Combining bacterial-immunotherapy with therapeutic antibodies: A novel therapeutic concept

Vaccine 30 (2012) 2786–2794

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Combining bacterial-immunotherapy with therapeutic antibodies: A novel therapeutic concept Ulrike Klier a,1 , Claudia Maletzki a,1 , Bernd Kreikemeyer b , Ernst Klar a , Michael Linnebacher a,∗ a b

Department of General, Vascular, Thoracic and Transplantation Surgery, Section of Molecular Oncology and Immunotherapy, University of Rostock, 18055 Rostock, Germany Institute of Medical Microbiology, Virology and Hygiene, University of Rostock, 18055 Rostock, Germany

a r t i c l e

i n f o

Article history: Received 8 December 2011 Received in revised form 17 January 2012 Accepted 21 January 2012 Available online 13 February 2012 Keywords: Bacterial-antibody conjugates Innate immune response Antitumoral effects Autologous T cell stimulation

a b s t r a c t Immunotherapeutic strategies become more and more important for cancer treatment. Therapeutic monoclonal antibodies (mAbs) like Panitumumab binding and blocking the EGF-receptor are in routine clinical use for the treatment of colorectal carcinoma (CRC). Also, bacterial therapy proved beneficial for experimental treatment of different tumor entities. The latter has been attributed to an activation of the immune system. Here, we describe a combination of both immunotherapeutic approaches in order to develop a novel targeted therapy for CRC. The therapeutic mAbs Trastuzumab and Panitumumab were conjugated to heat-inactivated bacteria expressing protein A or protein G. The potential of the conjugates was tested in comparison to the single components both in vitro and in vivo using a panel of patient-derived CRC cell lines. Antitumoral effects observed in vitro were strictly dependent on the presence of bacteria. Generally, effects could be enhanced by the addition of human lymphocytes. Detailed analysis of effector cells in autologous and allogeneic long-term stimulated lymphocyte cultures revealed the predominance of NK-cell-like cytolytic effectors. Reactivity was observed both against CRC target cells but also against the NK cell target K562. Similarly, in a subsequent in vivo study we observed substantial tumor growth delay accompanied by an increase in circulating NK cells. Contrary to this, the monotherapy with mAb alone caused only marginal effects and the treatment with bacteria was comparable to the mock-treated control. These data demonstrate successful targeting of CRC by bacteria/mAb conjugates. This novel concept may be interesting for future clinical approaches. Additionally, it illustrates the effectiveness of NK cells for cancer immunotherapy. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Immunotherapy is formed as a novel concept for cancer therapy in addition to the classical treatment regimens like resection, chemotherapy or irradiation. Until today, many different immune therapeutic strategies have been developed. The administration of therapeutic monoclonal antibodies (mAb) is one of the few approaches that have entered the clinics. These include the human mAb Panitumumab, which is accredited for monotherapy of EGFRoverexpressing metastasized colorectal cancers (CRCs), harboring wildtype Kras and Trastuzumab, a humanized mAb which is applied for Her2/neu expressing breast carcinomas. MAbs specifically bind

Abbreviations: OD, optical density; HI, heat-inactivated; mAbs, monoclonal antibodies. ∗ Corresponding author at: Section of Molecular Oncology and Immunotherapy, Department of General Surgery, University of Rostock, Schillingallee 35, D-18057 Rostock, Germany. Tel.: +49 381 494 6013; fax: +49 381 494 6002. E-mail address: [email protected] (M. Linnebacher). 1 These authors contributed equally. 0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2012.01.071

to tumor antigens, block signal cascades thereby inhibiting tumor cell proliferation or inducing apoptosis. Furthermore, they activate the innate as well as the adaptive immune system [1]. Via the Fc-fragment of mAbs, both complement dependent lysis and antibody dependent cellular cytotoxicity (ADCC), the latter typically triggered by NK cells, can be activated. NK cell-induced tumor cell apoptosis leads to antigen uptake and subsequent cross presentation by APCs inducing tumor specific cytotoxic T cell responses [2]. Especially for Trastuzumab, an additional way for activating tumor specific CD8+ T cells was described. MAb receptor binding is followed by internalization, intracellular degradation, loading of HLA class I molecules and an activation of Her2/neu specific cytotoxic T cells [3]. Another immunotherapeutic approach focuses on unspecific immune stimulation against tumors using bacteria or their components. We and others recently re-examined the potential of vital, lysed or lyophilized bacteria in regard to their effects on tumor growth in vivo [4–8]. We could show that bacteria are potent stimulators of the innate immune system and this is often followed by specific immune activation finally triggering immunological memory responses [6,7]. To improve specific targeting, bacterial toxins

