To target NK cells against non-Hodgkin’s lymphoma, we constructed a bispecific diabody (BsDb) with reactivity against both human CD19 and FcγRIII (CD16). Bacterially produced CD19 × CD16 BsDb specifically interacted with both CD19+ and CD16+ cells and exhibited significantly higher apparent affinity and slower dissociation from the tumor cells than from effector cells. It was able to induce specific lysis of tumor cells in the presence of isolated human NK cells or nonfractionated PBLs. The combination of the CD19 × CD16 BsDb with a previously described CD19 × CD3 BsDb and CD28 costimulation significantly increased the lytic potential of human PBLs. Treatment of SCID mice bearing an established Burkitt’s lymphoma (5 mm in diameter) with human PBLs, CD19 × CD16 BsDb, CD19 × CD3 BsDb, and anti-CD28 mAb resulted in the complete elimination of tumors in 80% of animals. In contrast, mice receiving human PBLs in combination with either diabody alone showed only partial tumor regression. These data clearly demonstrate the synergistic effect of small recombinant bispecific molecules recruiting different populations of human effector cells to the same tumor target.

Non-Hodgkin’s lymphoma (NHL)4 encompasses a heterogeneous group of hematological malignancies of B and T cell origin occurring in blood, lymph nodes, and bone marrow, which frequently disseminate throughout the body (1). NHL is one of the few malignancies that has increased in frequency more than the increase in population, with ∼53,000 new cases occurring annually in the United States (2). The most common forms of NHL are derived from the B cell lineage. While NHL can be treated with reasonable success at early and intermediate stages, the results of conventional chemotherapy and radiation in advanced stages remain disappointing. This particularly holds true for the prevalent low grade lymphomas. A fairly large number of patients relapse, and most remissions cannot be extended beyond the minimal residual disease. This discouraging situation has stimulated the search for alternative therapeutic strategies, such as activation of host immune mechanisms using bispecific Abs (BsAbs) (3). The BsAb makes a bridge between the tumor cell and the immune effector cell, followed by triggering the cytotoxic responses that include perforin and granzyme release, Fas-mediated apoptosis, and cytokine production. Since NHLs typically express one or more B cell markers, e.g., CD19 or CD20, these markers can be used to redirect effector cells toward malignant B cells. Although normal B cells will also be destroyed, they are repopulated from stem cells lacking the targeted Ags. To mediate redirected lysis, a BsAb must bind a target cell directly to a triggering molecule on the effector cell (4). The best-studied cytotoxic triggering receptors are multichain signaling complexes such as TCR/CD3 on T cells, FcγRIIIa (CD16) on NK cells, and FcγRI (CD64) and FcαRI (CD89) expressed by monocytes, macrophages, and granulocytes (3, 5). BsAbs directed to the TCR/CD3 complex have the potential to target all T cells regardless of their natural MHC specificity. To date, different forms of the CD19 × CD3 BsAb have been generated and used in a number of in vitro and in vivo therapeutic studies (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). These BsAbs have been mainly produced using rodent hybrid hybridomas (7, 8, 9) or by chemical cross-linking of two mAbs (6). However, the human anti-murine Ab response and release of inflammatory cytokines are the major drawbacks of BsAb derived from rodent mAbs in clinical use (18, 19). Recent advances in recombinant Ab technology have provided alternative methods for constructing and producing BsAb molecules (20, 21). For example, CD19 × CD3 single-chain variable fragment of Ab (scFv)-scFv tandems have been produced in mammalian cells (15). Alternatively, recombinant BsAbs can be formed by noncovalent association of two single-chain fusion products consisting of the VH and VL domains of different specificity in an orientation preventing intramolecular pairing with the formation of a four-domain heterodimer diabody (12, 22) or an eight-domain homodimer tandem diabody (17, 23). The two Ag binding domains have been shown by crystallographic analysis to be on opposite sides of the diabody such that they are able to cross-link two cells (24). Bispecific diabodies (BsDbs) are potentially less immunogenic than quadroma-derived BsAb and can be easily produced in bacteria in relatively high yield (16, 25). We have previously shown that CD3 × CD19 BsDbs are more effective than quadroma-derived BsAb in mediating T cell cytotoxicity in vitro against tumor cells (12, 16) and that they had similar antitumor activities in vivo (16, 17).

The aim of the present study was to target another subset of lymphocyte effectors, NK cells, against CD19-positive tumor cells. NK cells are one component of innate immunity and have the ability to both lyse target cells and provide an early source of immunoregulatory cytokines. Human NK cells comprise ∼15% of all lymphocytes and are defined phenotypically by their expression of CD56 and lack of expression of CD3 (26). The majority (∼90%) of human NK cells express CD56 at low density (CD56dim) and FcγRIII (CD16) at a high level (27). An effective CD16-mediated cytotoxicity induced by BsAb and BsDb has been documented for Hodgkin’s lymphoma (28, 29). To develop a similar approach for NHL, we constructed a recombinant anti-human CD19 × CD16 BsAb in a diabody format and examined its Ag-binding and antitumor activities both in vitro and in vivo.

