Published OnlineFirst August 4, 2017; DOI: 10.1158/2326-6066.CIR-16-0236 Research Article Cancer Immunology Research Using Antigen-Specific B Cells to Combine Antibody and T Cell–Based Cancer Immunotherapy Kerstin Wennhold1, Martin Thelen1, Hans Anton Schloßer€ 1,2, Natalie Haustein1, Sabrina Reuter1, Maria Garcia-Marquez1, Axel Lechner1,3, Sebastian Kobold4, Felicitas Rataj4, Olaf Utermohlen€ 5, Geothy Chakupurakal1, Sebastian Theurich1,6, Michael Hallek7, Hinrich Abken7,8, Alexander Shimabukuro-Vornhagen1, and Michael von Bergwelt-Baildon1 Abstract Cancer immunotherapy by therapeutic activation of T cells stimulation with IL21, IL4, anti-CD40, and the specificanti- has demonstrated clinical potential. Approaches include gen. Combined treatment of tumor-bearing mice with antigen- checkpoint inhibitors and chimeric antigen receptor T cells. specific CD40-activated B cells and antigen-specificplasma Here,wereportthedevelopmentofanalternativestrategyfor cells induced a therapeutic antitumor immune response result- cellular immunotherapy that combines induction of a tumor- ing in remission of established tumors. Human CEA or NY- directed T-cell response and antibody secretion without the ESO-1–specific B cells were detected in tumor-draining lymph need for genetic engineering. CD40 ligand stimulation of nodes and were able to induce antigen-specific T-cell responses murine tumor antigen-specific B cells, isolated by antigen- in vitro, indicating that this approach could be translated into biotin tetramers, resulted in the development of an antigen- clinical applications. Our results describe a technique for the presenting phenotype and the induction of a tumor antigen- exploitation of B-cell effector functions and provide the ratio- specific T-cell response. Differentiation of antigen-specific nale for their use in combinatorial cancer immunotherapy. B cells into antibody-secreting plasma cells was achieved by Cancer Immunol Res; 5(9); 730–43. Ó2017 AACR. Introduction majority of developmental efforts focus on the induction of T cell–mediated antitumor immunity. Antibodies against immune Cancer immunotherapy has demonstrated clinical success in checkpoint molecules such as PD-1 or CTLA-4 have demonstrated the treatment of hematologic and solid malignancies (1). The therapeutic efficacy (2). Furthermore, direct genetic engineering of T cells such as introduction of chimeric antigen receptors, which allows for MHC-unrestricted tumor-directed T-cell activation, has 1Cologne Interventional Immunology (CII), Department I of Internal Medicine, also shown clinical activity in defined indications (3). Here, we 2 University Hospital of Cologne, Cologne, Germany. Department of General, report an alternative approach, devoid of challenges resulting Visceral and Cancer Surgery, University Hospital of Cologne, Cologne, Germany. from genetic engineering and able to induce specific T-cell activity 3Department of Otorhinolaryngology, Head and Neck Surgery, University Hos- pital Cologne, Germany. 4Center of Integrated Protein Science Munich (CIPSM) by vaccination. and Division of Clinical Pharmacology, Medical Clinic and Policlinic IV, University CD40 activation of normal B cells improves antigen presen- Hospital Munich, Munich, Germany, Member of the German Center for Lung tation (4, 5), leading to induction of a specificna€ve or memory þ þ Research. 5Department for Medical Microbiology, Immunology and Hygiene, CD4 (6–8) and CD8 (4, 5) T-cell response. In contrast to DC 6 University Hospital of Cologne, Cologne, Germany. Laboratory for Cancer- vaccines, CD40-activated B cells (CD40B cells) can be greatly Immuno-Metabolism, Department I of Internal Medicine, University Hospital of expanded from small amounts of peripheral blood (4, 5, 9). Cologne, Germany. 7Department I of Internal Medicine, University Hospital of Cologne, Cologne, Germany. 8Center for Molecular Medicine Cologne, University These CD40B cells are home to tumor-draining lymph nodes of Cologne, Cologne, Germany. (TDLN; ref. 10) and induce antitumor immunity in mice (11, Note: Supplementary data for this article are available at Cancer Immunology 12). However, so far only B cells of unknown and polyclonal fi Research Online (http://cancerimmunolres.aacrjournals.org/). speci city have been used for CD40 activation and tumor targeting, disregarding the two main advantages of B cells: the M. Thelen and H.A. Schloßer€ contributed equally to this article. high affinity of the specific B-cell receptor (BCR) for its antigen A. Shimabukuro-Vornhagen and M. von Bergwelt-Baildon contributed equally to and the ability to produce large amounts of specific antibodies. this article. Development of CD40 activators that can be produced in GMP Corresponding author: Kerstin