense, the change in CSF albumin (not the ratio) is the biomarker of interest and the comparisons there between placebo and dosage arms show a treatment effect and a positive reduction of CSF albumin.

We also want to point out the malice in the chart included in the supplement as seen below.

The petition referred to multiple past studies where the CSF/Albumin ratio was reported in the range 4 -9. This is, in reality, 0.004 – 0.009 (without the 1000x factor to make QAlb read more easily in clinical charts). They placed Cassava’s value of ~0.25 alongside this in an attempt to magnify the difference. This is extremely misleading and can easily be shared on social media to propagate misinformation. The supplement conveniently omits mentioning the use of optical density in the paper. As a result, the above misleading chart is completely lacking context.

The petition claims that CSF IL-6 baseline does not match clinically observed ratios

The petitioner states that “In other AD studies, CSF IL-6 levels are 1-5 pg/mg.” That’s actually a typo and should be “pg/ml.” But let’s assume they meant 1-5 pg/ml. They further state that the baseline IL-6 levels for Cassavas’ studies are 33-34 pg/ml and are far outside the norm (they volunteered to provide references on request). Why are the values so different? It seems as if all the “Exemplary published studies” referenced in the petitioner’s supplement used ECLIA (Electro-chemiluminescence Immunoassay) in the measurement of CSF IL-6 levels whereas Wang et al used ELISA (Enzyme Linked Immunosorbent Assay). It was even stated in Dr. Wang’s publication, “Although these assays are not directly comparable, values from each are reported in pg/mL.” In essence, they are correct in pointing out that the values are drastically different. But what they fail to point out, however, is that they were measured using different methods. It is a continuation of the same theme of misleading comparisons made with seemingly incriminating charts.

Gruber et al., showed elevated CSF IL-6 levels for a variety of conditions. These levels were measured using ELISA. [2][3]

·   Neuro-lupus (71.40±5.89pg/mL)

·   CNS infection (374.24±92.61 pg/mL)

·   Other inflammatory, neurological conditions (33.92±29.36 pg/mL)

·   Controls without inflammatory CNS disease (4.35±3.00 pg/mL)

Note that the CSF IL-6 baseline values measured by Dr. Wang et al fit well within the inflammatory, neurological conditions range above. Again – both methods being ELISA.

We acknowledge that ECLIA may be a superior method over ELISA, but “Currently, enzyme-linked immunoassay (ELISA) is the most widely used approach for the detection of AD core biomarkers in CSF. However, these ELISA methods often show considerable inter and intra-lab variability that prevents the use of standard cut-off values and precludes the wide use of CSF biomarkers in clinical practice.” (reference). Regardless of the CSF IL-6 baseline values, or how they were obtained, it is crucial to note that it is the change from baseline that is important. And that is what was measured in the Phase 2b study.

The petition claims that CSF sTREM2 baseline does not match clinically observed levels

Similarly, the petitioner claims that the sTREM2 levels obtained by Cassava Sciences are far lower than other published studies. Another simple literature search can find studies with measured CSF sTREM2 levels even lower than Cassava’s, in the range of 172.5 to 305.4 pg/ml. (Heslegrave et al. Molecular Neurodegeneration (2016) 11:3)

Concern #5 Apparent False and/or Misstatements in Cassava’s Phase 3 SPA Request

We did not find any deliberate misstatements. There were some errors, but minor errors do not constitute fraud and do not in any way call into question the safety of Simufilam or its potential efficacy. In fact, publishing all this data and facing scrutiny is what has set up Cassava for a more robust rollout of its phase 3 trials. 

Authors conclusion

Remi Barbier’s recent fireside chat clarified a few things. Phase 3 trials are on track, and he confirmed that cognition and biomarker improvements are not tainted and show real treatment effects. He stated that while the results are encouraging, clear links have not been established between cognition and biomarkers. We appreciate him for volunteering to provide transparency on a chain of transfer of data relating to the upcoming 12-month results of the Open Label study. This shows confidence and will serve to reassure investors. We eagerly await those 12-month results. In a recently released corporate presentation, it was specified that there is a < 10% drop rate for patients transitioning to the CMS study – this is a very low rate that shows patients are willing to risk being put on placebo. 