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have been fused to ligands selectively binding receptors on tumor cells. The internalization of the receptor ligand complex finally leads to tumor cell death [9,10]. Dohlsten and Forsberg described a fusion protein generated by combining the Fab fragment of a tumor antigen recognizing mAb and a bacterial superantigen. Application of this recombinant protein induced significant tumor mass reduction and increased the number of tumor-infiltrating lymphocytes in an animal model [11,12]. Hence, this strategy combines excellent tumor cell-binding properties of therapeutic mAbs with the powerful cytotoxicity of bacterial antigens. In the present study, we followed the idea of combined mAb-directed tumor targeting and bacterial immunotherapy for treatment of experimental CRC. Inactivated bacteria, which proved their antitumoral efficiency in a previous study [13], were here conjugated to therapeutic mAbs. Antitumoral effects were examined both in vitro and in vivo. Our in vitro analyses revealed that bacteria alone often had the best antitumoral effects followed by the combination therapy whereas mAb alone had no or only marginal effects. Long term T cell stimulations against tumor cells evoked primarily NK cell activation by bacteria/mAb conjugates. Most important, systemic application of bacteria/mAb conjugates substantially affected tumor growth in vivo. This antitumoral effect was contributable to an effective immune stimulation. The latter finding suggests successful tumor targeting in vivo. Our model thus provides a solid basis for further evaluations on the concept of therapeutic mAbs-guided targeting of bacteria to tumors. This strategy may be of special interest for the treatment of metastasis difficult to access.

2. Materials and methods 2.1. Tumor cell lines, lymphocyte preparation and culture media The CRC cell lines HROC18, HROC60, HROC39 (all three MSS, Kraswt , Brafwt ), and HROC24 (MSI, Kraswt , Brafmut ) were established in our lab from patients subsequent to operation. Molecular characterization was performed as described before [14]. The CRC cell line HCT116 (MSI, Krasmut , Brafwt ) and the erythroleukemia cell line K562 were obtained from the German collection of cell cultures (DSMZ; Braunschweig, Germany). Cells were maintained in full medium: DMEM/HamsF12 supplemented with 10% fetal calf serum (FCS), glutamine (2 mmol/l) and antibiotics (medium and supplements were purchased from PAA, Cölbe, Germany). Peripheral blood lymphocytes (PBLs) were either obtained from healthy volunteers or from patients following Ficoll density-gradient centrifugation.

2.2. Bacteria, culture conditions and antibody conjugation The bacterial strains of the species Staphylococcus aureus (ATCC25923, Phillips, RN4220, COWAN I and SS3294), Streptococcus canis (GGS679, DSM20715, DSM20716), Streptococcus dysgalactiae spp. equisimilis (296743), Streptococcus pyogenes M49 strain 591, and Serratia marcescens were all from the research strain collection of the Institute of Medical Microbiology, Virology and Hygiene. Strains with either ATCC or DSM numbers were originally purchased from the American Type Culture Collection (ATCC, USA) or the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany), respectively. Except the preliminary test for binding affinity of mAbs to different bacteria strains subsequent in vitro analyses were exclusively performed with the strains S. aureus COWAN I (S. aureus) and the S. dysgalactiae spp. equisimilis (SDSE).

