Using Antigen-Specific B Cells to Combine Antibody and T Cell− Based Cancer Immunotherapy

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. 2017 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 2017 American Association for Cancer Research. murine and human B cells with a defined specificity can be

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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) by using an anti-murine CD34-bioti- Antigen-presentation assay nylated antibody (BioLegend). Cells were cultivated at a concen- Murine APCs were incubated with OVA-protein at a concentration of 0.25 106 cells/mL in VLE-RPMI medium (Biochrom) tration of 75 nmol/L for 24 hours prior to incubation with T cells. supplemented with 5% FBS, 50 mmol/L ß-mercaptoethanol and Human APCs were incubated with CEA-protein at a concentration 500 U/mL of murine GM-CSF (Immunotools) and 1 U/mL of 100 nmol/L for 1 hours at 37C prior to incubation with T cells. murine IL4 for 7 days. For maturation, medium was supplemen- APCs were irradiated once with 26 Gy. T cells were stained with 10 ted with 1 mg/mL anti-mouse CD40L antibody (HM40-3, Acris mmol/L CFSE. Murine APCs were mixed at a ratio of 1:1 with Antibodies). negatively isolated T cells from spleens of OT-I or OT-II mice. Human APCs were mixed at a ratio of 1:1 with autologous Generation of CD40B cells negatively isolated T cells from PBMCs. Cocultures were incubat- Cell culture with feeder cells for activation and expansion of B ed for 5 days. Subsequently, T-cell proliferation was assessed by cells was performed as described previously (15). CD40L-expres- flow cytometry. sing tmuCD40L HeLa feeder cells were kindly provided by Clem- ens Wendtner (Klinikum Schwabing, Munich, Germany). Briefly, Fluorospot analysis B cells were seeded at a density of 1 106 cells/mL on lethally Negatively isolated T cells (0.2 106) from PBMCs were irradiated feeder cells. Cell passaging of feeder cells was done cocultured together with 0.1 106 protein-pulsed (0.1 mmol/L) twice per week. B cells were cultures in DMEM medium (Life CD40B cells on precoated anti-human IFNg FITC fluorospot plates Technologies) supplemented with 580 mg/mL glutamine, 10% (Mabtech). AIM-V medium was supplemented with 0.1 mg/mL fetal bovine serum, 1% HEPES, 1% MEM and 15 mg/mL genta- anti-CD28. An antibody to CD3 (Mabtech) was used as positive

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control in a dilution of 1:1000. Fluorospot analysis was performed Panc02-OVA was cultivated as previously described (16). For on an AID reader 20 hours later. tumor formation, cells were harvested and resuspended at a concentration of 4 106 cells/mL or 20 106/mL 1 PBS, Quantification of antibodies and affinity determination respectively. Cells (100 mL) were injected s.c. into the right flank For quantification of OVA-specific antibodies, assays were of mice. Tumor size was determined daily from day 7 after performed according to the sandwich-ELISA protocol from Bio- inoculation by measuring tumor diameter in two dimensions Legend. In brief, high-binding plates were incubated with OVA- using a vernier caliper. The tumor volume was calculated using the 2 protein at a concentration of 20 mg/mL. Anti-OVA IgG1, IgG2a following formula: Tumor volume ¼ 0.5 (length width ) mm. (BioLegend), and IgE (Novus Biologicals) served as standards. Mouse detection antibodies were obtained from BioLegend and T-cell depletion applied at a concentration of 100 ng/mL. Avidin-horseradish Five and 25 days after tumor challenge, mice received either a peroxidase (BioLegend) was used at a dilution of 1:1,000. The mixture of each 250 mg of monoclonal anti-CD4 clone affinity of IgG1 OVA-specific antibodies was determined by pre- YTS191.1 and clone YTA3.1 or 500 mg of monoclonal anti- incubating supernatants of differentiation cultures with decreas- CD8cloneYTA169.4.Efficient depletion was controlled by ing concentrations of soluble OVA for 1 hours. Competition taking 100 mL blood from the tail vein 5 days after antibody þ þ ELISAs were performed as described above. injection and determining the percentage of CD4 or CD8 T cells by flow cytometry. Antibody-dependent cytotoxicity assay EG.7 lymphoma cells (0.3 105) were labeled with CFSE and Statistical analysis subsequently cocultured with 3 105 splenocytes isolated from Significant differences were calculated by a two-tailed t-test, spleens of C57BL/6 mice by Pancoll. The percentage of dead one-way ANOVA, or two-way ANOVA where appropriate using þ þ tumor cells was quantified by staining for 7AAD CFSE cells in GraphPad Prism Software. P values of statistical significance were flow cytometry analyses. NK cell activation in these cultures was marked with asterisks as indicated in figure legends. Mean values þ þ measured by determining the percentage of NK1.1 CD3 CD69 and standard deviations (SD) were calculated from at least three cells. independent experiments.

