Use of MPA Specificity Data in Regulatory Submissions

Integral Molecular hosted the webinar “Towards IND: Specificity Profiling of Antibody Therapies Using the Membrane Proteome Array” on June 10, 2021. The following is the video transcript for a portion of our webinar focused on using MPA data for regulatory submissions presented by Dr. Diana Norden. Dr. Norden is a Senior Communications Scientist at Integral Molecular, and lead author of a book chapter focused on evaluating off-target binding of biotherapeutics during preclinical development, published in Translational Medicine: Optimizing Preclinical Safety Evaluation of Biopharmaceuticals. CRC Press:2021.

Introduction (26:47)

And I'm excited to introduce our next speaker, Diana Norden, our Senior Research & Communications Scientist, who’s going to talk about how you can take MPA data to the next level in your regulatory submissions. 

Thank you, Dan. So, in my presentation, I have two small sections. 

Using Membrane Proteome Array Data for Regulatory Submissions (27:10) 

First, I will go over specificity profiling for FDA’s Investigational New Drug (or IND) application, and do a quick comparison of traditional tissue cross-reactivity studies with the MPA. And we hope that this is of interest to many of our listeners today. We know that right around half of our customers indicate that they anticipate using MPA data in their IND filings, hopefully, this is of interest for our listeners also. Then I will go over a quick case study, or project, that we did with Cabaletta Bio which shows how the MPA was adapted to screen specificity of their novel cell therapy and how it ultimately led to a successful IND filing. 

Specificity Analysis is Required for Safety (27:50) 

Specificity is just one component that is necessary in the overall preclinical safety assessments for any new biotherapeutic. The FDA has adopted two guidelines that describe biotherapeutic safety testing. This is “FDA’s Points to Consider...” and the ICH S6 guidelines. Both of these describe the need to assess off-target binding or cross-reactivity, and thus, IND applications require some sort of specificity analysis. Traditionally, tissue cross-reactivity studies have been used to screen for off-targets, but in more recent years cell array data, such as the MPA, are now frequently used in IND filings as well. 

What are Tissue Cross-reactivity Studies? (28:34) 

I mentioned tissue cross-reactivity studies, so I should just go over briefly what they are. They are immunohistochemical screening assays used to identify the binding of test biologics in different human tissues. The test article is screened across 37 different human tissues and positive staining is evaluated by a pathologist. There are two main outcomes that a TCR study can give you—it’s the target distribution and the target epitope binding. The target distribution is more of an assessment of overall toxicity. It tells you which tissues express the target, which is then used to monitor for tissues with potential toxicity associated with your therapeutic. The target distribution has also been used in selection of a relevant species for toxicity studies because the species should have a similar target expression profile as humans. In addition to TCRs there are many other methods available to determine target binding that can then assist with species selection as well.  

The target epitope binding is used for the specificity analysis. Here, unexpected binding patterns could be due to an actual off-target—a cross-reactive target—or also an off-target organ reactivity. This would be if you find staining in a tissue that you weren’t expecting to see the target. In some cases, it can be obvious to determine if you actually have an off-target binder, and in other cases, this unexpected staining pattern needs to be further investigated either by a pathologist or in follow-up studies.  

Recognized Limitations of Tissue Cross-reactivity Studies (30:16)

Even though they’ve been used many years now since about the 80s, there have come to be many recognized limitations of tissue cross-reactivity studies. Here I will just go over a few. TCR studies unfortunately come with frequent false positive staining. In this image to the right, I show some examples of cross-reactivity and non-specific binding. Unintended targets can bind either directly to the CDR region or really just anywhere on an antibody in a more non-specific manner. This more frequently occurs in TCR studies just by the nature of the IHC method used on tissues, and also glass slides which have a significantly higher background and rates of false positives. 

A main limitation is that the binding target is not identified, so in cases of expected staining patterns, this could actually include an off-target and you wouldn’t know it. In cases of unexpected staining pattern, the identity of the off-target is not known, which makes it really difficult to perform any follow-up studies of the off-target. TCR studies also use fixed tissues, and as Rachel mentioned, the non-native proteome tested is not always ideal. And results don’t correlate to in vivo toxicity—this is a major limitation. By now there are abundant examples and case studies found throughout the literature where TCR assays and in vivo toxicity studies don’t correlate. Because of these limitations, in their latest update to the “Points to Consider...” guidance, the FDA stated that appropriate newer technologies should be employed as they become available and validated. 

Advantages of the Membrane Proteome Array Over Tissue Cross-reactivity (32:00)

So how does the MPA compare and have advantages over TCR studies? The main one is the known target specificity; the MPA will tell you the identity of the off-target, which can then allow for follow-up studies to see if there is any toxicity associated with that off-target, for example using approaches that Dan discussed. The MPA has high sensitivity: the MPA uses an overexpression system, flow cytometry, has low background, is highly quantitative and has low rate of false positives. The TCR has high false-positive rates, as I mentioned, both due to native Fc receptors that can bind to test articles and native IgG that can then bind to your secondary antibody to give high background and false positives. Again, the native target reactivity is an advantage—the MPA uses unfixed cells versus TCR studies use fixed and frozen tissues. And the MPA is highly quantitative and has a significantly shorter turnaround time compared to TCR studies.  