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Bacteria from overnight cultures were inoculated into fresh medium and further cultivated to mid-log growth phase in brain heart infusion (BHI) or on BHI agar (Oxoid Unipath, Wesel, Germany). Bacteria were adjusted to an OD600 = 0.5 referring to approximately 1.4 × 108 (S. aureus) and 1.2 × 108 cfu/ml (SDSE), respectively. Bacteria were heat-inactivated (=HI bacteria) for 1 h in a water bath at 75 ◦ C. Inactivation was verified by plating samples on BHI agar. Prior to treatment of tumor cells, bacteria were diluted 1:10 with the appropriate medium. Binding of HI bacteria to the therapeutic monoclonal antibodies (mAbs) Trastuzumab (Herceptin® ) and Panitumumab (Vectibix® ) was achieved by 30 min incubation at 4 ◦ C prior to the experimental treatment. To test binding stability, bacteria were incubated with FITC-labeled mAb (1 ␮g; FluoroTag FITC Conjugation Kit, Sigma–Aldrich, Deidenhofen, Germany) for 30 min at 4 ◦ C. Thereafter, a 96-well U-bottom plate was prepared with heat inactivated human or murine serum (10 ␮l/well). Samples without serum were used as negative controls. Bacteria/mAb conjugates were added and incubated for several time periods (0–48 h). Binding stability was examined using a FACSCalibur Flow Cytometer (BD Pharmingen, Heidelberg, Germany). 2.3. Flow cytometry of tumor cells and lymphocytes For expression analysis of EGF and Her2/neu tumor cells (5 × 105 ) were incubated with 1 ␮g of mAbs (30 min, 4 ◦ C). After two washing steps, cells were stained with FITC labeled secondary goat anti-human Ab (1:200; Bethyl laboratories, Montgomery, USA) and incubated 30 min at 4 ◦ C. Cells treated without primary antibody were used as negative control. For phenotypic analysis of human lymphocytes, 5 × 105 cells were washed and stained with the directly FITC- or PE-labeled mAb against CD3, CD4, CD8, CD16 and CD25 (Immunotools, Friesoythe, Germany) for 30 min at 4 ◦ C. Cells were washed twice and resuspended in 200 ␮l PBS. Negative controls were stained with the appropriate isotypes. Cells were analyzed by flow cytometry. 2.4. Live/Dead cytotoxicity assay The Live/Dead assay is based on a two color system using calcein-acetoxymethylester (AM; Invitrogen, Darmstadt, Germany), for detection of viable cells and Ethidium HomodimerI (Invitrogen), a cell-impermeable, red fluorescent dye that stains dying or dead cells. HROC18, HROC39, or HROC60 cells (1 × 105 ) and HCT116 cells (2.5 × 104 ) were seeded in 24-well plates and incubated over night. Thereafter, therapeutic substances were added as described in Section 2.2. Untreated cells served as living cell control, methanol-treated cells (70%, 30 min) as dead cell control. Medium was removed 24 or 48 h, and cells were washed with PBS. Calcein-AM (4 mM) and Ethidium Homodimer-I (1 mM) were added and incubated for 45 min (37 ◦ C, 5% CO2 ). Cells were measured using the multi-well plate reader Tecan Infinite® 200 (Tecan, Crailsheim, Germany). Calcein-AM was detected by extinction/emission of 495/515 nm and Ethidium Homodimer-I of 535/636 nm. Data of at least three independent experiments each performed in duplicates are presented. 2.5. Co-culture experiments and MLC (mixed lymphocyte culture) stimulation The contribution of the immune system on direct tumor cell killing was examined by co-culture experiments using PBLs with or without inactivated bacteria, mAbs or bacteria/mAbs conjugates.

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A 100

Panitumumab

positive cells in %

90

C 1000

SDSE

80

without serum human serum

800

70

murine serum

mean

60 50 40 30 20

600 400 200

10

0

0 HCT116

HROC18

HROC24

HROC39

HROC60

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8

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48

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A us re au S.

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25 au 92 re 3 us S. Ph au ill ip re s us S. R N au 42 re 20 us C S. ow au a re n us I S. SS ca 32 ni s 9 4 D SM S. 20 ca 71 ni 5 s S. G G ca S ni 67 s 9 D SM S. 20 py 71 og 6 en es SD M 49 SE st ra in S. 5 91 m ar ce sc en s

0

mean

mean

4

time in hours

Fig. 1. Flow cytometric analyses. (A) Cell surface expression of EGFR and Her2/neu on CRC cell lines using therapeutic mAbs Panitumumab (EGFR) and Trastuzumab (Her2/neu). Data are given as % positive cells. (B) Binding of mAbs to different bacterial strains. (C) Binding stability of mAb to selected bacteria in the presence or absence of human and murine serum. Data are given as mean. Results show data of three independent experiments (mean ± SD).

Experiments and data acquisition were performed as described before [13]. MLC experiments were conducted using PBLs and ␥-irradiated tumor cells (60 Gy) at a final effector to target cell ratio of 4:1 (1 × 106 T cells and 0.25 × 106 tumor cells/well) in T cell medium (IMDM containing 10% FCS, 2 mmol/l glutamine and antibiotics), supplements (1:100) and IL-7 (10 IU/ml). T cells were re-stimulated every 7 days; IL-7 was replaced by IL-2 (100 IU/ml) from day 28 on [15]. 2.6. IFN- ELISpot Specific immune responses were assessed by a standard interferon-␥ ELISpot (Mabtech, Nacka Strand, Sweden). The assay was performed with 5 × 103 stimulated PBMCs and 1 × 104 target cells. Experiments were performed in triplicates, spots were counted manually under a microscope and the percentage of reactive T cells was calculated. Remaining workflow was conducted as previously described [16]. 2.7. FACSOTOX assay Lytic activity of effector cells was determined in a flow cytometric cytotoxicity assay (FACSOtox). Target cells (5 × 103 ) were stained with 10 ␮M CMFDA and stimulated PBMCs were added at different E:T cell ratios (2:1, 6:1, 20:1, and 60:1). For detection of NK-cell activity, K562 cells were used as additional targets. All further steps were performed as previously described [16]. 2.8. Calcein-AM release assay Calcein-AM release was examined according to the protocol described by Neri et al. with only minor modifications [17].