In vivo immunization of na€ve mice Results þ C57BL/6 or Luc mice were immunized i.p. with 100 mLof20 Murine antigen-specific B cells show a class-switched and mmol/L OVA-Protein in PBS þ Incomplete Freund's Adjuvant activated phenotype (IFA; Sigma Aldrich) for generation of OVA-specific B cells. For To test our hypothesis that tumor antigen-specific B cells can be immunization with cell subsets, APCs were exogenously loaded used for cancer immunotherapy, we chose a mouse model that with 10 mmol/L OVA protein for 1 hour at 37C. uses OVA as tumor antigen. We first established a method for detecting and enriching antigen-specific B cells with antigen- In vivo cytotoxicity assay biotin tetramers (Fig. 1A; ref. 17). After immunizing mice with As target cells, lymphocytes were isolated from spleens of OVA in IFA, the OVA-specific B-cell population made up 2% of þ þ C57BL/6 mice by Pancoll and labeled with either 2 mmol/L CFSE splenic CD19 B220 B cells compared with 0.60% in non- or 20 mmol/L CFSE. The 20 mmol/L CFSE population was pulsed immunized mice (Fig. 1B). Enrichment by antistreptavidin þ þ with 10 mmol/L OVA-peptide (SIINFEKL) for 1 hour at 37 C. A 1:1 microbeads resulted in 85% pure CD19 B220 B cells of which mixture of low and high CFSE lymphocytes were then injected i.p. 65% were OVA specific. into immunized mice on day 21. Twenty-four hours later, spleens These OVA-specific B cells (OVA-B cells) showed higher levels were removed and analyzed for killing of peptide-pulsed target of activation markers CD86, MHCI and MHCII compared with cells. The ratio of unpulsed versus pulsed (RatioUP) target cells non-OVA-specific B cells of the same mice (Fig. 1C). OVA-B cells was determined by dividing the percentage of CFSE low cells by upregulated the expression of surface IgG1 and downregulated fi the percentage of CFSE high cells. Speci c lysis was calculated by expression of IgD and IgM (Fig. 1D), indicative of an activated and the following formula: class-switched phenotype. These results were not restricted to fi ¼ % Speci c lysis (1 (RatioUP Negative Control/RatioUP OVA-B cells because we obtained similar purities and activation Immunized) 100. profiles for other antigens such as keyhole limpet hemocyanin (KLH)-specific and tyrosinase-related protein 2 (TRP-2)-specificB In vivo migration cells (Supplementary Fig. S1). þ þ Murine CD19 B cells or CD40B cells from Luc mice were þ injected i.v. into syngeneic mice. Detection of Luc B cells was Vaccination with antigen-specific CD40B cells induces an performed by injecting 7.5 mg D-Luciferin in 250 mL 1 PBS i.p. antigen-specific T-cell response Imaging was performed in the Xenogen IVIS 200 (Perkin Elmer). Next, we investigated whether antigen-specific B cells provide Mice were constantly kept under narcosis with 1.5–4% Isofluran superior T-cell stimulation compared with nonspecific B cells. at 37 C. Bioluminescence pictures were analyzed with the Living Theoretically, antigen-specificity would have several advantages Image Software (Perkin Elmer). because it enables B cells to take up antigen via the specific BCR whereas antigen uptake by polyclonal nonspecific B cells occurs Tumor challenge primarily by pinocytosis (18). BCR affinity is directly proportional þ The tumor cell line E.G7 was kindly provided by Tomo Saric to the capacity of B cells to present antigen to CD4 T cells (19). (University Hospital Cologne, Germany). The tumor cell line Furthermore, antigen uptake by the BCR leads to changes in the

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Figure 1. Antigen-specific B cells purified from immunized mice show a class-switched and activated phenotype. A, Biotinylated soluble antigen forms tetramers with fluorescently labeled streptavidin. Via cross-linking of the BCR, tetramers can be used for staining and purification of antigen-specific B cells. B, Splenocytes from mice were stained via antigen tetramers for OVA-B cells among the CD19þ B220þ B-cell population in nonimmunized mice and before and after purification in OVA-immunized mice. C, The mean fluorescent intensity (MFI) of the activations markers CD86, CD80, MHCI, and MHCII in OVA-specific B cells from immunized mice were determined by flow cytometry and normalized to values of nonspecific (non-OVA) B cells. D, The percentage of immunoglobulins IgG1, IgM, and IgD expressing CD19þ B cells in OVA-specific and nonspecific (Non-OVA) B cells from immunized mice were determined by flow cytometry. Bar charts show mean values þ SD of three independent experiments. Significant differences calculated with a two-tailed t test are marked by an asterisk. ns, not significant; , P 0.02; , P 0.005; , P 0.0001. Histograms show representative analyses compared with an isotype control.