The extracellular domain (ECD) of human FcγRIII (CD16) was a gift from Dr. G. P. Adams (Fox Chase Cancer Center, Philadelphia, PA). Human embryonic kidney (HEK) 293 cells stably transfected with human CD16B cDNA (293-CD16) were provided by Dr. R. E. Schmidt (Department of Clinical Immunology, Medical School Hannover, Hannover, Germany). Human CD19+ cell lines JOK-1 and Raji as well as CD3+ cell line Jurkat were from the cell line collection of the German Cancer Research Center. CD19 × CD3 BsDb was previously described (12, 16).

The genes coding for VH16-VL19 and VH19-VL16 hybrid scFvs were constructed by exchange of the anti-CD3 VH and VL genes in plasmids pHOG3-19 and pHOG19-3 (12) for their anti-human CD16 counterparts (29) using NcoI/HindIII and HindIII/XbaI restriction sites, respectively. The expression plasmid pKID19 × 16 containing dicistronic operon for cosecretion of two hybrid scFv was constructed by ligation of the BglII/XbaI restriction fragment from pHOG16-19 comprising the vector backbone and the BglII/XbaI fragment from pHOG19-16. CD19 × CD16 BsDb was produced in Escherichia coli XL1 Blue (Stratagene, La Jolla, CA) and was isolated from bacterial periplasmic extract and culture medium by ammonium sulfate precipitation, followed by immobilized metal affinity chromatography (IMAC), essentially as described for CD19 × CD3 BsDb (12). The final purification was achieved by ion exchange fast performance liquid chromatography on a Mono-Q HR 5/5 column (Amersham Pharmacia, Freiburg, Germany) in 20 mM Tris-HCl, pH 8.5, with a linear 0–1 M NaCl gradient. The fractions containing BsDb were concentrated with simultaneous buffer exchange for PBS containing 50 mM imidazole, pH 7.0, using an Ultrafree-15 centrifugal filter device (Millipore, Eschborn, Germany). Analysis of molecular forms of purified recombinant protein was performed by size exclusion fast performance liquid chromatography on a calibrated Superdex 200 HR 10/30 column (Amersham Pharmacia) as described previously (23).

Kinetic constants of interaction of CD19 × CD16 BsDb with ECD of human FcγRIII were determined by SPR using the BIAcore 2000 biosensor system (Biacore, Uppsala, Sweden). For immobilization on a streptavidin-coated sensor chip SA (Biacore), the CD16 ECD was biotinylated according to a modified protocol of the ECL protein biotinylation module (Amersham Pharmacia). As a negative control, biotinylated porcine tubulin was used. The biotinylated Ags diluted in HBS-EP buffer (10 mM HEPES, 0.15 M NaCl, 3 mM EDTA, and 0.005% polyoxyethylenesorbitan; Biacore) at a concentration of 10 μg/ml were applied to a sensor chip at a flow rate of 5 μl/min for 4 min, resulting in immobilization of 800 resonance units of CD16 ECD and 900 resonance units of tubulin. All SPR measurements were conducted at a flow rate of 20 μl/min in HBS-EP at 25°C. Analyses were performed at eight BsDb concentrations from 6.25–800 nM. Each injected sample (100 μl) was in contact with immobilized Ag for 5 min. The dissociation was followed for 10 min. After each cycle the surface of the sensor chip was flushed with the buffer. Kinetic constants were calculated according to a 1/1 (Langmuir) binding model using BIAevaluation version 3.0 software (Biacore).

The human CD19+ B cell line JOK-1 and 293-CD16 cells were used for flow cytometry experiments performed as previously described (12). In brief, 5 × 105 cells in 50 μl RPMI 1640 medium (Life Technologies, Eggestein, Germany) supplemented with 10% FCS and 0.1% sodium azide (referred to as complete medium) were incubated with 100 μl BsDb preparation for 45 min on ice. After washing with complete medium, the cells were incubated with 100 μl of 10 μg/ml anti-c-Myc mAb 9E10 in the same buffer for 45 min on ice. After a second washing cycle, the cells were incubated with 100 μl FITC-labeled goat anti-mouse IgG (Life Technologies) under the same conditions as before. The cells were then washed again and resuspended in 100 μl of a 1 μg/ml solution of propidium iodide (Sigma-Aldrich, Taufkirchen, Germany) in complete medium to exclude dead cells. The fluorescence of stained cells was measured using a FACScan flow cytometer (BD Biosciences, Mountain View, CA). Mean fluorescence (F) was calculated using CellQuest software (BD Biosciences), and background fluorescence was subtracted. Equilibrium dissociation constants (Keq) were determined as previously described (30) by fitting the experimental values to the Lineweaver-Burk equation 1/F = 1/Fmax + (Keq/Fmax)(1/[BsDb]) using the software program PRISM (GraphPad Software, San Diego, CA).

