Development of Novel, Highly Cytotoxic Fusion Constructs Containing Granzyme B: Unique Mechanisms and Functions

Rosenblum, Michael G.; Barth, Stefan

doi:10.2174/138161209788923958

Cited by 33 publications

(29 citation statements)

References 75 publications

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“…Targeting the HER1, HER2, and VEGF pathways with antibodies is now considered an essential component in the clinical therapeutic management of a variety of tumor types (37). There are also numerous groups developing antibody conjugates for the directed delivery of highly cytotoxic payloads such as small molecules-auristatin, caliecheamycin, or maytansinoids (38)(39)(40)(41), protein toxins, that is, RTA, PE, and gelonin (22,42,43), or other cytotoxic human enzymes such as serine proteases (44). The current study shows a unique Fn14 receptor-targeted antibody-toxin conjugate designated ITEM4-rGel that is shown to be capable of specifically delivering rGel to Fn14-expressing tumor cells in vitro and in vivo.…”

Section: Discussionmentioning

confidence: 99%

Targeted Therapy of Fn14-Positive Breast Tumors Using a TWEAK-Cytotoxin Fusion Protein or Noncovalent Complex

Winkles¹,

Brown²

2010

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Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. We found that these fusion proteins were difficult to purify from E. coli due to aggregation problems. In addition, when unaggregated protein was tested for its ability to kill Fn14-positive breast cancer cells the cytotoxic effect was not impressive (see Annual Report). Another approach listed in this Aim was to express and purify a protein A-Fc binding domain:rGel fusion protein, mix it with an IgG-Fc:TWEAK fusion protein (provided by Dr. Schneider, Univ. of Lausanne) and then add the noncovalent complex to cells. We did see some cytotoxicity on MDA-MB-231 breast cancer cells when these proteins were added at either a 3:1 or 2:1 ratio (see Annual Report). However, this approach, which requires mixing two proteins together, would probably not be applicable to patient treatment because the non-covalent complex may dissociate during IV administration or during transit in the bloodstream. Also, during the course of these studies a manuscript was published indicating that TWEAK can bind a monocyte/macrophage protein named CD163 (3). The identification of a second TWEAKbinding cell surface protein (besides Fn14) suggested to us that using TWEAK to direct toxins to Fn14+ tumor cells may not be the best strategy. An alternative strategy is to use an Fn14 extracellular domain-specific monoclonal antibody to deliver toxins into Fn14+ cells, and these studies are described below. REPORT DATEWe have developed two types of Fn14-targeted immunotoxins, both of which are based on the anti-Fn14 monoclonal antibody named ITEM4. This antibody was provided to us by Dr. Yagita (Juntendo University, Tokyo, JP). In our initial experiments, ITEM4 was chemically conjugated to gelonin. This ITEM4-rGel conjugate binds the Fn14 receptor and has excellent cytotoxic activity on many tumor cell lines, including breast cancer cell lines. This work has been published (4) and therefore will not be described in detail here. The manuscript is included in the Appendix. Chemical conjugates are not ideal for patient therapy because of their bulky size and other properties; therefore, we next constructed several single-chain immunotoxins. The structur...

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Section: Discussionmentioning

confidence: 99%

Targeted Therapy of Fn14-Positive Breast Tumors Using a TWEAK-Cytotoxin Fusion Protein or Noncovalent Complex

Winkles¹,

Brown²

2010

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“…While remaining difficulties to scale up protein production to levels required for treatment of larger cohorts of experimental animals may be overcome by utilizing optimized expression systems in yeast, insect and mammalian cells [24,58,65], also intrinsic features of GrB need to be addressed that may adversely affect availability of GrB fusions at the tumor site. With a calculated pI around 10, GrB is a highly basic protein with a positively charged surface.…”

Section: Target Cell Specificity Of Granzyme B Fusion Proteinsmentioning

confidence: 99%

“…Bacterial toxins such as Pseudomonas exotoxin A or diphtheria toxin (DT) have endogenous endosome escape activity, which can be readily employed for cytosolic delivery of recombinant ETA-and DT-based toxins [7]. While certain GrB fusion proteins are obviously able to reach the cytosol to some degree on their own [24], this does not appear to be as efficient as desirable, and GrB fusion proteins targeted to EGFR or ErbB2 were shown to be trapped in endosomal vesicles after receptor-mediated uptake. They require an exogenously provided endosomolytic activity like chloroquine to induce cell death at low concentrations [23,38].…”

