Elsevier

Biomaterials

Volume 178, September 2018, Pages 158-169
Biomaterials

Elimination of melanoma by sortase A-generated TCR-like antibody-drug conjugates (TL-ADCs) targeting intracellular melanoma antigen MART-1

https://doi.org/10.1016/j.biomaterials.2018.06.017Get rights and content

Abstract

Most tumor-associated proteins are located inside tumor cells and thus are not accessible to current marketed therapeutic monoclonal antibodies or their cytotoxic conjugates. Human leukocyte antigen (HLA) class I can present peptides derived from intracellular tumor-associated proteins and somatically mutated proteins on the cell's surface, forming an HLA/peptide complex as tumor-specific antigens for T cell receptor (TCR) recognition. Therefore, HLA-mediated presentation of intracellular tumor antigen peptides provides a viable way to distinguish tumor cells from normal cells, which is important for broadening antigen selection, especially for antibody-drug conjugates (ADCs) regarding their highly cytotoxic payload. We applied sortase A-mediated conjugation to develop TCR-like ADCs (i.e., EA1 HL-vcMMAE) targeting intracellular MART-1 protein, a melanocyte-differentiating antigen specific for metastatic melanomas, via the cell surface HLA-A2/MART-126-35 peptide complex. Homogenous EA1 HL-vcMMAE (drug to antibody ratio of 4) efficiently eliminated melanoma cells in xenograft mouse models with no obvious toxicity at the therapeutic dosage. Trametinib, an MEK inhibitor serving as an HLA expression enhancing agent, augmented the TL-ADCs’ efficacy both in vitro and in vivo by upregulating MART-126-35 peptide presentation, thus providing a strategy for overcoming the limitation of antigen presentation level for TL-ADCs. Hence, our findings validate the strategy of using sortase A-generated TL-ADCs to target tumor-specific intracellular proteins, with or without agents present, to increase presenting TCR epitope peptides.

Introduction

Antibody drug conjugates (ADCs), which combine monoclonal antibody specificity with highly potent chemical, cytotoxic drugs, has recently emerged as a fast-growing and promising biopharmaceutical class. Upon binding with target antigens, ADCs deliver cytotoxic cargo into tumor cells via antigen-mediated internalization, followed by enzyme-catalyzed (e.g., cathepsin B) cytotoxic drug release in late lysosomes [1]. To date, all marketed ADCs target extracellular areas of membrane proteins, which can also exist on normal cells. For example, HER2, the target protein of T-DM1, was found on heart cells in addition to breast cells, leading to cardiotoxicity of HER2-related antibodies and ADC therapies [2]. Therefore, highly tumor-specific proteins could be ideal targets for ADC therapy. If targeted, intracellular proteins should assist ADCs in distinguishing tumor cells from normal cells, since most tumor-specific antigens are intracellular proteins (e.g., KRAS) [3].

Major histocompatibility complex (MHC) class I, also known as human leukocyte antigen (HLA) class I, can bind peptides derived from intracellular proteins in the endoplasmic reticulum and present peptides on the cell surface as antigens [4,5]. Thus, antigen presentation by MHC provides an attractive strategy for antibodies to target intracellular tumor-specific proteins, particularly for MHC I epitopes. For example, many MHC I epitopes, such as MART-1, GP100, WT1, the hepatitis B viral (HBV) envelope, p53 and PRAME, have been exploited as antitumor targets for monoclonal antibody-based immunotherapy and CAR-T therapy [[6], [7], [8], [9], [10], [11], [12]]. One T-cell receptor-mimic (TCRm) IgG antibody targeting the HLA-A2/WT1 peptide complex was shown to be promising in vivo antitumor activity [8,10,13]. Because of its internalization upon antibody binding, the MHC I/peptide complex could also be targeted by antibody-drug (toxic protein) conjugates, which require entry into tumor cells to exert activity [14].

Although considerable efforts have been made for TCR-like antibodies to target the MHC I/peptide complex (TCR epitope) [8,12,15], few studies have evaluated the in vivo activities of antibody-drug (small molecule) conjugates targeting TCR epitopes. It remains unclear whether a small epitope peptide could determine the in vivo specificity of TCR-like ADCs, whether low cell surface presentation of the MHC I/peptide complex limits in vivo efficacy of TL-ADCs and how this potential limitation can be overcome by different approaches. To solve these problems, the melanocyte differentiation antigens (e.g., MART-1), which are specific for the melanocyte lineage, were used as targets to develop TCR-like antibody-drug conjugates. MART-1 was only found in melanocytes and melanocyte-related tissues and was highly expressed in 99% of primary and metastatic melanomas [16]. HLA-A2, which presents MART-126-35, is one of the most abundant MHC I molecules in vivo, indicating that an HLA-A2/MART-1 peptide (EAAGIGILTV) complex may serve as an ideal target for melanomas [17,18]. Thus, we developed TL-ADCs targeting the HLA-A2/MART-126-35 peptide complex by sortase A-mediated site-specific conjugation to maintain antibody binding affinity. We also evaluated the possibility of using trametinib to improve MART-1 peptide presentation and of combining it with TL-ADCs during antitumor therapy (Fig. 1).

