VDP-38: ApoptoSENS - Senolytic CAR-NK cells

One-liner: Funding proposal for SENS Research Foundation’s ApoptoSENS project to develop CAR-NK cells to precisely and safely eliminate senescent cells.

Longevity Dealflow WG team

Scientific evaluation : Sebastian Brunemeier, Tim Peterson, anonymous reviewer (professor), anonymous reviewer (industry professional), Jason Colasanti, Aaron King
Business evaluation : Sebastian Brunemeier, Tim Peterson and anonymous reviewer (professor), anonymous reviewer (industry professional)
Shepherd : Laurence Ion
Other squad members : Jason Colasanti, Tuan Dinh, Ines Simo
Sourced by : Laurence Ion

Project PI : Dr. Amit Sharma

Simple Summary

Over time, a subset of cells throughout the body experience damage and enter a state of senescence. Cellular senescence is the irreversible arrest of proliferation, and senescent cells secrete proinflammatory factors that lead to tissue dysfunction. Selective elimination of these senescent cells has been shown to ameliorate hallmarks of aging in both mice and humans. However, current methods to eliminate senescent cells are non-specific and may have off-target effects. As a result, Amit Sharma’s team is developing Chimeric Antigen Receptor Natural Killer (CAR-NK) cells to precisely and safely eliminate senescent cells in vivo.

Problem

When cells become damaged, they may enter a state of cellular senescence where they no longer function properly and secrete proinflammatory factors. The immune system’s ability to eliminate senescent cells diminishes with age, resulting in their accumulation throughout the body. This accumulation leads to dysfunction in many different tissue types and accelerates aging. Current approaches to eliminate senescent cells are non-specific and their mechanisms of action are largely unknown. Also, due to the highly heterogeneous nature of senescent cells, including the existence of senescent cells with beneficial effects, there is currently a lack of knowledge regarding cell surface markers to identify populations of harmful senescent cells.

Opportunity

The current focus on senescence as a therapeutic target was prompted by the discoveries that markers of cellular senescence accumulate with aging and accumulation is delayed by interventions that increase healthspan and lifespan, removal of senescent cells increases healthspan in progeroid mouse models, and transplantation of a relatively small number of senescent cells into previously healthy animals provokes multisystem dysfunction similar to what is seen in aged animals. Furthermore, it has been shown that the correlation of senescent cell accumulation with disease extends to humans. Since senescent cells are highly heterogeneous in both their molecular biology and their physiological function, targeted strategies are needed that ideally preserve senescent cells in beneficial contexts while eliminating effects that are detrimental.

Broadly speaking, current therapies can be broken down into the major categories of senomorphic (targeting SASP, i.e., senescence-associated secretory phenotype, components) and senolytic (targeting senescent cells) drugs. While the first senolytics were developed using a bioinformatically informed approach aimed at disrupting SCAPs and other pro-survival networks, the class has expanded to take advantage of additional senescence features and enhance immune-mediated clearance. Most notably, characterization of senescent cells has revealed unique markers that serve as senescence-associated self-antigens, and these can be co-opted for immune system-mediated senolytic activity and clearance.

A recent study took advantage of this approach using chimeric antigen receptor (CAR) T cells targeted against the urokinase-type plasminogen activator receptor (uPAR) in a mouse model (doi: 10.1038/s41586-020-2403-9). Cytotoxic CAR T cells were able to selectively clear uPAR-expressing senescent cells in vitro and in vivo. CAR T cell-mediated clearance of senescent cells led to survival and histopathologic benefit in murine models of both carbon tetrachloride- and diet-induced liver fibrosis, suggesting the feasibility and potential of this clearance strategy. However, supratherapeutic CAR T cell dosing is associated with a proinflammatory cytokine profile, weight loss and hypothermia, suggesting that careful attention would need to be paid to initial dosing strategies, particularly in geriatric populations where a compromised immune system together with ongoing chronic inflammation may restrict the therapeutic window. Additionally, CAR T cell approaches in humans involve initial immune system suppression with considerable attendant morbidity and are expensive. Although senolytic CAR T cells appear to be self-limiting, their expansion and contraction occurs over a period of days, which is a risk in the case of an acute to subacute adverse reaction. Overall, this strategy is highly promising given its preclinical efficacy and senescent cell specificity, but future optimization and testing is necessary before clinical administration.

See review here: Strategies for targeting senescent cells in human disease | Nature Aging

Despite the pre-clinical promises of targeting senescent cells and/or SASP, clinical trials in humans thus far have been less encouraging. Nevertheless, significant interest remains in the general approach.

