BEST PRACTICES FOR ANTIBODY SPECIFICITY TESTING FOR IND SUBMISSIONS

John (00:07):
Hello and thank you for attending today’s webinar. Best Practices for Antibody Specificity Testing for IND Submissions presented by Integral Molecular and Fierce Biotech. I’m John and I’ll be moderating and hosting this webinar today. Now, before we get started, just a few housekeeping notes for everyone. To learn more about our speakers today, head on over to the speaker bio window on your screen. Additional resources are available in the handouts window on your screen. Now, if you close one of these windows and need to reopen it, just click on its name in the top navigation bar.

(00:42):
This webinar is also being recorded and it will be available on demand within 24 hours. And finally, there will be a Q&A session at the end of today’s webinar. If you have any questions for our speakers, you can enter them anytime in the submit questions window. Okay. Now I’d like to introduce our speakers for today, Jonathan Richards, application scientists at Integral Molecular, and Riley Payne, Senior Director of Scientific Operations for the MPA Group at Integral Molecular. Okay, Jonathan, the floor is yours.

Jonathan Richards (01:15):
Great. Thank you very much. Thank you for the introduction. My name is Jonathan Richards and today with my colleague Riley Payne we’ll be discussing the best practices for antibody specificity testing for IND submissions. I’d like to tell you a little bit about our company first. Our membrane protein experience is really what sets us apart. We have 20 plus years of membrane protein experience, specifically with antibody discovery platforms and characterization technologies against difficult to drug targets such as GPCRs, transporters, ion channels, and viral Envelope proteins.

(01:45):
We’re trusted by over 500 companies and partner with some of the largest biotechs in the world with data being used by over 400 publications for IND filings, patents, and manuscripts. Today I’ll be discussing specificity profiling using the membrane proteome array or MPA, breaking that down into the MPA’s role in drug development, the technology and pipeline, involvement in the FDA’s ISTAND program, and customer case studies. My colleague Riley Payne will be discussing how specific are antibodies really breaking that down into mechanisms of antibody off-target binding, analysis of preclinical, and clinical stage antibodies, case studies comparing the MPA to TCR results followed by an interactive Q&A.

(02:30):
We’re all here because the antibody therapeutics market is growing rapidly. With over 300 monoclonal antibodies, or MAbs, entering clinical trials in 2022 and 2023. Interestingly, there’s been an increase in the diversity of the formats of these different antibodies. And the pipeline has really demonstrated that, with advancements in CAR T-cell therapies and antibody drug conjugates or ADCs. Monoclonal antibodies are typically described as exquisitely specific, demonstrating some sort of lock-and-key mechanisms and binding only unique epitopes.

(03:05):
While this is true, a recent publication by Norden et al. in 2024 demonstrated using the MPA technology that one in three monoclonal antibodies tested are polyspecific. Now this polyspecificity is really demonstrated by binding to proteins that are unanticipated. And this off-target binding is really a major source of side effects and clinical failures leading to drug attrition. The FDA does require specificity testing prior to clinical studies in humans and this landscape for this specificity guidance is really evolving. So let’s take a closer look at that evolving landscape. In 1997, the FDA published the points to consider in manufacturing and testing of monoclonal antibody products for human use.

(03:49):
In that publication, they really state that specificity testing should use tissue cross-reactivity, TCR, or appropriate newer technologies as they become validated. If we take a closer look at what TCR really entails, TCR studies are often conducted with immunohistochemistry or immunofluorescence assays using human tissue. This is somewhat an outdated assay and has many limitations. Since it’s often performed with fixatives, it’s not really quantitative, time-consuming, and very expensive. Some other alternatives are protein arrays which are typically conducted in a blotting technique using immunohistochemistry or immunofluorescence.

(04:26):
These are also somewhat difficult to quantify and can also be time-consuming and expensive. This is really led to a dissatisfaction by toxicologists, seen in this paper here, where the majority of toxicologists believe that tissue cross-reactivity studies have not impacted the design of toxicology studies and have never really seen a correlation of TCR with preclinical or clinical toxicity. Now this evolving landscape has now changed and we see this rapidly changing. In 2024, the FDA published the considerations for development of Chimeric Antigen Receptor T-cell products, specifically naming protein arrays as an appropriate specificity testing method.

(05:08):
This has now opened the door for the FDA to re-evaluate outdated specificity testing technologies specifically through the ISTAND program, which is a new program that has rolled out so that we can qualify new drug development tools such as cell-based protein arrays. The FDA has really laid out a roadmap to create actionable specificity data that’s biologically relevant, quantitative, comprehensive, and so they can be trusted by the IND. Now the MPA actually checks all of those boxes. Here we can see how the MPA checks those boxes. The MPA is a 6,000 protein array using natively expressed human membrane proteins expressed in HEK293 cells.