Section snippets

Cell lines and reagents

Metastatic melanoma cell line MEL 624.38, a clone of melanoma cell line MEL 624, was a gift from Professor Suping Ren (Department of Immunohematology, Beijing Institute of Transfusion Medicine, Beijing, China) [19]. W6/32, SK-MEL 28, and MCF-7 cells were cultured in DMEM, MEL 624.38 and K562 were cultured in RPMI-1640 and T2 was cultured in IMDM. All cell culture media were supplemented with 10% FBS (Gibco). Triple glycine-modified toxins Gly3-val-cit-PAB-MMAE and MAL-val-cit-PAB-MMAE were

Generation and characterization of TL-ADCs (EA1 HL-vcMMAE)

EA1 antibody and its C-terminally tagged (EA1 HL) antibody were successfully expressed by CHO-K1 cells, and W6/32 mouse antibody was secreted by W6/32 hybridoma. These antibodies were purified by protein A antibody affinity chromatography.

Our previous study demonstrated that GGG-vcMMAE cannot be conjugated to an LPETG-tagged antibody light chain [21]; thus, we added the flexible linker, GGGGS, between the light chain C-terminus and the LPETG tag. After sortase A-mediated conjugation, TL-ADCs

Discussion

Peptide-loaded MHCs (pMHCs) have become promising tumor-specific targets for antibody-based immunotherapy (e.g., CAR-T, IgG) since MHCs can present peptide fragments or a single mutated peptide from intracellular tumor-associated proteins, which remarkably broadens antigen selection and determines the pMHCs' high specificity as immunotherapy targets. For example, the MHC in complex with the oncogenic protein WT1 peptide and an EGFR peptide harboring an L858R mutation were targeted by antibodies

Author Contributions Statement

J.L. and L.Q.P. designed all experiments and prepared all the TL-ADCs samples. J.L. and Y.W. did the in vivo animal experiments. J.L. and S.S.W. did the in vitro evaluation of TL-ADCs. D.D. and Z.Y.S. did the MS analysis of the TL-ADCs and presented peptides. J.Z., Y.C.X. and Y.Z. made the antibody expression constructs and established stable CHO cell lines. Z.Z. and L.Q.P. prepared the tissue samples and did the serological analysis. J.L., L.Q.P. and S.Q.C. discussed the results and wrote the

Competing financial interests

The authors declare no competing financial interests.

Acknowledgements

We thank Prof. Suping Ren for the gift of MEL 624.38 cell line. Funding: This work was supported by the State Key Program of National Natural Science of China (Grant No. 81430081), the National Key Research and Development Program of China ‘Precision Medicine Research’ (Grant No. 2017YFC0908602), the National Natural Science Foundation of China (Grant No. 81502971), and the Fundamental Research Funds for the Central Universities (Project No. 2016QNA7024).

References (40)

  • T. Dao et al.

    Targeting the intracellular WT1 oncogene product with a therapeutic human antibody

    Sci. Transl. Med.

    (2013)
  • C. Ji et al.

    Targeted delivery of interferon-α to hepatitis B virus-infected cells using T-cell receptor-like antibodies

    Hepatology

    (2012)
  • T. Dao et al.

    Therapeutic bispecific T-cell engager antibody targeting the intracellular oncoprotein WT1

    Nat. Biotechnol.

    (2015)
  • D. Li et al.

    Development of a t-cell receptor mimic antibody against wild-type p53 for cancer immunotherapy

    Cancer Res.

    (2017)
  • A.Y. Chang et al.

    A therapeutic T cell receptor mimic antibody targets tumor-associated PRAME peptide/HLA-I antigens

    J. Clin. Invest.

    (2017)
  • S. Rafiq et al.

    Optimized T-cell receptor-mimic chimeric antigen receptor T cells directed toward the intracellular Wilms Tumor 1 antigen

    Leukemia

    (2016)
  • C. Peters et al.

    Antibody–drug conjugates as novel anti-cancer chemotherapeutics

    Biosci. Rep.

    (2015)
  • H. Liu et al.

    Targeting alpha-fetoprotein (AFP)–MHC complex with CAR T-cell therapy for liver cancer

    Clin. Cancer Res.

    (2017)
  • C. Barrow et al.

    Tumor antigen expression in melanoma varies according to antigen and stage

    Clin. Cancer Res.

    (2006)
  • E. Jäger et al.

    Peptide-specific CD8+ T-cell evolution in vivo: response to peptide vaccination with Melan-A/MART-1

    Int. J. Cancer

    (2002)
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