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Antibody-bound Viral Antigens

Posted in Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Immunotherapy, tagged anti-viiral cytotoxic T-cell (CTL) response, cytotoxic T-lymphocytes, Immunotherapy, targeting cancer on May 8, 2016| Leave a Comment »

Antibody-bound Viral Antigens

Reporter: Larry H. Bernstein, MD, FCAP

The following presentations are closely related to other similar pieces, except that this uniquely envisions the release and presentation of antibody-bound VIRAL antigens in targeting cancer cells. The approach compares the lifelong immunity conferred by immunity to herpesvirus to immunosuppression of cancer targets by high affinity cytotoxic T-lymphoctes targeting the cancer in vivo.

Abstract 2893: Engineered release and presentation of antibody-bound viral antigens: A highly specific and novel immunotherapeutic approach to target cancer in vivo

David G. Millar, Laura Morton, Manuela Carvalho Gaspar, …, Guy E. Pratt, and Mark Cobbold

Cancer Res Oct 1, 201474; 2893 http://dx. doi.org:/10.1158/1538-7445.AM2014-2893

Harnessing the power of adaptive immunity to combat cancer has been a long-term goal of translational immunotherapy. Tumor-specific immunity, where present, is typically at low frequency and affinity with compromised effector function. By contrast, immunity against persistent herpesviruses in man is characterised by high affinity cytotoxic T-lymphocytes (CTL) at high frequency with potent effector function. Furthermore, the immunosubversive mechanisms employed by herpesviruses show striking parallels to tumors, yet the associated anti-viral immunity limits these to life-long asymptomatic infections.

We reasoned that the delivery of immunodominant viral peptide epitopes to the tumor surface might facilitate passive-loading of peptides into empty MHC class-I molecules, effectively mimicking viral infection, rendering tumors susceptible to lysis by anti-viral immunity.

To address this we developed a new class of targeting antibodies: APEC (Antibody Peptide Epitope Complexes) that are able to deliver an antigenic payload at the cell surface through proteolytic release of covalently-coupled peptide antigens. As a proof-of-concept we used clinically-validated antibodies cetuximab (anti-EGFR) and rituximab (anti-CD20) to develop APECs that are able to target human tumors.

We screened 15 HLA-A*0201+ EGFR-expressing NCI-60 cell lines, CD20+ lymphoma cell lines, 20 primary CD20+ CLL tumor samples and four healthy B-cells against a library of 190 cetuximab-APECs (cAPEC) or rituximab (rAPEC) incorporating the immunodominant cytomegalovirus (CMV) pp65495-503 epitope and candidate protease cleavage sites following co-incubation with CMV-specific CTL (CMV-CTL). The most effective cAPEC and rAPEC were those incorporating MMP2, MMP9, Cathepsin B and Cathepsin D protease recognition domains. Very few (2/190) rAPEC were able to redirect CMV-CTL against healthy cells. Heterogeneity was observed for primary CLL tumors but a limited number of rAPEC were effective in all cases (5/190).

Mechanistic studies demonstrated that: (i) peptide loading occurred at the cell surface, (ii) required the expression of target antigens at the cell surface and (iii) T-cell recognition could be inhibited by unconjugated antibody (92%) or by incubation with protease inhibitors (83%).

T-cell specificity was examined using rAPEC treated tumor targets co-incubated with various HLA-matched effector T-cell populations. No activation of CD4+ was observed including CD4+CD25hi regulatory T-cell populations. Incubation with CD8+ T-cells revealed that only pp65495-503-specific CD8+ T-cells engaged with APEC-treated tumor cells. Lastly, xenograft studies using EGFR+ and CD20+ tumor cell lines demonstrated efficacy of both cAPEC and rAPEC to eliminate tumors in vivo by redirecting anti-viral CTL.

These data indicate that APECs represent a powerful new approach to combat cancer.

Citation Format: David G. Millar, Laura Morton, Manuela Carvalho Gaspar, Punam Mistry, Hugo De La Peña, Ricky Joseph, Sarah Penny, Oliver C. Goodyear, Margaret Goodall, Guy E. Pratt, Mark Cobbold. Engineered release and presentation of antibody-bound viral antigens: A highly specific and novel immunotherapeutic approach to target cancer in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2893. doi:10.1158/1538-7445.AM2014-2893

Re-directing the anti-viral T cell response towards cancer

David Millar¹, Hugo de la Pena¹, Laura Morton¹, Manuela Carvalho-Gaspar¹, Punam Mistry¹, Oliver Goodyear¹, Mark Cobbold¹

¹University of Birmingham, Birmingham, UK http://conference.ncri.org.uk/abstracts/2013/abstracts/B63.htm

Background Therapeutic antibodies are widely used but very rarely curative in the setting of either haematological or solid-organ malignancies. In order to address this lack of efficacy, we asked the question: how can clinically relevant antibodies be empowered to enhance their clinical efficacy? We reasoned that by engaging a potent T cell response, such as anti-viral T cells, we could re-direct this effective immune response to target malignant cells.

Method Immunogenic viral epitopes were conjugated to clinically relevant antibodies. These conjugated antibodies could then be used as a delivery vehicle and we hypothesised that we could release viral antigens only in close proximity to the surface of the tumour cell. Using this novel therapeutic entity – termed an antibody-peptide epitope conjugate (APEC) – T cells could recognise and kill the labelled tumour cells. We developed APECs based on the anti-CD20 monoclonal antibody Rituximab for B cell neoplasms and the EGFR-specific antibody Cetuximab to treat patients with solid tumours.

Results Target cells labelled with an APEC conjugated with immunogenic peptide results in strong T cell recognition of the target cell and cytotoxicity. Importantly, our novel therapeutic agent also has the ability to differentially target tumour cells whilst sparing healthy cells of the same lineage.

Early ex vivo work using Chronic Lymphocytic Leukaemia (CLL) patient cells has shown the ability of Rituximab APEC to re-direct anti-viral T cells towards primary CLL cells. Furthermore, early in vivo murine models using the Cetuximab APEC have demonstrated efficacy of the APEC with tumour clearance seen up to 32 days post-APEC treatment.

Conclusion In summary, this novel approach promises to provide existing therapeutic antibodies with a new mechanism of action that allows them to engage with highly potent T cells in an antigen-specific manner. Further in vivo work is on-going with the aim of pushing this work toward the clinic.

APEC antibodies

cyclic di-nucleotides

CTLA4 and PD1 antibodies

STING

STING pathway activators

NK cell immunotherapies

T cell activation

CRC Surface Phosphopeptides

tumor — Surface MHC

Tumor-specific neoantigens

CD8+ T-cells

O-GlcNAcylation