Cell surface retention assays were performed at 37°C under conditions preventing internalization of cell surface Ags as previously described (31), except that the detection of retained diabody was performed using anti-c-Myc mAb 9E10, followed by FITC-labeled anti-mouse IgG. The kinetic dissociation constant (koff) and t1/2 values for dissociation of BsDb were deduced from a one-phase exponential decay fit of experimental data using GraphPad PRISM.

Human PBMCs were isolated from the blood of healthy donors by Ficoll (Sigma-Aldrich) density gradient centrifugation. For cytotoxicity assays in vitro, cultures of PBMC were grown overnight in RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated FCS (Life Technologies), 2 mM glutamine, and recombinant human IL-2 (25 U/ml; Eurocetus, Amsterdam, The Netherlands). For animal experiments, PBLs were preactivated in vitro by overnight incubation with immobilized mAb OKT3 (anti-human CD3), soluble mAb 15E8 (anti-human CD28), and recombinant human IL-2 (20 U/ml). The NK cells were negatively enriched from human PBMCs by immunomagnetic depletion of human T cells, B cells, and myeloid cells using the NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) to a purity of up to 90%, as estimated by FACS analysis, and were not additionally stimulated.

The efficacy of the diabodies in mediating tumor cell lysis by human PBLs or NK cells was determined using the JAM test (32). The CD19-expressing Burkitt’s lymphoma cell line Raji was used as the target cell. For the cell kill assay, 105 effector cells were mixed in round-bottom microtiter plates with 104 target cells labeled with [3H]thymidine in 100 μl medium plus 50 μl diabody sample. After incubating the plate at 37°C in 5% CO2 for 4 h, the cells were harvested, and radioactivity was measured with a liquid scintillation beta counter (LKB, Wallach, Germany). Cytotoxicity related to the apoptosis-induced DNA fragmentation was calculated as % specific killing = (SE)/S × 100, where E is experimentally retained labeled DNA in the presence of killers (in cpm), and S is retained DNA in the absence of killers (spontaneous). The synergistic effect of BsDbs in vitro was analyzed using PBLs from three healthy donors using four different E:T cell ratios. Each measurement was performed in triplicate. For each E:T cell ratio, the paired groups of results were compared by a paired t test using GraphPad PRISM.

SCID mice were obtained from Charles River (Sulzfeld, Germany) and kept under specific pathogen-free conditions at the Central Animal Facilities of the German Cancer Research Center. In each experiment cohorts of five animals were used to permit accurate comparisons among differently treated groups. Mice were irradiated (300 rad) and received i.p. injections of 10 μl anti-asialo-GM1 mAb (WAKO, Neuss, Germany) according to the manufacturer’s suggestions. One day later, 107 Raji cells were injected s.c. dorsolaterally. Treatment was started after the tumors reached a size of 5 mm in diameter (day 0). On days 0, 7, and 15 the animals received i.v. injections of either PBS (control group) or 5 × 106 preactivated human PBLs. Four hours after each PBL injection either PBS or Ab combinations were administered via the tail vein. These combinations included 50 μg CD19 × CD3 BsDb plus 25 μg mAb 15E8, 50 μg CD19 × CD16 BsDb alone, or 25 μg CD19 × CD3 BsDb together with 25 μg CD19 × CD16 BsDb and 25 μg mAb 15E8. Tumor size was measured using a caliper every second day. Animals were followed until the s.c. tumors reached a maximal tolerated size of 15 mm in diameter and were killed by cervical dislocation. The days of sacrifice were recorded and were used for survival time analysis. The surviving animals were followed up to 60 days after the first treatment. For statistical evaluation, the follow-up duration of the tumor treatment experiment was 30 days (end of experiment). The median survival times were estimated by the method described by Kaplan and Meier (33). Differences between survival curves were compared using a log-rank test (34).

To target human NK cells against malignant B cells, we constructed a small recombinant molecule with dual specificity for both the human B cell surface Ag CD19 and ECD of FcγRIII (CD16). The scFv Ab fragments derived from hybridoma HD37 (35) and A9 (36) were used to create CD19 × CD16 BsDb (Fig. 1). BsDb is a heterodimer formed by noncovalent association of two hybrid scFvs consisting of the VH domain from one Ab connected by a short linker to the VL domain of another Ab. E. coli cells containing the plasmid pKID19 × 16 for simultaneous expression of both components of the BsDb were grown and induced under conditions favoring their dimerization (23). Recombinant molecules were isolated by IMAC from crude periplasmic extracts and culture medium. Due to the higher expression of the VH19-VL16 hybrid scFv, the samples of IMAC-purified heterodimeric diabody contained a significant amount of VH19-VL16 monomers and putative homodimers. The final separation of bispecific molecules was achieved by ion exchange chromatography. Purified BsDb was mainly in a dimeric form with an Mr of ∼60 kDa as demonstrated by gel filtration on a Superdex 200 column (Fig. 2,A). In contrast, the nonfunctional VH19-VL16 molecules were mainly monomeric with an Mr of 30 kDa (Fig. 2,A). SDS-PAGE analysis demonstrated that the BsDb could be resolved into two protein bands corresponding to the calculated Mr of 28,730 for VH16-VL19 scFv and 29,460 for VH19-VL16 scFv (Fig. 2 B).