In summary, we find the fraud allegations in the citizen petition are filled with malice as they continue to attempt to discredit Simufilam and Cassava. The allegations do not explain how phase 2 clinical results show improvements in cognition scores measured across multiple clinical trial sites and are obfuscating the blinding processes put in place. 

We are confident that the FDA will reject the allegations in the petition.

Note: In the event of any verifiable errors, we will take steps to update the post. Please add comments on the blog to help us ensure the accuracy of our findings. Thank you!

Science Integrity – Part 1

Dr. Elizabeth Bik is a renowned science consultant who runs a blog and a twitter account where she discusses manipulated images in scientific publications. Her work is widely respected in the scientific community. Her twitter bio reads

Scientific discussions should not be held in the courtroom

In this post we will examine her recent actions with respect to Cassava Sciences. Below are some of her first posts on the subject;

Source: twitter

Dr. Bik was quick to validate the petition, in particular the fraud claims pertaining to inconsistent western blots. She later posted her comments on pubpeer while giving the authors a chance to make any corrections. The contributors of this blog article agree with her concerns on these topics.

Next she followed it up with a blog post.

Source: twitter

The papers in question are at least a decade old and within 2 business days of posting the initial tweet, she was already talking dollars involved and tagging cassava investors with her unproven allegations. To be fair, her tweet above was in response to Cassavas initial fact/fiction response which was dismissive of the petition. Even so, one would expect Dr. Bik to hear back from the scientist(s) in question instead of using company PR as a reason to insinuate fraud against scientists and scare investors. Note that Dr. Bik has this disclosure on her blog page.

Source: https://scienceintegritydigest.com/2021/08/27/cassava-sciences-of-stocks-and-blots/

Question to Dr. Bik: What was the motive behind that tweet?

Later we were dismayed by Dr. Bik not following up on many requests to examine the high resolution western blots on the patent by Dr. Wang. She also chose not to engage or update her blog when she received responses like the ones below – instead choosing to highlight the threats from abusive trolls on twitter.

Source: twitter

Her next twitter post was a new blog post that highlighted another of the original petition’s charges on Cassava Sciences which was the poster presentation at AAIC. Meanwhile, that ticker symbol she is highlighting everyday in her tweets, is taking a hit causing shorts to pile on and heavy losses for knowledgeable investors. Note that we think her work on the blog article about the poster was unprofessional and didn’t meet the high standards she is renown for. We will be commenting on her work on the poster and plasma ptau in upcoming posts.

Source: twitter

Overall it was obvious that a scientific discussion was being held in Dr. Bik’s courtroom where she seemed to be judge, jury and executioner. First she piled on with short interests questioning a company she barely knew much about. Next she had no problem with her work being used to supplement the petition that questioned the integrity of the Cassava team and Dr. Wang. And finally while ignoring Dr. Burns, she also complained about how women of science like her were being downplayed by techbros.

We have more questions for Dr. Bik.

  • Can Dr. Bik say with conviction that she is a neutral arbiter and not an interventionist supporting the fraud claims?
  • Can she declare that her associations with Adam Feurestein, Prof. Rob Howard Prof. Lon Schneider and other critics did not cloud her view of Cassava Sciences?
  • Does she support the intent of the petition asking for halting of drug trials based on unproven allegations from some of her work?
  • Can she call out Adam Feurestein for his stat news article which engaged in veiled sexism and questioned the credentials of an accomplished woman scientist?
Authors conclusion

The authors of this blog acknowledge Dr. Bik’s renowned expertise in spotting errors/issues and falsifications in academic journal publications. However with her actions surrounding the allegations on Cassava Sciences, we question her motives and invite her to engage in a balanced scientific discussion with us the authors of this blog on any concerns regarding the company. We also challenge her to hold herself to the high standards she has set.

The Hyper Growth Story

Any statements on hyper growth must be prefaced by answers to the accusations on the petition

Presence of a Whistleblower

This one is obvious – there wasn’t any. The petitioner used the term once and that too in the title of the petition. It was merely a ploy to garner increased attention. They being short on the stock, this smells of manipulation and we hope the SEC investigates them for inducing hysteria.