Given the success of CAR T cells (see example above), particularly in the cancer field, the team here has identified unique surface markers on senescent cells for targeted removal.

Using a commercially available protocol, the investigators have found several cell surface markers of IMR-90 cells made senescent by ionizing radiation or doxorubicin treatments that are not present on healthy cells. To date, they have focused on 5 top candidates, of which SENS-1, -2 and -3 have been further vetted, with SENS-3 perhaps looking the most promising (data of Figure 3). In the proposal here, priority senescent cell markers will be more extensively validated using additional cell model paradigms (stressors), as well as in mouse models (cross-species validation). Validated surface markers will be used to generate monoclonal antibodies, which will then be confirmed for specificity against the target antigen. Appropriate CAR NK cells will be created to further establish proof of concept for their approach of selective removal of senescent cells in their various model systems.

Intellectual Property

A patent regarding methods to develop the CAR-NK cells is intended to be filed. They already have filed patents for specific surface-marker antibodies and subsequent reagents that allow selective targeting of senescent cells.

Team

Dr. Amit Sharma: Project Lead at SENS Research Foundation, PhD in Biotechnology (https://www.linkedin.com/in/amit-sharma-1613b189/)

Dr. Amit Sharma was awarded a Master’s degree in Biomedical Sciences from Delhi University, India. He received his PhD in 2009 in Biotechnology from University of Pune for his work demonstrating microRNA regulation of cytokines involved in allergic inflammation in mouse models. Dr. Sharma’s postdoctoral research at the Buck Institute, Novato, California involved investigating novel molecular regulatory pathways in regards to genotoxic stress and cellular senescence in invertebrate and mammalian models.

Dr. Sharma has joined SENS Research Foundation as Group Lead in the Senescence Immunology Research Group (“ImmunoSENS”). His research focus involves studying how aging and senescence affects the immune system and his research group will also investigate strategies to harness the immune system in mitigating deleterious effects of senescent cells with translational focus.

Budget

They are requesting the following budget each year, for a total of two years. Overhead cost is 10%, which includes rent and admin support.

Lab equipment and reagents:                           $40,000
Tech services:                                        $60,000
(monoclonal antibody generation, cloning and expression lentiviral vectors, and CAR-NK cell generation) 
Postdoctoral fellow salary:                           $90,000
10% of the PI salary:                                 $25,000
----
Total for 1 year:                                     $215,000
Total for 2 years:  $215,000 x 2 =                    $430,000
----
Paid by SRF:                                          $200,000

Paid by VitaDAO:                                      $230,000
10% indirect (overhead):                              $23,000  

TOTAL amount requested (including overhead):          $253,000

Highlights

  • Project lead has a strong scientific background in senescence
  • SENS Research Foundation provides strong support and track record of successful spin-outs (eg Underdog Pharma)
  • Used multiple senescence-inducing methods in their experiments
  • Removal of senescence cells is an attractive therapeutic approach to combat aging and promote longevity
  • The study here aims at identifying better targeting agents for the surface of senescent cells, a logical strategy given the current success of using CAR T cells.
  • In vitro data provides additional proof of concept, consistent with prior work using CAR T cells, that targeting senescent cells, in this case with CAR NK cells (see questions below), is feasible.

Risks

  • Based on their preliminary in vitro data, non-senescent cells seem to also be killed by this approach, albeit at lower rates than senescent cells. (i.e. killing 40-50% senescent cells and 10-25% healthy cells)
  • p16 seems to be the only marker used for tracking senescence. Given the heterogeneity of senescence, that may be limiting in terms of applicability. Additional markers are needed to confirm senescence.
  • Although referred to, there is no formal plan to validate their markers using human biospecimens. That seems to be a critical missing element looking forward to potential value.

Longevity WG scientific evaluation digest:

The 4 senior reviewers have expressed a vote in agreement to fund this proposal. Here is the digest:

Quantitative reviews:

To quantify the level of conviction, they have provided a score on a scale of 1-5 (with 5 being the highest).
The average score was 4.04/5

Brief qualitative review summaries:

1)

This is one of the most translationally relevant projects in our pipeline, especially with regard to future pharma interest – they love NK cell therapies and they’re increasingly aware of senolytics. SENS is a strong mission-aligned partner and we have the potential to collaborate with them much more in the future. The team is well qualified to carry out the work, and it is likely to result in Composition of Matter IP around the CAR-NK sequence. Overall it’s a great fit for us.