FIGURE 1.

Schematic representation of operon encoding CD19 × CD16 BsDb in plasmid pKID19 × 16 and protein model of BsDb. The locations of wild-type lac promoter/operator (p/o), ribosome binding sites (rbs), pelB leader sequences (pelB), c-Myc epitope (c-myc), hexahistidine tag (His6), and stop codon (stop) are indicated. The amino acid sequence of the peptide linker between VH and VL domains is shown below the drawing. Carboxyl termini (COOH), linkers (L), and CD16 and CD19 Ag binding sites are indicated on the schematic model of BsDb.

FIGURE 1.

Schematic representation of operon encoding CD19 × CD16 BsDb in plasmid pKID19 × 16 and protein model of BsDb. The locations of wild-type lac promoter/operator (p/o), ribosome binding sites (rbs), pelB leader sequences (pelB), c-Myc epitope (c-myc), hexahistidine tag (His6), and stop codon (stop) are indicated. The amino acid sequence of the peptide linker between VH and VL domains is shown below the drawing. Carboxyl termini (COOH), linkers (L), and CD16 and CD19 Ag binding sites are indicated on the schematic model of BsDb.

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FIGURE 2.

Analyses of purified recombinant Abs. A, Elution profiles of CD19 × CD16 BsDb (solid line) and hybrid VH19-VL16 scFv (dotted line) from a calibrated Superdex 200 gel filtration column. B, Twelve percent SDS-PAGE under reducing conditions. Lane 1, Mr markers (kilodaltons, Mr in thousands); lane 2, VH19-VL16 scFv; lane 3, CD19 × CD16 BsDb. The gel was stained with Coomassie.

FIGURE 2.

Analyses of purified recombinant Abs. A, Elution profiles of CD19 × CD16 BsDb (solid line) and hybrid VH19-VL16 scFv (dotted line) from a calibrated Superdex 200 gel filtration column. B, Twelve percent SDS-PAGE under reducing conditions. Lane 1, Mr markers (kilodaltons, Mr in thousands); lane 2, VH19-VL16 scFv; lane 3, CD19 × CD16 BsDb. The gel was stained with Coomassie.

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The association and dissociation rate constants for the anti-CD16 moiety of CD19 × CD16 BsDb were measured by SPR using biotinylated CD16 ECD as an Ag. BsDb exhibited a fairly high off-rate from the CD16-coated sensor chip, thus making regeneration of the biosensor surface unnecessary. The calculated off- and on-rate constants were 2.3 × 10−2 and 2.7 × 104 s−1 M−1, respectively, resulting in a Kd of 8.5 × 10−7 M (Table I). A nearly identical affinity constant was deduced from the evaluation of steady state binding levels (Table I).

Table I.

Affinity and binding kinetics of CD19 × CD16 BsDb

Agkon (M−1s−1)koff (s−1)at1/2 (min)bKd (M)cKeq (M)d
JOK-1 cells (CD19+ND 1.1 × 10−3 10.6 ND 6.1 × 10−9 
293-CD16 cells (CD16+ND 3.2 × 10−3 3.6 ND 3.9 × 10−8 
CD16 ECD 2.7 × 104 2.3 × 10−2 0.5 8.5 × 10−7 8.7 × 10−7 
Agkon (M−1s−1)koff (s−1)at1/2 (min)bKd (M)cKeq (M)d
JOK-1 cells (CD19+ND 1.1 × 10−3 10.6 ND 6.1 × 10−9 
293-CD16 cells (CD16+ND 3.2 × 10−3 3.6 ND 3.9 × 10−8 
CD16 ECD 2.7 × 104 2.3 × 10−2 0.5 8.5 × 10−7 8.7 × 10−7 
a

Off-rate constants were either deduced from JOK-1 and 293-CD16 cell surface retention experiments or measured together with the on-rate constant (kon) by SPR using immobilized biotinylated CD16 ECD.

b

The t1/2 values for dissociation of diabody-Ag complexes were deduced from the ratio ln2:koff.

c

Affinity constants were calculated directly from the ratio koff:kon.

d

Equilibrium dissociation constants were deduced either from Lineweaver-Burk plots shown on Fig. 3 A or from the steady state analysis of SPR data.

