Targeted clearance of senescent cells using an antibody-drug conjugate against a specific membrane marker

A wide range of diseases have been shown to be influenced by the accumulation of senescent cells, from fibrosis to diabetes, cancer, Alzheimer’s and other age-related pathologies. Consistent with this, clearance of senescent cells can prolong healthspan and lifespan in in vivo models. This provided a rationale for developing a new class of drugs, called senolytics, designed to selectively eliminate senescent cells in human tissues. The senolytics tested so far lack specificity and have significant off-target effects, suggesting that a targeted approach could be more clinically relevant. Here, we propose to use an extracellular epitope of B2M, a recently identified membrane marker of senescence, as a target for the specific delivery of toxic drugs into senescent cells. We show that an antibody–drug conjugate (ADC) against B2M clears senescent cells by releasing duocarmycin into them, while an isotype control ADC was not toxic for these cells. This effect was dependent on p53 expression and therefore more evident in stress-induced senescence. Non-senescent cells were not affected by either antibody, confirming the specificity of the treatment. Our results provide a proof-of-principle assessment of a novel approach for the specific elimination of senescent cells using a second generation targeted senolytic against proteins of their surfaceome, which could have clinical applications in pathological ageing and associated diseases.

Introduction

Senescence is an irreversible proliferation arrest and a key restriction mechanism to prevent the propagation of damaged cells^1,2. However, the progressive accumulation of senescent cells with time has been associated with loss of tissue homeostasis, and is known to contribute to the functional impairment of different organs typically seen in ageing³. Recently, it has been shown that it also plays an important role in fibrosis⁴ and tumour progression⁵, and that it may be involved in cataracts, obesity, diabetes, Alzheimer’s and Parkinson’s diseases, arthritis, atherosclerosis and many other age-related conditions^6,7. This supports the hypothesis that senescence is an antagonistically pleiotropic process, with beneficial effects in the early decades of life of the organism (in development⁸, tissue repair⁹ and as a tumour suppressor mechanism^10,11,12,13) but detrimental to fitness and survival at later stages, after the percentage of senescent cells in tissues reaches a critical threshold^14,15. This is thought to be mediated, at least in part, by the secretion of a series of growth factors, chemokines and cytokines, collectively known as the senescence-associated secretory phenotype (SASP)^16,17.

Consistent with this view, it has been reported that clearing senescent cells from tissues has a protective effect against cancer^5,18 and the onset of age-related pathologies^19,20. Because of this, great interest has been placed in a recently discovered group of drugs that can preferentially kill senescent cells, collectively known as senolytics, which have been shown to increase healthspan and lifespan of mice²¹ with attenuation of age-related dysfunctions like emphysema²², hepatic steatosis²³, lung fibrosis²⁴, osteoporosis²⁵, osteoarthrisis²⁶, cardiac regeneration dysfunctions²⁷, cognitive memory impairments²⁸ or Alzheimer disease²⁹ in different in vivo models. Recently, senolytics, were shown to also decrease the number of senescent cells in humans³⁰ and alleviate the symptoms of idiopathic pulmonary fibrosis³¹. Despite these important advances, the translational potential of senolytics is still limited, due to its multi-target nature^32,33. Although other compounds of potential interest are being investigated^34,35,36,37, some of them of natural origin^{34,38,39,40,41,42}, a more specific strategy would be desirable in order to reduce the side effects and increase the efficiency of these drugs.

In this context, targeted senolytics are emerging as a promising alternative. For instance, it has recently been shown that toxic nanoparticles activated by the presence of β-galactosidase can eliminate senescent cells in vitro and in vivo, confirming the feasibility of the approach⁴³. We propose that the senescent surfaceome, the specific profile of membrane proteins differentially upregulated in senescent cells, could be used to this end even more effectively. Using mass spectrometry, we identified a number of markers highly expressed in the plasma membranes of senescent cells in response to the activation of one of the two main pathways of induction of the phenotype (p53/p21 or 16)^44,45. Moreover, we showed that molecularly imprinted nanoparticles (nanoMIPs), nanostructured polymeric particles that can recognise and bind a target molecule thus acting like “plastic antibodies”⁴⁶, can bind to an extracellular epitope and thus detect and kill senescent cells⁴⁷, providing the first evidence that the senescent surfaceome can be used to design targeted senolytics⁴⁸…