Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Jun 1.
Published in final edited form as: J Immunol. 2015 Apr 24;194(11):5305–5311. doi: 10.4049/jimmunol.1402517

B7H6-specific bispecific T cell engagers (BiTEs) lead to tumor elimination and host anti-tumor immunity1,2

Ming-Ru Wu *, Tong Zhang *, Albert T Gacerez *, Tiffany A Coupet *, Leslie R DeMars †, Charles L Sentman *
PMCID: PMC4433849  NIHMSID: NIHMS676854  PMID: 25911747

Abstract

Substantial evidence showed that T cells are the key effectors in immune-mediated tumor eradication. However, most T cells do not exhibit anti-tumor specificity. In this study, a bispecific T cell engager (BiTE) approach was utilized to direct T cells to recognize B7H6+ tumor cells. B7H6 is a specific ligand for the NK cell activating receptor, NKp30. B7H6 is expressed on various types of primary human tumors, including leukemia, lymphoma, and gastrointestinal stromal tumors (GISTs), but it is not constitutively expressed on normal tissues. In this study, data show that B7H6-specific BiTEs direct T cells to mediate cellular cytotoxicity and IFN-γ secretion upon co-culturing with B7H6+ tumors. Furthermore, B7H6-specific BiTE exhibited no self-reactivity to pro-inflammatory monocytes. In vivo, B7H6-specific BiTE greatly enhanced the survival benefit of RMA/B7H6 lymphoma bearing mice through perforin and IFN-γ effector mechanisms. In addition, long term survivor mice were protected against a RMA lymphoma tumor re-challenge. The B7H6-specific BiTE therapy also decreased tumor burden in murine melanoma and ovarian cancer models. In conclusion, B7H6-specific BiTE activates host T cells and has the potential to treat various B7H6+ hematological and solid tumors.

Keywords: NK cell, BiTE, lymphoma, melanoma, ovarian cancer

Introduction

Immunotherapy holds the promise of being an effective means to treat cancer. Evidence shows that T cells are the key effectors in tumor cell recognition and destruction (1). There are multiple immunotherapy strategies that aim to harness T cell effector functions, such as chimeric antigen receptor (CAR) T cells therapy, cancer vaccines, bi-specific T cell engagers (BiTE), and immunological checkpoint inhibitors. BiTE strategies utilize protein engineering techniques to create fusion proteins with dual specificity that lack a fragment crystallizable (Fc) region. Its common format is a fusion protein consisting of a tumor recognition single chain variable fragment (scFv) linked in tandem with another scFv specific to T cell CD3ε (2). T cells re-directed by a BiTE can form immunologic synapses with tumor cells, perform serial killing, secrete cytokines, and are activated in the absence of a co-stimulatory signal (3, 4).

There are several intrinsic advantages of BiTE therapy. BiTE therapy confers anti-tumor specificity to T cells without having to genetically engineer T cells and therefore avoids the costs and time for ex-vivo T cell manipulation. So far, BiTEs have shown potent therapeutic efficacy in treating non-Hodgkin lymphoma and acute lymphoblastic leukemia (5, 6). This strategy can potentially target any tumor cell surface antigen as long as an antibody can be generated. There are several BiTEs in pre-clinical and clinical trials which can target multiple tumor types (7, 8). However, many BiTEs target tumor associated antigens (TAAs), such as epidermal growth factor receptor, human epidermal growth factor receptor 2, epithelial cell adhesion molecule, carcinoembryonic antigen, which can be expressed by multiple types of normal tissues. This expression pattern may potentially cause on-target off-tumor side effects.

NK cells recognize a panel of ligands specifically expressed on multiple tumor types with activating receptors, such as NKG2D, NKp30, NKp44, and NKp46 (9, 10). This broad tumor targeting activity of NK cells makes these receptors an attractive part for generating tumor recognizing BiTE proteins with the potential to exhibit a very broad tumor targeting capacity (11). To develop an effective NK cell receptor based-BiTE therapy, we created a novel BiTE based on the specificity of NKp30. NKp30, a NK cell activating receptor which mediates anti-tumor immunity in various tumor types (12, 13). One of its ligands, B7H6 is expressed on about 20% of human tumor cell lines and is also expressed on several types of primary human tumors (14). RNA analysis showed that several subsets of human primary lymphoma, leukemia, ovarian cancer, brain tumors, breast cancers, renal cell carcinomas, and various sarcomas potentially expresses high amount of B7H6. In addition, B7H6 mRNA is not found on 48 normal tissues under steady state conditions (14). NKp30 is a pseudogene in Mus musculus, and a B7H6 ortholog is missing in mice (14, 15). In this study, we describe a novel B7H6-specific BiTE which recognizes B7H6. In this study, we showed that an B7H6-specific BiTE directs T cells to mediate cytotoxicity and IFN-γ secretion against B7H6+ tumor cells. B7H6-specific BiTE therapy enhanced the survival of lymphoma bearing mice and decreased tumor burden of melanoma and ovarian cancer bearing mice. These data suggest that B7H6-specific BiTE therapy can potentially be beneficial for treating various tumors.

Material and Methods

Mice

C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD). Mice were used in experiment at the age of 6–12 weeks old. All experiments were conducted according to Dartmouth College's Institutional Animal Care and Use Committee.