Since the CD16 target Ag could be present in many orientations on the BIAcore chip, and some of its epitopes might be masked or destroyed due to biotinylation, the Kddetermined by SPR may not accurately reflect the binding of BsDb to the surface of effector cells. Besides, the Ag-binding properties of the anti-CD19 moiety of the CD19 × CD16 BsDb could not be characterized by SPR because of the lack of free CD19. Therefore, the apparent equilibrium (Keq) and off-rate (koff) constants were also determined for binding to cell surface-expressed CD19 and CD16 by flow cytometry. The flow cytometry experiments demonstrated a specific interaction of CD19 × CD16 BsDb with both CD19+ JOK-1 cells and 293-CD16 cells expressing ECD of human FcγRIII on their surface. The deduced Keq value for binding to JOK-1 cells was 6.5-fold lower than that for CD16-expressing cells (Fig. 3,A and Table I). To investigate the biological relevance of the differences in direct binding experiments, the in vitro retention of the BsDb on the surface of both CD19+ and CD16+ cells at 37°C was determined by flow cytometry (Fig. 3,B). CD19 × CD16 BsDb had a relatively short retention half-life (t1/2) on 293-CD16 cells (3.6 min) and a 3-fold longer t1/2 on the surface of CD19+ JOK-1 cells, thus correlating well with the lower CD16 binding affinity deduced from direct binding experiments (Table I). To determine whether the CD19 activity of BsDb is influenced by the second moiety of the bispecific molecule, we used CD19 × CD3 BsDb as a control in all flow cytometry experiments. Direct binding and cell surface retention on CD19+ JOK-1 cells were practically indistinguishable for both BsDbs. The calculated Keq and t1/2 values were 5.7 nM and 10.8 min, respectively, for CD19 × CD3 BsDb, and 6.1 nM and 10.6 min for CD19 × CD16 BsDb. These results indicate that the second specificity present in the BsDb molecule does not significantly affect the affinity for binding to CD19.

FIGURE 3.

Flow cytometric analysis of CD19 × CD16 BsDb binding to CD19+ JOK-1 cells (▴) and CD16+ 293-CD16 cells (○). A, Lineweaver-Burk analysis of fluorescence dependence on BsDb concentration. B, In vitro cell surface retention assay. Values are expressed as a percentage of the initial mean fluorescence intensity.

FIGURE 3.

Flow cytometric analysis of CD19 × CD16 BsDb binding to CD19+ JOK-1 cells (▴) and CD16+ 293-CD16 cells (○). A, Lineweaver-Burk analysis of fluorescence dependence on BsDb concentration. B, In vitro cell surface retention assay. Values are expressed as a percentage of the initial mean fluorescence intensity.

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The ability of the CD19 × CD16 BsDb to induce tumor cell lysis by redirecting NK cell-mediated cytotoxicity was investigated using a JAM test, which is based on measuring DNA fragmentation in the target cell as a result of apoptosis (32). The death of CD19+ Raji cells in the presence of freshly prepared PBLs from a healthy donor was specifically triggered by CD19 × CD16 BsDb in a dose-dependent manner, resulting in 45% of specific killing at a BsDb concentration of 5 μg/ml and an E:T cell ratio of 50:1 (Fig. 4,A). Substitution of PBLs by NK cells isolated from the blood of the same donor further increased the cytotoxic effect of CD19 × CD16 BsDb up to 60% under the same conditions (Fig. 4,B). To examine the cytotoxic potential of different effector cell populations retargeted by BsDb, we used PBLs from three healthy donors in combination with CD19 × CD16 BsDb, CD19 × CD3 BsDb, or both of them. We observed more tumor cell killing for each donor using a diabody combination than for any BsDb alone, although the absolute values of specific killing differed according to the donor. For example, at an E:T cell ratio of 25:1, CD19 × CD16 BsDb alone, CD19 × CD3 BsDb alone, and a combination of both resulted in 2.1, 10.6, and 26.3% specific killing for donor 1; 37.3, 30.8, and 39.4% for donor 2; and 20.2, 21.7, and 41.4% for donor 3, respectively. For analyzing the results we used a paired t test, which compares two paired groups and calculates the t ratio, p value, and confidence interval based on the differences between each set of pairs. The results shown in Fig. 4 C demonstrated that both BsDbs possessed fairly similar cytotoxic activities when used alone and exhibited much higher activities when used together. There was no significant difference between the values of specific killing obtained using each BsDb alone (p = 0.1528). In contrast, the killing curve for the diabody combination differed significantly from those for CD19 × CD16 and CD19 × CD3 BsDb alone (p = 0.0068 and 0.0408, respectively).

FIGURE 4.

BsDb-mediated lysis of CD19+ Raji cells by human PBLs or NK cells at different E:T cell ratios. A and B, Dose-dependent lysis of tumor cells by PBLs (A) or NK cells (B) in the presence of CD19 × CD16 BsDb at concentrations of 0.5, 1, and 5 μg/ml. C, Lysis of tumor cells by human PBLs mediated either by CD19 × CD16 BsDb or CD19 × CD3 BsDb alone at a concentration of 5 μg/ml or by combination of both BsDb at a concentration of 2.5 μg/ml. Experiments were performed in triplicate; bars represent SDs of measurements for three different donors.

FIGURE 4.

BsDb-mediated lysis of CD19+ Raji cells by human PBLs or NK cells at different E:T cell ratios. A and B, Dose-dependent lysis of tumor cells by PBLs (A) or NK cells (B) in the presence of CD19 × CD16 BsDb at concentrations of 0.5, 1, and 5 μg/ml. C, Lysis of tumor cells by human PBLs mediated either by CD19 × CD16 BsDb or CD19 × CD3 BsDb alone at a concentration of 5 μg/ml or by combination of both BsDb at a concentration of 2.5 μg/ml. Experiments were performed in triplicate; bars represent SDs of measurements for three different donors.

