Antibody-mediated delivery of viral epitopes to tumors harnesses CMV-specific T cells for cancer therapy

Abstract

Several cancer immunotherapy approaches, such as immune checkpoint blockade and adoptive T-cell therapy, boost T-cell activity against the tumor, but these strategies are not effective in the absence of T cells specific for displayed tumor antigens. Here we outline an immunotherapy in which endogenous T cells specific for a noncancer antigen are retargeted to attack tumors. The approach relies on the use of antibody–peptide epitope conjugates (APECs) to deliver suitable antigens to the tumor surface for presention by HLA-I. To retarget cytomegalovirus (CMV)-specific CD8+ T cells against tumors, we used APECs containing CMV-derived epitopes conjugated to tumor-targeting antibodies via metalloprotease-sensitive linkers. These APECs redirect pre-existing CMV immunity against tumor cells in vitro and in mouse cancer models. In vitro, APECs activated specifically CMV-reactive effector T cells whereas a bispecific T-cell engager activated both effector and regulatory T cells. Our approach may provide an effective alternative in cancers that are not amenable to checkpoint inhibitors or other immunotherapies.

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Fig. 1: CMV-CTLs are found at high frequencies in patients with cancer and recognize antigen-modified cancer cells.
Fig. 2: Engineering APECs to deliver peptides to the surface of tumor cells.
Fig. 3: APEC modulation of antigenicity in vivo.

Data availability

Data presented in this study are available in the article, Supplementary Information or from the corresponding author on reasonable request.

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Acknowledgements

D.G.D.’s work was supported through NIH grant no. R41-CA213678, the Proton Beam/Federal Share Program and MGH ECOR. We thank P. Huang, S. Roberge and T. Hbatmu for the maintenance of gnotobiotic animal colonies and experimental assistance. This work was supported by NCI program project grant nos. P01-CA080124 and R01-CA208205 (to R.K.J. and D.F.), NCI grant nos. R35-CA197743 and U01-CA 224348 (to R.K.J.) and also in part by the Ludwig Center at Harvard. The NIH Tetramer Facility is supported by contract HHSN272201300006C from the National Institute of Allergy and Infectious Diseases, a component of the National Institutes of Health in the Department of Health and Human Services.

Author information

D.G.M., R.R.R., K.K., N.G., J.C., S.Z., T.N., W.W.H., S.A., K.J., I.C., F.S., J.M.H., K.S., L.T.M., S.S., L.W., R.J. and E.M. performed the experiments and analyzed the data. A.K. synthesized peptides and provided intellectual input regarding peptide chemistry. A.B., L.W.E., R.B.C., A.N.H. and S.I.P. provided human samples and intellectual input. R.K.J., D.F. and D.G.D. provided support and assisted in the design of in vivo experiments and intellectual input. D.G.M. and M.C. designed the experiments and wrote the manuscript.

Correspondence to Mark Cobbold.

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Competing interests

R.K.J. received an honorarium from Amgen and consultant fees from Ophthotech, SPARC, SynDevRx and XTuit. R.K.J. owns equity in Enlight, SPARC and SynDevRx, and serves on the Board of Directors of XTuit and Boards of Trustees of Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund and Tekla World Healthcare Fund. D.G.D. received consultant fees from Bayer and BMS and has research grants from Bayer, Merrimack, Leap, Exelixis and BMS. M.C. and D.G.M. are named inventors on patent application no. WO 2012/123755, which is licensed to Revitope Oncology. D.G.M. and M.C. own equity in Revitope Oncology. M.C. holds equity in Gritstone Oncology. M.C. received consultant fees from Merck Laboratories. M.C. is now an employee of AstraZeneca.

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Integrated supplementary information

Supplementary Fig. 1 Predicted affinities of HLA-bound peptides.

In silico prediction of peptides eluted from HLA molecules.

Supplementary Fig. 2 Presence of empty HLA molecules at the surface of tumor cell lines that can be loaded with exogenous CMV peptide with tumor cell cytotoxicity.

(a) Ten tumor cell lines labelled with antibodies specific for peptide-loaded HLA (red line) and empty HLA molecules (blue line) compared with control stained cells (black line). Percentages shown are percentage cells positive for empty HLA molecules (HC10) staining. All cell lines tested were 100% positive for peptide-loaded HLA (W6/32). Staining was repeated (n=3) in selected cell lines. (b) Five HLA-A2+ cell lines lysed by peptide-specific CMV-CTL and no lysis of HLA-A2- cell line (Caki-2) (n=3 independent samples). Data represented as mean and error bars represent standard error of the mean.

