Development of a Novel Humanized Single Chain Antibody-Streptococcal Superantigen-Derived Immunotherapy Targeting the 5T4 Oncofetal Antigen

Superantigens (SAgs) are microbial toxins that cross-link T cell receptors with major histocompatibility complex (MHC) class II (MHC II) molecules leading to the activation of large numbers of T cells. Herein, the development and preclinical testing of a novel tumour-targeted SAg (TTS) therapeutic built using the streptococcal pyrogenic exotoxin C (SpeC) SAg and targeting cancer cells expressing the 5T4 tumour-associated antigen (TAA) was described. 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, a humanized single-chain variable fragment (scFv) antibody to recognize 5T4 (scFv5T4) was engineered. Specific targeting of scFv5T4 was verified. SpeCD203A used to scFv5T4 maintained the ability to activate and induce immune cell-mediated cytotoxicity of colon cancer cells. Using a xenograft model of established human colon cancer, it was demonstrated that the SpeC-based TTS was able to control the growth and spread of large tumours 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 \u27next-generation\u27 TTSs for cancer immunotherapy

Control of established colon cancer xenografts using a novel humanized single chain antibody-streptococcal superantigen fusion protein targeting the 5t4 oncofetal antigen

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 \u27next-generation\u27 TTSs for cancer immunotherapy. © 2014 Patterson et al

Functionality of SpeC mutants and fusion proteins for human PBMC proliferation and cytotoxicity <i>in vitro</i>.

<p>A) SpeC proteins were used to compare scFv5T4 alone, scFv5T4::SpeC_Y15A/D203A and subsequently scFv5T4::SpeC_D203A in the uptake of ³H-thymidine as a measure of PBMC proliferation after 4 day incubation (n = 5). B–C) Dose-dependent SpeC-mediated PBMC cytotoxicity of scFv5T4::SpeC_D203A was determined by comparing SpeC controls, scFv5T4 alone and scFv5T4::SpeC_Y15A/D203A after 48 h incubation by using FACS analysis of WiDr (panel B), measuring percent cancer cell death with 7AAD-exclusion staining (n = 3) and ⁵¹Cr-release to measure the specific cytotoxic potential (panel C) when incubated with increasing effector∶target ratios and ⁵¹Cr-labeled HT-29 cancer cells. Data shown (mean ±SEM) is from four independent human donors each done in triplicate. *p<0.05, ***p<0.001, compared to the inactive SpeC_Y15A/D203A control protein.</p

Specific targeting of scFv5T4.

<p>A) Histograms demonstrating surface binding of the indicated antibodies, either commercial mAb5T4 or generated scFv5T4 (empty curves), to 5T4 TAA on colorectal cancer cell line HT-29 measured by FACS. The shaded curves show the IgG2b isotype control (top panel) or streptavidin-FITC alone (bottom panel). B) Visualization of commercial mAb5T4, or scFv5T4, targeting of HEK293 cells transfected with empty vector (pCMV6-XL5) or pCMV6-XL5::5T4 by fluorescence microscopy. Representative images taken at 400× magnification. C) Visualization of HEK293 transfected with pEGFP-N1 or pEGFP-N1::5T4 and incubated with scFv5T4::mRFP1. Same field of view photographs were taken under phase contrast, and green and red fluorescent filters at 100× magnification.</p

SpeC-based TTS therapy of established HT-29 colon cancer.

<p>A) Schematic illustration of the xenograft solid tumor model experimental timeline. NSG mice with established (3 week) intraperitoneal human HT-29 tumors were injected once with human PBMC intraperitoneally, followed by 8 daily intravenous injections of scFv5T4::SpeC_D203A, or individual controls (2 µM/kg/injection). B) Primary tumor size was evaluated throughout the experimental timeline by external caliper measurements. Twenty-eight days post-final injection, final tumor volume was measured (panel C) and metastatic score (panel D) evaluated. All groups contained n = 4, with exception of saline alone control (n = 3). *p<0.05, **p<0.005. Gross pathology and metastases in representative NSG mice with HT-29 tumors treated with scFv5T4::SpeC_Y15A/D203A or scFv5T4::SpeC_D203A. The primary tumor is labeled with a triangle and metastases are labeled with arrows.</p

Generation of the scFv5T4::SpeC_D203A fusion protein and control reagents.

<p>A) Schematic illustration representing the components of the generated fusion protein constructs. The protein consists of the generated 5T4-targeted humanized single chain variable fragments, V_H and V_L (grey bar), genetically fused to streptococcal superantigen SpeC (blue bar) either containing an alanine substitution at residue D203 or an additional alanine substitution at residue Y15. All constructs were generated to contain a C-terminal biotin tag. The purified recombinant proteins are shown by SDS-PAGE (panel B), and detected by Western blot analysis by streptavidin-IRDye800 (panel C).</p

Control of Established Colon Cancer Xenografts Using a Novel Humanized Single Chain Antibody-Streptococcal Superantigen Fusion Protein Targeting the 5T4 Oncofetal Antigen

<div><p>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 (SpeC_D203A) that demonstrated a pronounced reduction in mitogenic activity, yet this mutant could still induce immune cell-mediated cancer cell death <i>in vitro</i>. 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. SpeC_D203A 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 <i>in vivo</i>. 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.</p></div