Cell culture and cell lines

Anti-B7H6 hybridoma was described previously (16). The anti-mouse CD3ε hybridoma 145.2C11, K562 was obtained from American Type Culture Collection (Manassas, VA). The B3Z T cell hybridoma was obtained from Dr. Nilabh Shastri (University of California at Berkley). Mouse T cell lymphoma line RMA, melanoma cell line B16F10, and ovarian cancer cell line ID8 have been described previously (17–19). Mouse T cell lymphoma line RMA/B7H6, melanoma cell line B16F10/B7H6, ovarian cancer cell line ID8/B7H6 were generated by retrovirus transduction of their parental line RMA, B16F10, or ID8 cells, respectively, using dualtropic retroviral vectors containing the human B7H6 gene according to protocols previously described (17). RMA, RMA/B7H6, B16F10, B16F10/B7H6, and K562 were cultured in RPMI 1640, supplemented with 10% heat-inactive FBS (Atlanta Biologicals, Lawrenceville, GA), 10mM HEPES, 0.1mM non-essential amino acids, 1mM sodium pyruvate, 100U/mL penicillin, 100ug/mL streptomycin, and 50uM 2-ME. ID8, ID8/B7H6 were cultured in DMEM with a high glucose concentration (4.5g/L) containing the same supplements. 293F cells (Life Technology, Carlsbad, CA) were cultured in Gibco® FreeStyle 293™ Expression Medium (Life Technology) on an orbital shaker shaking at 120rpm. Primary human ovarian cancer samples were obtained from Dartmouth-Hitchcock Medical Center after surgery with informed consent. Cancer samples were mechanically disrupted and red blood cells were lysed with ACK lysis buffer (0.15M NH4Cl, 10mM KHCO3, 0.1mM EDTA, pH 7.4). Primary ovarian cancer cells were cultured for two days before used for functional assay. To stimulate PBMCs with lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), or interleukin-1β (IL-1β), human cells from cell cones obtained from leukapheresis (Dartmouth-Hitchcock Medical Center Blood Donor Center) were cultured in 24 well plates at a cell density 3×106 cells/well in complete RPMI 1640 at 37°C and 5% CO2 for 48 h with or without the following stimulation, LPS (1µg/mL; Sigma-Aldrich, Saint Louis, MO), TNF-α (100ng/mL; PeproTech, Rocky Hill, NJ), or IL-1β (1ng/mL; PeproTech).

Design and Construction of B7H6-specific and MICA-specific BiTEs

The anti-B7H6 scFv was constructed by fusing VH [aa 1–134] and VL [aa 23–129] region of an anti-B7H6 hybridoma 47.39 (16) with a 15 amino acid glycine (G)-serine (S) linker (G4S)3 linker (3 repeats of GGGGS). Anti-human CD3ε scFv was constructed by fusing VH [aa 20–138] and VL [aa 23–128] region of an anti-human CD3ε hybridoma OKT3 with (G4S)3 linker. Anti-mouse CD3ε scFv was constructed by fusing VH [aa 20–135] and VL [aa 21–128] region of an anti-mouse CD3ε hybridoma 145.2C11 with (G4S)3 linker. All the fragments mentioned above were PCR amplified using cDNA derived from individual hybridoma with a high-fidelity DNA polymerase Phusion (New England Biolabs, Beverly, MA, USA). All oligos for PCR were synthesized by Integrated DNA Technologies (Coralville, IA) or Sigma-Genosys (Woodsland, TX). Human version B7H6-specific BiTE was constructed by fusing anti-B7H6 scFv with OKT3 scFv via a (G4S)3 linker. Murine version B7H6-specific BiTE was constructed by fusing anti-B7H6 scFv with 145.2C11 scFv via a G4S linker. A histidine tag (6 repeat of histidine) was added to the C-termini of both constructs to facilitate protein purification. The construct of human B7H6-specific BiTE was further cloned into a CMV promoter based expression vector. The construct of murine B7H6-specific BiTE was cloned into the expression vector pCEP4 (Life Technology). The MICA-specific BiTE is generated by fusing a scFv that recognize MICA with OKT3 scFv via a (G4S)3 linker.

Production and purification of B7H6-specific BiTEs

For production of B7H6-specific BiTEs, a suspension of growing 293F cells cultured in Gibco® FreeStyle 293™ Expression Medium were transfected with B7H6-specific BiTE DNA constructs by 40kD PEI (Polyscience Inc, Warrington, PA). Transfection was done by gently mixing 293F cells with DNA and PEI at a final concentration of 2×107 cells/mL, 12.5ug/mL DNA, 25ug/mL PEI and letting it shake on a orbital shaker at 37°C at 120 rpm for 3h. After 3h, the whole mixture was diluted with Gibco® FreeStyle 293™ Expression medium at a 1:9 ratio for a final cell concentration of 3×106 cells/mL. Valproic acid (Sigma-Aldrich) was added to the culture to a final concentration of 2mM. The culture was maintained in 37°C shaking at 120rpm for 4 days and cell-free culture supernatant was harvested. The supernatant was mixed with 4× Ni column binding buffer (1.2M NaCl, 200mM NaH2PO4, 80mM, Imidazole pH=7.4) and loaded onto a HisTrap HP column (GE Healthcare, Waukesha, WI). The column was washed with 10 column volumes of Ni column binding buffer (0.3M NaCl, 50mM NaH2PO4, 20mM Imidazole, pH=7.4) and elution was performed with 20 column volumes of elution buffer (0.3M NaCl, 50mM NaH2PO4, pH=7.4) with a linear gradient of Imidazole from 250mM-500mM. Eluted fractions were collected and examined by SDS-PAGE. The fractions with B7H6-specific BiTEs were further combined and buffer exchanged to PBS by 30kD molecular weight cutoff Amicon ultrafiltration column (EMD Millipore, Billerica, MA). The final protein solution was filtered sterile with a 0.22µm syringe filter (EMD Millipore). The quantity and purity of BiTE was accessed by SDS-PAGE, followed by staining with SYPRO orange (Life Technology). Concentration of BiTEs was quantified by using Image J software (NIH, Bethesda, MD) and compared to an ovalbumin protein standard curve.

Flow cytometry

To confirm human B7H6-specific BiTE binds to T cell CD3ε and B7H6 simultaneously, human T cells were stained with various amount of human B7H6-specific BiTE (1ng-1000ng), followed by staining with DyLight650 conjugated-soluble B7H6. All samples were preincubated with human Cohn fractions (Sigma-Aldrich) to reduce non-specific binding. Samples were analyzed by Accuri C6 flow cytometer (BD Biosciences, San Jose, CA). Data analysis was done by Accuri (BD Biosciences) or FlowJo software (TreeStar Inc, Ashland, OR).