Section: Intracellular Routing and Cytosolic Delivery Of Granzyme Bmentioning

confidence: 99%

Selective Induction of Cancer Cell Death by Targeted Granzyme B

2013

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Abstract:The potential utility of immunotoxins for cancer therapy has convincingly been demonstrated in clinical studies. Nevertheless, the high immunogenicity of their bacterial toxin domain represents a critical limitation, and has prompted the evaluation of cell-death inducing proteins of human origin as a basis for less immunogenic immunotoxin-like molecules. In this review, we focus on the current status and future prospects of targeted fusion proteins for cancer therapy that employ granzyme B (GrB) from cytotoxic lymphocytes as a cytotoxic moiety. Naturally, this serine protease plays a critical role in the immune defense by inducing apoptotic target cell death upon cleavage of intracellular substrates. Advances in understanding of the structure and function of GrB enabled the generation of chimeric fusion proteins that carry a heterologous cell binding domain for recognition of tumor-associated cell surface antigens. These hybrid molecules display high selectivity for cancer cells, with cell killing activities similar to that of corresponding recombinant toxins. Recent findings have helped to understand and circumvent intrinsic cell binding of GrB and susceptibility of the enzyme to inhibition by serpins. This now allows the rational design of optimized GrB derivatives that avoid sequestration by binding to non-target tissues, limit off-target effects, and overcome resistance mechanisms in tumor cells.

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“…We have developed a series of hCFPs using effector domains based on the human serine protease granzyme B [24], human ribonucleases (RNases) such as angiogenin [25], human kinases such as death-associated protein kinase 2 (DAPK2) [26], and the microtubule-associated proteins (tau) [27]. Further examples of human effector domains include death receptor ligands and pro-apoptotic members of the BCL-2 family [16,28]. Several hCFPs have already been tested to confirm their selective activity and efficiency in vitro and in vivo [16,29].…”

Section: Introduction: From Classical Immunotoxins To Human Cytolyticmentioning

confidence: 99%

Engineered Versions of Granzyme B and Angiogenin Overcome Intrinsic Resistance to Apoptosis Mediated by Human Cytolytic Fusion Proteins

Cremer

Hehmann-Titt²,

Schiffer

et al. 2015

Resistance to Targeted Anti-Cancer Therapeutics

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The use of therapies based on antibody fusion proteins for the selective elimination of tumor cells has increased markedly over the last two decades because the severe side effects associated with conventional chemotherapy and radiotherapy are reduced or even eliminated. However, the initial development of immunotoxins suffered from a number of drawbacks such as nonspecific cytotoxicity and the induction of immune responses because the components were non-human in origin. The most recent iteration of this approach is a new class of targeted human cytolytic fusion proteins (hCFPs) comprising a tumor-specific targeting component such as a human antibody fragment fused to a human effector domain with pro-apoptotic activity. Certain tumors resist the activity of hCFPs by upregulating the intracellular expression of native inhibitors, which rapidly bind and inactivate the human effector domains. Higher doses of the hCFPs are, therefore, required to improve therapeutic efficacy. To circumvent these inhibitory processes, novel isoforms of the enzymes granzyme B and angiogenin have been designed to increase their intrinsic activity and reduce their interactions with native inhibitors resulting in more potent hCFPs that can be applied at lower doses. This chapter summarizes the basic scientific knowledge that can facilitate the rational development of human enzymes with novel and beneficial characteristics, including the ability to avoid neutralization by native inhibitors

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Development of Novel, Highly Cytotoxic Fusion Constructs Containing Granzyme B: Unique Mechanisms and Functions

Cited by 33 publications

References 75 publications

Targeted Therapy of Fn14-Positive Breast Tumors Using a TWEAK-Cytotoxin Fusion Protein or Noncovalent Complex

Targeted Therapy of Fn14-Positive Breast Tumors Using a TWEAK-Cytotoxin Fusion Protein or Noncovalent Complex

Selective Induction of Cancer Cell Death by Targeted Granzyme B

Engineered Versions of Granzyme B and Angiogenin Overcome Intrinsic Resistance to Apoptosis Mediated by Human Cytolytic Fusion Proteins

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