Target cells were resuspended in complete medium and stained with calcein-AM (15 ␮M; 30 min; 37 ◦ C) with occasional shaking. After two washes in complete medium the cells were adjusted to a concentration of 104 cells/ml. The test was performed in v-bottom 96-well microtiter plates with E:T ratios ranging from 60:1 to 2:1 in triplicate, and with at least six replicate wells for spontaneous (only target cells in complete medium) and maximum release (target cells with 2% Triton X-100). Each well contained 1 × 103 to 3 × 104 lymphocytes in complete medium and 5 × 102 labeled target cells. After incubation at 37 ◦ C in 5% CO2 for 4 h, 75 ␮l supernatant was harvested and transferred into new plates. Samples were measured with an excitation/emission of 495/515 nm. Data were expressed as arbitrary fluorescent units (AFU) and were calculated as follows: [(test release − spontaneous release)/(maximum release − spontaneous release)] × 100. 2.9. In vivo tumor models and treatment regimen Experiments were performed on female 8–10-week old mice (Charles River, Fa. Wiga, Sulzfeld, Germany) weighting 18–20 g. All animals were fed standard laboratory chow and given free access to water. Trials were performed in accordance with the German legislation on protection of animals and the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council; NIH Guide, vol.25, no. 28, 1996). NMRI nu/nu mice were challenged with 5 × 106 HROC24 cells. Mice with established subcutaneous (s.c.) tumors received four intravenous injections of 109 HI bacteria (S. aureus), Panitumumab (50 mg/kg bw) or bacteria/mAb conjugates twice a week (n = 9–12 per group). As control, tumor-carrying mice received equivalent volumes of PBS (saline, n = 9 per group). Tumor growth was routinely controlled at least twice a week and tumor volume was estimated according to the formula: V = width2 × length × 0.52.

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Tumor carrying mice were sacrificed at day 21 or when they became moribund before the tumor volume reached 2000 mm3 . At the end of each experiment, blood was collected from the animals of all groups for further analysis. For analyses of lymphocytes from murine blood, 25 ␮l of the samples were diluted 1:1 with PBS and samples were stained with FITC or PE-conjugated rat anti-mouse mAbs: CD11b, NK1.1 (Immunotools) and CD11c (Miltenyi Biotec, Bergisch-Gladbach, Germany). Subsequently, erythrocytes were lysed (FACS Lysing solution, BD Pharmingen). Cells were washed and conducted to flow cytometry as described. 2.10. Statistical analysis All values are expressed as mean ± SD. After proving the assumption of normality, differences between controls and treated animals were determined by using the unpaired Student’s t-test. If normality failed, the nonparametric Mann–Whitney U-Test was applied. The tests were performed by using Sigma-Stat 3.0 (Jandel Corp., San Rafael, CA). The criterion for significance was set to p < 0.05. 3. Results 3.1. Antibody binding on tumor cells Our primary aim was to improve microbial-based immunotherapy by using targeted strategies. Determining the molecular status of tumor cells may predict applicability and thus potential effectiveness of targeted mAb therapies. In this regard, applicability of EGFR-targeting mAbs is dependent on wildtype Kras and Braf. Consequently, the mutational status of Kras, Braf and other tumor associated antigens was tested in our collection of freshly established CRC cell lines. From 15 cell lines screened, the following three microsatellite stable lines were chosen for in vitro analyses based on their mutational profile: HROC18, HROC60 and HROC39 (all three Kraswt , Brafwt ). However, to test mAb targeting in cells where EGFR-signaling is disrupted, two other cell lines were included in some experiments: the well established cell line HCT116 (Krasmut , Brafwt ) and the freshly established cell line HROC24 (Kraswt , Brafmut ). As conducted by flow cytometry, varying results were observed for EGF receptor expression with HROC24 and HCT116 showing strongest Ab binding. In contrast to that, all five cell lines expressed high levels of the Her2/neu receptor (each >95%; Fig. 1A). The cell wall proteins A and G of the bacterial species S. aureus and Streptococcus possess the ability to bind the Fc region of human immunoglobulins. In order to identify the best combination for subsequent application of the tumor-targeting bacteria/mAb immunotherapy, different bacterial strains were first incubated with therapeutic mAbs and binding affinity was determined by flow cytometry (Fig. 1B). Based on best antibody binding properties, S. aureus and SDSE were chosen for all subsequent analyses. Next, we investigated the stability of mAb-binding to bacteria in the presence of murine and human sera. A slight decrease in fluorescence intensity was detected after 1 h of incubation. Although we observed differences when comparing murine and human serum concerning their ability to squeeze mAb from bacteria, the general binding of the bacteria–mAb was stable up to 48 h (Fig. 1C). Obviously human serum was more effective in replacing mAb from SDSE than from S. aureus and vice versa for murine serum. These data provided the basis for subsequent determination of the therapeutic potential using a combined approach of targeted bacteria/mAb conjugates.