In vitro cytotoxicity assay

In vitro cytotoxicity was measured by LDH release assay. OKT3 and IL-2 activated human T cells or concanavilin A activated murine splenocytes were incubated with tumor cells at a E:T ratio 5:1 (105:2×104) in triplicate wells in 96 well V-bottom plates at 1ug/mL concentration of B7H6-specific BiTEs. Six hours later, cell-free culture supernatant was harvested and LDH release was determined by CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega, Madison, WI) following the manufacturer’s protocol. Specific lysis was calculated as: percentage of specific lysis = [(experimental O.D value - effector spontaneous O.D value - target spontaneous O.D value) / (target maximum O.D value - target spontaneous O.D value)] × 100. Media O.D. values were subtracted from all samples.

Cytokine production by T cells

OKT3 activated and cultured for 5 to 12 days, human T cells were co-cultured with tumor cells at an E:T=1:1 ratio (105:105) in triplicate wells with various concentrations (0.98ng/mL-1000ng/mL) of human B7H6-specific BiTE in 96 well V-bottom plates. To test B7H6-specific BiTE reactivity to primary ovarian cancer cells, OKT3 activated human T cells were co-cultured with primary ovarian cancer cells at an E:T=1:1 ratio (105:105) in triplicate wells with 250ng/mL of human B7H6-specific BiTE in 96 well V-bottom plates. To test B7H6-specific BiTE reactivity to pro-inflammatory monocytes in PBMCs, human T cells were co-cultured with LPS, TNF-α, or IL-1β stimulated PBMCs at an E:T = 1:1 ratio (105:105) in triplicate wells with 250ng/mL of human B7H6-specific BiTE in 96 well V-bottom plates. Cell-free medium was collected after 24h and IFN-γ concentration determined by Duoset IFN-γ ELISA kit (R & D Systems, Minneapolis, MN). To generate murine T cells for in vivo injection in the ovarian tumor model, murine splenocytes were activated with concanavalin A (1µg/mL) and cultured with recombinant human IL-2 (25U/mL) for 4 to 6 days. For in vitro experiments, concanavalin A activated murine splenocytes were co-cultured with tumor cells at an E:T ratio 5:1 (105:2×104 for B16F10/B7H6) or 1:1 (105:105 for RMA, RMA/B7H6) with various concentrations (7.8ng/mL-500ng/mL) of murine B7H6-specific BiTE. Cell-free medium was collected after 24h and IFN-γ concentration determined by ELISA.

Quantitative RT-PCR

Total RNA of unstimulated, LPS, TNF-α, or IL-1β stimulated PBMCs were extracted with RNeasy Mini Kit (QIAGEN Inc, Valencia, CA). Complementary DNA (cDNA) synthesis was performed using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). 5ng of cDNA was used as the template for quantitative RT-PCR with iQ SYBR Green Supermix (Bio-Rad). Primer pairs sequences for PCR are as following: IL-6F: 5'-ACTCACCTCTTCAGAACGAATTG-3', IL-6R: 5'-CCATCTTTGGAAGGTTCAGGTTG-3', B7H6-F: 5'-GACCCTGGGACTGTCTACCA, B7H6-R: 5'-ATAGGCCACCAATGAATGGA-3', GAPDH-F: 5'-CTCCTGTTCGACAGTCAGCCGC-3', GAPDH-R: 5'-TGACCAGGGGCCCAATACGACC-3'. IL-6 and B7H6 mRNA fold induction was calculated by −2ΔΔCt method.

Treatment of tumor bearing mice with murine B7H6-specific BiTE

RMA/B7H6 lymphoma cells (105 cells) were injected i.v. into B6 mice on day 0. Murine IgG or murine B7H6-specific BiTE (10µg) was injected i.v. on days 3, 5, and 7. Mice were closely monitored and euthanized when moribund signs were observed. As a melanoma model, B16F10/B7H6 cells (106 cells) were injected s.c. into B6 mice in their shaved right flank on day 0. Mice were treated with murine IgG or murine B7H6-specific BiTE (10µg) i.v. on days 5, 7, 9, and 11. Tumors were measured every other day using a caliper and tumor area was calculated. Mice were sacrificed when tumor area reached 200mm2. As an ovarian cancer model, ID8/B7H6 cells (5×106 cells) were injected i.p. into B6 mice on day 0, and the mice were treated with murine IgG or murine B7H6-specific BiTE (10µg) i.p. (days 7, 9, and 11). In some groups, additional concanavilin A activated and expanded murine T cells (5×106 cells, i.p.) were injected at the time of the first BiTE administration on day 7. The number of solid tumors on the peritoneal wall and free tumor cells in the i.p. wash was quantified on day 50.

Statistical analysis

One-way ANOVA, Mann-Whitney U test, or student's t-test were used to analyze differences between groups, as appropriate, and p values < 0.05 were considered statistically significant. Kaplan-Meier survival curves were plotted and analyzed using GraphPad Prism software (GraphPad Software, San Diego, CA).

Results

Human B7H6-specific BiTE binds to T cell CD3ε and tumor cell B7H6 simultaneously and triggers T cells effector responses against B7H6+ tumors

Two B7H6-specific BiTEs, one that binds to human CD3 and another that binds to murine CD3, were constructed by fusing anti-B7H6 scFv with anti-human and anti-murine CD3ε scFv, respectively (Fig. 1A). SDS-PAGE analysis under reducing and non-reducing conditions showed human and murine B7H6-specific BiTEs were expressed as monomers with the expected molecular weight of 56Kd and 55Kd, respectively (data not shown). To confirm that human B7H6-specific BiTE preserved the binding specificity of both parental scFvs, human T cells (CD3ε+, B7H6−, NKp30−) were stained with human B7H6-specific BiTE, followed by DyLight650-conjugated soluble B7H6 protein. Only when the B7H6-specific BiTE bound to both human T cell CD3ε and soluble B7H6 simultaneously was there a DyLight650 signal. We observed a dose dependent increased staining intensity with increasing amounts of the B7H6-specific BiTE protein (Fig. 1B). Cytotoxicity assays showed that the human B7H6-specific BiTE triggered T cell cytotoxicity against two B7H6+ tumors, RMA/B7H6 and K562, but not a B7H6− tumor (Fig. 1C). Human B7H6-specific BiTE also triggered dose-dependent IFN-γ production by T cells when co-cultured with RMA/B7H6 or K562 tumor cells (B7H6+ tumors), but not RMA tumor cells (B7H6− cells) (Fig. 1D). In contrast, a non B7H6-specific BiTE (MICA specific) did not trigger human T cells against B7H6+ tumor cells, but only against MICA+ tumor cells (K562) (Fig. 1E).