Wennhold, University Hospital of Cologne, grade(13,14)hasovercomeanobstaclefortheuseofCD40B € 50937 Koln, Germany. Phone: 0049-221-4784489; Fax: 0049-221-4785912; cells in a clinical trial. We have thus hypothesized that a CD40B E-mail: [email protected] cellular vaccine based on tumor antigens could represent a doi: 10.1158/2326-6066.CIR-16-0236 promising approach for cancer therapy. Here, we report that Ó2017 American Association for Cancer Research. murine and human B cells with a defined specificity can be 730 Cancer Immunol Res; 5(9) September 2017 Downloaded from cancerimmunolres.aacrjournals.org on September 25, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst August 4, 2017; DOI: 10.1158/2326-6066.CIR-16-0236 Antigen-Specific B Cells for Cancer Immunotherapy enriched and used for bifunctional cancer immunotherapy that micin. Recombinant murine IL4 (50 U/mL; Immunotools) was combines B cell–mediated T-cell activation and antibody-medi- freshly added. ated antitumor effects. Cell culture with soluble CD40 ligand was performed as described previously (14). Human B cells were seeded at a  6 Materials and Methods density of 1 10 cells/mL in IMDM medium (Life Technol- ogies) supplemented with 10% heat-inactivated human AB Blood samples and mice serum (Thermo Fisher Scientific), 5 mg/mL human insulin For buffy coat preparations donors gave their consent. All Actrapid (Novo Nordisk Pharma GmbH), and 15 mg/mL gen- experiments were approved by our institutional ethical board. þ tamicin. Recombinant human IL4 (50 U/mL; Immunotools) C57BL/6 mice were purchased from JANVIER Labs. Luc C57BL/6 was added freshly. On days 0 and 5, the medium was supple- mice were kindly provided by Robert Zeiser (University Hospital mented with cross-linked human CD40L (Miltenyi Biotec): Freiburg, Germany). OT-I and OT-II mice were purchased Recombinant CD40L (0.5 mg/mL) was preincubated with fi from The Jackson Laboratory. Mice were housed under speci c cross-linking antibody (10 mg/mL) for 30 minutes at room pathogen-free conditions. All animal experiments were approved temperature. On days 7 and 11, cells were reseeded as described by our regional animal care committee. above. Isolation of lymphocytes Flow cytometry Human peripheral blood mononuclear cells (PBMC) or Cell phenotypes were evaluated using the following fluoro- murine splenocytes were isolated using Pancoll density-gradient phore-conjugated antibodies: human B cells with CD19, CD80, centrifugation (Pan-Biotech). B cells were enriched using immu- CD86, CD138, IgD, IgG1, IgM (BioLegend), CD20 (Life Tech- nomagnetic selection with human or murine CD19 microbeads nologies), HLA-DR and HLA-A2 (eBioscience); murine B cells (Miltenyi Biotec) according to the manufacturer's protocol. with CD62L, CD80, CD86, CD138, CCR7, CXCR4, CXCR5, IgD, Murine T cells were purified by using EasySep Mouse T-cell IgG1, IgM, H2Kb, streptavidin (BioLegend), B220 and CD19 (Life Enrichment Kit (Stem Cell Technologies) according to the man- Technologies); murine T cells with CD3e, CD4, CD8, CD25 ufacturer's protocol. (BioLegend) and SIINFEKL-H2Kb Tetramer (Glycotope Biotech- nology). Murine DCs with CD11b, CD11c, CD80, CD83, and Isolation of antigen-specific B cells CD86 (BioLegend). Foxp3 was stained with Foxp3 Fix/Perm Antigens were biotinylated by using the EZ-Link NHS-Biotin Buffer set and anti-mouse Foxp3 antibody (BioLegend). Data Reagent (Thermo Scientific) according to the manufacturer's were collected on a Gallios flow cytometer (Beckman Coulter) protocol. Ovalbumin (OVA) and KLH were purchased from and analyzed using FlowJo (TreeStar) or Kaluza (Beckman Sigma Aldrich, Hepatitis B surface antigen (HBV)-protein, and Coulter) software. TRP-2 protein were purchased from Abcam, recombinant human protein CEA was purchased from Sino Biological and recombi- Generation of antibody-secreting plasma cells nant human protein NY-ESO-1 was purchased from OriGene. For in vitro generation of antibody-secreting plasma cells, B cells Antigen-specific B cells were enriched by labeling with 0.2 mg/mL from mice were resuspended at a concentration of 1  106 cells/ biotinylated antigen and subsequent selection using the EasySep mL DMEM medium (Life Technologies) supplemented with Biotin Selection Kit (Stem Cell Technologies) according to the different stimuli, including IL4 (1 U/mL, Immunotools), IL21 manufacturer's protocol. (50 ng/mL, Immunotools), antibody to mouse CD40 (1 mg/mL, Acris Antibodies), and OVA-biotin tetramers (0.2 mg/mL OVA- Generation of mature dendritic cells Biotin þ 0.5 mg/mL streptavidin-PE, BioLegend). Cells were incu- þ Murine CD34 progenitor cells were purified from bone mar- bated for 72 hours. row of mice by positive selection with EasySep Biotin Selection Kit (Stem Cell Technologies)