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To examine whether the synergistic effect of CD19 × CD16 BsDb and CD19 × CD3 BsDb could also be observed in vivo, we established a xenotransplant model of the Raji Burkitt’s lymphoma in SCID mice. Raji cells after s.c. injection gave rise to locally growing tumors. Treatment was started when the tumors reached a size of 5 mm in diameter. On days 0, 7, and 15 cohorts of five mice received i.v. either PBS (control group) or in vitro preactivated human PBLs. Four hours after each PBL inoculation, the mice were treated with no Ab, with CD19 × CD16 BsDb alone, or with CD19 × CD3 BsDb in combination with anti-human CD28 mAb 15E8 administered as a tail vein injection. The fifth animal group received the combination of CD19 × CD16 BsDb, CD19 × CD3 BsDb, and mAb 15E8. The total amount of injected BsDb was the same in all Ab-treated groups (50 μg (∼1 nmol)/animal). None of the animals in the control groups receiving PBS or PBLs alone showed any tumor suppression and developed tumors larger than 1.5 cm in diameter in <3 wk (Fig. 5). There was no significant difference between tumor growth in mice receiving PBS and mice receiving activated PBLs alone, which indicated that under the conditions used, any allogeneic reaction of the effector cells toward the tumor could be ignored. The animals were sacrificed when the tumors reached the maximum tolerated size of 15 mm in diameter. Sacrifice dates were recorded, and median survival was calculated for each group (Fig. 6). The median survival times were not significantly different in the control groups receiving PBS and human PBLs alone at 21.5 and 23 days, respectively (p = 0.4469).

FIGURE 5.

Treatment of SCID mice bearing human Burkitt’s lymphoma xenografts. The mice received PBS (□), preactivated human PBLs alone (▪), or preactivated human PBLs followed 4 h later by the administration of CD19 × CD3 BsDb plus mAb 15E8 (○), CD19 × CD16 BsDb alone (•), or CD19 × CD16 BsDb in combination with CD19 × CD3 BsDb and mAb 15E8 (▴). Tumor size was measured every second day. Tumor growth curves of individual animals up to 30 days of the experiment are presented.

FIGURE 5.

Treatment of SCID mice bearing human Burkitt’s lymphoma xenografts. The mice received PBS (□), preactivated human PBLs alone (▪), or preactivated human PBLs followed 4 h later by the administration of CD19 × CD3 BsDb plus mAb 15E8 (○), CD19 × CD16 BsDb alone (•), or CD19 × CD16 BsDb in combination with CD19 × CD3 BsDb and mAb 15E8 (▴). Tumor size was measured every second day. Tumor growth curves of individual animals up to 30 days of the experiment are presented.

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FIGURE 6.

Survival of SCID mice bearing human Burkitt’s lymphoma xenografts. The mice received PBS (□), preactivated human PBLs alone (▪), or preactivated human PBLs followed 4 h later by the administration of CD19 × CD3 BsDb plus mAb 15E8 (○), CD19 × CD16 BsDb alone (•), or CD19 × CD16 BsDb in combination with CD19 × CD3 BsDb and mAb 15E8 (▴).

FIGURE 6.

Survival of SCID mice bearing human Burkitt’s lymphoma xenografts. The mice received PBS (□), preactivated human PBLs alone (▪), or preactivated human PBLs followed 4 h later by the administration of CD19 × CD3 BsDb plus mAb 15E8 (○), CD19 × CD16 BsDb alone (•), or CD19 × CD16 BsDb in combination with CD19 × CD3 BsDb and mAb 15E8 (▴).

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In contrast to control groups, all mice receiving BsDb demonstrated significant tumor regression. The animals receiving three injections of CD19 × CD3 BsDb in combination with anti-CD28 mAb displayed a minimal tumor size on days 12–15, when three of five mice were tumor free. Afterward, the tumors began to reappear and grew progressively in two animals (Fig. 5). One animal remained tumor-free until the end of monitoring (day 60 after the first treatment). Similar results were obtained for mice receiving CD19 × CD16 BsDb alone. In this group all animals also demonstrated tumor regression until days 15–20, when two mice were tumor free. Afterward, however, the tumors started to grow again, with comparable rates in all animals in this group (Fig. 5). The median survival times calculated for the groups receiving CD19 × CD3 BsDb plus mAb 15E8 and CD19 × CD16 BsDb alone were not significantly different (33 and 32.5 days, respectively; p = 0.67), but were significantly different from those in the control groups (p < 0.01).