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3.2. Conjugates of bacteria and antibodies have no stronger cytotoxic effects on tumor cells in vitro than bacteria alone In previous analyses, we could show that avitalized bacteria exhibited substantial cytotoxic effects in vitro when applied directly on human tumor cells [13]. Here, we wanted to test our hypothesis that the cytotoxicity of HI bacteria should be boosted by the addition of therapeutic mAbs targeting surface molecules of tumor cells. The mAbs alone had no impact on tumor cell viability (Fig. 2A). As can also be depicted from Fig. 2A, treatment with HI bacteria led to an increase of dead cells. Strongest effects were observed for HCT116 cells after 24 h (S. aureus 10 ± 3%, SDSE 19 ± 11%). Moreover, conjugates did not mediate stronger effects on the tumor cells. Comparable results were obtained for all CRC cell lines tested (HCT116, HROC18, HROC39, and HROC60). Exemplary results are displayed for HCT116 and HROC18. 3.3. Addition of lymphocytes boosts antitumoral effect of bacteria and of bacteria/mAb conjugates MAb directed against Her receptors can have effects on tumor cells (i) by disturbing receptor-mediated signaling, (ii) by complement-mediated tumor cell lysis and (iii) by cell-mediated tumor cell attack. The latter is the consequence of immune cell recognition of antibody-marked target cells. Since the Fc region of the conjugates is blocked by binding to bacteria, the mechanism of ADCC is unlikely. In order to investigate, whether immune mediated antitumoral mechanisms are active in our in vitro system, we next performed a series of in vitro co-culture experiments adding immunocompetent cells and therapeutic components (mAb, inactivated bacteria and the conjugates) to tumor target cells (Fig. 2B). MAbs Panitumumab and Trastuzumab both had only marginal effects on tumor cells with the exception of HROC60, where Trastuzumab seemed to stimulate tumor cell growth after 72 h incubation (Fig. 2B, lower panel). Antitumoral effects were, however, boosted by the addition of conjugates with a maximum of approximately 70% reduction of tumor cell numbers (Fig. 2B). When compared to the effects of HI bacteria alone, one can however conclude that these effects are due to unspecific stimulation of lymphocytes by bacterial components. Generally, effects were observed for all three cell lines, with a trend toward highest susceptibility of HCT116 cells. 3.4. Long-term stimulation in allogeneic and autologous settings Clinical treatment of cancer patients with biologicals is performed repetitively. To simulate this situation in vitro, we tested the effect of weekly stimulations with conjugates on immune cells. Because no differences were observed between Panitumumab and Trastuzumab in the experiments described above, this long-term stimulation was performed solely with Trastuzumab. First, PBLs of healthy donors were stimulated against tumor cells in allogeneic settings. Exemplarily, we present data obtained for HROC18. Flow cytometric phenotyping of outgrowing bulk cultures stimulated with conjugates revealed generation of activated T helper cells (CD3+ /CD4+ /CD25+ ). Comparable results were obtained for the stimulation with SDSE alone. On the contrary, lymphocytes stimulated with S. aureus alone were dominated by CD3+ /CD8+ /CD25+ T cells and a small fraction of CD16+ /CD56+ NK cells. Bulk cultures stimulated with mAb alone exhibited a phenotype comparable to the control (Table 1A). Thereafter, stimulated lymphocytes were tested by IFN-␥ELISpot assay. Data revealed a well defined response of S. aureus stimulated T cells against HROC18 cells but no effects of cells stimulated with SDSE, mAb or the conjugates (Fig. 3A). Subsequent