2)

Strengths: Novel target molecules have been identified with sound supporting preliminary data. Targeting of senescent cells is an attractive therapeutic approach for a range of age-related diseases and relevant to the DAO mission. The application of CAR NK cells is of growing interest as a therapeutic strategy, presenting opportunity. Strong scientific team within a supporting research environment.
Weaknesses: The main issue will be whether their new target molecules will provide increased specificity in the clearance of bad senescent cells over good cells. That poses risk, but needs to be determined. At present, limited plan to confirm efficacy of targets in human biospecimens, although a brief mention is noted.

3)

The project is a solid idea presented by a solid team. However, it’s in a preliminary stage with a lot of unknowns relative to other projects.

Pros:

  • SENS is mission aligned with VitaDAO
  • PI has solid expertise
  • CAR-NK cells and antibodies are valuable assets

Cons:

  • It’s unclear whether in vitro senescence markers will have the same importance in vivo
  • It’s unclear whether the team has generated CAR-NK before. Feasibility of making the patentable material is unknown.
  • It’s unclear what will determine when CAR-NK cells vs. antibodies will be prioritized.
  • The in vivo disease model has not yet been identified. This could be substantial work to find the best model.

4)

I believe we should fund this project. The project is led by an experienced PI, and the team is well suited to carry out the work. The negotiations have re-focused the project on the generation of useful IP, which there would likely be interest in commercializing (from VCs or Pharma) if the work proves to be successful. The creation of CAR NK cells that lead to the selective killing of senescent cells is certainly aligned with the mission of VitaDAO, advancing longevity research. There are, however, still risks - the project is early in its development, so there is some risk that no valuable IP will be generated, even in the 2 year timeframe. Really impressive PI and work.

  • Agree
  • Agree with revisions (please comment)
  • Disagree

0 voters

A more in-depth description is available internally (in the dealflow funnel - Sign in - Airtable).

If you don’t have access feel free to DM me.

1 Like

IP sharing negotiations are in progress, as well as finalizing the senior reviewer’s evaluation and further questions (under NDA). A Gantt chart is also coming up.

If negotiations go well, before phase 3, I’ll post the conviction level and summary reviews.

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Regarding the risks:

  • Point 1: Yes, but they might find a way of reducing it or in the next iteration.

  • Point 3: This seems the most critical point to me. I don’t have an understanding of this. Any idea on the likelyhood that it works with humans as well?

What’s the expected timeline to see any early results and end results? Isn’t quite clear to me

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FYI to the more lay audience, most proposals will have solid team and a reasonably important scientific topic. Meaning, most of this write up is extraneous information. (This is not intended to insult the hard work that went into this write up. Of course, I sincerely thank those involved. My comment is simply meant to inform on what will matter to those in the know.)

Because all else is basically equal, what separates a good project from a bad project is the approach. Meaning, the experiments themselves and how the team will generate IP. In this case, the weakness in the approach is that there isn’t a focus on the maximally valuable IP. Methods patents aren’t valuable. What’s valuable are novel compositions. The expectation should be that the very first activity and only activity should be to create CAR NK cells or antibodies that lead to the selective killing of cells that harbor the SENS-1,2,3 antigens. The determination of whether the prioritization is on NK cells or an antibody should be based on a thorough assessment of their pros and cons.

Another issue is we can’t spend $500K on this. There’s no legitimate reason the IP can’t be generated for half as much like other projects we’re funding.

I’m strongly in favor of this project but these issues should be addressed before we commit to funding.

3 Likes

Promising proposal and love the concise writeup!

Absolutely agree and thanks for raising these points here! How would you go about it to include this in the proposal, potentially discussing the approach for patentability as well as budget before onchain proposal?

Absolutely agree and thanks for raising these points here! How would you go about it to include this in the proposal, potentially discussing the approach for patentability as well as budget before onchain proposal?

In general we are trying to standardize deals without existing IP into two milestones with fixed amounts*.

  1. Milestone 1: $100K to generate the IP
  2. Milestone 2: $100K to validate the IP

The important thing to remind the team is that the Milestone 2 payment won’t be given unless the IP gets generated during Milestone 1. As long as this framework is in the on-chain proposal each time we should be good.

CC’ing @longevion @tylergolato for transparency.

*indirects not to exceed 20%

1 Like

Agreed with Tim on this – focusing on the ‘deliverable’ of CoM IP on the CAR/mAb sequence should be the priority.

We may be able to reduce the budget a bit, but keep in mind that the 500k is over 2 years, other proposals have often been for 1 year.