(05:51):
We quantify this binding in unfixed cells by flow cytometry and we’re the only platform to use unfixed cells at each step of the process. This is really a paralleled sensitivity using a quantitative method of flow cytometry. Let’s examine the key features of the MPA. The MPA library was designed based off the same human tissues that are used in tissue cross-reactivity. With over 34 human tissues. Taking a bioinformatic approach we’re able to identify about 94% coverage of the human membrane proteome. These membrane proteins include GPCRs, ion channels, single- and multi-pass transmembrane proteins, as well as some protein heterocomplexes In our library.

(06:34):
Again, we have 6,000 of these human membrane proteins expressed in HEK293 cells. Additionally, we’ve rolled out another library, the secreted protein library. This is really an interesting library to examine because the secreted proteins can cause off-target effects leading to drug attrition and some clinical trial issues. These secreted proteins can also be related to pharmacokinetics. So we rolled out this new protein library using 1,200 secreted proteins that are now tested in the most biologically relevant manner possible, in an ELISA.

(07:07):
Importantly, for all of our testing metrics, we’ll be using unfixed cells. And I think that this is the most important aspect of a cell-based protein array because using unfixed cells will create an expression of native epitopes. And we know that the actual proteins that will be expressed properly will be biologically relevant and will be confirmationally similar to human expression. In fixed cells we see altered epitopes mainly caused by cross-linking of proteins and denaturing epitopes, which can then lose interactions that are biologically relevant.

(07:40):
This really increases the chance of false positives and most importantly, false negatives. We conducted a proof-of-concept experiment here. On the MPA we conducted two different experiments. First our standard workflow using unfixed cells and then another workflow using the same exact protocol, but introducing fixative prior to screening. In both of these experiments, our on-target epitope remained intact, and we were able to identify CD19 binding in both of these conditions. Importantly, the off-target epitope, the off-target interaction that was identified in these unfixed environments was not able to be recapitulated when using fixed cells.

(08:21):
This really means that this off-target epitope was altered due to fixation and then in a fixed condition we see this false negative, which is very, very important and can be dangerous moving forward in a clinical trial because this can cause severe adverse events. Another large highlight of our protein array is that we have an alternative cell line that can overcome this high background. Let’s take an example of B7-H3 monoclonal antibody and we run that on the human protein array using HEK293 cells which have high endogenous expression of B7-H3, we now lose that target binding in the noise where we cannot even with an over expression model overcome this high background expression. This then makes screening impossible.

(09:08):
Since we have an alternative cell line, avian cell line QT6, which have low or no expression of B7-H3, we are able to still move forward with screening in the cell line where we express our 6,000 human membrane proteins, which makes screening possible where we were able to demonstrate binding to B7-H3 with relatively low background noise and we did not identify any other off-targets since we had the sensitivity and reliability of our controls using this alternative cell line. Now importantly, our membrane protein array is compatible with all biotherapeutic modalities. We don’t have monoclonal antibodies listed here because we’ve tested thousands of them.

(09:48):
So I want to highlight some of the other biotherapeutic modalities that we are compatible with. We’re compatible with scFvs, bispecific antibodies, nanobodies, peptides, as well as antibody drug conjugates, CAR-Ts, and even DARPins, as well as a host of more biotherapeutic modalities. We have a wide range of detection methods which makes us compatible with these different modalities. This is not an exhaustive list, but if you have any questions, I’m more than happy to answer them in the Q&A about specific detection methods, but these typically cover most of the detection methods that we deal with on the MPA.

(10:22):
Now let’s talk about the specific MPA process. So for us to do MPA specificity testing, we just need about 500 micrograms of your test article in a tube. When we receive that at our facility in Philadelphia, we’ll conduct first an assay setup experiment to create an optimized cell line and antibody drug concentration to move forward to screening. We’re testing in parallel across two different cell lines in four different concentrations using positive and negative controls to then identify low background and binding to your known target so that we can then move forward and screen using that concentration and cell line in our 6,000 proteins.

(11:02):
Our second experiment here is the library screen using the 6,000 proteins at the concentration identified in the assay setup. And here we’re really trying to identify reproducible binding to the target as well as potential off targets in the screen. And we’re taking those reproducible binding events and then moving them forward to a hit validation stage, which is a 4.4-fold titration to analyze each protein that demonstrated reproducible binding in the screen, and then either to rule them out as potential false positives or artifacts or to really validate these off targets as novel binding interactions.

(11:37):
If we do identify a novel binding interaction, we do have some post-project services that we can offer because often we are asked what is next when we see something that binds that is unexpected? Well, we have an Off-Target Risk Analysis that we can do in-house, which provides additional statistical confidence and relative binding strength by increasing the replicates and titration used, including biological and technical replicates, normalizing our protein expression to be able to calculate Bmax and EC50, as well as analyzing the epitope accessibility with immunofluorescence.

