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Control of established colon cancer xenografts using a novel humanized single chain antibody-streptococcal superantigen fusion protein targeting the 5T4 oncofetal antigen.

ABSTRACT: Superantigens (SAgs) are microbial toxins that cross-link T cell receptors with major histocompatibility class II (MHC-II) molecules leading to the activation of large numbers of T cells. Herein, we describe the development and preclinical testing of a novel tumor-targeted SAg (TTS) therapeutic built using the streptococcal pyrogenic exotoxin C (SpeC) SAg and targeting cancer cells expressing the 5T4 tumor-associated antigen (TAA). To inhibit potentially harmful widespread immune cell activation, a SpeC mutation within the high-affinity MHC-II binding interface was generated (SpeCD203A) that demonstrated a pronounced reduction in mitogenic activity, yet this mutant could still induce immune cell-mediated cancer cell death in vitro. To target 5T4+ cancer cells, we engineered a humanized single chain variable fragment (scFv) antibody to recognize 5T4 (scFv5T4). Specific targeting of scFv5T4 was verified. SpeCD203A fused to scFv5T4 maintained the ability to activate and induce immune cell-mediated cytotoxicity of colorectal cancer cells. Using a xenograft model of established human colon cancer, we demonstrated that the SpeC-based TTS was able to control the growth and spread of large tumors in vivo. This required both TAA targeting by scFv5T4 and functional SAg activity. These studies lay the foundation for the development of streptococcal SAgs as 'next-generation' TTSs for cancer immunotherapy.

SUBMITTER: Patterson KG

PROVIDER: S-EPMC3988171 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Control of established colon cancer xenografts using a novel humanized single chain antibody-streptococcal superantigen fusion protein targeting the 5T4 oncofetal antigen.

Patterson Kelcey G KG Dixon Pittaro Jennifer L JL Bastedo Peter S PS Hess David A DA Haeryfar S M Mansour SM McCormick John K JK

PloS one 20140415 4

Superantigens (SAgs) are microbial toxins that cross-link T cell receptors with major histocompatibility class II (MHC-II) molecules leading to the activation of large numbers of T cells. Herein, we describe the development and preclinical testing of a novel tumor-targeted SAg (TTS) therapeutic built using the streptococcal pyrogenic exotoxin C (SpeC) SAg and targeting cancer cells expressing the 5T4 tumor-associated antigen (TAA). To inhibit potentially harmful widespread immune cell activation ...[more]

PMID: 24736661

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Project description:5T4 oncofetal molecules are highly expressed during development and upregulated in cancer while showing only low levels in some adult tissues. Upregulation of 5T4 expression is a marker of loss of pluripotency in the early differentiation of embryonic stem (ES) cells and forms an integrated component of an epithelial-mesenchymal transition, a process important during embryonic development and metastatic spread of epithelial tumors. Investigation of the transcriptional changes in early ES differentiation showed upregulation of CXCL12 and down-regulation of a cell surface protease, CD26, which cleaves this chemokine. CXCL12 binds to the widely expressed CXCR4 and regulates key aspects of development, stem cell motility and tumour metastasis to tissues with high levels of CXCL12. We show that the 5T4 glycoprotein is required for optimal functional cell surface expression of the chemokine receptor CXCR4 and CXCL12 mediated chemotaxis in differentiating murine embryonic stem cells and embryo fibroblasts (MEF). Cell surface expression of 5T4 and CXCR4 molecules is co-localized in differentiating ES cells and MEF. By contrast, differentiating ES and MEF derived from 5T4 knockout (KO) mice show only intracellular CXCR4 expression but infection with adenovirus encoding mouse 5T4 restores CXCL12 chemotaxis and surface co-localization with 5T4 molecules. A series of chimeric constructs with interchanged domains of 5T4 and the glycoprotein CD44 were used to map the 5T4 sequences relevant for CXCR4 membrane expression and function in 5T4KO MEF. These data identified the 5T4 transmembrane domain as sufficient and necessary to enable CXCR4 cell surface expression and chemotaxis. Furthermore, some monoclonal antibodies against m5T4 can inhibit CXCL12 chemotaxis of differentiating ES cells and MEF which is not mediated by simple antigenic modulation. Collectively, these data support a molecular interaction of 5T4 and CXCR4 occurring at the cell surface which directly facilitates the biological response to CXCL12. The regulation of CXCR4 surface expression by 5T4 molecules is a novel means to control responses to the chemokine CXCL12 for example during embryogenesis but can also be selected to advantage the spread of a 5T4 positive tumor from its primary site.

Project description:BACKGROUND: The major virulence factors determining the pathogenicity of streptococcal strains include M protein encoded by emm and emm-like (emmL) genes and superantigens. In this study, the distribution of emm, emmL and superantigen genes was analyzed among the streptococcal strains isolated from the patients of acute pharyngitis. METHODS: The streptococcal strains were isolated from the throat swabs of 1040 patients of acute pharyngitis. The emm and emmL genes were PCR amplified from each strain and sequenced to determine the emm types. The dot-blot hybridization was performed to confirm the pathogens as true emm nontypeable strains. The presence of eleven currently known superantigens was determined in all the strains by multiplex PCR. RESULTS: Totally, 124 beta-hemolytic streptococcal strains were isolated and they were classified as group A streptococcus (GAS) [15.3% (19/124)], group C streptococcus (GCS) [59.7% (74/124)] and group G streptococcus (GGS) [25.0% (31/124)]. Among 124 strains, only 35 strains were emm typeable and the remaining 89 strains were emm nontypeable. All GAS isolates were typeable, whereas most of the GCS and GGS strains were nontypeable. These nontypeable strains belong to S. anginosus [75.3% (67/89)] and S. dysgalactiae subsp. equisimilis [24.7% (22/89)]. The emm and emmL types identified in this study include emm12.0 (28.6%), stG643.0 (28.6%), stC46.0 (17.0%), emm30.11 (8.5%), emm3.0 (2.9%), emm48.0 (5.7%), st3343.0 (2.9%), emm107.0 (2.9%) and stS104.2 (2.9%). Various superantigen profiles were observed in typeable as well as nontypeable strains. CONCLUSIONS: Multiplex PCR analysis revealed the presence of superantigens in all the typeable strains irrespective of their emm types. However, the presence of superantigen genes in emm and emmL nontypeable strains has not been previously reported. In this study, presence of at least one or a combination of superantigen coding genes was identified in all the emm and emmL nontypeable strains. Thus, the superantigens may inevitably play an important role in the pathogenesis of these nontypeable strains in the absence of the primary virulence factor, M protein.

Dataset Information

Control of established colon cancer xenografts using a novel humanized single chain antibody-streptococcal superantigen fusion protein targeting the 5T4 oncofetal antigen.

Publications

Control of established colon cancer xenografts using a novel humanized single chain antibody-streptococcal superantigen fusion protein targeting the 5T4 oncofetal antigen.

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