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Figure 2. Antigen-specific CD40B cells induce a specific T-cell response. A, Purified OVA-B cells were stimulated with the CD40 ligand and analyzed for the expression of different activation markers. The MFI was normalized to values of the same population before stimulation. Histograms show representative analyses compared with an isotype control. B, In antigen-presentation assays, T cells from OT-II or OT-I mice were cocultured with different unpulsed or protein-pulsed APCs in a ratio 1:1, i.e., polyclonal (Poly) CD40B cells, OVA-CD40B cells, and CD40L-matured dendritic cells (CD40 DC). T-cell proliferation was determinedbya decrease of CFSE intensity as shown in representative CFSE plots. C–E, Mice were immunized with protein-pulsed APCs, i.e., CD19þ-enriched polyclonal CD40B cells (Poly), CD19þ-enriched OVA-CD40B cells (OVA), or DCs. PBSþ IFA served as negative control (Neg). C, The specific lysis of peptide-pulsed target cells was determined by a decrease of the CFSE signaling in flow cytometry analyses. D, CD3þ CD8þ T cells from vaccinated mice were stained for the presence of OVA peptide-specific T cells. E, Specific lysis after injection of different types of protein-pulsed APCs at different concentrations. Bar charts show mean values þ SD of four independent experiments. Significant differences calculated with (A þ E) a two-tailed t test (B–D) or ordinary one-way ANOVA with multiple comparisons are marked by an asterisk. ns, not significant; , P 0.02; , P 0.0035; , P 0.0006; , P 0.0001.

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þ antigen processing machinery that facilitate the intracellular traf- pulsed purified OVA-CD40Bs with both antigen-specific CD4 þ ficking of antigen and MHC-class II molecules and the generation and CD8 T cells from OT-II or OT-I mice, respectively, increased of peptide-MHCII complexes (20). proliferation of T cells compared with polyclonal CD40B cells First, OVA-B cells were incubated on CD40L-expressing feeder (Fig. 2B). Induction of T-cell proliferation by OVA-CD40B cells cells to induce activation and to enhance their antigen-presenting was similar to that of conventional professional APCs, as exem- capacity. After 24 hours, B cells were analyzed for the expression of plified by anti-CD40 matured dendritic cells (DCs). Maturation of activation markers. OVA-CD40B cells showed higher expression DCs was confirmed by flow cytometry (Supplementary Fig. S2). T- of activation markers such as CD80, CD86, MHCI and MHCII cell stimulation by OVA-CD40B cells or DCs resulted in a similar (Fig. 2A) compared with unstimulated controls. degree of T-cell activation that was higher than that achieved by To confirm the enhanced antigen-presenting function of OVA- polyclonal B cells (Supplementary Fig. S3). CD40B cells, their ability to stimulate an antigen-specific T-cell To further characterize the capacity of OVA-CD40B cells to response was investigated in vitro. Cocultures of OVA protein- induce specific T-cell responses, we next assessed their antigen-

Figure 3. CD40B cells migrate to secondary lymphoid organs in mice. A, OVA-CD40B cells (1 106) from Lucþ mice were injected i.v. into tumor-free mice. 12 hours (12 h), 36 hours (36 h), or 5 days (5 d) after injection, mice were analyzed for the presence of CD40B cells by imaging in the IVIS 200 system. B, Polyclonal or C OVA-CD40B cells from Lucþ mice were injected into E.G7 lymphoma–bearing mice. 36 hours or 5 days after injection, mice were analyzed for presence of CD40B cells in the spleen and lymph nodes (front view) or in tumor areas (side view, "¼ tumor site) by imaging in the IVIS 200 system. Representative pictures out of three experiments are shown. D, Expression of the migratory markers CCR7, CXCR4, CXCR5, and CD62L on OVA-B cells from Lucþ mice was analyzed by flow cytometry before (d0) and after (d3) CD40L stimulation. The MFI was normalized to values of the same population before stimulation. Bar charts show mean values þ SD of four independent experiments. Significant differences calculated with a two-tailed t test are marked by an asterisk. ns, not significant; , P 0.05; , P 0.004. Histograms show representative analyses compared with an isotype control.

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Figure 4. Stimulation of OVA-B cells with IL21 leads to PC differentiation. OVA-B cells (OVA) of immunized mice or na€ve B cells of nonimmunized mice (Polyclonal) were stimulated with the indicated stimuli for three days. A, Cells were analyzed by flow cytometry for expression of CD138. B, The amount of anti-OVA IgG1 was determined by ELISA. Bar charts show mean values þ SD in three independent experiments. Significant differences calculated with two-way ANOVA are marked by an asterisk. , P 0.02; , P 0.01. C, Anti-OVA IgG2b, IgG3 and IgM titers in supernatants of cultures were determined by ELISA. Polyclonal B cells were treated with LPS and served as positive control. Mean values of three independent experiments of absorption at 450 nm at different 10-fold dilutions are shown. D, The affinity of anti-OVA IgG1 antibodies in supernatants of cultures that were stimulated with OVA þ IL4 þ IL21 þ

anti-CD40 was determined by calculating the LogIC50 from competition ELISAs with decreasing concentrations of soluble OVA (10-fold dilution). Shown are mean values of 10 independent experiments. E and F, Supernatants of OVA-B-cell cultures that were stimulated with OVA þ IL4 þ IL21 þ anti-CD40 were incubated with splenocytes from C57BL/6 mice and OVA-expressing EG.7 cells. EG.7 cells þ splenocytes were used as negative controls (Neg. Control). E, The percentages of CD69þ activated NK1.1þ CD3 cells were determined by flow cytometry after 3 hours. Values were normalized to internal control without EG.7 target cells. Shown are mean values þ SD of five independent experiments. Significant differences calculated with two-tailed t test are marked by an asterisk. , P 0.0001. F, The percentage of 7AADþ EG.7 cells was determined by flow cytometry after 24 hours. 2% DMSO were used as positive control; an irrelevant IgG1 antibody was used as additional negative control. Shown are mean values þ SD of five independent experiments. Significant differences calculated with one-way ANOVA are marked by an asterisk. , P 0.0001.