Figure 1. Design, production, and functionality of B7H6-specific BiTEs.

Figure 1

Open in a new tab

(A) A schematic diagram of the human and murine B7H6-specific BiTE proteins. (B) Human T cells (CD3ε+, NKp30−, B7H6−) were stained with B7H6-specific BiTE protein (1ng-1000ng) followed by soluble B7H6-DyLight650. Data shown are representative histograms of staining intensity. (C) OKT3 activated human T cells were co-cultured with tumor cell lines at an E:T ratio of 5:1 (105:2×104) with or without human B7H6-specific BiTE (1ug/mL). Six hours after co-culturing, culture supernatant was harvested and specific lysis was determined by LDH release assay. The data shown are mean +SD of triplicate wells, and are representative data from 3 different human donors. (D) OKT3 activated human T cells were co-cultured with B7H6− (RMA) or B7H6+ (RMA/B7H6, K562) tumor cells at an E:T of 1:1 (105:105) at the indicated concentrations of human B7H6-specific BiTE protein. Cell-free medium was collected after 24h and IFN-γ concentration determined by ELISA. The data shown are mean +SD of triplicate wells, and are representative of data from 3 different human T cell donors. (E) OKT3 activated human T cells were cultured with B7H6 negative (RMA, B16F10), B7H6 positive (RMA/B7H6, B16F10/B7H6), and B7H6 & MICA positive (K562) tumor cell lines at an E:T of 4:1 (105:2.5×104) with B7H6-specific BiTE or a MICA-specific BiTE. Cell-free medium was collected after 24h and IFN-γ concentration determined by ELISA. Statistical analysis compares the IFN-γ value of B7H6 positive tumor cells (RMA/B7H6 or K562) to B7H6 negative tumor cells (RMA) at each BiTE concentration. An ** indicates p < 0.005; *** indicates p < 0.0001.

Human B7H6-specific BiTE triggers T cells to react to primary human ovarian cancer but not pro-inflammatory monocytes

B7H6 is known to be expressed on primary human lymphoma, leukemia, and GIST(13, 14). Analysis of the Oncomine gene array data shows that B7H6 mRNA is also found to be over-expressed in several other types of primary human tumors, such as ovarian cancer, brain tumors, breast cancers, renal cell carcinomas, and various sarcomas (Wu et al., Gene Therapy, 2015, in press). When co-culturing human T cells with the BiTE protein and primary ovarian cancer cells, IFN-γ production was observed and this activity could be blocked by pre-incubating the tumor cells with anti-B7H6 antibodies (Fig. 2A). Besides tumor cells, B7H6 has been reported to be expressed on circulating pro-inflammatory monocytes in a subset of severe sepsis patients or on monocytes stimulated with Toll-like receptor (TLR) agonists or pro-inflammatory cytokines in vitro (20). However, we did not observe B7H6-specific BiTE triggering T cells to lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), or interleukin 1β (IL-1β) stimulated monocytes in PBMCs (Fig. 2B). In addition, flow cytometry analysis did not show specific binding of the B7H6 antibody on PBMCs or stimulated PBMCs (data not shown). Furthermore, the LPS, TNF-α, and IL-1β used in the experiments triggered a significant IL-6 mRNA upregulation but did not increase expression of B7H6 mRNA in these activated PBMCs (Fig. 2C).

Figure 2. B7H6-specific BiTEs trigger T cells to produce IFN-γ to primary human ovarian cancer but not pro-inflammatory monocytes.

Figure 2

Open in a new tab

(A) Activated human T cells were co-cultured with primary human ovarian cancer cells or K562 at an E:T ratio of 1:1 in the presence of murine IgG or B7H6-specific BiTE (250ng/mL). Blocking experiments were done by pre-incubating tumor samples with 10ug of murine IgG or anti-B7H6 mAbs for 45min before co-culturing. Cell-free medium was harvested after 24h and IFN-γ concentration determined by ELISA. Data shown are mean +SD of triplicate wells, and are representative of two independent experiments with different T cell donors. (B) Activated human T cells were co-cultured with unstimulated PBMCs or LPS, TNF-α, or IL-1β stimulated autologous PBMCs in the presence of murine IgG or B7H6-specific BiTE protein. Blocking experiments were done by pre-incubating target cells with 5ug of murine IgG or anti-B7H6 mAbs for 45min before co-culturing. Cell-free medium was harvested after 24h and IFN-γ concentration determined by ELISA. Data shown are representative of four different PBMC donors. (C) Unstimulated, LPS, TNF-α, or IL-1β stimulated PBMCs were harvested after 4h of stimulation. The amounts of IL-6 and B7H6 mRNA in each sample were measured by quantitative real-time PCR. The amount of mRNA in the unstimulated PBMCs is set to 1. Data shown are representative of two different PBMC donors. An * indicates p < 0.05; ** indicates p < 0.005; *** indicates p < 0.0001.

A murine B7H6-specific BiTE redirects primary murine T cells to kill B7H6+ tumor cells and secrete IFN-γ

To test whether a murine B7H6-specific BiTE triggered effector responses from murine T cells, B7H6+ tumor cells were co-cultured with murine T cells and increasing amounts of the murine activating B7H6-specific BiTE protein. Cytotoxicity assays showed that murine B7H6-specific BiTE triggered murine T cells to specifically kill B7H6+ tumors (Fig. 3A). In addition to direct tumor cytotoxicity, cytokines produced by T cells are also known to play important roles for in vivo therapeutic efficacy of immunotherapy (21–23). The murine B7H6-specific BiTE elicited a robust IFN-γ production by murine T cells when co-cultured with B7H6+ tumor cells (B16F10/B7H6) but not B7H6− tumor cells (RMA, B16F10, ID8) (Fig. 3B). The murine B7H6-specific BiTE also elicited a robust dose-dependent IFN-γ production by murine T cells when co-cultured with B7H6+ tumor cells (RMA/B7H6, B16F10/B7H6) but not a B7H6− tumor cell (RMA) (Fig. 3C).

