In situ delivery and production system of trastuzumab scFv with Bifidobacterium

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Abstract

A monoclonal antibody targeting human epidermal growth factor receptor-2 (HER2), trastuzumab has become a standard treatment for HER2-positive breast cancer. Recent advancements in antibody engineering have enabled the efficient generation of the trastuzumab single-chain variable fragment (scFv).

In this study, we genetically engineered Bifidobacterium, a bacterial strain shown to accumulate safely and selectively in hypoxic tumor sites by intravenous (iv) injection, to express and secrete the trastuzumab scFv. The recombinant scFv bound to cell surface HER2 and inhibited in vitro growth of HER2-positive human cancer cells. Moreover, iv-injected recombinant bacteria specifically localized and secreted trastuzumab scFv in xenografted human HER2-positive tumors and consequently inhibited tumor growth.

The development and results of this novel in situ delivery and production system for trastuzumab scFv with Bifidobacterium represents a promising avenue for future application in cancer treatment.

Introduction

Great advances are being made in translational research on molecular-targeting anti-cancer drugs [1]. Regarding the treatment of breast cancer, trastuzumab, a monoclonal antibody targeting human epidermal growth factor receptor-2 (HER2), has become one of the standard therapeutics for HER2-positive tumors [2]. In spite of the anti-tumor effectiveness of antibody medicines, however, side effects remain problematic [3] and high drug prices are a source of concern for both patients and public health care budgets alike [4].

Given such circumstances, we have been developing an insitu delivery and production system (iDPS) for anti-cancer drugs that selectively function at tumor sites by making use of Bifidobacterium, a well known non-pathogenic obligate anaerobe and major component of normal human intestinal flora [5]. We earlier reported that the bacteria selectively localized and proliferated in the hypoxic microenvironment of solid tumors after intravenous (iv) administration to tumor-bearing animals [6], [7].

The premise of iv injection of bacteria in vivo has been criticized as irrational because it would appear to induce artificial bacteremia and septicemia, even if the bacteria were non-pathogenic. However, we have shown that such treatment of tumor-bearing animals with the non-pathogenic obligate anaerobic bacterium Bifidobacterium longum (B. longum) is safe and rational as a selective delivery tool to solid cancers [6], [7]. In fact, APS001F, a recombinant Bifidobacterium that carries cytosine deaminase converting 5FC (Flucytosine) to 5FU (5 Fluorouracil), has been already trialed in a phase 1 clinical study in combination with orally administered 5FC [6], [7] without serious adverse effects.

In the present study, we examined a novel iDPS model for the trastuzumab single-chain variable fragment (scFv) using Bifidobacterium. The development of such small antibodies as scFv has been an attractive topic in recent protein engineering [8]; active scFv, including bispecific T cell engager antibodies, has been produced and reported to improve antitumor efficacy [8], [9].

An expression vector for the trastuzumab scFv was constructed using the sequences corresponding to the light and heavy chains of trastuzumab. We first prepared genetically engineered Escherichia coli (E. coli) to express biologically active trastuzumab scFv. Then, we established trastuzumab scFv-expressing/secreting strains of B. longum (H1 and H2) for our iDPS. We assessed the function of trastuzumab scFv secreted by B. longum, such as binding activity to HER2, and further evaluated the selective accumulation of iv-administered B. longum in tumors and their anti-tumor activity. Based on our results, the potential clinical application of iDPS trastuzumab scFv in cancer therapy will be discussed.

Section snippets

Reagents and cell lines

AlexaFluor®488- or 594-conjugated anti-His-tag antibodies were purchased from MEDICAL & BIOLOGICAL LABORATORIES (Nagoya, Japan). SK-MEL-28, SK-BR-3, BT-474, and NCI-N87 cell lines were obtained from American Type Culture Collection (VA, USA). The cells were maintained in commercially recommended media under standard cell culture conditions of 37°C and 5% CO2.

Establishment of trastuzumab scFv-secreting Bifidobacterium strains

The nucleotide sequence of the trastuzumab scFv cDNA used in E. coli (Supplement) was optimized to the codon of Bifidobacteria via the

Establishment of Bifidobacterium H1 and H2 strains secreting trastuzumab scFv

Shuttle vectors for expression/secretion of the trastuzumab scFv were constructed for B. longum (Fig. 1a) by modifying the above-described E. coli plasmid (Supplement). The characterization of the recombinant trastuzumab scFv produced by E. coli are shown in the Supplement Figures. The clonal representatives of the genetically engineered B. longum transformed with plasmids were named H1 and H2, respectively.

Secreted trastuzumab scFv was purified from the culture supernatants of H1 and H2 by the

Acknowledgments

This study was supported by a Grant-in-Aid for Scientific Research (KAKENHI) for Scientific Research C (15K08022), grants from the Kobayashi Foundation for Cancer Research and Anaeropharma Science, Inc. and a kind donation to iDPS research from Dr. Tsuneo Baba, a former instructor of S. Taniguchi.

References (17)

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