Optimizing a Lupus Autoantibody for Targeted Cancer Therapy

Noble, Philip W.; Chan, Grace; Young, Melissa R.; Weisbart, Richard H.; Hansen, James E.

doi:10.1158/0008-5472.can-14-2278

Cited by 26 publications

(46 citation statements)

References 29 publications

(34 reference statements)

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“…BRCA2 and PTEN are both known to play roles in DSB repair, and cells deficient in either of these proteins have been reported to have elevated levels of unrepaired DNA damage (4,5,29,35,36). Previous publications have reported that 3E10 is synthetically lethal with BRCA2-deficiency as well as PTEN-deficiency (26,37). Based on this, we elected to assess chemosensitization to Etoposide in BRCA2-deficient PEO-1 cells and PTEN-deficient U251 cells.…”

Section: Resultsmentioning

confidence: 99%

“…This property may further contribute to an increased therapeutic window, further highlighting the clinical potential of this unusual cell-penetrating antibody. Recent work by Noble et al suggests that engineering recombinant 3E10 molecules to comprise multiply valent scFvs can substantially boost activity (37). In this regard, the work presented here not only identifies 3E10 as a novel RAD51 inhibitor but also may help to guide further modifications to the 3E10 scFv to tune its DNA and RAD51 binding properties as a means to further increase efficacy for cancer therapy.…”

Section: Discussionmentioning

confidence: 99%

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A cell-penetrating antibody inhibits human RAD51 via direct binding

Turchick¹,

Hegan²,

Jensen³

et al. 2017

Nucleic Acids Research

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RAD51, a key factor in homology-directed repair (HDR), has long been considered an attractive target for cancer therapy, but few specific inhibitors have been found. A cell-penetrating, anti-DNA, lupus autoantibody, 3E10, was previously shown to inhibit HDR, sensitize tumors to radiation, and mediate synthetic lethal killing of BRCA2-deficient cancer cells, effects that were initially attributed to its affinity for DNA. However, as the molecular basis for its ability to inhibit DNA repair, we report that 3E10 directly binds to the N-terminus of RAD51, sequesters RAD51 in the cytoplasm, and impedes RAD51 binding to DNA. Further, we generate separation-of-function mutations in the complementarity-determining regions of 3E10 revealing that inhibition of HDR tracks with binding to RAD51 but not to DNA, whereas cell penetration is linked to DNA binding. The consequences of these mutations on putative 3E10 interactions with RAD51 and DNA are correlated with in silico molecular modeling. Taken together, the results identify 3E10 as a novel inhibitor of RAD51 by direct binding, accounting for its ability to suppress HDR and providing the molecular basis to guide pre-clinical development of 3E10 as an anti-cancer agent.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A cell-penetrating antibody inhibits human RAD51 via direct binding

Turchick¹,

Hegan²,

Jensen³

et al. 2017

Nucleic Acids Research

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“…Murine di-scFv with C-terminal Myc and His6 tags was previously designed and evaluated using a combination of in vitro and in vivo studies [9]. Di-scFv lacks an Fc, and therefore efforts to humanize di-scFv focused on VL and VH domains.…”

Section: In Silico Humanization Of Di-scfvmentioning

confidence: 99%

“…Previously a divalent 3E10 single chain variable fragment with improved DNA binding affinity (3E10 (D31N) di-scFv), hereafter referred to as di-scFv was evaluated [9]. Di-scFv lacks the unnecessary 3E10 Fc region that has potential to contribute to non-specific toxicity.…”

Section: Introductionmentioning

confidence: 99%

“…In conclusion, we have designed several humanized variants of 3E10 (D31N) di-scFv that have potential for application as monotherapy or conjugates for targeted nuclear drug delivery. KEYWORDS 3E10; di-scFv; autoantibody; DNA damage repair; synthetic lethality tumors and as a drug delivery ligand [9,10], but due to C-terminal Myc and His6 tags is unsuitable for human use. Herein, we report the design, expression and evaluation of a panel of novel humanized di-scFv variants that lack any tags with a view to utilizing them as novel anticancer agents or drug delivery ligands.…”