Case Study: How the Membrane Proteome Array was Used to Enable Successful IND Filing for a Novel Cell Therapy (33:12)

Those are some advantages of the MPA, and next I will go over a study we did with Cabaletta Bio a couple of years ago to show how the MPA was used to enable a successful IND filing for their novel cell therapy.  If you’re interested in this project or in Cabaletta Bio or this specific study, it is published in Journal of Clinical Investigation (JCI) just last year. 

Cabaletta Bio is a biotech company that has developed novel CAAR T therapies to be used for the treatment of autoimmune diseases. Their therapy is very similar to traditional CAR T-cells. Traditional CAR T-cells express an antibody fragment that then binds to a target on cancer cells, which leads to activation of the CAR T, and the cancer cell will be destroyed. These auto-antigen CAR T-cells that Cabaletta makes—they're very similar in that they express the same costimulatory signaling domains, but instead of an antibody fragment, they express an autoantigen that is associated with autoimmune disease. Upon binding to an autoantibody receptor—it’s basically flipped—it binds to the antibody on the B cell, then the CAAR T cell becomes activated and kills the pathogenic B cell. As with any T-cell therapies, and especially CART- cells, they require absolute specificity. Any off-target binding can lead to CAAR T activation, cytokine release, and potentially misdirected cell killing. Therefore, they really needed to have a thorough specificity analysis. 

Problem: Lack of Validated Assays to Test Specificity of Cell Therapies (34:43)

The problem that Cabaletta faced is that there is a lack of validated assays to test the specificity of cell therapies. There are numerous limitations of in vivo preclinical safety and specificity testing. There is a lack of good models—these are human cells that need to recognize human proteins, so the species-specific difference really plays a huge role. Any off-target won’t be recognized because, again, it’s in the wrong species. And further complicating the issue is that there is no specific guidance by the FDA to evaluate the specificity of T-cell therapies.  

I should say that for CAR T-cells, people have used tissue cross-reactivity studies, but there are limitations to this as well. It is difficult to adapt an IHC method for the CAR, either scFv or ectodomain. There is low sensitivity, difficult interpretation, and low-affinity interactions potentially aren’t detected in the IHC method, but in vivo they could potentially lead to CAR T activation. Cabaletta was hoping to use the MPA because of its versatile screening modalities, its high sensitivity, and the fact that binding will be tested against the human membrane proteome.  

Solution: Assay Development for DSG3-CAART Specificity Screening (35:55)

We were able to work with Cabaletta and modify their screening therapeutic into an Fc-fusion and then use that for screening. In this image, I show the auto-antigen for this particular project, which was called DSG3. This shows the DSG3-CAAR in this complete form and how we were able to modify it into an Fc-fusion protein with the Fc-domain. We did some extensive assay development for this project in our assay setup portion using different conditions, and ultimately, we were able to show target identification with high signal, low background, and we also had an isotype-matched control. This gave Cabaletta Bio confidence in going forward with the complete MPA and allowed for high-throughput assessment of any binding interactions. 

Solution: Membrane Proteome Array Screen of DSG-CAART (36:50)

On the MPA, this protein showed specific binding.  It showed binding to the correct target—in this case, a protein called Px44, which was spiked in as a positive control. The MPA did show minimal binding to an off-target, but here again, knowing the identity of the off-target was crucial. They performed validation and functional studies, which then showed no CAAR T-cell activation from the off-target, and thus eliminating any cross-reactivity concerns. So, we had a successful MPA screen of their T-cell therapeutics.

Impact: FDA Granted IND (37:24)

And as a result, this extensive specificity analysis proved to be sufficient in addressing cross-reactivity and the FDA did not request any TCR to be performed. Cabaletta is now currently doing a Phase I clinical trial of this therapy—it’s enrolling participants. We were very excited that the MPA data was able to be used to support this first-in-human novel cell therapy. And there’s this reference if you’re interested more in this study (Lee et al. 2020).  

Standard Membrane Proteome Array Platform for Biotherapeutic Specificity Screening (37:56)

Just to wrap up, the MPA is a standardized platform for biotherapeutic specificity screening. It’s compatible will all biotherapeutic modalities. Most of what we do is MAbs (monoclonal antibodies)—I’ve shown you one study here where we did cell therapy. We’ve also done gene therapies, and we’ve even done 1 or 2 small molecules on the MPA. A lot of this comes from the versatility in the MPA and its screening options—it's expressed in live cells, so we can look for binding, activation, transduction, or infection all with high sensitivity. The MPA has proven to be highly successful–we've screened over 1,000 MAbs, and now we also offer the Enhanced Validation and off-target decision support, as Dan discussed. We have a team of expert scientists who will work with you to have the MPA included in your IND filing.  

If you want to start a project with us, you can contact us directly, or we’re also available through these vendors and CROs. That wraps up this portion of the webinar.