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HCT116 p=0.400

p=0.998

p=0.423

p=0.288

p=0.87

p=0.846

p=0.879

u

i an /T

ra

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ur eu

s

p=0.917

st

35 30 25 20 15 10 5 0

SD

dead cells in %

A

48h

HROC18 p=0.565

p=0.451

p=0.100

p=0.365

p=0.153

1,0

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/T

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ra

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p=0.843

p=0.217

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dead cells in %

p=0.803

1,5

1,0

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HROC60

72h

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HROC18

S. aureus Panitumumab Trastuzumab S. aureus/Panitumumab S. aureus/Trastuzumab

HROC60

HCT116

HROC18

HROC60

SDSE Panitumumab Trastuzumab SDSE/Panitumumab SDSE/Trastuzumab

Fig. 2. Antitumoral potential of bacteria, mAbs and conjugates toward CRC cells. (A) Quantitative analysis of direct cytotoxicity toward tumor cells. Tumor cells were treated for 24 and 48 h with HI bacteria, mAbs or a combination of both. Killing efficiency against HCT116 (left panel) and HROC18 cells (right panel) was determined by fluorimetric ethidium homodimer staining. Numbers of dead cells were calculated after correction for untreated cells. Results show data of three separate experiments. Values are given as the mean ± SD. (B) Co-culture experiments. Tumor cells were co-cultured with PBLs of four different healthy donors in the presence of HI bacteria, mAbs or a combination of both for 24 h (upper panel) and 72 h (lower panel). Thereafter, numbers of viable cells were quantified by flow cytometry using microsphere beads as internal calibrator. Cells treated with PBLs alone were set as 1 and all other data were given as x-fold increase. Experiments were performed in duplicates (mean ± SD).

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Table 1A Flow cytometric phenotyping of HROC18 stimulated lymphocyte culture. d28

CD3+

CD3+ CD4+

CD4+ CD25+

CD3+ CD8+

CD8+ CD25+

CD16+ CD56+

Control S. aureus S. equisimilis Trastuzumab S. aureus/Trastuzumab S. equisimilis/Trastuzumab

95.45 88.38 91.83 94.02 92.04 89.32

79.91 22.40 67.16 81.23 61.52 67.43

12.51 8.35 25.70 11.51 19.03 17.58

14.03 66.78 23.09 13.06 28.09 23.75

2.48 50.40 13.00 3.48 20.05 14.48

5.76 8.04 1.56 2.16 2.85 5.39

Presented data are taken from d28 of T cell culture (% positive cells are given).

Table 1B Flow cytometric phenotyping of HROC39 stimulated lymphocyte culture. d28

CD3+

CD3+ CD4+

CD4+ CD25+

CD3+ CD8+

CD8+ CD25+

CD16+ CD56+

Control S. aureus S. equisimilis Trastuzumab S. aureus/Trastuzumab S. equisimilis/Trastuzumab

69.22 43.86 58.08 82.06 96.38 96.33

46.21 12.19 21.12 63.82 20.88 12.80

36.78 9.10 13.23 51.19 19.73 10.68

17.23 15.54 27.20 13.04 68.61 78.62

11.09 9.14 12.73 7.27 31.99 36.22

4.69 43.16 22.00 6.61 2.65 2.87

Presented data are taken from d28 of T cell culture (% positive cells are given).

accomplishments of functional analyses were only feasible with S. aureus stimulated cells. Lymphocytes in the remaining settings had reduced growth capacities which prevented further analyses. However, the cytolytic effects of S. aureus stimulated lymphocytes are unlikely tumor-specific since high reactivity against the classical NK cell target K562 was also observed (Fig. 3B). Additionally, these tests were performed in a complete autologous setting using HROC39 tumor cells and lymphocytes of the autologous patient (Table 1B). Here, repetitive stimulation of lymphocytes with bacteria alone revealed an increase in CD16+ /CD56+ NK cells. Similar to the allogeneic setting, stimulation with the mAb was comparable to the control (Table 1B). However, bulk cultures stimulated with conjugates were dominated by CD3+ /CD8+ /CD25+ cells. Despite

3.5. Targeted bacteria/mAb therapy in vivo Although only minor effects of mAb/bacteria conjugates were observed in vitro we decided to test our hypothesis on tumor targeting by bacteria/mAb conjugates in vivo.

B 3

HROC18

2

1 0

70 60

cytotoxicity (%)

activated cell in %

A

suggesting T cell character of these activated cells, functional testing again revealed mainly NK cell-like reactivity (Fig. 3C and D). Recognition and killing of K562 cells was confirmed for both bacteria/mAb conjugates and S. aureus, or SDSE stimulated lymphocytes. This is of particular interest since NK cell proportions ranged from <5 up to >40% in the bulk cultures. Hence, repetitive stimulation of lymphocytes with HI bacteria generally seems to favor the induction of highly activated NK cells.

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Fig. 3. Analyses of allogeneic or autologous PBLs stimulated with HI bacteria, mAbs or conjugates. ELISpot and cytotoxicity assay of HROC18 (A and B) or HROC39 (C and D) stimulated lymphocytes. (A and C) ELISpot assay. The specific IFN-␥ production of cells stimulated against tumor cells after 28 days is shown. Percentage of IFN-␥ releasing cells was calculated by the number of spots and the total number of cells analyzed (5 × 103 ). Values are given as the mean ± SD from three replicate wells. (B and D) Analysis of cytotoxic potential with (B) calcein release assay and (D) FACSOtox at day 49 of stimulation.