Figure 3. Murine B7H6-specific BiTE redirect T cells to specifically kill B7H6+ tumor cells and secrete IFN-γ.

Figure 3

Open in a new tab

(A) Murine splenocytes were activated with concanavalin A (1µg/mL) and cultured with recombinant human IL-2 (25U/mL) for 4 to 6 days to generate murine T cells. Murine T cells were co-cultured with B7H6− tumor cells (RMA), or B7H6+ (RMA/B7H6, B16F10/B7H6, or ID8/B7H6) tumor cells at an E:T ratio of 5:1 for 6h. Specific lysis was quantified by LDH release assay. The data shown are mean +SD of quadruplicate wells and are representative of two independent experiments. (B) Murine T cells were co-cultured with B7H6− tumor cells (RMA, B16F10, ID8) or B7H6+ (B16F10/B7H6) tumor cells at an E:T ration of 4:1(105:2.5×104) at a concentration of 500ng/mL B7H6-specific BiTE. Cell-free medium was harvested after 24h and IFN-γ concentration determined by ELISA. The data shown are mean +SD of triplicate wells and are representative of two independent experiments. (C) Murine T cells were co-cultured with indicated tumors at an E:T ratio of 5:1 (B16F10/B7H6) or 1:1 (RMA, RMA/B7H6) at various concentrations of murine B7H6-specific BiTE (7.8ng/mL–500ng/mL). Cell-free medium was harvested after 24h and IFN-γ concentration determined by ELISA. The data are shown as mean +SD of triplicate wells and are representative of two independent experiments. Statistical analysis compares the IFN-γ value of B7H6 positive tumor cells (RMA/B7H6 or B16F10/B7H6) to B7H6 negative tumor cells (RMA) in each BiTE concentration. An * indicates p < 0.05; ** indicates p < 0.005; *** indicates p < 0.0001.

The B7H6-specific BiTE mediates therapeutic efficacy against lymphoma, melanoma, and ovarian cancer in vivo

The therapeutic efficacy of the murine B7H6-specific BiTE in clinically relevant murine tumor models was investigated. In a T cell lymphoma model, B6 mice injected with 105 RMA/B7H6 cells i.v. on day 0 were treated with 10ug of msIgG or murine B7H6-specific BiTE i.v. on days 3, 5, and 7. Data showed that murine B7H6-specific BiTE greatly improved the survival of tumor-bearing mice, and about half (40% to 60%) of the animals became long-term survivors (Fig. 4A & 4D). When this BiTE therapy was administered to a RMA lymphoma model, where tumor cells did not express B7H6, no therapeutic efficacy was observed. This finding indicated that BiTE indeed must bind to tumor cells to confer therapeutic efficacy (Fig. 4B). Furthermore, the long-term, tumor-free surviving mice were protected against tumor growth when challenged s.c. with RMA tumor cells (B7H6− cells), suggesting that this therapy elicited a broader host immunity against RMA tumor cells (Fig. 4C). To determine the role of host effector molecules in BiTE efficacy, the RMA/B7H6 tumor model was assessed using perforin-deficient or IFN-γ-deficient mice as hosts. The lack of either perforin or IFN-γ in the host resulted in a loss of protection compared to immune intact mice treated with the B7H6-specific BiTE protein. These data indicated that therapeutic efficacy was dependent on host perforin and IFN-γ activity (Fig. 4D), which is consistent with a role for BiTEs to activate T cell killing and cytokine production and inhibit tumor growth.

Figure 4. Murine B7H6-specific BiTE mediate therapeutic efficacy against lymphoma in a perforin and IFN-γ dependent manner.

Figure 4

Open in a new tab

(A) RMA/B7H6 cells (105 cells) were injected into mice i.v. on day 0. Murine IgG or B7H6-specific BiTE (10µg) was injected i.v. on days 3, 5, and 7. Kaplan-Meier survival curves are shown. Data shown are pooled results from two independent experiments (n = 12 for both groups). (B) RMA cells (105 cells) were injected into mice i.v. on day 0. Murine IgG or anti-B7H6 BiTE (10µg) was injected i.v. on days 3, 5, and 7. Kaplan-Meier survival curves are shown. (n = 6). (note the survival curves for both treatments were the same). (C) Long-term surviving mice from (A) were rechallenged with 2×104 RMA (B7H6−) s.c. on the right shaved flank and tumor area (mm2) was measured. Data shown are representative of two independent experiments (n = 5). Error bars represent SEM. (D) Wild-type B6, Perforin-deficient, and Ifn-γ-deficient mice were injected with 105 RMA/B7H6 cells i.v. on day 0. Murine IgG or B7H6-specific BiTE (20µg) was injected i.v. on days 3, 5, and 7. Kaplan-Meier survival curves are shown. Data shown are pooled results from two independent experiments (n = 11 or 12). An * indicates p < 0.05; ** indicates p < 0.005; *** indicates p < 0.0001.

To examine the therapeutic efficacy of the B7H6-specific BiTE in a melanoma model, mice were injected with B16F10/B7H6 s.c. on day 0 and treated with murine IgG or B7H6-specific BiTE i.v. on days 5, 7, 9, and 11. These data showed that systemic murine B7H6-specific BiTE administration significantly decreased the rate of tumor growth (Fig. 5A). The same BiTE protein administered to mice bearing a B16F10 melanoma, which did not express B7H6, did not confer therapeutic efficacy (Fig. 5B). As a model for ovarian cancer, mice were injected with ID8/B7H6 cells i.p. on day 0 and treated with murine IgG or B7H6-specific BiTE i.p. (days 7, 9, and 11) with or without additional T cells (5 × 106 T cells) on day 7. The number of solid tumors on the peritoneal wall and the number of free tumor cells in the i.p. wash were quantified on day 50. The data showed that murine B7H6-specific BiTE therapy significantly decreased overall ovarian cancer tumor burden in these tumor-bearing mice (Fig. 5C). These data demonstrated that B7H6-specific BiTE therapy mediates therapeutic efficacy against both hematological malignancy and solid tumors in vivo.