Survival was significantly improved in the group receiving the combination of CD19 × CD16 BsDb, CD19 × CD3 BsDb, and anti-CD28 mAb, where four of five animals had no palpable tumors after the second injection (day 12, Fig. 5). These mice remained disease free during the entire experiment (30 days) and even 60 days after the first treatment. Compared with the other treatment groups this result was statistically significant (CD19 × CD3 BsDb plus mAb 15E8, p < 0.05; CD19 × CD16 BsDb, p < 0.01) and was extremely significant in comparison with control groups (p < 0.001). These in vivo data clearly confirm the synergistic antitumor effect of CD19 × CD16 BsDb and CD19 × CD3 BsDb, which recruit different populations of human effector cells to the same tumor target.

Although chemotherapy and radiation therapy can induce clinical remissions in patients with NHL, this group of malignancies remains a therapeutic challenge due to frequent lymphoma relapse and chemotherapy resistance. The malignant cells not destroyed by cytotoxic therapy appear to be responsible for treatment failure. These remaining tumor cells, referred to as minimal residual disease, are major targets for immunotherapeutic strategies, which include retargeting the cellular effector systems, such as T lymphocytes, NK cells, or myeloid cells by BsAbs. Others and we ourselves have previously demonstrated the antitumor efficacy of CD19 × CD3 BsAbs in vitro (9, 12, 15), in animal models (16, 17), and in phase I clinical trials (18, 19). However, CD3-based immunotherapy requires additional stimulation of the T cell population via a signal delivered by a distinct coreceptor (37). Unlike T cells, FcR-bearing cellular mediators of innate immunity, e.g., NK cells, monocytes, macrophages, and granulocytes, tend to exist in constitutively activated states and do not need additional (pre-) stimulation (4). In the present report we have generated for the first time the CD19 × CD16 BsAb in a diabody format and investigated the potential of combinatory immunotherapy by retargeting both human NK cells and T cells to the same tumor site.

The CD19 × CD16 BsDb was produced in bacteria in a soluble functional form by cosecretion of two hybrid scFv fragments encoded by a dicistronic operon. Unlike the previously constructed CD19 × CD3 and CD30 × CD16 BsDbs (12, 29), nonequal amounts of the hybrid scFvs were found to be expressed in bacteria. Surprisingly, the scFv19–16 consisting of anti-CD19 VH connected to the anti-CD16 VL through a short 10-aa linker did not appear to form any homodimer and remained monomeric. It could, therefore, be easily separated from functional BsDb by ion exchange chromatography or size exclusion chromatography. CD19 × CD16 BsDb specifically interacted with cell surface-bound CD19 with an affinity in the nanomolar concentration range, nearly identical with that of the previously generated CD19 × CD3 BsDb. In contrast, BsDb binding to CD16 was 6.5-fold weaker. Characterization of the binding kinetics revealed that the half-life of retention was fairly short, 3.6 min and 30 s for CD16-positive cells and recombinant CD16, respectively (Table I). The discrepancy in koffvalues obtained in cell surface retention experiments and by SPR measurements could be explained if the epitope recognized by the anti-CD16 moiety of CD19 × CD16 BsDb was either presented differently in recombinant CD16 ECD or was partially masked by biotinylation of the Ag. Due to its rapid off-rate, CD19 × CD16 BsDb would be expected to bind transiently to effector cells, thus allowing it to engage many FcγRs in successive rounds of ligation, triggering, and dissociation. Such relatively strong binding to a target tumor cell and weaker binding to an effector cell may have certain advantages for tumor therapy. For example, low affinity for FcγRIII may reduce the toxicity caused by the binding and potential triggering by BsDb of peripheral blood cells expressing FcγRIII.

Besides the Ab fragments derived from the anti-human CD16 hybridoma A9 (Ref. 29 and this study), an scFv NM3E2 of the same specificity isolated from a human scFv phage display library was successfully used for making recombinant bispecific molecules (38, 39). The binding characteristics of the CD19 × CD16 BsDb to immobilized CD16 ECD appeared to be fairly similar to those of scFv NM3E2 when measured by SPR. Although SPR can be successfully used for ranking Abs of the same specificity (see, for example, the comparison of HER2/neu × CD16 (scFv)2 in Ref. 39), we clearly demonstrated here that the affinity values deduced from BIAcore measurements should be interpreted with caution, especially when dealing with Abs against cell surface Ags.

In vitro experiments demonstrated that CD19 × CD16 BsDb was able to effectively recruit human PBLs for killing CD19-positive lymphoma cells in a concentration-dependent manner. Using enriched human NK cells instead of PBLs led to further augmentation of BsDb-mediated lysis of tumor cells. The lysis of lymphoma cells by human PBLs could also be mediated by CD19 × CD3 BsDb (12, 16). In the present report we clearly demonstrated a synergistic effect in vitro of bispecific molecules recruiting different effector cells (NK and T cells) against the same tumor Ag.

