Combining intracellular antibodies to restore function of mutated p53 in cancer

Abstract

TP53 is a tumor suppressor gene that is mutated in 50% of cancers, and its function is tightly regulated by the E3 ligase, Mdm2. Both p53 and Mdm2 are localized in the cell nucleus, a site that is impervious to therapeutic regulation by most antibodies. We identified a cell-penetrating lupus monoclonal anti-DNA antibody, mAb 3E10, that targets the nucleus, and we engineered mAb 3E10 to function as an intranuclear transport system to deliver therapeutic antibodies into the nucleus as bispecific single chain Fv (scFv) fragments. Bispecific scFvs composed of 3E10 include PAb421 (3E10-PAb421) that binds p53 and restores the function of mutated p53, and 3G5 (3E10-3G5) that binds Mdm2 and prevents destruction of p53 by Mdm2. We documented the therapeutic efficacy of these bispecific scFvs separately in previous studies. In this study, we show that combination therapy with 3E10-PAb421 and 3E10-3G5 augments growth inhibition of cells with p53 mutations compared to the effect of either antibody alone. By contrast, no enhanced response was observed in cells with wild-type p53 or in cells homozygous null for p53.The TP53 tumor suppressor gene is considered the "guardian" of the genome, and mutations in this gene are found in 50% of cancers. 1 p53 is self-regulating by inducing the synthesis of Mdm2, an E3 ligase that leads to the destruction of p53. 2 In addition to several p53-independent functions of Mdm2, overexpression of Mdm2 has oncogenic activity as a result of inactivating the tumor suppressor function of p53. 3 Therapy to restore the function of p53 remains a significant challenge in cancer therapy. Small molecules have been the mainstay of cancer therapy, and small molecules have been developed to restore the function of mutant p53 4 and to inhibit Mdm2. 5 However, small molecules often demonstrate off-target binding resulting in undesirable side effects. By contrast, antibodies have exquisite specificity but are largely reserved for targeting molecules present in the circulation or sites on cell membranes, as most antibodies do not penetrate into living cells.Our laboratory identified a lupus anti-DNA antibody, mAb 3E10, which penetrates into living cells and localizes to the nucleus without apparent harm. 6 We engineered the Fv fragment of 3E10 to optimize cell penetration and developed this antibody as an intracellular transport system to deliver other antibodies to the cell nucleus as bispecific single-chain Fv (scFv) fragments. 7 In previous studies it was shown that the function of some cells with p53 mutations could be restored by microinjection of mAb PAb421, an anti-p53 antibody that binds and restores structural conformation of mutant p53. 8 However, there has not been a satisfactory therapeutic approach to deliver mAb PAb421 into the nucleus of cancer cells. In an attempt to achieve this goal, we developed 3E10 cell penetrating antibody as a bispecific scFv with the Fv fragment of PAb421 (3E10-PAb421) to transport PAb421 into cancer cells with p53 mutations. This approach was effective in tissue cultur...

Peptide Sharing Between Viruses and DLX Proteins: A Potential Cross-Reactivity Pathway to Neuropsychiatric Disorders

“…In such a scenario, the passage in the fetus of maternal memory B cells (Vernochet et al, 2005 , 2007 ) against epitopes shared between the pathogen(s) and DLX proteins might induce an immune response targeting the developing nervous systems, where DLX proteins are expressed early (see Figure 1 ). Cellular damage from (auto)antibodies targeting intracellular antigens, like the DLX family of TFs may be, not only plays a pathogenic role in a variety of autoimmune diseases (Racanelli et al, 2011 ) but even represents a promising therapeutic strategy for cancer treatment (Weisbart et al, 2012 ; Wang et al, 2015 ; Chan et al, 2016 ). The notion that autoantibodies can penetrate living cells is not new.…”

Targeted Intracellular Delivery of Antibodies: The State of the Art

“…As phage display technology allows rapid and relatively easy generation of antibodies to almost any antigen of interest ( Vaughan et al, 1996 ; Colwill and Graslund, 2011 ; Frenzel et al, 2017 ), intrabodies aimed at many different proteins, including their splice variants and specific post-translational modification sites ( Koo et al, 2014 ; Chirichella et al, 2017 ), have been developed and shown to be rather efficient in vitro and sometimes in vivo ( Amici et al, 2016 ). The most widely utilized targets include cancer-linked antigens ( Van Impe et al, 2013 ; Chan et al, 2016 ; Alirahimi et al, 2017 ; Amici et al, 2016 ), neurodegenerative-disease-related antigens ( Lynch et al, 2008 ; Joshi et al, 2012 ; Butler and Messer, 2011 ), toxins ( Tremblay et al, 2010 ; Alzogaray et al, 2011 ), viral infection (e.g., HIV or HCV) – related antigens ( Matz et al, 2014 ; Boons et al, 2014 ; Ashour et al, 2015 ; Amici et al, 2016 ), and miscellaneous antigens, mostly for protein function studies, within the cell ( Van Audenhove et al, 2013 ). The therapeutic potential of intrabodies against cancer ( Amici et al, 2016 ), Huntington’s disease ( Southwell et al, 2009 ), and Alzheimer’s disease ( Ryan et al, 2010 ) has been already demonstrated in mouse models of these diseases.…”

Single domain antibodies for the knockdown of cytosolic and nuclear proteins

“…Fusion of peptides and proteins to reach the cytoplasm is often not very efficient because of inefficient endosomal escape . However, a cell penetrating anti‐DNA antibody localizes to the nucleus through a nucleoside salvage pathway and is used as a delivery vehicle and combined with specific targeting scFvs …”

“…110 However, a cell penetrating anti-DNA antibody localizes to the nucleus through a nucleoside salvage pathway and is used as a delivery vehicle and combined with specific targeting scFvs. 90 Some highlights referring to cell-penetrating peptides should be mentioned. Highly efficient delivery was achieved using a cell-penetrating peptideadaptor system built of TAT-calmodulin fusion and a fusion consisting of calmodulin binding site and cargo.…”

“…Additionally, the nucleus‐targeted intrabody inhibited tumor activity of HPV16‐positive tumor cells in two pre‐clinical mouse models. Interestingly, two bispecific scFv fragments have been constructed that (1) comprises a scFv that targets the nucleus and a scFv fragment that restored the function of mutated p53 and (2) comprises the intracellular nuclear transport scFv fragment and a scFv fragment that binds Mdm2 and prevents destruction of p53 by Mdm2 . All three scFv fragments were constructed from corresponding hybridoma clones.…”