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confidence: 99%

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Re-engineering and evaluation of anti-DNA autoantibody 3E10 for therapeutic applications

Rattray

Dubljevic

Rattray

et al. 2018

Biochemical and Biophysical Research Communications

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A key challenge in the development of novel chemotherapeutics is the design of molecules capable of selective toxicity to cancer cells. Antibodies have greater target specificity compared to small molecule drugs, but most are unable to penetrate cells, and predominantly target extracellular antigens. A nuclear-penetrating anti-DNA autoantibody isolated from the MRL/lpr lupus mouse model, 3E10, preferentially localizes to tumors, inhibits DNA repair, and selectively kills cancer cells with defects in DNA repair. A murine divalent single chain variable fragment of 3E10 with mutations for improved DNA binding affinity, 3E10 (D31N) di-scFv, has previously been produced in P. pastoris and yielded promising pre-clinical findings, but is unsuitable for clinical testing. The present study reports the design, expression and testing of a panel of humanized 3E10 (D31N) di-scFvs, some of which contain CDR substitution. These variants were expressed in a modified CHO system and evaluated for their physicochemical attributes and ability to penetrate nuclei to selectively cause DNA damage accumulation in and kill cancer cells with DNA repair defects. Secondary structure was conserved and most variants retained the key characteristics of the murine 3E10 (D31N) di-scFv produced in P. pastoris. Moreover, several variants with CDR substitutions outperformed the murine prototype. In conclusion, we have designed several humanized variants of 3E10 (D31N) di-scFv that have potential for application as monotherapy or conjugates for targeted nuclear drug delivery.

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Post-genomic platform for development of oligonucleotide vaccines against RNA viruses: diamond cuts diamond

et al. 2022

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The coronavirus pandemic has starkly demonstrated the need to create highly effective vaccines against various viral diseases. The emerging new platforms for vaccine creation (adenovirus vectors and mRNA vaccines) have shown their worth in the fight against the prevention of coronavirus infection. However, adenovirus vectors and mRNA vaccines have a serious disadvantage: as a rule, only the S protein of the coronavirus is presented as an antigen. This tactic for preventing infection allows the ever-mutating virus to escape quickly from the immunity protection provided by such vaccines. Today, viral genomic databases are well-developed, which makes it possible to create new vaccines on a fundamentally new post-genomic platform. In addition, the technology for the synthesis of nucleic acids is currently experiencing an upsurge in demand in various fields of molecular biology. The accumulated experience suggests that the unique genomic sequences of viruses can act as antigens that trigger powerful humoral and cellular immunity. To achieve this effect, the following conditions must be created: the structure of the nucleic acid must be single-stranded, have a permanent 3D nanostructure, and have a unique sequence absent in the vaccinated organism. Oligonucleotide vaccines are able to resist the rapidly changing genomic sequences of RNA viruses by using conserved regions of their genomes to generate a long-term immune response, acting according to the adage that a diamond cuts a diamond. In addition, oligonucleotide vaccines will not contribute to antibody-dependent enhanced infection, since the nucleic acid of the coronavirus is inside the viral particle. It is obvious that new epidemics and pandemics caused by RNA viruses will continue to arise periodically in the human population. The creation of new, safe, and effective platforms for the production of vaccines that can flexibly change and adapt to new subtypes of viruses is very urgent and at this moment should be considered as a strategically necessary task.

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Optimizing a Lupus Autoantibody for Targeted Cancer Therapy

Cited by 26 publications

References 29 publications

A cell-penetrating antibody inhibits human RAD51 via direct binding

A cell-penetrating antibody inhibits human RAD51 via direct binding

Re-engineering and evaluation of anti-DNA autoantibody 3E10 for therapeutic applications

Post-genomic platform for development of oligonucleotide vaccines against RNA viruses: diamond cuts diamond

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