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presenting function in vivo using an in vivo cytotoxicity assay. Mice cytes were injected as target cells into immunized mice. The in vivo- þ were immunized i.v. with 5 106 protein-pulsed APCs, i.e., specific lysis by antigen-specific CD8 T cells was calculated by polyclonal CD40B cells, OVA-CD40B cells or anti-CD40 matured determining the ratio between unpulsed and pulsed target cells in þ DCs. On day 21, CFSE OVA-peptide pulsed syngeneic lympho- spleens. The specific lysis was higher in mice immunized with

Figure 5. Combinational therapy induces antitumor immunity. A–C, On days 7 and 15, after Panc02OVA tumors became palpable, mice were treated i.v. with 0.1-2 106 APCs, i.e., polyclonal CD40B cells (Poly CD40B), OVA-CD40B cells (OVA CD40B), DCs, OVA-specific PCs alone (OVA PC) or in combination with OVA-CD40B cells (OVA CD40Bþ OVA PCs), PBSþIFA as negative controls (neg. control). A, Growth curves show mean increase of tumor volume in mm3 of one representative experiment out of three experiments with 3–6 mice per group. Significant differences calculated with two-way ANOVA are marked by an asterisk. , P 0.03. B, Survival curves show means of one representative experiment out of three with 3–6 mice per group. Significant differences calculated with a log-rank test are marked by an asterisk. ,p 0.02. C, Serum samples of mice were taken before tumor challenge, before treatment, and 7 days after the last treatment. ELISA for OVA-specific IgG1 antibodies was performed. Significant differences calculated with two-way ANOVA are marked by an asterisk. , P 0.009; , P 0.0001. D and E, On days 5 and 25 after Panc02OVA challenge, mice were treated with 500 mg of anti-CD4 (a-CD4) or anti-CD8 (a-CD8). 0.1–2 106 OVA-CD40B cells (OVA CD40B) or PBS as negative controls were injected on days 7 and 15 after tumors became palpable. D, Growth curves show mean increase of tumor volume in mm3 of one representative experiment with 4–6 mice per group. Tumor growth is shown as 0.5 times the product of (length diameter x width diameter2). Significant differences calculated with two-way ANOVA are marked by an asterisk. , P 0.04; , P 0.002. E, Survival curves show means of one representative experiment with 4–6 mice per group.

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OVA-CD40B cells compared with mice immunized with poly- tion by CD40L resulted in further upregulation of CCR7 and clonal CD40B cells (Fig. 2C). Immunization with OVA-CD40B CD62L on OVA-B cells. CXCR4 expression did not vary over cells and DCs resulted in a similar degree in specific lysis. the observed period, whereas CXCR5 was reduced by 64.61% Spleens of vaccinated mice were stained for the presence of 13.59% (Fig. 3D). This expression pattern is consistent with þ þ OVA-peptide specific CD3 CD8 T cells (Fig. 2D). Mice vacci- homing to T-cell-rich areas in the secondary lymphoid organs nated with OVA-CD40B cells had higher percentages of OVA- (23, 24). þ specific CD8 T cells than mice that were treated with polyclonal þ CD40B cells or negative controls. There was no difference in CD8 Antigen-specific B cells stimulated by IL21 and anti-CD40 T-cell induction compared with DC-based vaccinations. The effect differentiate into plasma cells þ was specific to CD8 T cells and did not lead to compensatory The classic effector function of B cells is the secretion of þ þ þ induction of CD4 CD25 Foxp3 Tregs (Supplementary Fig. S4). antibodies upon differentiation into plasma cells (PC). We rea- The induction of antigen-specific T-cell immunity was dose- soned that the combination of this effector function with antigen- dependent, as increasing the injected number of APCs for vacci- presentation offers additional advantages for cancer immuno- nation also resulted in an enhanced specific lysis in all three therapy. To induce differentiation into antibody-secreting PCs, we different APCs (Fig. 2E). These results demonstrate that antigen- developed a protocol in which antigen-specific B cells were specific CD40B cells are potent inducers of antigen-specific T-cell stimulated with a combination of cytokines and a BCR agonist. responses. PC differentiation was confirmed by analysis of CD138 expression (Fig. 4A). We obtained the most efficient differentiation Antigen-specific CD40B cells migrate to secondary lymphoid when stimulating OVA-B cell cultures with OVA, IL4, IL21, and organs and to the tumor site anti-CD40 (44.24% 0.76). IL21 and anti-CD40 were crucial, To further understand the nature and dynamics of B- and T-cell because stimulation without either of the two stimuli resulted in þ þ encounters, the in vivo migration kinetics of luciferase (Luc ) decreased percentages of CD138 PCs. Based on these results, þ CD40B cells were assessed. OVA-specific Luc CD40B cells OVA-B cells were stimulated with the combination of OVA- appeared in the spleen within 12 hours after injection and then protein, IL4, IL21, and anti-CD40 to produce PCs for subsequent migrated to the abdominal lymph nodes within the next 5 days in vivo experiments. Treatment of na€ve polyclonal B cells also þ (Fig. 3A). resulted in differentiation into CD138 PCs; however, matura- In mice with OVA-expressing E.G7-lymphoma, polyclonal tion of polyclonal B cells to PCs was less efficient than for OVA-B CD40B cells homed to the spleen, but preferentially accumulated cells. To further characterize these OVA-specific PCs, we next in TDLN relative to abdominal lymph nodes (Fig. 3B). OVA- assessed their ability to produce OVA-specific antibodies. In þ CD40B cells migrated to the spleen and TDLN over a period of 36 accordance with the observed changes in CD138 B cells, hours (Fig. 3C). In contrast to polyclonal CD40B cells, 1/10 of OVA-B cells secreted OVA-specific IgG1 antibodies when treated injected OVA-CD40B cells appeared at the tumor site where they with the cytokine cocktail (6.68 ng/mL 0.34; Fig. 4B). In þ still could be detected on day 5. The majority of these Luc cells addition, antibodies of the subclasses IgG2b, IgG3, or IgM was detectable in the spleen and TDLN. (Fig. 4C) could be detected after stimulation with the cytokine Encounter of APCs and T cells is regulated by chemokine combination. Polyclonal B cells did not secrete OVA-specific gradients in T-cell areas of secondary lymphoid organs (21). antibodies (Fig. 4B and C). Therefore, we studied the chemokine receptor pattern of OVA- The affinity of these OVA-specific IgG1 antibodies in super- CD40B cells and could indeed confirm that they display a natants of cultures was determined by competition ELISAs. Secret- pattern of chemokine receptors that is consistent with migra- ed OVA-specific IgG1 antibodies had a higher affinity for tion to secondary lymphoid organs (10, 22). CXCR4 and CCR7 the soluble antigen than IgG1 clone TOSG1C6 (Biolegend), a were upregulated in OVA-B cells compared with non–OVA-B commercially available standard antibody (LogIC50: 1.397 vs. cells of the same mice (Supplementary Fig. S5). On the other 1.523; Fig. 4D). In cocultures with splenocytes and OVA-expres- hand, CXCR5 and CD62L were not significantly altered in sing E.G7 cells, the secreted antibodies induced NK cell OVA-B cells. Compared with an unstimulated control, activa- activation (Fig. 4E) and killing of tumor cells (Fig. 4F), thereby