(12:11):
Importantly, we have many other services within our company that can help support your drug discovery programs or specificity testing. We have significant IND support within our platform. We can include many different controls including isotype controls or mono-specific monovalent arms. We have CDR scanning as well as epitope mapping services included in our platform. We are the discoverers of the lipoparticles technology as well as we have many other platforms and teams within our company that can help support your programs. Externally, we have many connections that can help with production services and even primary cell binding as a off-target risk analysis following an MPA project.

(12:50):
Now, many of you’re probably wondering, so what about regulatory submissions for the MPA? Well, there have been over 100 IND filings that have been successfully submitted to the FDA that include MPA data. And importantly we know that there have been successful IND filings without tissue cross-reactivity, as shown here on a project where we completed for Cabaletta Bio where we demonstrated binding to the known target and even identified an off-target, which we were able to de-risk with Cabaletta Bio and still move this forward into clinical trials. Importantly, we have very competitive pricing and timelines where we’re able to complete this entire package in four weeks.

(13:29):
Importantly, the MPA is currently in review as an FDA-approved drug development tool or DDT. As I mentioned before, the MPA was the first technology entered into the ISTAND program, which is going to be a program to accelerate new technologies to be used in clinical testing. The proposed context of use of the MPA is to be a replacement for TCR, making this TCR data this roadblock to moving forward to clinical trials that making that optional. And we know that once qualified, any sponsor will be able to use these types of methodologies and the FDA will accept this data. We’ve been involved in this ISTAND program for approximately three years now and we’re very close to the finish line, getting great feedback from the FDA and submitting our full qualification plan.

(14:17):
We predict that this will be accepted or at least receiving large amounts of feedback by the end of 2025 or early 2026, which would then make MPA a qualified DDT and would then be accepted by the FDA without any review or question of the program. Now let’s quickly move into some case studies to identify other uses of the MPA technology. So we can use the MPA technology in lead selection. Here we’re showing four different monoclonal antibodies that are all targeting Claudin-6. Claudin-6 is expressed in cancers and not normal adult tissue, and there is a large specificity challenge where Claudin-6 differs from widely expressed Claudin-9 by only three extracellular residues.

(15:03):
Now, this is extremely important and makes specificity testing difficult, but the MPA was able to overcome this challenge and produce quality results through a lead selection process where you can see here IM171 in the red box was the only binder that demonstrated specificity to Claudin-6 without any validated other off-targets. You can see the other three molecules actually demonstrated binding to some of the Claudin family members, which are included in the MPA screen. And Context Therapeutics move forward with IM171 and is currently moving towards the clinic. Another example is a novel CAR T-cell therapy for solid tumors where the therapeutic is Claudin-18.2 TAC T-cell.

(15:45):
And using the 2024 CAR T-cell guidance, we are screening the VHH-GFP fusion on the MPA to identify binding to Claudin-18.2. Claudin-18.2 VHH bound a known target with no off-target binding identified, which included about 28 Claudin family members. The FDA granted this IND with no tissue cross-reactivity study reported. The MPA is really the leading solution to antibody specificity. We know that we’ve tested over 2000 monoclonal antibodies as well as many other biotherapeutics in our platform and our customers have published in very high-impact journals with this data. We’re trusted by over 150 companies, including the list that you can see here as well as many others.

(16:30):
We’re currently under review by the FDA for qualification as a drug development tool, which is very important and a very exciting aspect for the specificity testing and the evolving landscape. Finally, to hit home the important aspects of the MPA, we know that the MPA has been used in 100+ IND submissions and that data has been accepted by the FDA, EMA and NMPA. We’re the only platform that’s screening on unfixed cells to ensure that native protein conformation. Compatible with all biotherapeutics including MAbs, scFvs, VHH, bispecifics, peptides, ADCs, CAR-T cell therapies and more. We have 6,000 membrane proteins and 1200 secreted proteins in our library making us the largest library available for cell-based protein arrays and the most comprehensive specificity testing available.

(17:23):
We’ve received direct input from the FDA through the ISTAND program for MPA plus IND compliance and we’ve received great feedback. We predict that we will be qualified as a drug development tool very shortly. And we think that this will be at a very exciting development to the specificity testing space. Finally, we have extremely competitive pricing and extremely competitive timelines so that we can produce this data for IND-ready data packages in only a four-week. So now I want to pass this off to my colleague Riley Payne, who will finish the discussion with how specific are antibodies really.

Riley Payne (18:01):
Thank you, Jonathan. Now with all of these new insights, we really wanted to take a more exhaustive scientific approach to answer the question of how specific are antibodies, clinical antibodies, really? In the next few slides I’ll discuss some of the mechanisms of antibody off-target binding, give you an analysis of preclinical and clinical stage antibodies, and then talk about some case studies comparing the results of our MPA platform to the TCR, tissue cross-reactivity, studies that Jonathan mentioned. So first, how is antibody polyspecificity even possible on a molecular level?

(18:41):
Well, there’s some principles we can talk about that can contribute to this, including molecular mimicry where a critical epitope is mirrored in an unrelated protein, CDR plasticity where flexibility in the paratope in native confirmations can accommodate more than one antigen, or even differential engagement where multiple paratopes exist on a single antibody, essentially creating polyspecificity just with the existing CDRs. One really good example of this that we found in our work is a molecule whose target was SLC2A4, a glucose transporter, found in skeletal muscle cells.

(19:25):
Now unexpectedly we found that Notch1, a widely expressed membrane protein that’s highly unrelated to this, less than 7% sequence identity, showed off-target binding with this molecule. Further investigation revealed to us that this shared LGXXGP peptide really was comprising an off-target epitope that exhibited some combination of molecular mimicry and CDR plasticity and led to a really unexpected and potentially dangerous result for this molecule if it were to go into clinical trials. More importantly, this off-target binding can and does lead to toxicity.

(20:11):
One example is the molecule camrelizumab, which has been shown to have off-target binding against the program death receptor one in immune cells. Interestingly, this also showed off-target reactivity to VEGFR2 found in the vascular. The consequence: severe hemangioma in phase one clinical trials leading to the end of this molecule in its pipeline. These adverse events were almost certainly caused directly by binding and activation of VEGFR2. In a second example, the molecule ABT-736 whose target is amyloid beta oligomers for the treatment of Alzheimer’s-related dementia.

(20:58):
Well, this had an unexpected off-target to monkeys cinnămolgus, and human platelet factor four. The consequence this was lethal to the monkeys in these toxicity studies and was immediately pulled from the clinical pipeline. PF-4 has been known to be involved with heparin-induced thrombocytopenia and humans and it almost certainly would’ve caused severe risks to these clinical trial patients. In other words, we really need better tools to evaluate specificity and improve safety. So just to harken back to something Jonathan mentioned.

(21:40):
This really reinforces why the FDA is pushing to use new technologies or anything else that can give us new insight into these risks. Protein arrays have been named as an appropriate method, and today I hope I’ll convince you that they are superior in many ways to what is currently out there, particularly tissue cross-reactivity studies. So back to our research project. How specific are MAbs really? As Jonathan mentioned last year we published a study based on just our own internal pipeline. It was a retrospective summary of around the last 250 antibodies that were tested on our platform of which 83, 33%, showed polyspecificity, in other words, off-target binding.

(22:33):
This doesn’t include binding to nonspecific interactions including lectin proteins or Fc-receptors and is really specific binding to a target that was unexpected. We wanted to take this a step further and say, well, what’s going on downstream of us? To do this we chose 83 clinical stage biosimilars, which included those that had been withdrawn from clinical testing, those that had entered phase two or three and even those that were approved and on the market currently. They were screened on our platform for off-target binding and we were able to compare these results to some publicly available specificity data, primarily tissue cross-reactivity.

(23:21):
So the results were surprising even to us. We noticed a high prevalence of polyspecificity even in clinical-stage MAbs. Overall of the 83 molecules tested, 15 ,or 18%, still showed polyspecificity or off-target binding in our assay. Even more importantly, these off-target rates do indicate that polyspecificity is a major factor in drug attrition. The number does go down as things proceed in the clinic whereas higher in those withdrawn lower in phase two and three. But shockingly even of approved biologic drugs, 15% still demonstrated polyspecificity and unpredicted off-targets on our platform. This really surprised us.

(24:18):
And it kind of raises the question here. Aren’t these molecules already tested for this? This is not a new problem in clinical development, and this is where tissue cross-reactivity studies really come into play. I’ll review a few case studies that demonstrate the power of the MPA platform relative to the current technology. First case study. Off-targets can be masked in the TCR. One way that this can happen is really illustrated in this example where the target is a plasma membrane protein expressed in myeloid cells. However, the off-target that was discovered in the MPA is another unrelated membrane protein, but is expressed on the same cell type as the target.

(25:05):
In other words, it’s an off-target, on-tissue interaction. The biologic license application stated that staining was consistent with a known expression profile of the target, and this is one example of where TCR cannot discriminate between this particular interaction. The second example is where off-targets are essentially buried in the TCR. And what I mean by this is, in this example, the target is expressed on the plasma membrane and intracellular vesicles. However, it has low, but very broad, expression in essentially almost all human tissues. The application stated that staining again was consistent with a known expression profile, but essentially in the absence of a good negative control tissue the TCR was limited in its ability to identify the off-target.

(26:04):
Because the MPA expresses each protein in the array individually, these off-targets are revealed by our platform. Finally, there are simply the ones that we can’t quite explain. There are many instances where a target is a plasma membrane protein, in this case for this molecule expressed on lymphocytes, which can be a difficult tissue to assess using the TCR technology. And again, the application stated that staining was consistent with a known expression profile in lymphocytes. However, our platform revealed two additional off-targets that were not discovered by the TCR for unknown reasons. So just to put everything in review here.

(26:57):
Our membrane protein array precisely identifies on a genetic level what these off-target proteins are. TCR, on the other hand, could only identify your target tissue and not the protein involved in the actual epitope. Native confirmations, as Jonathan really highlighted, is critical to identifying biologically relevant interactions and we are the only platform out there that does all of our work in unfixed cells. Tissue cross-reactivity cells and tissues are heavily treated. Epitope retrieval is done, but they can be fixed and frozen many times throughout the process.

(27:40):
We also have comprehensive membrane protein representation, including the SPL and MPA over 7,000 membrane proteins. Where variable expression of these proteins in target tissues is always a sensitivity issue in TCR studies, we’re able to do quantitative analysis with high sensitivity and a low false positive rate, where TCR is essentially a qualitative assessment with low sensitivity and is more prone to false positives. Also, as Jonathan mentioned, our platform is compatible with almost all biotherapeutic modalities including MAbs, scFvs, VHHs, CAR-T binding domains, et cetera.

(28:24):
And finally, the TCR really cannot do this particularly with some of the newer modalities like CAR-T molecules or multivalent proteins. And all important also is the timeline and the price. We are significantly cheaper than a TCR study and the timeline is significantly shorter. In about a month we can turn over a study like this and help our customers identify potential off targets for their clinical pipeline. So just to review the main points of our presentation today. Recent data and case studies really challenge the assumption of absolute and exquisite antibody specificity.

(29:11):
Our most recent study reveals up to one in three antibody drugs and additionally two out of three of those throughout the clinical pipeline occur across all phases of antibody development and is likely a major cause of drug attrition and potentially even side effects of approved drugs. And we really feel improved specificity testing can improve safety and approvals. The MPA and SPL platforms provide detailed specificity data for more confident antibody drug development, which has really become a priority for the FDA. And they’ve demonstrated this by showing the specificity data generated from our platform it’s been accepted in over 100 submissions by both the FDA, EMA and NMPA. So with that, I’d like to thank you for being here today and I’ll open the floor to any questions.

John (30:25):
All right, thank you. Now it is time for our live Q&A portion of today’s webinar, so if you have not submitted any questions, please do so at this time. All right. Jonathan and Riley, first question goes to you, Jonathan. How do you ensure proteins are expressed?

Jonathan Richards (30:44):
Yeah, that’s a great question. So all of our proteins in our library have a V5 tag. And we’re able to measure the expression of all of our proteins using this tag. We periodically screen our entire library so that we can ensure consistent expression of these proteins. And we know that we’re doing this in unfixed cells, so they’re native confirmation and most biologically relevant. So we really want to confirm that these are expressed well and then historically that that expression stays consistent over time.

John (31:15):
Right. Another question for you Jon, is the initial MPA screening also performed on unfixed cells?

Jonathan Richards (31:23):
Yeah, that’s a great question. And that’s really what separates our platform apart from any other cell-based array. All of our steps that we do as highlighted in the MPA workflow are all done in unfixed conditions. So we know that they are the most biologically relevant and that these are the most similar produced proteins to what is seen in vivo.

John (31:45):
Another question in here. What is included in the IND package? Can it be sent to the FDA as is?

Jonathan Richards (31:53):
Yes. Yeah, so we do have two packages within the MPA. They are a very similar workflow and their report structure is really what makes the difference. So we received direct feedback from the FDA and have styled the FDA/IND-level report to really give every single piece of data that the IND wants to see. So it’s very easy to slot that IND-level report right into your submission. And it has all of the information as well as additional audit support and technical support that may be necessary when conducting these IND submissions with MPA data.

John (32:34):
Another question coming in. Does human proteome target binding change for an ADC pre and post conjugation? Do you test both formats?

Jonathan Richards (32:47):
Yeah. So we have done both formats. I always highly recommend testing the final biotherapeutic as well as the unconjugated form. It’s typically related to the type of ADC that we’re working with, but we have done both unconjugated and conjugated really relating then to what the payload function is doing because we want these testings to be done in a biologically safe environment as well. So we have done unconjugated and conjugated. And of course we can follow up on another call to talk more about the specifics of the particular ADC that we would like to test.

John (33:26):
All right. Thank you for that answer. Riley, question in for you. Can MPA data replace TCR data only for CAR T-cell therapies or can it be a replacement for all antibody based therapies?

Riley Payne (33:40):
Yeah, thanks John. Good question. Well, the CAR T guidance is the one that specifically lists the protein arrays as a viable alternative to TCR. Many of our customers who had success relying on MPA for normal antibodies or multiple modalities other than just CAR T.

John (34:03):
Question for Jon. And by the way, the Q&A window is open for anyone still watching. Just to submit your questions there in that Q&A window if you do have one. Jon, can you expand on what it means to be in the ISTAND program? What would a DDT designation mean for your customers?

Jonathan Richards (34:22):
Yeah. Yeah. The ISTAND program is a really exciting program and leads into the roadmap that the FDA is leading to change specificity testing for the better. There’s a lot of developments with tissue cross-reactivity and drug attrition in clinical trials. And the ISTAND is really a position so that we can accelerate the submissions for new drug discovery tools. And the drug discovery tool, really, once we have that designation, it will just streamline the process of the submission of the MPA results into an IND package. So we really are excited about this. We’ve been in this program for a couple years and have tailored that IND level report based on the feedback we’ve received from the FDA, which has been extremely positive overall. But this ISTAND program is very, very exciting and we know that we are actively moving through this process to become a new drug development tool.

John (35:20):
All right, see if I get this question correct for you, Riley. Do any of the approved antibodies that show polyspecificity in your essay also show an adverse events that could be explained by the off-target specificity?

Riley Payne (35:35):
Yeah. This is an excellent question and it kind of really gets at the core question of the study I talked about. Many of the ones we plan on testing in our pretty exhaustive review of clinical MAbs do have various side effects and things like that. I think one of the first analyses we’ll do on that end is to really understand any correlations between the off-targets we discovered from that panel and that. But we are really working hard now to answer that question that really gets at the core of our research question.

John (36:11):
Excellent. A lot of great questions coming in. Keep those questions moving in. So it looks like this one’s for you, Jon. Does the human proteome array correct in any way for relative expression levels or is this used post-identification to determine risk of that particular off-target binder?

Jonathan Richards (36:30):
So as I mentioned, we have all of our proteins tagged with a V5 tag. So we do have the kind of internal database to understand this expression. We really use this internally as a QC and QA metric to make sure our library is expressing and is consistently expressing over time. We’re not normalizing all 6,000 proteins, but we do know that they all fall within a relatively close range in a full difference above our background signal. So we cannot make very strong claims about relative binding strength in this high-throughput screening, but rather we’re using this as a tool that can identify and rank particular proteins within our 6,000 that really need to be followed up on and identified as potential issues in polyspecificity.

John (37:16):
This one’s for Riley. For secreted proteins, are those also screened using a cell-based system?

Riley Payne (37:24):
Yeah, that’s another great question. All of the proteins that we test in the array are produced in human cells to ensure everything in terms of glycosylation, post-translational modifications are correct, but the screen itself is performed using an ELISA approach on those secreted proteins directly.

John (37:46):
Okay. Thank you for that answer. Jonathan, when would a study like this typically performed an antibody development: after a lead is selected prior to GLP talks, prior to IND submission or in a later phase development?

Jonathan Richards (38:02):
The short answer there is that we like to promote specificity testing early and often. So I would highly recommend that the MPA can be used for lead selection or even earlier than lead selection to identify if there are any potential risks for your pool of antibodies. We can really identify a pathway that we can help with your lead selection, your antibody development, but we also have the IND package so that we can really demonstrate this in a metric that can submitted directly to the IND once you have selected your lead. So again, I would say testing for polyspecificity as well as polyreactivity should occur early and often throughout your drug discovery programs.

John (38:48):
Another question for you. What are the advantages of using unfixed cells for antibody specificity testing?

Jonathan Richards (38:55):
Yeah, that’s a really great question and one that I personally think is such a great unique advantage of our platform. The ability to test using unfixed cells really creates a situation where we can express proteins in their most biologically relevant manner. And we know that these proteins are similar in their development to being biologically relevant, have similar confirmations to what is seen in the human condition. And if we do introduce fixatives, the likelihood of increasing our false positives and more importantly our false negatives rates increases dramatically.

(39:31):
We know that these can change epitopes as well as paratopes altering those via some cross-linking. And this as shown in one of our case studies really cause there to be drugs that are moving forward that do have polyspecificity that then fall out in clinical trials due to there being severe adverse events related to those off-target binding events. So testing in unfixed cells should be done in any types of specificity assays and can really help to move forward drugs through the clinical trial process in a much smoother manner.

John (40:04):
Jon, another one for you. Talking about false positives or false negatives, how are those?

Jonathan Richards (40:10):
Yeah. So we have a really rigorous QC team. We conduct a lot of testing on our library constantly and we’re always improving. False positives are relatively easier to quantify. And we do have a relatively low reporting criteria in our validation stages. That’s to really increase the sensitivity of the assay because, again, we’re using this as a metric to rank potential interactions that need to be further investigated. And we do have a risk analysis package that we can do that in-house or you can do that back within your company.

(40:44):
But I think false positives it’s one, always better to kind of prioritize over-reporting rather than under-reporting. Because missing a potential interaction and having a false negative is what’s really going to down the line cause this drug to fail in clinical trials. False negatives are a little bit harder to quantify, but we do know that within our process when we conduct sequencing of all of our proteins, when we use the expression of our V5 tag, when we have co-transfection controls, we are able to really reduce the likelihood of false negatives to the highest degree. And I think that’s one thing that we’re really proud of and really stand by scientifically of the MPA platform.

John (41:24):
Fantastic. Riley, here’s a question in for you. If you choose not to do TCR then how do you know which tissues your antibody binds to?

Riley Payne (41:35):
Yeah, that’s a really interesting question, John. So the beauty of this is today there’s so many online freely accessible sources of this information. We use databases like the Human Protein Atlas or UniProt. Once you’ve genetically identified that target it’s really easy to look through these databases and find out which tissues, which cell lines? All of these things are expressed in and that can really inform further decisions on what the potential risk of these off targets are and what effects that might have in the clinic.

John (42:11):
All right. Question in for you. It looks like Jonathan. How should MAbs be produced for optimal assay?

Jonathan Richards (42:21):
Yeah. So we do have some recommendations as well as companies that we can recommend for antibody production. We highly recommend to send us the final drug product as closely as possible to what will be the final drug product, can be related then to the detection method. But for a monoclonal antibody, again, we would use an anti-FC to detect that molecule, but we always recommend including the most highly pure sample is possible so that we can be very sensitive in trying to detect any off targets as well as a relatively concentrated sample so that we can use a single sample over the course of multiple experiments. And if there are sent multiple aliquots, it can be something that can be used for each experiment of the assay. And we just want those to be from the same lot from the same production facility that we would be moving forward with to take these drugs into the next stages of the development.

John (43:23):
All right. Excellent. Once again, keep those questions coming in on the Q&A panel on your screen. Submit question window. This question in for you Riley. Do you do serum profiling?

Riley Payne (43:40):
Another good one. So we don’t do serum profiling in the traditional sense, however, our SPL array answers many of the same questions. We’ve included, as we mentioned, over 1,000 secreted proteins. These include and we do specifically test for proteins that are at a high level, things like serum albumin and things like that. And that can often yield some of the same information as serum studies as well as genetic identification of what those targets are. So while we don’t do that directly, we really can give a lot of that same information and even more detail using the SPL platform.

John (44:25):
Thank you for that. Jon, a question for you. Do you assume that the MPA needs to or will performed under GLP like the TCR?

Jonathan Richards (44:35):
So currently the MPA is not performed under GLP conditions. And from the feedback from the ISTAND program and the FDA, we are taking under consideration the potential for us to become GLP compliant. We’ve actually started to change some of our facilities and work with our QC team to create a potential option to be able to quickly move to GLP compliance. But as of now we have not received any information that would suggest that that would need to be changed even if we do receive the DDT designation from the FDA.

John (45:14):
Another one for you, Jon. How sensitive is the MPA platform?

Jonathan Richards (45:19):
That’s a great question. Within our assay set up conditions, we’re able to identify an optimal concentration that your molecule could be screened at. And in that phase we really are assessing any interactions with the cells that are endogenous expression on HEK293 cells or QT6 cells. So the more pure and the better sample that we see, we see better sensitivity. So it’s a case by case basis. But I can say that we have detected interactions using our flow cytometry platform as low as single digit micromolar affinities.

(45:58):
And we’ve received that information after we’ve completed this assay. And we get great feedback from our customers so that we can quantify those types of numbers. But really the use of flow cytometry in unfixed cells gives us that ability to really detect very, very weak interactions. And this is of course an over expression system using higher concentrations than potentially are what would be biologically relevant. So we really have that ability to be extremely sensitive and to identify very weak interactions using this platform.

John (46:33):
Thank you for that. For you, Jon. What happens if my antibody specificity screen indicates validated off-targets?

Jonathan Richards (46:42):
Yeah. Yeah, that’s always a really great question and we provide a lot of support for when there are novel binding interactions. So one of my roles is to really get onto project calls and to really speak with you and your team so that we can identify next steps. The one really great advantage of the MPA is if we do identify an off target or a novel binding interaction, there’s a very clear path forward, because we can tell you exactly what that protein is, the sequence of that protein. We then can offer in-house experiments, which we title our Risk Analysis experiments to just provide more statistical reliability to those results.

(47:22):
So potentially if you see a weak interaction in our validation stages and you want to move forward and try to better understand that interaction, we can conduct an expanded titration using an eight point titration range and multiple biological and technical replicates to then increase that statistical confidence in this reporting of the off-target. In those experiments, we can also quantify Bmaxes and EC50s and we even do immunofluorescence to try to understand the availability of that protein within the cells.

(47:57):
And using those types of experiments as well as working with other companies that can perform primary cell assays can really help to strengthen the data package. So even though we may identify an off target, we still could move forward and identify a plan that we could either de-risk that target or use publicly available literature to really say about the availability or the relevance of that target. And we’ve done that for packages that have actually moved forward into clinical trials where we identified an off target. It was actually de-risked through a pretty intensive process and moved forward and is currently, I believe, in phase two clinical trials.

John (48:38):
Right. Thank you for that answer. Riley, here’s a question for you. For the case studies where TCR missed an off target, were there any safety concerns found from those off targets?

Riley Payne (48:51):
Yeah. Again, this is very akin to the question regarding the clinical antibodies that we’ve tested on our platform. In addition to the ones I’ve mentioned where TCR did miss some targets, we really want to look retrospectively from our data, compare it to the TCR data that was submitted for the IND study and try to kind of trace this question back. Our hypothesis is that yes, this is one of the causes of safety concerns not only during clinical trials, but even for approved drugs, missed targets from TCR. Hopefully our investigation will confirm or deny that hypothesis, but stay tuned for our paper and we hope to address that.

John (49:43):
Very good. Jonathan, when or at what stage should I screen my therapeutics for off targets?

Jonathan Richards (49:52):
Yeah, that’s definitely a great question. And again, I would say early and often. I think identifying a potential polyreactivity or polyspecificity during your lead selection phase can help inform about which protein or which of your biotherapeutics would be the best one to move forward in your lead selection phase. So I think that that’s a really important stage. And then as you modify or change any of these leads, you can retest them on the MPA to make sure that none of those changes caused any types of polyspecificity alterations due to those mutations or those changes to final biotherapeutics. And then even after lead selection, it’s a great platform that you can use to give that final piece of evidence stating the specificity and the selectivity of your molecule within the human proteome array in which we cover about 94% of that protein array.

John (50:46):
Another one for you. What is the turnaround time for standard project and MPA+IND project?

Jonathan Richards (50:53):
Yeah, great question. So we do accept all sorts of projects on a rolling basis, so there’s no lead time or there’s no wait to get into our queue. And we can perform our MPA+IND studies in four weeks. So we can provide you with quantifiable specificity testing data within four weeks for an IND submission. And then for our standard projects that has a little bit more of a range in which we would say those types of projects can fit into a four to eight week time window depending on the amount of molecules that we’re receiving at one time.

John (51:31):
All right. Excellent. Thank you for that answer. This one for you Riley. What’s on the horizon for next generation development of MPA? What avenues are you exploring for even closer biological relevance of this assay?

Riley Payne (51:50):
One of my favorite things to talk about. I’ll try to give a couple examples really specific to the question. Jonathan already alluded to our Risk-A [Risk Analysis] product line, which is really intended to take these validated off-targets and give our customers more information on not just the biological relevance, but again, the relative binding strength, cellular localization of the epitopes, all of these kind of things. So a lot of our downstream validation that really comes with the MPA-IND product is meant to inform that.

(52:26):
In terms of pushing that concept upstream into the screen, one huge innovation we’ve made, I would say in the past year or so, is to include a number of what we call heterocomplexes in the array. You can have every membrane protein in the proteome, but so many of these exist in complexes or require chaperones. I think we’ve really leveraged our, Integral Molecular’s, knowledge of membrane protein expression with our array technology. So we can cover a lot of these multi-subunit complexes that are really important for clinical discovery, some neuronal complexes, things like TCR and those kind of technologies that really allowed us to assemble what are the biologically relevant structures on the cell surface and in these cells.

John (53:18):
All right. Thank you for that. For you, Jon. Can we readily find the identity of all 6,000 membrane proteins and 1,200 secreted proteins?

Jonathan Richards (53:31):
Yeah, we’re more than happy to share the identity of our library. We’re very open about the proteins that we include so that we can be very transparent with what’s included. So the 6,000 membrane proteins as well as the 1,200 secreted proteins can be provided in our comprehensive protein list. I would recommend going on our website and setting up a short pre-project call. And we’re more than happy to share our slide deck for any sorts of calls as well as our full protein list so you can see if your particular target or your family of targets is included.

(54:09):
We also have the ability to include customized proteins within our library, which is very important. So if we do share that list with you and your known target is not on that list, we have worked with many customers to include their specialized proteins as well as some unique binding characteristics and events within our library. So we’re more than happy to share the identity of the 7,200 proteins in our library and work with you to create customized assays and really get at the entire point of the specificity testing.

John (54:45):
Ton of great questions that came in today. If we didn’t happen to be able to answer your question, we’ll do our best to follow up after today’s webinar, but that is all the time we have for today. As a reminder, a recording of this webinar will be available within 24 hours. Jon and Riley, thank you. Thank you for everyone who tuned in today and listened to this presentation. Thank you for joining us today. And we look forward to seeing you next time. Have a great day.