There is an itemized budget/Gantt chart available under NDA.

CAR-NK cells are hot in biopharma today and it would be relatively attractive to VC and biopharma partners.

Ultimately, for some projects, it’s not possible to reduce the costs or operate more leanly. There are simply some costs that cannot be cut – and the budget in the data room seems quite reasonable.

That said, if VitaDAO does not want to pay the entire bill, SENS said they have the capital to finance the project as well, so we could split the project and retain a smaller proportion of the upside as per the licensing terms / IP co-ownership. There is a way to find a deal structure that is agreeable to both parties.

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As someone who is intimately familiar with writing budgets and executing on them, budgets are much more flexible than people claim. We have to move away from the teams dictating the budget and putting that under VitaDAO’s control. Research is commoditized to point that nobody can claim to have expensive special sauce.

If you see room to ‘economize’ the current budget, by all means, let’s propose it to the team.

You just tell them here’s the number we’ll give you. You don’t ask for a budget.

This has not been clinically demonstrated in humans right? Other than lowering of “surrogate” markers of aging? Am I missing something?

Are there any strategies to lower/mitigate the killing of 10-25% of healthy cells?

Nevertheless, a great project! Great write-up! Exciting stuff

After negotiations, we’re lowering the amount requested:

Overhead cost goes down to 10%.

Total for 1 year:                                     $215,000
Total for 2 years:  $215,000 x 2 =                    $430,000
----
Paid by SRF:                                          $200,000

Paid by VitaDAO:                                      $230,000
10% indirect (overhead):                              $23,000  

TOTAL amount requested (including overhead):          $253,000

Other terms included in the exclusive license agreement:

  • SRF gets a board observer seat for 7 years in any resulting NewCo that will license this technology
  • There will be time limit on the license to ensure that the technology moves forward in a timely fashion: either we out-license by 2026 or have an IND filed
1 Like

Great proposal! I just have a couple of questions.

  1. Is the plan to develop iPSC-derived NK cells? or do you plan to engineer each patient’s autologous NK cells?2
  2. Are there any concerns regarding off-target effects? will the CAR-NK cells incorporate an “off-switch”?

I know this may be a bit down the line but still something we should keep in mind.

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Their responses to your questions, @kdl:

This has not been clinically demonstrated in humans right? Other than lowering of “surrogate” markers of aging? Am I missing something?

Yes, the effect of selective removal of senescent cells on the hallmarks of aging has only been demonstrated in preclinical models. It hasn’t been possible to test in humans so far because we really did not yet have safe tools to test this idea.
Yes, several senolytics have shown great promise and progress has been made but something like CAR-NKs based on surface makers will have much higher specificity (in theory or at least based on our experiences with cancer clinical trials). But I think the larger question that is being asked here is if removal of senescent cells in humans will actually improve lifespan. I am reasonably confident that removal of senescent cells will have a positive impact on inflammation (or rather inflammaging) and that should generally improve general health span. CAR-NKs will provide a great opportunity to test this idea.

Are there any strategies to lower/mitigate the killing of 10-25% of healthy cells?

All the data presented here is done with primary cells and even in a dish of non-senescent control cells we do expect to see a proportion of senescent or senescent like cells which will express NK cell ligands, this is an artifact of cell culture model so some degree of killing in non-senescent cells is expected. However, we are planning to build in some fail-safe in CAR design to circumvent some of those potential downsides. We also plan on higher specificity by making dual CAR (ie CAR mediated cytotoxicity only activated in cells that express two antigens not one). Also, the next step in a couple of years from now is to test our CAR-NKs in preclinical models, which is more optimal to address this very question.

1 Like

Their responses to your questions, @ines:

  1. The biggest advantage of using NK cells is that allogenic transplantation of these cells are relatively and frequently shown to be safe in cancer patients. Having said that, it may be possible to make a bank of haplo matched CAR-NKs that covers the MHC diversity in humans. This will make CAR-NK vastly more price effective.
  2. There are potential off-target effects that can be mitigated by choosing the right senescent antigen which is not expressed in non-senescent cells. We are going to validate these targets in human primary cells from young and old humans by co-staining with senescent makers. Even though NK cells are relatively short-lived, for added security, we will incorporate a kill switch in our CAR design. We can (and may) increase stringency by making CAR design such that it only kills targets if two antigens are expressed not one. We have recently hired an excellent scientist with decades of experience with CAR engineering who will be doing this project.

This proposal is now live for a community vote on Snapshot.