Figure 5. B7H6-specific BiTE protein mediates therapeutic efficacy in melanoma and ovarian cancer.

Figure 5

Open in a new tab

(A) B16F10/B7H6 (106 cells) were injected into mice s.c. on the shaved right flank on day 0, and the mice were treated with 10µg murine IgG or B7H6-specific BiTE i.v. (days 5, 7, 9, and 11). Tumor area was measured. Error bars represent SEM. Data shown are pooled results from two independent experiments (n = 12). (B) B16F10 (106 cells) were injected into mice s.c. on the shaved right flank on day 0, and the mice were treated with 10µg murine IgG or anti-B7H6 BiTE i.v. (days 5, 7, 9, and 11). Tumor area was measured. Error bars represent SEM (n = 6). (C) ID8/B7H6 cells (5×106 cells) were injected into mice i.p. on day 0, and the mice were treated with 10ug murine IgG or B7H6-specific BiTE i.p. (days 7, 9, and 11) with or without concanavalin A-activated and cultured T cells (5×106 cells, i.p. on day 7) as an additional source of effector T cells. The number of solid tumors on the peritoneal wall and suspension tumors in the i.p. wash were quantified on day 50. Data shown are pooled results from three independent experiments. Dots represent values from individual mice (n = 9 to 16). Mann-Whitney U test was used for statistical analysis. An * indicates p < 0.05; ** indicates p < 0.005.

Discussion

Bispecific antibodies (TriomAb format: Catumaxomab, Ertumaxomab; BiTE format: Blinatumumab) which trigger T cell effector functions have demonstrated promising therapeutic efficacy in several clinical trials (7). Regardless of the format, most bispecific antibody designs focus on targeting TAAs, such as epidermal growth factor receptor, human epidermal growth factor receptor 2, epithelial cell adhesion molecule, carcinoembryonic antigen, CD19, or CD20. These TAAs are upregulated by multiple tumor types, which enables targeting of antigen expressing tumors. However, these TAAs are also found on many normal tissues and cell types. This non-tumor exclusive expression pattern is likely to compromise the specificity of the treatment and may result in severe on-target off-tumor self reactivity. Self reactivity is a major concern for all targeted therapies. Due to the intrinsic self-amplifying characteristic of immune response, self-reactivity of BiTE therapy may be severe. Treatment with an anti-CD19 BiTE (Blinatumumab) results in elimination of peripheral B cells and B cell progenitors, which gradually recovers after treatment is completed (5, 6). This treatment also caused reversible symptoms in the central nervous system (5, 6).

Studies suggest that NKp30 plays an important role in mediating tumor immunosurveillance in several clinical settings. In acute myeloid leukemia, leukemic blasts actively down regulate NKp30 expression on NK cells to evade immune surveillance, and a natural cytotoxicity receptor (NCR)dull phenotype on NK cells correlates with poor prognosis (12). Gastrointestinal stromal tumor (GIST) patients expressing immunostimulatory isoforms of NKp30 have better prognosis than those patients with an immunosuppressive isoform (13). These observations highlighted the importance of NKp30 recognition and control of various tumor types and justify the approach of targeting B7H6, a cell surface NKp30 ligand, with BiTE therapy.

B7H6 is expressed on about 20% of human tumor cell lines and is also expressed on a subsets of primary leukemia, lymphoma (14). In addition, mRNA expression data suggest B7H6 is over-expressed on some ovarian cancers, breast cancers, brain tumors, renal cell carcinomas, and various sarcomas. B7H6 mRNA is not found on 48 normal tissues under steady state conditions (14). It has been reported that B7H6 can be induced on circulating pro-inflammatory monocytes in a sub-group of patients suffering severe sepsis or on monocytes after treatment in vitro with TLR agonists (20). However, we did not observe B7H6 expression on pro-inflammatory monocytes using the 47.39 mAb, which is used as the basis for the B7H6-specific scFv in this BiTE. Furthermore, the B7H6-specific BiTE did not trigger T cells to react to activated pro-inflammatory monocytes (Fig. 4B). These data suggest that B7H6 is a highly tumor specific antigen.

The data showed that B7H6-specific BiTEs can be readily produced by a mammalian cell expression system as monomers, which is consistent with others BiTEs in the literature (11, 24). It maintains the specificity of both scFvs and triggered T cells to mediate robust T cell effector mechanisms against B7H6 positive tumors but not negative tumors. BiTE treatment prolonged survival and most mice become long-term survivors. Treatment with the B7H6 specific BiTE was able to decrease tumor burden in two models of solid tumors. Solid tumors are often more difficult to treat than hematopoietic tumors. Solid tumors generally have a lower accessibility for T cells and BiTEs due to high local interstitial fluid pressure (25). BiTEs have a short circulating half-life (26), and to maintain optimal BiTE concentration, continuous i.v. pumps or slow release formulations can be used (5, 6). BiTEs should bind to T cells rapidly, so the absence of free BiTE protein in serum may not reflect the presence of BiTEs on patient T cells. The microenvironment in solid tumors can be immunosuppressive (27). To overcome low accessibility, one could actively enhance vascular and tumor permeability by administering tumor-infiltrating peptides (28) simultaneously with BiTE therapy. One could also utilize immunological check point blockade strategies, such as anti-CTLA4 or anti-PD1 mAbs, to revert exhausted tumor infiltrating lymphocytes and to enhance T cell infiltration (29, 30). These strategies and others may enhance BiTE therapeutic efficacy against solid tumors.