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tumor volume[mm 3]

A

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3000

4. Discussion

control Panitumumab

2500

S. aureus combination

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* 0 0

4

7

11

18

21

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70 CD11b

positive cells [%]

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20 10 0 control

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S. aureus

combination

Fig. 4. In vivo analyses of systemic treatment of HROC24 CRC xenografts. (A) Growth kinetics of HROC24 tumors in NMRI nu/nu mice after systemic therapy. Therapy was performed by repetitive local application of therapeutic agents twice a week (n = 9–12 mice per group). Control animals received equivalent volumes of saline (n = 9). (B) Flow cytometric analyses of peripheral blood lymphocytes of treated and control mice. Values are given as mean ± SD; *p < 0.05 vs. Panitumumab; t-test; # p < 0.05 vs. control; t-test.

We chose the xenograft model of HROC24 CRC cells in athymic nude mice. The tumor cell line was chosen since it carries a Braf mutation and shows resistance toward Panitumumab in vitro (own unpublished results). Thus, effects of the mAb alone were expected to be absent or low. Moreover, our in vitro data on bacteria/mAb conjugates hint toward NK cells as the main mediators of antitumoral activity. Athymic nu/nu mice lack functional T cell activity, but possess high numbers of circulating NK cells. Based on our initial idea of combining bacteria and mAb for targeted therapy, we focused on systemic rather than local injection. Mice with established HROC24 tumors received twice a week applications of HI bacteria, mAb or conjugates to a total of four applications. The conjugate treatment induced a substantial delay in tumor growth, already after the first therapy cycle. Tumors tended to keep growing very slowly, finally resulting in tumor sizes less than half of those of untreated animals (Fig. 4A). Unexpected from the in vitro results, Panitumumab therapy also affected HROC24 tumor growth. In contrast, systemic application of bacteria alone had no impact on tumor growth. Tumor sizes were comparable to controls. In subsequent flow cytometric analyses, we were able to correlate these findings with immunological parameters. Mice treated with conjugates had massively increased numbers of circulating NK cells (29.4 ± 3.9% vs. control 14.2 ± 4.2%). Moreover, levels of dendritic cells and monocytes rose after therapy (Fig. 4B). When comparing with single agent therapy using bacteria or mAbs, in vivo application of bacteria/mAb conjugates had a stronger impact on tumor growth. Although we did not induce complete remission, results obtained so far may provide a basis for further promoting the idea of bacteria/mAb tumor targeting in vivo.

In this study, we aimed at improving the concept of microbial based cancer immunotherapy by using conjugates of mAb and avitalized bacteria for tumor targeted therapy. The principle concept is not totally new. Combined targeted therapies have proven successful in some experimental studies [9,11,12]. This strategy is based on the idea that specific tumor cell-binding properties of therapeutic mAb can be combined with cytotoxic and immunogenic properties coming from the bacterial side. Bacterial Protein A and G, expressed by the selected Staphylococcal and Streptococcal strains, strongly binds to the Fc region of human Abs in general [18,19] and of the therapeutic mAbs Panitumumab and Trastuzumab in particular. However, this procedure blocks the mAbs Fc region and thus immunological effects, i.e. antibody dependent-cellular cytotoxicity and complement activation. Since no effects of mAb on colorectal tumor cells were observed in vitro, this disadvantage might be ignored. In previous studies on bacterial immunotherapy we demonstrated direct effects of inactivated bacteria on tumor cells [13]. To extend these findings, we examined the impact of mAbs, bacteria and their conjugates on tumor cells. Taking advantage of our unique collection of patient-derived and molecularly characterized colorectal cancer cell lines, we selected several lines with high expression levels of EGFR and HER2/neu. However, significant growth inhibition after exposure to EGFR or HER2/neu targeting mAbs was not achieved. This was surprising since besides the high levels of receptor expression on the cell surface, all but one cell line were wildtype for Kras and Braf and thus should at least be susceptible for mAbs targeting EGFR [20,21]. In the present study, heat inactivated bacteria were used since application of vital bacteria would be very controversial in regard to future clinical applications. On the one hand, live bacteria directly act on tumor cells and mediate release of necrotic tumor debris into the systemic circulation [7]. This facilitates initiation of cytotoxic T cell responses [22,23]. On the other hand, live bacteria possess the potential risk of unpleasant side effects like systemic toxicity. Excessive neoplastic cell destruction may trigger the so-called “tumor lysis syndrome”. This phenomenon sometimes occurs in patients with bulky, rapidly proliferating tumors strongly responding to treatment and thereby complicating patients’ treatment [24–26]. By inactivation, the immune stimulating character of bacteria is preserved [4,6,8,13,27,28]. In line with this previously validated fact, we here observed boosted antitumoral effects in an in vitro co-culture setting resembling aspects of a competent immune system [29,30]. However, sufficient tumor cell killing was dependent on the presence of avitalized bacteria, while the mAbs alone were not able to stimulate effective antitumoral immune responses. Consequently, antitumoral effects were due to unspecific immune stimulation by bacterial components and thus support recent results of other groups [28,31,32]. NK and NK-like cells are the main tumor cell attacking cell types as revealed in long term stimulation experiments. There, lymphocyte stimulations against tumor cells were exclusively successful when bacteria were present. The failure of the settings avoid of bacteria may hint toward the tumor cells’ natural immunosuppressive capacity [33,34]. These data underline the immune stimulating properties of bacteria [6,7,35] and we hypothesize that bacterial therapy even has the potential to overcome established tumorinduced tolerance [27]. Another unexpected finding was the sharp phenotypic difference in the lymphocyte in vitro cultures. Allogeneic stimulation with bacterial/mAb conjugates mainly induced CD4+ T cells whereas in the autologous setting CD8+ T and/or NKT cells were predominant. Therefore, combining tumor cells with bacteria/mAb

