Survivin Monoclonal Antibodies Detect Survivin Cell Surface Expression and Inhibit Tumor Growth In Vivo

Abstract

Purpose: Survivin is an inhibitor of apoptosis protein (IAP) that is highly expressed in many cancers and represents an attractive molecule for targeted cancer therapy. Although primarily regarded as an intracellular protein with diverse actions, survivin has also been identified in association with circulating tumor exosomes.Experimental Design: We have reported that active, specific vaccination with a long peptide survivin immunogen leads to the development of survivin-specific CD8-mediated tumor cell lysis and prolongation of survival in tumor-bearing mice. In addition to cellular antitumor responses, circulating anti-survivin antibodies are detected in the serum of mice and human glioblastoma patients following vaccination with the survivin immunogen.Results: Here we demonstrate that survivin is present on the outer cell membrane of a wide variety of cancer cell types, including both murine and human glioma cells. In addition, antibodies to survivin that are derived from the immunogen display antitumor activity against murine GL261 gliomas in both flank and intracranial tumor models and against B16 melanoma as well.Conclusions: In addition to immunogen-induced, CD8-mediated tumor cell lysis, antibodies to the survivin immunogen have antitumor activity in vivo Cell-surface survivin could provide a specific target for antibody-mediated tumor immunotherapeutic approaches. Clin Cancer Res; 24(11); 2642-52. ©2018 AACR.

Conflict of interest statement

R.A. Fenstermaker and M.J. Ciesielski are co-inventors listed on patents regarding the survivin vaccine and are co-founders of MimiVax, LLC, which has licensed such patents from Roswell Park Cancer Institute. The other authors report no conflicts of interest.

©2018 American Association for Cancer Research.

Figures

Figure 1

(A) Antibody panel raised following vaccination of mice with survivin peptide (SVN53-67/M57) present in SurVaxM. Relative binding of different hybridoma clones to immobilized survivin peptide (ELISA) is displayed with indicated amount of supernatant volume (MFI = mean fluorescence index). (B) Western blot of whole cell lysates from human embryonic kidney 293T cells with indicated antibodies demonstrating the dominant 16.5 kD survivin species. (C) Western blot of purified recombinant GST or GST-survivin proteins detected by the indicated antibodies (GST, glutathione-S-transferase). (D) Slot blots detecting filter-immobilized KLH carrier protein and scrambled, mutant and wild type survivin peptides with indicated survivin antibodies. (E) Competition blocking of monoclonal antibodies pre-incubated with peptides (left) and then used to detect filter-immobilized, wild type SVN53-67 peptide. (F) AlphaLISA assay performed to determine relative affinity of purified 2C2 and 30H3 antibodies forSVN53-67 peptide.

Figure 2

(A) Flow cytometric detection of cell-surface survivin in unfixed (i.e. non-permeabilized) human U87 and A1207 glioma cells. Purified anti-survivin 2C2 mAb was used to stain cells with or without pre-incubation with SVN53-67 peptide, or a scrambled peptide to assess specificity through blocking. (B) Loss of cell surface survivin binding by 2C2 in non-permeabilized A1207 cells treated with proteinase K or trypsin (left panel) and detection of intracellular survivin in protease-stripped and permeabilized cells (right panel). (C) Flow cytometric measurement of cell-surface survivin (red) staining compared to IgG control (black) in non-permeabilized cancer cells lines, including: MCF7 (human breast carcinoma), HeLa (human cervical carcinoma), Jurkat (human T cell leukemia), B16f1 (mouse melanoma), HT-29 (human colon carcinoma), Raji (human Burkitt’s lymphoma), PC3 (human prostate carcinoma), A2780 (human ovarian carcinoma) cells and GL261 (murine glioma cells). (D) Immunofluorescence microscopic detection of total (permeabilized) and cell-surface (non-permeabilized) survivin in human (U87) and murine (GL261) glioma cells with 2C2, 30H3 and commercially available 60.11 anti-survivin mAbs.

Figure 3

(A) Photograph of gradient separation in U87 cells with arrow indicating low density lipid raft band corresponding to survivin-containing fraction #4. Western blot detection of lipid raft-associated proteins and survivin in subcellular fractions derived from U87 (B) and A1207 (C) glioblastoma cells. (D) Transiently expressed recombinant c-Myc-tagged human survivin in association with lipid raft protein fractions in HEK293T cells detected with an anti-c-Myc-tag antibody (9E10). (E) Imagestream imaging flow cytometric visualization of co-localized bound cholera toxin B (CT-B) and survivin in lipid rafts.

Figure 4

Survivin immunohistochemistry with 2C2 mAb in human cancers and normal tissues with indicated staining patterns, including: (A) glioblastoma (nuclear), (B) normal brain (negative), (C) invasive ductal breast carcinoma (nuclear), (D) lobular breast carcinoma (nuclear), (E) normal breast tissue (negative), (F) typical carcinoid tumor (apical), (G) clear cell renal cell carcinoma (membranous and cytoplasmic), (H) normal kidney (nuclear), (I) hepatocellular carcinoma (membranous and cytoplasmic), (J) normal liver (negative), (K) medullary thyroid carcinoma (cytoplasmic), and (L) pheochromocytoma (cytoplasmic). Images are shown at 400x.

Figure 5

Subcutaneous (A–D and F) and intracranial (E) tumor models in C57BL/6 (A, B, E and F) and nude (C and D) mice with GL261 glioma (A, C, E and F), and B16f1 melanoma (B and D) cells. Mice were given the indicated treatments once every 5 days beginning 3 days following tumor cell implantation (A, C n = 8–10 per group; B, D n = 5 per group). Subcutaneous tumors (A–D and F) were measured and tumor volumes were calculated as described. (E) Mice with GL261 intracranial tumors were treated with PBS (control), nonspecific IgG, mAb 2C2 at a dose of 10 or 20 μg, or SurVaxM vaccine (n = 11–17 per group) and survival was determined by the Kaplan-Meier method. Median survival time and range; control=22 days (range 17–31 days), nonspecific IgG = 19 days (range 17–27 days), mAb 2C2 = 28 days (range 21–58+ days), SurVaxM = 45 days (range 21–120+ days). (F) Mice with GL261 flank tumors were treated with conjugated survivin vaccine (SVN53-67/M57-KLH; SurVaxM), pooled antiserum derived from non-tumor-bearing mice that had been vaccinated with SurVaxM, or nonspecific IgG control antibody (n = 4 per group). Statistical significance in A–D and F was assessed using the Wilcoxon matched-pairs test; *p<0.05; **p<0.0014. Statistical significance in E was assessed using the Logrank Mantel-Cox test **p=0.0041 and ***p<0.0001.

Figure 6

Monoclonal antibody 2C2 produces FcγRIV activation in luciferase reporter assays (A) Jurkat lymphoma cells as both effector cells and reporter targets. Jurkat cells have cell-surface survivin expression (Figure 2C) leading to antibody (2C2) dose-dependent activation of luciferase reporter via mFcγRIV. Dilutions of murine anti-survivin mAb (2C2) or anti-IgG were added to effector cells and luminescence was measured. (B) Blocking of Fc-mediated activation of luciferase in Jurkat effector cells by SVN53-67 peptide binding to 2C2 compared to scrambled peptide. (C) U87 human glioma cells engage mAb 2C2 with activation of FcγRIV in Jurkat effector cells (RLU = relative luminescence units). (D) Growth curve of Gl261 cells, in vitro, exposed to 10μg of indicated antibody over 11 days.