Secreted Antibody/Granzyme B Fusion Protein Stimulates Selective Killing of HER2-overexpressing Tumor Cells

Abstract

HER2/ErbB2, an epidermal growth factor receptor-related tyrosine kinase, is overexpressed on the surface of a variety of tumors of epithelial origin, such as breast tumors, ovarian carcinomas, and gastric tumors, and therefore constitutes an ideal therapeutic target (1-3). Among the many advances in the development of HER2-targeted cancer therapy over the past decade (4 -7), antibody-based strategies (e.g. herceptin (trastuzumab) (8 -10), intracellular antibody (11, 12), and fusion proteins for targeted delivery of various classes of therapeutic agents (13-17)) are especially notable. Our previous studies exploited the in vitro and in vivo cytotoxicity of immunotoxins by generating a new class of antigen-specific killer cells (18,19), but this approach may well be limited in its clinical applications because of antigenicity of heterogeneous toxin protein. Hence, our attention turned to human endogenous proteins, especially those acting as executioners during apoptosis, which in principle should be optimal candidates for triggering the death of tumor cells in an intrinsic physiological manner.Granzymes are a family of granular serine proteinases produced by cytotoxic T lymphocytes and natural killer cells, with a triad of key residues, histidine, aspartate, and serine, conserved at the catalytic site (20, 21). One member of the family, granzyme B (GrB), 1 plays an essential role in granule-mediated killing. GrB is originally synthesized as zymogen and activated by removal of the amino-terminal signal peptide and an acidic dipeptide after lymphocyte receptor recognition (21). The active GrB (GrBa) is then released from leukocyte granules by exocytosis, entering target cells to cause extensive proteolysis at specific amino acid residues (22, 23). GrB-mediated apoptosis involves the caspase-dependent pathway, direct nuclear entry, and caspase-independent pathways (24 -28). The potential of GrB therapy to induce multiple apoptotic pathways makes this approach to tumor cell killing especially attractive.In our most recent previous study, chimeric immunocasp-3 protein was shown to have a potent selective antitumor effect both in vitro and in vivo (29). This result led us to another targeted pro-apoptotic fusion protein, designated immunoGrB, which consisted of a single-chain anti-HER2 antibody, e23sFv (30, 31), a Pseudomonas exotoxin A (PE) translocation domain (32-35), and active GrB (GrBa) (21). The recombinant immunoGrB molecule was predicted to bind to tumor cells overexpressing HER2 and be internalized, such that endosomal cleavage would give rise to the active granzyme B domain that would consequently translocate into the cytosol to induce tumor cell death (18,19,29). Here we evaluate the antitumor activity of immunoGrB and immunoGrB-secreting lymphocytes in both in vitro and in vivo models. EXPERIMENTAL PROCEDURESConstruction of Expression Plasmids-The human GrB gene was obtained by reverse-transcription PCR from total RNA derived from healthy human peripheral blood mononuclear cells (PBMCs) following stimulatio...

HER2-targeting recombinant protein with truncated Pseudomonas Exotoxin: A translocation domain efficiently kills breast cancer cells

“…Three PEA II domain variants, C-terminally truncated PEA II domain (PEA253-358 aa), both C-terminally and N-terminally truncated PEA II domain (PEA275-358 aa), and one short sequence containing only a furin cleavage site (PEA275-280 aa) were successfully fused to a single-chain antibody against HER2(e23sFv) 13,14 and constitutively active granzyme B. The recombinant genes were subcloned downstream and in frame with DNA encoding a signal sequence (MKHLWFFLLLVAAPRWVLS) in the expression vector pCMV.…”

“…A signal sequence was also introduced in the constructs to ensure that all the recombinant proteins were secreted. 13 We monitored translocation activities indirectly by measuring cytotoxicity of ImmunoGrB fusion protein as well as directly by immunofluorescence.…”

“…8,9 Although earlier studies used PEA fragment from amino acids 253-412 or 253-364 for protein translocation, these data suggested that amino acids 253-358 is sufficient for PEA translocation. [10][11][12][13] In order to determine the minimum domain length required for translocation, a set of deletions in the PEA translocation domain were introduced into the previously constructed ImmunoGrB, 13 which is composed of a single-strand antibody against HER2, translocation domain from PEA, and active granzyme B. A signal sequence was also introduced in the constructs to ensure that all the recombinant proteins were secreted.…”

Targeted induction of apoptosis by chimeric granzyme B fusion proteins carrying antibody and growth factor domains for cell recognition

“…There the translocation domain of a bacterial toxin was included in the molecule to achieve cytoplasmic delivery of an active GrB fragment in target cells. 35 In the presence of chloroquine, GrB-5 and GrB-T were able to specifically kill target cells with IC 50 values in the picomolar to nanomolar range, whereas nontarget cells were not affected at considerably higher concentrations. These cytotoxic activities of the chimeric GrB derivatives are comparable to those of Pseudomonas exotoxin A fusion proteins which employ the same cell targeting domains.…”

“…In another approach, the problem to produce a functional tumor-specific GrB fusion protein was solved by coincubating tumor cells with engineered T cells releasing such a molecule. 35 We have followed a different strategy expressing the ErbB2-specific GrB-5 and EGFR-specific GrB-T molecules as secreted proteins in the yeast P. pastoris, thereby bypassing the need for refolding or in vitro activation, and resulting in large amounts of active recombinant proteins after a single purification step. Attachment of the C-terminal antibody and growth factor domains did not affect enzymatic activity of GrB.…”