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conjugates may be a good stimulator of autologous cytotoxic T cell responses in vitro. However, functional tests revealed that the CD8+ T cells induced in the autologous stimulations were not strictly tumor specific. As an explanation for this, either stimulatory signals were absent or unknown factors inhibited T cell functionality [36,37]. Similar to previous findings, we observed mainly NK-like reactivity [13]. Moreover, it is conceivable that part of the CD8+ cells are NKT cells able to kill target cells in an NKG2D-dependent manner. NKT are different from conventional ␣/␤ T cells and play a role in tumor rejection but additionally in protection against infections [38]. Following activation, NKT cells secrete high levels of IFN-␥ and mediate NK-like cytolytic effector function in the absence of TCR recognition. Besides their potential to directly kill CD1d+ cells, killing of the classical NK cell target K562, being CD1d− , was described, too [39]. This line of argumentation may best explain our observations of unspecific NK cell activity, evidenced by lysis of K562, and massive IFN-␥ secretion upon specific stimulation. Finally, we wanted to address the question whether the bacteria/mAb conjugates have the potential to control tumor disease in vivo. With regard to the in vitro results, we decided to use an athymic nude mouse model. These mice are T cell deficient; possess high levels of NK cells and allow for efficient engraftment with human tumors. The cell line HROC24 was chosen as tumor model since it carries a Braf mutation and consequently shows strong resistance toward mAbs targeting EGFR in vitro (own unpublished results). Therefore, effects of the mAb alone were expected to be absent or low thus maximizing the window for detection of conjugates’ therapeutic effects. Systemic treatment of established HROC24 tumors with conjugates resulted in a notable tumor growth delay. This fits well with observations from a very recent study demonstrating TLR-agonist induced sensitivity toward cetuximab of Kras mutated tumor cells [40]. They additionally argue for successful mAb-directed tumor targeting of conjugates. Contrary to our expectations based on the in vitro data, single Panitumumab treatment also affected HROC24 tumor growth in vivo. Several reasons might explain this finding. Firstly, the applied doses in vitro differed from those given in vivo. Secondly, Panitumumab was injected repetitively in vivo, while it was only once given to the cell culture. Thirdly, since Panitumumab exerts their biological activity via multiple mechanisms, inhibition of tumor angiogenesis and thus tumor progression may be anticipated. Finally, several factors, yet to be identified, might have been missing in vitro, but were provided by the murine host that enabled tumor growth control. Somehow unexpectedly, the systemic application of bacteria had no effect on tumor growth. Bacteria were trapped in livers, but not detectable in tumors (data not shown). Lastly, a sharp raise in circulating NK cell numbers was exclusively observed after treatment with conjugates. Such massive NK cell increase has been described as part of the natural immune response toward bacterial infections [41]. These findings are particularly interesting since experiments were conducted on ectopic tumors. One might even expect stronger effects in patient-like animal tumor models with physiological blood supply [42,43]. However, this has to be addressed in subsequent studies. Taken together, we here provide evidence for successful tumor growth control by conjugates of inactivated bacteria and therapeutic mAbs. On the one hand our treatment strategy was not able to effectively stimulate tumor specific cytotoxic cells in vitro but on the other hand our data revealed the potential of NK cells in tumor control both in vitro and in vivo. The beneficial role of NK cells in controlling human malignancies is known from the clinics [44]. NK cells play an important role in tumor surveillance by both acting directly on tumor cells and regulating immune responses by bridging innate and adaptive immunity [45]. In our study, we created a

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