One-liner: This proposal asks for 253,000 USD to fund 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: https://www.nature.com/articles/s43587-021-00121-8

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.

For more information, please see the phase 2 proposal and discussion on Discourse.