One of the main goals of the present study was to compare the therapeutic efficacies in vivo of two recombinant bispecific molecules prepared in the same diabody format. The antitumor potency of CD19 × CD16 and CD19 × CD3 BsDbs was tested in a fairly stringent model of SCID mice bearing an established s.c. growing human B cell lymphoma (17). CD19 × CD3 BsDb was used together with an anti-CD28 mAb providing a costimulatory signal for the efficient activation of T cells (37). Cytotoxic T cells mediated by CD19 × CD3 BsDb possessed a somewhat higher antitumor activity than NK cells retargeted by CD19 × CD16 BsDb, although the observed difference was not significant. These results correlate with the observation that a combination of T cell-activating CD30 × CD3 and CD30 × CD28 BsAbs was more effective than CD30 × CD16 BsAb in a preclinical model of Hodgkin’s disease (40, 41). A combination of human PBLs with CD19 × CD16 BsDb, CD19 × CD3 BsDb, and anti-CD28 mAb led to the complete cure of an established Burkitt’s lymphoma in four of five tested animals. The demonstrated synergistic effect illustrates the favor of a combinatory immunotherapeutic approach exploiting different populations of effector cells.

Besides NK cells, CD19 × CD16 BsDb can recruit other effector cells, such as monocytes/macrophages, a subpopulation of which is CD16+ (5). CD16-directed BsAbs can also bind the GPI-linked isoform of FcγRIII (FcγRIIIb) on polymorphonuclear granulocytes (PMNs), which cannot trigger tumor cell killing. An analysis of the interactions of antitumor × anti-CD16 BsAbs with human neutrophils demonstrated that, on the one hand, the BsAb did not promote the lysis of target cells by PMNs, and, in contrast, PMNs did not inhibit BsAb-mediated cytotoxicity (42). Therefore, although human neutrophils may serve as a significant competitive binding pool of systemically administered CD16-directed BsAbs in vivo, the therapeutic potential of the targeted cytotoxicity properties of these BsAbs does not appear to be compromised. This was also confirmed by preclinical studies and phase I/II clinical trials of patients with refractory Hodgkin’s disease treated with HRS-3/A9 (CD30 × CD16) BsAb (28, 43).

In addition to the direct action of two BsDbs recruiting different effector cells to the tumor site, other factors may contribute to the observed synergistic effect. In vitro and in vivo studies suggest that NK cell-mediated and CTL-mediated cytotoxic systems regulate the functions of each other (44). Previously, we demonstrated that the CD19 × CD3 BsDb was able to activate T cells in the presence of CD19+ tumor cells (16, 23). T cell activation caused by antitumor × anti-CD3 BsAb and CD28 costimulation, followed by killing of malignant cells, are accompanied by the release of cytokines, such as TNF-α (cachectin), IFN-γ, IL-1β, IL-2, IL-6, IL-8, and IL-10 (45, 46). The CD56bright human NK cell subset proliferates profusely in response to low doses of IL-2 secreted by activated T cells and can, therefore, be selectively expanded (27). Importantly, since most of those expanded CD56bright NK cell populations express CD16, they can efficiently mediate Ag-dependent cellular cytotoxicity (47). Following activation, NK cells are able to migrate in response to additional CC and CXC chemokines expressed by tumor-infiltrating lymphocytes (48). Some chemokines also increase their cytolytic activity. Activated NK cells themselves produce XCL1, CCL1, CCL3, CCL4, CCL5, CCL22, and CXCL8 chemokines that can recruit other effector cells (49). In addition, CD56bright NK cells appear to have an intrinsic capacity for high level production of NK-derived immunoregulatory cytokines, including IFN-γ, TNF-β (lymphotoxin), IL-10, IL-13, and GM-CSF (27). It might well be that these cytokines exert a direct tumoricidal effect and may even attract further effector cells to the tumor site. In contrast, some findings indicate that activated T cells produce several factors that could inhibit NK cell activity, such as IL-4 and TGF-β, and therefore down-regulate and limit NK cell responses (44). The cellular mechanisms underlying the BsDb synergy observed in the present study may thus be quite complex. They will be addressed in additional experiments.

To date, most complications associated with using BsAb in clinical trials are caused by the murine origin of BsAbs. Most of the BsAb-treated patients develop a human anti-murine Ab response and/or suffer from toxicities caused by nonspecific cytokine release due to FcR binding of mouse Igs to cells of the monocyte/phagocyte system (18, 50). Although different application schemes might reduce murine BsAb immunogenicity to a certain degree (50), a better solution to this problem can be achieved by Ab engineering. The efficacy of the immune recruiting capacity of BsDbs (Refs. 16 and 29 and present study) suggests that they can be used to replace BsAbs for immunotherapy. The binding and pharmacokinetic properties of BsDb can be further improved by converting it to the tetravalent tandem diabody format (17, 23). This would facilitate larger clinical trials using repetitive cycles of sufficiently dosed Ab applications with more extensive cycles of treatments, since these recombinant molecules are less immunogenic and can be produced and purified at relatively low cost.

1

This work was supported by the German BioRegio Program (Grant BEO32/AZ12389).

4

Abbreviations used in this paper: NHL, non-Hodgkin’s lymphoma; BsAb, bispecific Ab; BsDb, bispecific diabody; ECD, extracellular domain; IMAC, immobilized metal affinity chromatography; PMN, polymorphonuclear granulocyte; scFv, single-chain variable fragment of Ab; SPR, surface plasmon resonance.

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