Figure 6. Human antigen-specific CD40B cells induce specific T-cell responses. A, PBMCs from vaccinated healthy donors were stained via antigen tetramers for HBV-B cells among the CD45þ CD20þ B-cell population in nonvaccinated donors and before and after purification in HBV-vaccinated donors. Plots show representative result of five independent experiments. B, Single cell suspensions from TDLN of patients with colorectal cancer (left) or gastric esophageal cancer (right) were stained via antigen tetramers (CEA and NY-ESO-1, respectively) for antigen-specific B cells among the CD45þ CD20þ B-cell population before cultivation with the CD40L and on day 11 of culture. Plots show representative results of three independent experiments. C, The MFI of the activation markers CD86, CD80, and HLA-DR were determined in HBV-specific B cells from vaccinated donors by flow cytometry. The MFI was normalized to values of nonspecific (non-HBV). Shown are means þ SD of 8 independent donors. D, Purified HBV-B cells were stimulated with the CD40 ligand and analyzed for expression of the activation markers CD80, CD86, and HLA-DR after 7 days. The MFI was normalized to values of the same population before stimulation. Shown are means þ SD of 5 independent donors. Significant differences calculated with a two-tailed t test are marked by an asterisk. ns, not significant; , P 0.016. E and F, CD19þ purified B cells from PBMCs or single cell suspensions from TDLN or tumors of patients with gastric esophageal cancer were cultivated for 14 days in the presence of the CD40L. Afterward, 0.2 106 negatively isolated T cells from PBMCs were cocultured with 0.1 106 protein-pulsed (þNYESO) or unpulsed (NYESO) CD40B cells. Unstimulated T cells served as negative controls (T cells only); T cells þ anti-CD3 served as positive controls. E, Means of IFNgþ spots per 0.2 106 (200K) T cells þ SD from low responders (left) and high responders (right) are shown. n ¼ 4andn ¼ 3, respectively. Significant differences calculated with a one-way ANOVA are marked by an asterisk. ns, not significant; , P 0.0083; , P 0.0008; , P 0.0001. Tumor samples were excluded from statistical analysis. F, Representative fluorospot pictures of IFNg signal in FITC are shown. Fluorospots with tumor-infiltrating B cells were performed with one high responding donor.