Supplementary Fig. 3 Assessment of peptide antibody ratio conjugation.

The number of peptides conjugated to each antibody was assessed by high pressure liquid chromatography (HPLC). Data from single experiment.

Supplementary Fig. 4 Screening 96 APECs in multiple tumor cell lines.

Eight tumor cell lines are labelled with cAPECs and cytokine release by peptide-specific T cells assayed.

Supplementary Fig. 5 Peptide specificity of ex vivo cultured T cell lines.

Flow cytometric analysis of ex vivo cultured T cell lines used for in vitro assays using HLA-peptide tetramers. Cell lines were cultured at various times and each cell line was analyzed for tetramer positive T cells once.

Supplementary Fig. 6 Analysis of empty MHC molecules using HC10 antibody staining.

Colo205 Tumor cells were assayed for the presence and decrease in the amount of empty MHC molecules at the cell surface by HC10 staining. Cells were acid-stripped to remove peptides from surface MHC molecules, lightly fixed or left untreated and incubated with either peptide or APEC before HC10 staining.

Supplementary Fig. 7 APEC binding to tumor cell surface essential for T cell recognition.

(a) Flow cytometric analysis of EGF-R and CD20 expression on MDA-MB-231 and JY tumor cell lines. Staining was repeated n=3. (b) Surface binding of APEC is required for antigenic reprogramming and can be inhibited by the pre-treatment with unconjugated antibody (n=3 independent samples). Data represented as mean and error bars represent standard error of the mean. (c) CD20+ tumor cells labelled with cAPEC or rAPEC (350nM) and demonstrating T cell activation only when bound by the anti-CD20 rAPEC. Peptide loaded (1μM) target cells were used to determine efficacy of T cells (n=3 independent samples). Data represented as mean and error bars represent standard error of the mean.

Supplementary Fig. 8 Flow cytometric analysis of T cell proliferation and regulatory T cells.

(a) Flow cytometric staining to allow analysis of T cell proliferation after treatment with free peptide (black), phytohaemagglutinin (PHA, red) or untreated T cells (blue) (data from single experiment). (b) Flow cytometric staining to allow analysis of CD4+ CD45RO FoxP3+ regulatory T cells (data from single experiment).

Supplementary Fig. 9 Expanding APEC to include peptides that bind to other HLA alleles.

APECs conjugated with CMV epitopes covering multiple HLA alleles are able to activate and trigger cytokine release of peptide-specific CMV-CTL(n=3 independent samples). Data represented as mean and error bars represent standard error of the mean.

Supplementary Fig. 10 Construction of APECs containing multiple T-cell epitopes (polytopes).

We tested whether it was possible to generate APECs that contained multiple T-cell epitope peptide payloads. (a) The initial concatemer design utilized linear peptides with tandem T-cell epitopes (NLV and RPH) juxtaposed by proteolytic cleavage sequences (-x-). Using T-cells against each epitope, both epitopes elicited T-cell responses (n=3 independent samples). Data represented as mean and error bars represent standard error of the mean. A second method (b) involved the mixed conjugation of peptides to antibodies. In this case the peptide payloads were the same as original APEC design, but two different peptides (with 7-different ratios) were conjugated onto a single APEC. These mixed APECs were able to activate the two different T-cell populations (NLV or RPH) but with varying potency. In a separate experiment three different ratios were tested against two different epitopes (NLV and ELK) which gave concordant results (n=3 independent samples). Data represented as mean and error bars represent standard error of the mean.. Lastly, we created a single APEC species that was conjugated to a branched peptide that contained multiple different cleavable peptides (c). These branched peptides were able to activate T-cell populations (n=3 independent samples). Data represented as mean and error bars represent standard error of the mean.

Supplementary Fig. 11 EBV-specific T cells can be re-directed to target tumor cells using APEC and CMV-CTL activation using different proteases to cleave APECs.

(a) Tumor cells treated with APEC conjugated with EBV-derived epitopes are recognised by EBV-specific T cells (n=3 independent samples). Data represented as mean and error bars represent standard error of the mean. Significance was determined by unpaired two-tailed t-test. (b) Multiple tumor types can be recognized by peptide-specific T cells after treatment with cAPEC containing ADAM28, MMP2, MMP9 or MMP14 cleavable peptides (n=3 independent samples). Data represented as mean and error bars represent standard error of the mean.

Supplementary Fig. 12 Tumor penetration of APEC in orthotopic breast cancer model.

Tumor-bearing NOD/SCID mice (n=5) were injected with either PBS or MMP14-cAPEC and tumors resected at timepoints up to 24h. Tumors were taken for immunofluorescence to stain for the presence of APEC.

Supplementary Fig. 13 In vivo T cell activation by APEC in orthotopic breast cancer model and the presence of peptide-specific T cells within tumors.

Breast cancer tumor-bearing mice were injected intratumorally with freshly isolated peptide-specific CMV-CTL and 24h post-injection, tumors were resected and T cells isolated. Flow cytometric analysis of intratumoral CD3+ T cells was undertaken for the presence of T cell activation markers (a) CD38 (n=2 independent samples) and (b) CD25 (n=2 independent samples). Data represented as mean and error bars represent standard error of the mean. (c) Gating strategy to select human T cells from the excised tumor sample. Firstly, CD45+ was used to gate human lymphocytes before live cells were gated using forward and side scatter. T cells were then gated using CD3. (d) Peptide-specific T cells were labelled using HLA-peptide tetramer complexes conjugated to phycoerythrin (PE) and the cells were co-labeled with CD8 APC (data from single experiment).

Supplementary Fig. 14 Generation of the anti-murine EGFR D1D4J APEC which has no effect on survival compared to control IgG as single agent therapy.

(a) Flow cytometry analysis of the murine colorectal cell line SL-4 demonstrates cells positive for both EGF receptor (D1D4J antibody) and the MHC class I molecule H2-kb (MHC I allele which presents the SIINFEKL peptide) (data from single experiment). (b) T cell function assay demonstrating the production and detection of lacZ by the activated B3Z T cell hybridoma after recognizing SL-4 cells exogenously labelled with SIINFEKL peptide (n=3 independent samples). Data represented as mean and error bars represent standard error of the mean. (c) SL-4 cell line labelled with a library of 35 D1D4J-APECs and lacZ production by B3Z T cells assayed. (d) Immunocompetent mice used in the SL4 colorectal cancer model treated with single agent D1D4J-APEC therapy demonstrated no difference in survival compared with mice treated with control IgG (n=10). Significance was determined by Mantel Cox test (two-sided).

Supplementary Fig. 15 APEC mechanism of action.

Proposed mechanism of action for APEC with antibody attachment to target antigen (1), release of virus-derived epitope at the cell surface (2). Released peptide loads into empty MHC class I molecule at the cell surface (3) and T cell lysis by the re-directed anti-viral immune response.

Supplementary information

Supplementary Information

Supplementary Figs. 1–15 and Tables 1–4.

Reporting Summary

Supplementary Video 1

Untreated breast cancer cells do not instigate CMV-CTL cytotoxicity. Real-time imaging of MDA-MB-231 cell line containing a red nuclear dye to determine cytotoxicity by NLV-specific CMV-CTLs over 40 h. Untreated MDA-MB-231 cells were not targeted by CMV-CTLs during co-culture.

Supplementary Video 2

Breast cancer cells exogenously labeled with CMV peptide are killed by peptide-specific CMV-CTL. Breast cancer cells (MDA-MB-231) were exogenously labeled with CMV peptide (NLVPMVATV) and co-incubated with peptide-specific CMV-CTLs. During co-culture, CMV-CTLs recognized the peptide displayed by the tumor cells and efficiently killed the breast cancer cells.

Supplementary Video 3

Cetuximab-labeled breast cancer cells are not recognized by CMV-CTLs. Breast cancer cells (MDA-MB-231) were labeled with the anti-EGF receptor antibody cetuximab and incubated with CMV-CTLs. There was no recognition of tumor cells by CMV-CTLs.

Supplementary Video 4

Negative control cetuximab-APEC-labeled breast cancer cells are not recognized by CMV-CTLs. A negative control cetuximab APEC, designed to prevent release of the CMV epitope, was used to label the breast cancer cell line MDA-MB-231. After co-incubation of labeled tumor cells with CMV-CTLs, tumor cells were not targeted for killing by T cells.

Supplementary Video 5

Breast cancer cells labeled with an MMP2-cleavable cetuximab-APECs are killed by peptide-specific CMV-CTLs. An MMP2-cleavable cetuximab APEC was used to label the breast cancer cell line MDA-MB-231. After co-incubation of labeled tumor cells with CMV-CTLs, tumor cell lysis occurred as T cells recognized the CMV epitope being presented at the cell surface by tumor cells.

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Millar, D.G., Ramjiawan, R.R., Kawaguchi, K. et al. Antibody-mediated delivery of viral epitopes to tumors harnesses CMV-specific T cells for cancer therapy. Nat Biotechnol (2020). https://doi.org/10.1038/s41587-019-0404-8

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