Dr. Wang’s papers and experiments (western blots, tissue images, experiment concerns)

We don’t know the truth here and don’t want to speculate. As Dr. Bik suggests, the original high resolution images could help alleviate any concerns and if there were errors, Dr. Wang may be able to correct them.

Our concern with the experiments being fudged are low because we were able to find complementary research by a team at Yale as described in this paper. Here they were able to determine that targeting elevated levels of aberrant Filamin A with PTI-125 reduced seizure activity by over 60% in mice when compared to saline. They ran several experiments like treating neonatal mice with a genome type that cause seizures with PTI-125 and also treating mice that were already experiencing seizures for many weeks. Results were extremely positive in all their experiments.

One can also read about research on Filamin A and diseases caused by mutations of the protein

Joe Springer, a Seeking alpha contributor also listed a host of complementary research here. Here in this blog, we are working on an article that will attempt to explain the MoA of the drug. Please subscribe for an update.

SavaDx poster

After corrections to the poster data, measurements done with the plasma assay showed a significant correlation between Simufilam use and p-tau181 decline. This matches the strong correlation that was also seen in the OLE study between cognition and p-tau181 decline.

The number of patients being < 64 is not a concern as they clearly marked the criteria for p-tau181 evaluation in the posters methods section. We also posted earlier on why the poster data is not relevant to phase 3 studies.

Independence of the CUNY lab

This particular point is prone to the most speculation. Here are some details on the study.

The masking requirements are clearly specified. Those concerned with the rigor of following these guidelines can read documents from the FDA here and here. Also the following excerpt gives more information.

Source: https://www.clinicalleader.com/doc/data-integrity-in-clinical-trials-key-concerns-of-the-fda-mhra-0001

Cassava has also confirmed that they use independent bio-statisticians to analyze the data obtained from primary and secondary outcomes. In the phase 2b study they used axiom real time metrics.

Cassava also released a press statement after the reanalysis of phase2b samples. It can be observed below that the blinding was maintained even though there is some confusion on the difference between outside labs and the academic lab. It would be nice if Cassava clarifies this.

Source: cassavasciences.com/news-releases
Concluding remarks on the petition

Our view is, the FDA verified all processes were followed correctly before granting SPA for phase 3 trials. We also think the petition will be dismissed in due course because of the obvious conflict of interest of the petitioners and the fact that safety concerns were never even raised (the primary purpose of a citizen petition).

One additional point to note is that in comparison with many other AD trials listed on clinicaltrials.gov, Cassava have been one of the few companies that has been diligent in updating the database with protocol data and results. They have invited the scientific community and investors to scrutinize those results. One must also note that many big bio pharmas tend to use their own affiliated labs and don’t even list them publicly.

Here is CEO Remi Barbier’s statement on the phase 3 trials,

Source: cassavasciences.com

Criticisms of the Open Label trial

Dr. Burns and her team of scientists have persistently reminded us that the OLE trial does not prove Simufilam is effective without confirmatory phase 3 studies and there will be risks. However the smart investor can see some positive signals from these trials. As discussed previously, the impressive cognition results at 9 months is in comparison with a placebo baseline from a meta analysis of over 20,000 patients with mild to moderate AD. Also it has been researched that the placebo effect for AD patients begins to decline after ~6 months. Even if placebo was in effect, showing improvements in cognition for 9 months, where decline is the norm is truly ground breaking. If we see these results repeat for the next 50 patients and at 12 months, one should really start to feel good even without the results of the p3 trials. Cassava have also initiated a double blind Cognition Maintenance Study with randomized placebo control that proceeds to extend the duration of patients getting treated with Simufilam in the OLE trial and we hope to see interim results from that study the first half of next year.

The Hyper-growth story

For a company that is being accused of misconduct and false claims, Cassava sure isn’t playing it slow and milking investor cash. Their cash spend is low, insiders haven’t sold, even bonus targets that were met weren’t awarded. All this to preserve cash for all the expensive trials they are running. They are also working hard and fast with the FDA to run multiple studies to collect as much data as possible for approval.