In conclusion, the data in this study show that B7H6-specific BiTEs directed T cells to mediate cytotoxicity and IFN-γ secretion against multiple B7H6 positive tumor cell lines. B7H6-specific BiTE therapy promoted the survival of lymphoma bearing mice in an IFN-γ and perforin dependent manner. To our best knowledge, this is the first direct evidence of IFN-γ and perforin mediating BiTE therapeutic efficacy in vivo. Furthermore, BiTE protein decreased tumor burden in melanoma and ovarian cancer bearing mice. The findings support the further development of B7H6-specific BiTE therapy for treatment of lymphoma, melanoma, and ovarian cancer.

Acknowledgments

The authors thank the staff of the Center for Comparative Medicine and Research in Dartmouth College for animal care and the National Cancer Institute Biological Resource Branch for providing recombinant human IL-2.

Disclosures

TZ and CS are inventors on a patent application covering the B7H6 BiTE described in this study. This technology has been licensed by Cardio3 Biosciences. This work is managed in compliance with the policies of Dartmouth College.

Abbreviations

BiTE

bispecific T cell engager

GIST

gastrointestinal stromal tumor

scFv

single-chain variable fragment

TAA

tumor associated antigen

Footnotes

1

Financial support: This work was supported by a grant from the NIH CA164178 and funds from the Center for Synthetic Immunity