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demonstrating their functional activity and suggesting a mecha- whereas mice additionally treated with CD40B cells stayed tumor nism for the observed antibody-mediated cytotoxicity. free until the end of the experiment. Taken together, these results support the value of coordinated exploitation of synergistic B-cell Antigen-specific CD40B cells synergize with antigen-specific effector functions for cancer immunotherapy. plasma cells for cancer therapy Next, the influence of a preventive immune intervention with Human antigen-specific B cells develop an APC phenotype tumor antigen-specific CD40B cells on tumor growth was inves- upon CD40L stimulation tigated. For this purpose, protein-pulsed APCs (0.1–2 106) were As a proof-of-principle for translation into clinical application, intravenously injected into mice thrice every 7 days. On day 28, we developed a protocol for isolation of human HBV-specificB immunized mice were inoculated s.c. with E.G7-lymphoma cells. cells (HBV-B cells) from vaccinated donors based on the method Tumor growth in mice treated with OVA-CD40B cells was reduced established for murine antigen-specific B cells (Fig. 6A). The þ þ and survival was prolonged compared with mice that were CD20 CD19 B-cell population consisted of 3% HBV-B cells. immunized with DCs or polyclonal CD40B cells. The combined Selection of HBV-B cells via HBV-biotin tetramers resulted in a 10- treatment with OVA-CD40B cells and PCs was superior in pro- fold enrichment of the antigen-specific B-cell population. In tecting mice from tumor challenge and delayed tumor growth addition, B cells specific for the two shared self/tumor antigens compared with mice that were immunized with DCs or OVA- CEA and NY-ESO-1 were also detected by this method in single- CD40B cells alone (Supplementary Fig. S6A and S6B). Survival cell suspensions of TDLN obtained from patients with colorectal (Supplementary Fig. S6C) was also prolonged. The presence of or gastric esophageal cancer, respectively (Fig. 6B). OVA-specific IgG1 antibodies in serum of treated mice was Purified HBV-B cells were characterized by flow cytometry. confirmed by ELISAs before and after vaccination (Supplementary Analogous to murine antigen-specific B cells, they showed an Fig. S6D). Mice treated with the combination of OVA-CD40B cells activated phenotype by upregulating the activation marker CD86 and PCs had more specific antibodies in their serum than mice and MHCII (Fig. 6C). Next, human antigen-specific B cells were treated with DCs. Mice that stayed tumor-free until day 50 after incubated with soluble CD40L to stimulate their antigen-present- the first tumor inoculation were rechallenged with E.G7 cells and ing function. On day 7 or 11 of culture, the phenotype of the B tumor growth was observed until day 84 (Supplementary cells was assessed. HBV-CD40B cells upregulated the activation Fig. S6E). Mice that were treated with OVA-CD40B cells alone markers CD80, CD86, and HLA-DR (Fig. 6D) compared with or in combination with PCs were protected from renewed tumor unstimulated cells. Like HBV-CD40Bs, CEA or NY-ESO-1-specific challenge. B cells also proliferated when activated with the CD40L (Fig. 6B). To extend our findings to other disease models and provide IFNg fluorospot analysis of cocultures of NY-ESO-1–specific evidence for therapeutic efficacy of our concept, mice were chal- CD40B cells from TDLN cells together with T cells of the same lenged s.c. with Panc02OVA tumor cells and treated twice with donor confirmed the immunostimulatory capacity of NY-ESO-1– 0.1–2 106 APCs as soon as the tumor became palpable. Tumors specific CD40B cells (Fig. 6E and Fig. 6F). Although all patients grew less in mice that were treated with the combination of OVA- showed NY-ESO-1–specific B cells in flow cytometry analyses, CD40B cells and PCs compared with controls (Fig. 5A). OVA- they showed variable T-cell responses in fluorospot analyses and CD40B cells alone reduced tumor growth as effectively as treat- thereby were classified as low and high responders. Within low ment with DCs. Three of four mice that were treated with the responders, the T-cell response induced by protein-pulsed CD40B combination of OVA-CD40B cells and PCs, and two of four mice cells from TDLN was higher than that induced by CD40B cells that were treated with either cell type alone remained tumor-free from PBMCs. In one donor, B cells were also isolated and until day 65 (Supplementary Fig. S7A). In contrast, tumors of cultivated from single cell suspensions of the tumor. These CD40B mice treated with DCs grew more slowly, but they grew out in four cells induced similar T-cell responses when pulsed with the of five mice. Accordingly, mice treated with OVA-CD40B cells and specific protein as B cells from TDLN or PBMCs in high responders PCs survived longer than controls (Fig. 5B). In mice treated with (Fig. 6F). Similar results were obtained in IFNg fluorospot anal- PCs alone or the combination with OVA-CD40B cells, the ther- yses with CEA-protein pulsed CD40B cells from patients with apeutic activity was positively correlated with the amount of OVA- colorectal cancer (Supplementary Fig. S8A and S8B). Antigen- specific IgG1 antibodies (Fig. 5C). To explore the mechanism of presentation confirmed our results obtained from fluorospot þ the induced antitumor immunity, mice were treated with CD4 or analyses by showing induction of T-cell proliferation (Supple- þ CD8 T cell–depleting antibodies on day 5 and 25 after tumor mentary Fig. S8C). inoculation. Efficient depletion of T cells was determined in blood samples (Supplementary Fig. S7B) before mice were either treated with OVA-CD40B cells or with PBS as negative control. Depletion Discussion þ of CD8 T cells resulted in increased tumor growth (Fig. 5D) and After decades of rather disappointing results, cancer immuno- reduced survival (Fig. 5E) compared with nondepleted mice. therapy has experienced a clinical breakthrough. Immune check- þ þ Depletion of CD4 T cells had contrary effects. CD4 T-cell point blockade with anti-CTLA4 or anti–PD-1 and CAR T-cell depletion resulted in reduced tumor growth and prolonged therapies have yielded favorable results in clinical trials (1). þ survival. This effect might be due to depletion of Foxp3 Tregs, Unfortunately, only about 20% of patients show a response to þ because Panc02-OVA tumors possess a high percentage of Foxp3 checkpoint blockade. However, approximately 75% of patients þ cells (12.54 % 1.4%) in the tumor-infiltrating CD4 T-cell who respond show a durable response (26). The remaining 25% compartment (Supplementary Fig. S7C and ref. 25), which was relapse because the tumor acquires resistance to immunotherapy þ also targeted by the CD4 T cell–depleting antibody (Supple- through several distinct mechanisms, including defects in þ mentary Fig. S7C). Tumors in CD4 T cell–depleted negative immune signaling or antigen presentation (27). Therefore, controls showed recurrent tumor growth from day 29 onward, despite advances in immunotherapy, there still is the need for

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Antigen-Speciﬁc B Cells for Cancer Immunotherapy

improvement. Complementary immunotherapeutic approaches improvement over conventional immunotherapeutic strategies may be useful. because it combines two distinct immunologic mechanisms. The present study reports a cellular immunotherapy based on Tumor-specific antibodies can mediate effective antitumor tumor antigen-specific B cells. The method combines the secretion immunity (40). In patients with cancer, the presence of certain of specific antibodies with the induction of T-cell immunity and antibodies against tumor antigens is associated with improved offers the possibility of translation to different kinds of cancer or survival (41). We demonstrated that the induced PCs secrete OVA- infectious diseases with a known antigen. specific antibodies of the major subclass IgG1 but also IgG2b, B cells play an important role in antitumoral immunity (28). IgG3, and IgM, which were functional and capable of inducing NK Whereas regulatory B-cell subsets can impair tumor immunity, cell–mediated cytotoxicity. All four subclasses were shown to be activated B cells can also contribute to immunosurveillance of induced by IL21 and anti-CD40 stimulation and represent a TH2- cancers (29). In animal models, the antitumoral activities of B polarized immune response (42). In addition, antigen-presenting cells are mediated by several immunologic mechanisms includ- B cells can efficiently induce T-cell responses against tumor ing antibody-dependent effects and stimulation of tumor-spe- antigen. Activated B cells are relatively resistant to inhibition by cific T-cell responses (30). We aimed to exploit the immuno- tumor-associated immunosuppressive molecules such as IL10, therapeutic potential of B cells by combining both their anti- TGF-b, and VEGF (43). Although some studies failed to demon- body-mediated and the T-cell stimulatory antitumoral effector strate an effect of therapeutic treatment with polyclonal CD40B mechanisms. cells on tumor growth (44, 45), we showed that antigen-specificB Published approaches on B cell–based immunotherapy cells are superior to nonspecific B cells and that combined (31, 32) have used polyclonal, and not antigen-specific, B lym- immunotherapy with tumor antigen-specific CD40B cells and phocytes. The role of antigen-specificity of B cells has been poorly PCs resulted in the remission of established tumors. The combi- studied, likely due to difficulties in isolation and characterization nation of two distinct antitumoral immune mechanisms may also of antigen-specific B cells (17, 33). As a consequence, isolation of prove to be more resistant to immune escape. Our strategy might antigen-specific B cells and use of their APC function have not be usefully combined with checkpoint blockade. been combined or tested in vivo. Here, we report the isolation and Although the murine studies provided a proof-of-principle for enrichment of both murine and human antigen-specific B cells by our bifunctional B cell–based vaccine platform, we also per- using antigen tetramers, and subsequent differentiation of these B formed additional experiments to evaluate the translational cells into APCs and antibody-producing PCs for use in cancer potential of this immunotherapeutic approach. We demonstrated immunotherapy. that antigen-specific B cells can be isolated from TDLN of cancer We demonstrated that OVA-CD40B cells were superior to poly- patients and that these tumor antigen-specific B cells can be clonal CD40B cells in the induction of a T-cell response resulting differentiated into potent APCs. Although lymphocytes are usu- from affinity of the BCR for its antigen (19). The antigen concen- ally more tolerant against shared self/tumor antigens, we show tration for pulsing of OVA-CD40B cells was 0.03% of that of the that CD40B cells from cancer patients induce strong T-cell minimal concentration necessary for presentation after fluid- responses against these antigens. These results are in line with phase pinocytosis of antigen, typical for antigen uptake by poly- earlier studies showing antigen presentation of cancer testis anti- clonalCD40Bcells(19). AlthoughDCshavebeenconsideredtobe gens and induction of T-cell responses with polyclonal CD40B more potent APCs than B cells (34), we found that OVA-CD40B cells isolated from PBMCs (4, 6). Nevertheless, in contrast to cells and DCs had a similar APC capacity in vitro and in vivo. TDLN, we could not detect tumor antigen-specific B cells in APCs must physically encounter T cells in order to induce PBMCs of cancer patients. Moreover, T-cell responses were lower immune responses. In vivo migration experiments confirmed when induced by protein-pulsed CD40B cells derived from homing of polyclonal and OVA-CD40B cells into the secondary PBMCs than from TDLN. We therefore conclude that TDLN are lymphoid organs of mice. After entry into lymphoid tissue, a more promising source of tumor antigen-specific B cells with CD40B cells accumulate both in B-cell follicles and at the edge regard to antigen-presentation and T-cell induction. Although of the T-cell zone (35), enabling them to interact with T cells. This tumor-infiltrating B cells did not stain for CEA or NY-ESO-1, they would constitute an advantage of CD40B cells over DCs, which showed induction of T-cell responses in fluorospot analysis. We have been shown to poorly migrate to secondary lymphoid conclude that tumor antigen-specific B cells were present, but organs after injection and to instead stay at the site of injection could not be stained with antigen-tetramers because the BCR of (36). In tumor-bearing mice, tumor antigen-specific CD40B cells tumor-infiltrating B cells was saturated with antigen. appeared in the tumor three days after injection. These results are Taken together, our findings demonstrate that the combination supported by studies that detected tumor antigen-specific anti- of cognate presentation of tumor antigens and production of bodies in human breast cancer (37), thereby suggesting that at tumor-reactive antibodies generates antitumor immunity that is least some of the tumor-infiltrating B cells are antigen-specific. At capable of inducing tumor remission. Our method allows simul- the same time, about 90% of injected CD40B cells stayed in taneous generation of tumor antigen-specific antigen-presenting TDLN, allowing interaction with T cells. B cells and antibody-producing plasma cells from small amounts Tumor-infiltrating B cells and antibody-secreting PCs play a of peripheral blood B cells. This study presents a proof-of-prin- crucial role in the context of tumor immunity. Tumor-infiltrating ciple in murine tumor models and provides a rationale for the B cells are associated with improved survival (38, 39). With the clinical evaluation of this B cell–based immunotherapeutic expectation to enhance the antitumor immune response, we platform. combined cellular and humoral immune functions of B cells by treating mice with a combinatorial immunotherapeutic approach Disclosure of Potential Conflicts of Interest using antigen-specific CD40B cells as APCs in combination with H. Abken is a consultant/advisory board member for Miltenyi Biotec. No antibody-secreting PCs. This dual approach seems to represent an potential conflicts of interest were disclosed by the other authors.

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Authors' Contributions Acknowledgments Conception and design: K. Wennhold, H. Abken, A. Shimabukuro-Vornhagen, We thank Holger Spiegel from the Fraunhofer IME in Aachen, Germany, for M. von Bergwelt-Baildon his expertise in antibody characterization. Development of methodology: K. Wennhold, O. Utermohlen,€ M. von Bergwelt-Baildon Grant Support Acquisition of data (provided animals, acquired and managed patients, K. Wennhold was supported by the Graduate Program for Pharmacology € provided facilities, etc.): K. Wennhold, M. Thelen, H.A. Schloßer, N. Haustein, and Experimental Therapeutics. This work was supported by grants from the Else S. Reuter, M. Garcia-Marquez, A. Lechner, S. Kobold, F. Rataj Kroner-Fresenius€ Stiftung, Koln€ Fortune and the German Ministry for Innova- Analysis and interpretation of data (e.g., statistical analysis, biostatis- tion, Science, Research and Technology of North Rhine-Westphalia. S. Kobold € tics, computational analysis): K. Wennhold, M. Thelen, H.A. Schloßer, was supported by the international doctoral program "i-Target: Immunotarget- N. Haustein, S. Reuter, M. Garcia-Marquez, A. Lechner, S. Kobold, ing of cancer" funded by the Elite Network of Bavaria, the Marie-Sklodowska- G. Chakupurakal, S. Theurich, H. Abken, A. Shimabukuro-Vornhagen, Curie "Training Network for the Immunotherapy of Cancer (IMMUTRAIN)," M. von Bergwelt-Baildon the Deutsche Krebshilfe, the Wilhelm Sander Stiftung, the Ernst Jung Stiftung, Writing, review, and/or revision of the manuscript: K. Wennhold, H.A. and the Else Kroner€ Fresenius Stiftung. € Schloßer, S. Kobold, F. Rataj, G. Chakupurakal, S. Theurich, M. Hallek, The costs of publication of this article were defrayed in part by the payment of H. Abken, A. Shimabukuro-Vornhagen, M. von Bergwelt-Baildon page charges. This article must therefore be hereby marked advertisement in Administrative, technical, or material support (i.e., reporting or organizing accordance with 18 U.S.C. Section 1734 solely to indicate this fact. data, constructing databases): K. Wennhold, M. Garcia-Marquez, O. Utermoh-€ len, M. von Bergwelt-Baildon Received September 9, 2016; revised May 30, 2017; accepted July 27, 2017; Study supervision: G. Chakupurakal, A. Shimabukuro-Vornhagen, M. von published OnlineFirst August 4, 2017. Bergwelt-Baildon

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Using Antigen-Specific B Cells to Combine Antibody and T Cell− Based Cancer Immunotherapy

Kerstin Wennhold, Martin Thelen, Hans Anton Schlößer, et al.

Cancer Immunol Res 2017;5:730-743. Published OnlineFirst August 4, 2017.

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