We fully believe Cassavas growth story can continue and don’t think anything has significantly changed except for the stock price becoming cheaper. We are buyers at these levels. Here are some of our predictions,

  • Continued cognition improvements in a majority of the 50 patients in the OLE trial at 12 months with data being released by early October
  • Phase3 trials begin recruiting mid september with large interest driven by word of mouth
  • A potential partnership deal that may be delayed because of all the bad press
  • Announcement of trials for new indications
  • Data release from more patients on the current OLE trial.
  • Break through therapy designation

Reason for caution

Our chief concern at this point is the extra scrutiny around the company and if there were any concerns from the FDA on how the blinding was maintained during pandemic times. We don’t have access to the end of phase 2 meeting minutes. However if Dr. Wang’s academic lab maintained blinding standards and there were no concerns raised by the FDA in those minutes then the concerns will shift to successful outcomes in the phase 3 trial.

In conclusion

We hope this article will switch you from the fear and uncertainty being propagated by short interests and instead look at the Cassava team in a new light. We hope the Cassava team are more diligent about how they present results and hope they use the extra scrutiny to make less errors while continuing to be transparent

Good luck to all!

AAIC Poster and Phase 3

The poster presentation at AAIC had some errors and this was exploited by the petition. We will discuss the errors at length on another post.

This post however is to inform readers that SavaDx or plasma p-tau end points were not part of the phase 2b trial and are not subject to the blinding requirements. Cassava independently chose to get their SavaDx test assay verified by correlating it with p-tau results from the commercial assay run by Quanterix. This was purely to show SavaDx as a valid biomarker while also showing how reduced altered FLNA helped with lowering p-tau181.

Any errors on the poster therefore should not impact phase 3 decisions by the FDA.

Dr. Burns at AAIC ’21

Alzheimer’s Association International conference (AAIC) was held on July 29th – Cassava Sciences presented 9 month cognition data for the first 50 patients enrolled in their open label study. Their slide deck was presented word for word by Dr. Lindsay Burns, SVP and Lead Scientist.

The company used the term encouraging and made no claims of efficacy while describing their results. However, that did not stop many from weighing in. Here are some social media criticisms posted by well known influencers –

Source: twitter

An excerpt from the Adam Feuerstein’s article above:

It’s not hard to see past Adam Feuerstein’s implied misogyny and lack of basic research on a publicly available study design. For the record, Dr. Burns, an author of multiple scientific papers, has the following background

Source: cassavasciences.com/management

Facing criticism for his remarks, Dr. Rob Howard, one of the quoted scientists on the stat+ article, doubled down and posted on his twitter feed.

Source: twitter

Dr. Howard ignored the questions surrounding his role in legitimizing the article while only responding to comments from fans and the harassment from reddit trolls. As for comments from users who asked him to take a look at the data and research – crickets.

Their criticism doesn’t change facts. Dr. Burns emphasized this was an open label study and only large phase 3 studies can prove efficacy. How is doing this overblown, uninterpretable or naive? Is being invited to present inappropriate? Before delivering the presentation, cassava had also announced two phase 3 studies.

It’s hard not to question the motives of these personalities and their backers when they engage in rhetoric. Note that their comments were picked up by various media articles to enable the “sell the news” effect that caused the stock to crater ~50%.

Some questions for these social media influencers,

  • Are companies that complete open label studies not allowed to present results to academia and investors?
  • Is your platform intended to foster progress with constructive criticism or plainly serve as a signal to short sellers and day traders?
  • Are you willing to be gracious and admit missteps when wrong?

There exists a world of folks with honorable reputations, who make questionable comments and never leave their echo chambers. They are mostly right but will never admit it when wrong. This blog hopes to critique these positions.

Author’s thesis on 9 month cognition release

The author of this piece acknowledges that Simufilam is not a proven drug. Preclinical studies on mice models and post mortem human brains have illustrated the mechanism of action. Phase 2 trials have shown encouraging results. The ongoing open label study is using a placebo baseline from a meta-analysis of over 20,000 patients with mild to moderate AD. Since a clinical hallmark of Alzheimer’s disease is decline in cognitive function, the improvements shown over 9 months, despite the placebo effect, is unheard of. The small sample size is the biggest concern and any investment should factor that in.

Biomarker results backing cognition were impressive. Here again it must be noted that biomarkers for Alzheimer’s disease is an active subject of research and the correlation between biomarkers and disease progression hasn’t been completely established. Cassava in their studies so far have found decreases in CSF p-tau 181 best correlates with cognition improvement.