References

  • 1.Dudley ME, Rosenberg SA. Adoptive-cell-transfer therapy for the treatment of patients with cancer. Nat Rev Cancer. 2003;3:666–675. doi: 10.1038/nrc1167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Baeuerle PA, Reinhardt C. Bispecific T-cell engaging antibodies for cancer therapy. Cancer Res. 2009;69:4941–4944. doi: 10.1158/0008-5472.CAN-09-0547. [DOI] [PubMed] [Google Scholar]
  • 3.Hoffmann P, Hofmeister R, Brischwein K, Brandl C, Crommer S, Bargou R, Itin C, Prang N, Baeuerle PA. Serial killing of tumor cells by cytotoxic T cells redirected with a CD19-/CD3-bispecific single-chain antibody construct. Int J Cancer. 2005;115:98–104. doi: 10.1002/ijc.20908. [DOI] [PubMed] [Google Scholar]
  • 4.Offner S, Hofmeister R, Romaniuk A, Kufer P, Baeuerle PA. Induction of regular cytolytic T cell synapses by bispecific single-chain antibody constructs on MHC class I-negative tumor cells. Mol Immunol. 2006;43:763–771. doi: 10.1016/j.molimm.2005.03.007. [DOI] [PubMed] [Google Scholar]
  • 5.Bargou R, Leo E, Zugmaier G, Klinger M, Goebeler M, Knop S, Noppeney R, Viardot A, Hess G, Schuler M, Einsele H, Brandl C, Wolf A, Kirchinger P, Klappers P, Schmidt M, Riethmuller G, Reinhardt C, Baeuerle PA, Kufer P. Tumor regression in cancer patients by very low doses of a T cell-engaging antibody. Science. 2008;321:974–977. doi: 10.1126/science.1158545. [DOI] [PubMed] [Google Scholar]
  • 6.Topp MS, Kufer P, Gokbuget N, Goebeler M, Klinger M, Neumann S, Horst HA, Raff T, Viardot A, Schmid M, Stelljes M, Schaich M, Degenhard E, Kohne-Volland R, Bruggemann M, Ottmann O, Pfeifer H, Burmeister T, Nagorsen D, Schmidt M, Lutterbuese R, Reinhardt C, Baeuerle PA, Kneba M, Einsele H, Riethmuller G, Hoelzer D, Zugmaier G, Bargou RC. Targeted therapy with the T-cell-engaging antibody blinatumomab of chemotherapy-refractory minimal residual disease in B-lineage acute lymphoblastic leukemia patients results in high response rate and prolonged leukemia-free survival. J Clin Oncol. 2011;29:2493–2498. doi: 10.1200/JCO.2010.32.7270. [DOI] [PubMed] [Google Scholar]
  • 7.Chames P, Baty D. Bispecific antibodies for cancer therapy: the light at the end of the tunnel? MAbs. 2009;1:539–547. doi: 10.4161/mabs.1.6.10015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Dhimolea E, Reichert JM. World Bispecific Antibody Summit, September 27–28, 2011, Boston, MA. MAbs. 2012;4:4–13. doi: 10.4161/mabs.4.1.18821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Lanier LL. NK cell recognition. Annu Rev Immunol. 2005;23:225–274. doi: 10.1146/annurev.immunol.23.021704.115526. [DOI] [PubMed] [Google Scholar]
  • 10.Kruse PH, Matta J, Ugolini S, Vivier E. Natural cytotoxicity receptors and their ligands. Immunol Cell Biol. 2014;92:221–229. doi: 10.1038/icb.2013.98. [DOI] [PubMed] [Google Scholar]
  • 11.Zhang T, Sentman CL. Cancer immunotherapy using a bispecific NK receptor fusion protein that engages both T cells and tumor cells. Cancer Res. 2011;71:2066–2076. doi: 10.1158/0008-5472.CAN-10-3200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Fauriat C, Just-Landi S, Mallet F, Arnoulet C, Sainty D, Olive D, Costello RT. Deficient expression of NCR in NK cells from acute myeloid leukemia: Evolution during leukemia treatment and impact of leukemia cells in NCRdull phenotype induction. Blood. 2007;109:323–330. doi: 10.1182/blood-2005-08-027979. [DOI] [PubMed] [Google Scholar]
  • 13.Delahaye NF, Rusakiewicz S, Martins I, Menard C, Roux S, Lyonnet L, Paul P, Sarabi M, Chaput N, Semeraro M, Minard-Colin V, Poirier-Colame V, Chaba K, Flament C, Baud V, Authier H, Kerdine-Romer S, Pallardy M, Cremer I, Peaudecerf L, Rocha B, Valteau-Couanet D, Gutierrez JC, Nunes JA, Commo F, Bonvalot S, Ibrahim N, Terrier P, Opolon P, Bottino C, Moretta A, Tavernier J, Rihet P, Coindre JM, Blay JY, Isambert N, Emile JF, Vivier E, Lecesne A, Kroemer G, Zitvogel L. Alternatively spliced NKp30 isoforms affect the prognosis of gastrointestinal stromal tumors. Nat Med. 2011;17:700–707. doi: 10.1038/nm.2366. [DOI] [PubMed] [Google Scholar]
  • 14.Brandt CS, Baratin M, Yi EC, Kennedy J, Gao Z, Fox B, Haldeman B, Ostrander CD, Kaifu T, Chabannon C, Moretta A, West R, Xu W, Vivier E, Levin SD. The B7 family member B7-H6 is a tumor cell ligand for the activating natural killer cell receptor NKp30 in humans. J Exp Med. 2009;206:1495–1503. doi: 10.1084/jem.20090681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Hollyoake M, Campbell RD, Aguado B. NKp30 (NCR3) is a pseudogene in 12 inbred and wild mouse strains, but an expressed gene in Mus caroli. Mol Biol Evol. 2005;22:1661–1672. doi: 10.1093/molbev/msi162. [DOI] [PubMed] [Google Scholar]
  • 16.Zhang T, Wu MR, Sentman CL. An NKp30-based chimeric antigen receptor promotes T cell effector functions and antitumor efficacy in vivo. J Immunol. 2012;189:2290–2299. doi: 10.4049/jimmunol.1103495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Zhang T, Lemoi BA, Sentman CL. Chimeric NK-receptor-bearing T cells mediate antitumor immunotherapy. Blood. 2005;106:1544–1551. doi: 10.1182/blood-2004-11-4365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Grundy MA, Zhang T, Sentman CL. NK cells rapidly remove B16F10 tumor cells in a perforin and interferon-gamma independent manner in vivo. Cancer Immunol Immunother. 2007;56:1153–1161. doi: 10.1007/s00262-006-0264-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Barber A, Zhang T, Sentman CL. Immunotherapy with chimeric NKG2D receptors leads to long-term tumor-free survival and development of host antitumor immunity in murine ovarian cancer. J Immunol. 2008;180:72–78. doi: 10.4049/jimmunol.180.1.72. [DOI] [PubMed] [Google Scholar]
  • 20.Matta J, Baratin M, Chiche L, Forel JM, Cognet C, Thomas G, Farnarier C, Piperoglou C, Papazian L, Chaussabel D, Ugolini S, Vely F, Vivier E. Induction of B7-H6, a ligand for the natural killer cell-activating receptor NKp30, in inflammatory conditions. Blood. 2013;122:394–404. doi: 10.1182/blood-2013-01-481705. [DOI] [PubMed] [Google Scholar]
  • 21.Barth RJ, Jr, Mule JJ, Spiess PJ, Rosenberg SA. Interferon gamma and tumor necrosis factor have a role in tumor regressions mediated by murine CD8+ tumor-infiltrating lymphocytes. J Exp Med. 1991;173:647–658. doi: 10.1084/jem.173.3.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Qin Z, Blankenstein T. CD4+ T cell--mediated tumor rejection involves inhibition of angiogenesis that is dependent on IFN gamma receptor expression by nonhematopoietic cells. Immunity. 2000;12:677–686. doi: 10.1016/s1074-7613(00)80218-6. [DOI] [PubMed] [Google Scholar]
  • 23.Zhang T, Barber A, Sentman CL. Chimeric NKG2D modified T cells inhibit systemic T-cell lymphoma growth in a manner involving multiple cytokines and cytotoxic pathways. Cancer Res. 2007;67:11029–11036. doi: 10.1158/0008-5472.CAN-07-2251. [DOI] [PubMed] [Google Scholar]
  • 24.Loffler A, Kufer P, Lutterbuse R, Zettl F, Daniel PT, Schwenkenbecher JM, Riethmuller G, Dorken B, Bargou RC. A recombinant bispecific single-chain antibody, CD19 × CD3, induces rapid and high lymphoma-directed cytotoxicity by unstimulated T lymphocytes. Blood. 2000;95:2098–2103. [PubMed] [Google Scholar]
  • 25.Heldin CH, Rubin K, Pietras K, Ostman A. High interstitial fluid pressure - an obstacle in cancer therapy. Nat Rev Cancer. 2004;4:806–813. doi: 10.1038/nrc1456. [DOI] [PubMed] [Google Scholar]
  • 26.Klinger M, Brandl C, Zugmaier G, Hijazi Y, Bargou RC, Topp MS, Gokbuget N, Neumann S, Goebeler M, Viardot A, Stelljes M, Bruggemann M, Hoelzer D, Degenhard E, Nagorsen D, Baeuerle PA, Wolf A, Kufer P. Immunopharmacologic response of patients with B-lineage acute lymphoblastic leukemia to continuous infusion of T cell-engaging CD19/CD3-bispecific BiTE antibody blinatumomab. Blood. 2012;119:6226–6233. doi: 10.1182/blood-2012-01-400515. [DOI] [PubMed] [Google Scholar]
  • 27.Shields JD, Kourtis IC, Tomei AA, Roberts JM, Swartz MA. Induction of lymphoidlike stroma and immune escape by tumors that express the chemokine CCL21. Science. 2010;328:749–752. doi: 10.1126/science.1185837. [DOI] [PubMed] [Google Scholar]
  • 28.Sugahara KN, Teesalu T, Karmali PP, Kotamraju VR, Agemy L, Greenwald DR, Ruoslahti E. Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs. Science. 2010;328:1031–1035. doi: 10.1126/science.1183057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Sakuishi K, Apetoh L, Sullivan JM, Blazar BR, Kuchroo VK, Anderson AC. Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity. J Exp Med. 2010;207:2187–2194. doi: 10.1084/jem.20100643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Curran MA, Montalvo W, Yagita H, Allison JP. PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors. Proc Natl Acad Sci U S A. 2010;107:4275–4280. doi: 10.1073/pnas.0915174107. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES