[CANCER RESEARCH 60, 4336 – 4341, August 15, 2000]
Advances in Brief
Cure of Burkitt’s Lymphoma in Severe Combined Immunodeficiency Mice by T Cells, Tetravalent CD3 ⴛ CD19 Tandem Diabody, and CD28 Costimulation1 Bjo¨rn Cochlovius,2 Sergey M. Kipriyanov,2,3,4 Marike J. J. G. Stassar, Jochen Schuhmacher, Axel Benner, Gerhard Moldenhauer, and Melvyn Little3 Recombinant Antibody Research Group [B. C., S. M. K., M. L.], Department of Tumor Progression and Immune Defense [M. J. J. G. S.], Department of Diagnostic and Therapeutic Radiology [J. S.], Central Unit Biostatistics [A. B.], and Department of Molecular Immunology [G. M.], German Cancer Research Center (DKFZ), D-69120 Heidelberg, Germany
Abstract To increase the valency, stability, and therapeutic potential of bispecific antibodies, we have constructed a tetravalent tandem diabody (Tandab) that is specific to both human CD3 (T-cell antigen) and CD19 (B-cell marker; S. M. Kipriyanov et al., J. Mol. Biol., 293: 41–56, 1999). It was generated by the functional dimerization of a single chain molecule that contained four antibody variable domains (VH and VL) in an orientation that prevented intramolecular pairing. Compared with a previously constructed heterodimeric CD3 ⴛ CD19 diabody, the Tandab exhibited a higher apparent affinity to both CD3ⴙ and CD19ⴙ cells and longer blood retention when injected into mice. Biodistribution studies in mice bearing Burkitt’s lymphoma xenografts demonstrated specific accumulation of the radioiodinated Tandab in a tumor site with tumor-to-blood ratios of 1.5, 8.1, and 13.3 at 3, 18, and 24 h, respectively. Treatment of severe combined immunodeficiency mice bearing established Burkitt’s lymphoma (5 mm in diameter) with human peripheral blood lymphocytes, Tandab, and antiCD28 MAbs resulted in the complete elimination of tumors in all of the animals within 10 days. In contrast, mice receiving human peripheral blood lymphocytes in combination with either the diabody alone or the diabody plus anti-CD28 MAbs showed only partial tumor regression. These data demonstrate that the CD3 ⴛ CD19 Tandab may be a promising tool for the immunotherapy of human B-cell leukemias and lymphomas.
have provided several alternative methods for constructing and producing BsAb molecules (7). But in contrast to native antibodies, most of the recombinant bispecific molecules have only one binding domain for each specificity. However, bivalent binding is an important means of increasing the functional affinity and possibly the selectivity for particular cell types carrying densely clustered antigens. To increase the valency, stability and therapeutic potential of recombinant bispecific antibodies, we constructed a novel tetravalent BsAb with Mr 113,000, described by us as a Tandab that is specific for both human CD3- and CD19-cell surface antigens (8). The CD3 ⫻ CD19 Tandab demonstrated higher avidity to target cells, increased potency to mediate tumor cell lysis by human PBL in vitro, increased stability in human serum, and longer blood retention compared with the bivalent CD3 ⫻ CD19 diabody (8). In this study, we have focused on the in vivo application of the CD3 ⫻ CD19 Tandab. The biodistribution, pharmacokinetic behavior, and antitumor activity of this molecule was analyzed in immunodeficient mice bearing s.c. growing Burkitt’s lymphomas. Materials and Methods
Production of Recombinant Protein. The bivalent CD3 ⫻ CD19 diabody as well as the tetravalent Tandab containing a 12-residue SL-linker were expressed in Escherichia coli RV308 induced in a rich 2YT medium containIntroduction ing 1 M sorbitol and 2.5 mM glycine betaine (8). The recombinant proteins were isolated from soluble periplasmic extracts by IMAC followed by ion-exchange 5 BsAbs have a significant potential for cancer therapy because they chromatography on a Mono S HR5/5 column (Pharmacia Biotech, Freiburg, can be used to retarget cytotoxic effector cells against tumor cells (1). Germany), as described previously (8, 9). Analysis of molecular forms of CTLs, for example, can be recruited for killing tumor cells if they are purified recombinant protein was performed by size-exclusion FPLC on a activated by a BsAb that binds both to the CD3 antigen associated calibrated Superdex 200 HR10/30 column (Pharmacia), as described previwith the T-cell receptor complex and to the target cell. Clinical studies ously (8). Analysis of Tandab Stability in Vitro. Homogeneous Tandab preparation showed tumor regression in patients treated with an anti-CD3 ⫻ antitumor BsAb (2). One of the best targets for bispecific antibodies on [1 mg/ml in PBSI (PBS containing 50 mM imidazole), pH 7.0] was sterilized malignant human B cells is CD19 (3). Thus far, bispecific antibodies by filtration through a Membrex 4CA filter with a void volume of 50 l and have mainly been produced using murine hybrid hybridomas (4) or by a pore size of 0.2 m (MembraPure, Lo¨rzweiler, Germany). Aliquots (90 l) were immediately prepared under sterile conditions and stored at 37°C. At chemical cross-linking (5). However, the immunogenicity of BsAbs given time points, the aliquots were removed from an incubator and centriderived from rodent monoclonal antibodies is a major drawback for fuged for 10 min to remove aggregated and precipitated material. Both soluble clinical use (6). Recent advances in recombinant antibody technology and precipitated protein was analyzed by 12% SDS-PAGE followed either by Coomassie staining or by Western blot analysis using anti-c-myc MAb 9E10 as Received 11/18/99; accepted 6/28/00. described previously (10). Fifty l of cleared material were used for analytical The costs of publication of this article were defrayed in part by the payment of page size-exclusion FPLC on a Superdex 200 HR10/30 column (Pharmacia). charges. This article must therefore be hereby marked advertisement in accordance with Radioiodination and Measurement of Stability in Vivo. Tandab was 18 U.S.C. Section 1734 solely to indicate this fact. 1 labeled with 125I using IODO-Gen (Pierce, Rockford, IL) as described previSupported by Deutsche Krebshilfe/Mildred Scheel Stiftung. 2 B. C. and S. M. K. contributed equally to this work. ously (8). The final specific activity of 125I-Tandab was 3.75 mCi/mg. Radio3 Present address: Affimed Therapeutics AG, Dr. Albert-Reimann Strasse 2, D-68526 labeled Tandab retained greater than 80% immunoreactivity, as evaluated in a Ladenburg, Germany. live-cell binding assay using CD19⫹/CD3⫺ JOK-1 and CD3⫹/CD19⫺ Jurkat 4 To whom requests for reprints should be addressed, at the Affimed Therapeutics AG, cells. For an analysis of stability in vivo, 200 l of PBSI containing 10 g of c/o BK Giulini Chemie, Dr. Albert-Reimann Strasse 2, D-68526 Ladenburg, Germany. human serum albumin and 5 g of 125I-Tandab were injected into the tail veins Email:
[email protected]. 5 The abbreviations used are: BsAb, bispecific antibody; MAb, monoclonal antibody; of male NMRI mice, each weighing ⬃40 g (8). At 10, 20, 40, and 90 min after %ID/g, percent injected dose per gram; SCID, severe combined immunodeficiency; injection, animals in triplicates were anesthetized, bled, and killed in accordTandab, tandem diabody; T:O, tumor:organ; PBL, peripheral blood lymphocyte; IMAC, ance with local animal protection laws. Blood (100 l) from each sacrificed immobilized metal affinity chromatography; FPLC, fast protein liquid chromatography; animal was pooled for each time point and was mixed with 15 l of heparin NK, natural killer; TCR, T-cell receptor. 4336
CURE OF B-CELL LYMPHOMA BY CD3 ⫻ CD19 TANDAB
(5000 IU/ml; Braun Melsungen AG, Melsungen, Germany) followed by sedimentation of cellular material. Ten l of cleared plasma were mixed with 25 l of 20% SDS and 12 l of 4⫻ Laemmli sample buffer (11). After boiling for 10 min, plasma samples (⬃5 ⫻ 104 cpm) were analyzed by 12% SDS-PAGE followed by autoradiography. Biodistribution Studies. All animal experiments were performed according to the guidelines of the German Cancer Research Center and the animalprotection laws of the Bundesland Baden-Wu¨rttemberg and the Federal Republic of Germany. Ten-week-old Rag2-deficient female mice were obtained from the Central Animal Facilities of the German Cancer Research Center. To reduce inherent NK cell activity, the animals were irradiated (300 rad) 1 day before tumor inoculation and, on every 5th day of the whole experiment, received i.p. injections of 25 g of MAb PK136 (anti-interleukin 2 receptor). Burkitt’s lymphoma Raji cells (108) in log phase were implanted s.c. dorsolaterally in the mice. After 3 weeks, when the tumors weighed 200 –500 mg, a 0.04% potassium iodide solution was placed in the drinking water to block thyroid accumulation of radioiodine. One day later, the mice were given injections of 200 l of PBSI containing 10 g of human serum albumin and 5 g of labeled antibody fragment in the tail vein. The animals in triplicate were anesthetized and killed in accordance with local animal protection laws at 1, 3, 6, 18, and 24 h after injection. The tumors and organs were removed, weighed, and counted in a gamma counter to determine the percentage of the injected dose localized per gram of tissue (%ID/g). The blood pharmacokinetics was evaluated by the two-phase exponential decay fit of blood retention data, performed using GraphPad Prism (GraphPad Software, San Diego, CA). Treatment of Burkitt’s Lymphoma in SCID Mice. The SCID mice were obtained from Charles River (Sulzfeld, Germany) and were 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) 1 day prior to tumor inoculation and received i.p. injections of 10 l of anti-asialo-GM1 antibody 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). At days 0, 7, and 15, the animals received i.v. injections of either PBS (control group) or 5 ⫻ 106 human PBLs that were preactivated in vitro by immobilized MAb OKT3 (anti-CD3), soluble MAb 15E8 (anti-CD28), and low-dose interleukin 2. Four-to-six h after each PBL injection, either PBS or 50 g of the CD3 ⫻ CD19 diabody, or the bispecific Tandab, or combinations of these with 25 g of anti-CD28 MAb 15E8, respectively, were administered via the tail vein. Tumor size was measured using a caliper every 2nd 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 100 days after the first treatment. Statistical Analysis. For statistical evaluation, the follow-up duration of the tumor-treatment experiment was 50 days (end of experiment). The survival times were estimated by the method described by Kaplan and Meier (12). Differences between survival curves were compared using a log-rank test (13). Tumor eradication was compared by Fisher’s exact test (14). To compare the tumor regression and tumor growth over time in the different treatment groups, a mixed-effects model for time-dependent data was used (15).
Results Production of Bispecific Tandab and Diabody. The genetically engineered bispecific constructs—the CD3 ⫻ CD19 diabody and Tandab—are schematically shown in Fig. 1. Diabody is a heterodimer formed by noncovalent association of two single-chain fusion products consisting of the VH domain from one antibody connected by a short linker to the VL domain of another antibody (9). In contrast, Tandab is a product of homodimerization of single-chain molecules comprising four antibody variable domains (VH and VL) of two different specificities in an orientation preventing Fv formation (8). E. coli RV308 cells containing either the plasmid pKID3 ⫻ 19 for simultaneous expression of components of bivalent CD3 ⫻ CD19 diabody (9) or pDISC6-SL for expression of the four-domain singlechain protein VH3-VL19-VH19-VL3 (8) were grown and induced
Fig. 1. Schematic representation of operons and protein models of diabody (A) and Tandab (B). The locations of promoter/operator (p/o), ribosome binding site (rbs), pelB leader (pelB), c-myc epitope (c-myc), hexahistidine tag (His6), and stop codon (stop) are indicated. On protein models, the carboxy termini (COOH), linkers (L or SL), and CD3 and CD19 antigen binding sites are indicated. The diabody orientation corresponds to the crystal structure described by Perisic et al. (16), and Tandab orientation is shown according to the molecular model of CD3 ⫻ CD19 Tandab (8).
under conditions favoring their dimerization (8). Bispecific molecules were isolated from crude periplasmic fractions in two chromatographic steps (IMAC, Mono S) with a purity greater than 95% (Fig. 2A). Size-exclusion chromatography on a calibrated Superdex 200 column revealed a tetrameric Tandab and demonstrated a predominantly dimeric form of the diabody with only a small proportion of putative tetramers (Fig. 2B). Stability of Tandab in Vitro. Previously we demonstrated that incubation of CD3 ⫻ CD19 Tandab in human serum at 37°C for prolonged periods of time did not cause any large decrease in antigen binding as would be expected for a dimer-monomer transition (8). To examine the putative dissociation of tetrameric Tandab into its dimeric single-chain-diabody components, we analyzed the molecular form of Tandab by size-exclusion chromatography after incubation in phosphate buffer at 37°C. Fig. 3A shows the presence of the only peak corresponding to tetrameric Tandab even after incubation for 24 h. No indication of the appearance of dimeric diabody-like molecules was found. The observed decrease in the amount of soluble Tandab was probably caused by aggregation, which is quite characteristic for single-chain antibody fragments (17). The products of Tandab degradation were found among precipitated material only after 10 h of incubation at 37°C (Fig. 3B). Stability of Tandab in Vivo. The 125I-labeled Tandab was administered i.v. to normal mice and serum samples were taken at intervals up to 1.5 h after injection when ⬃80% of protein-bound radioactivity
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CURE OF B-CELL LYMPHOMA BY CD3 ⫻ CD19 TANDAB
model of the Raji Burkitt’s lymphoma in SCID mice. Raji cells, when injected s.c., led to locally growing tumors. The treatment was started when the tumors reached a size of 5 mm in diameter. At days 0, 7, and 15, cohorts of five mice received i.v. either PBS (control group) or in vitro preactivated human PBLs. Four h after each PBL inoculation, the mice were treated either with no antibody or with 50 g (1 nmol) of the CD3 ⫻ CD19 bispecific diabody or 50 g (0.5 nmol) of the bispecific tetravalent Tandab administered as a tail vein injection. Additional groups received the same amounts of diabody or Tandab in combination with 25 g of antihuman CD28 MAb 15E8. All of the animals in the control groups receiving PBS or PBLs alone did not show any tumor suppression and developed tumors larger than 1.5 cm in diameter in less than 3 weeks (Fig. 6). There was no significant
Fig. 2. Analyses of purified diabody and Tandab. A, 12% SDS-PAGE under reducing conditions. Lane 1, Mr markers (kDa, Mr in thousands); Lane 2, Tandab; Lane 3, diabody. The gel was stained with Coomassie. B, elution profiles of Tandab (———) and diabody (. . . . . . ) from a calibrated Superdex 200 gel filtration column.
was already eliminated from the blood because of extravasation and kidney clearance (8). SDS-PAGE of plasma samples followed by autoradiography revealed that all radiolabel in blood plasma was associated with bands having the characteristic apparent molecular weight of four-domain single-chain antibody (Fig. 4). This indicates the absence of quick proteolytic degradation of Tandab in a blood stream. Biodistribution and Targeting. The in vivo targeting potential of the Tandab was assessed in Rag2 immunodeficient mice bearing s.c. CD19⫹ Raji tumors. Tumor, blood, and organ retention of radioiodinated Tandabs was determined at 1, 3, 6, 18, and 24 h after i.v. administration. One h after injection, the highest percentage of the injected dose localized per gram of tissue was found in kidneys, blood, and blood-rich organs such as lungs, liver, and spleen. The Tandab also displayed a rapid tumor uptake to a maximum of 6.77 ⫾ 1.48%ID/g at 3 h, which remained high at 6 h (Fig. 5). Blood activity revealed a rapid first-pass clearance ␣-phase with a t1/2␣ of 0.27 h and a slower -phase with a t1/2 of 2.8 h, quite similar to the previously observed pharmacokinetics of Tandabs in normal mice (8). Table 1 shows a summary of the biodistribution data for radioiodinated Tandabs, including T:O ratios. The kidney appears to be the principle organ of excretion, although the size of the Tandab (Mr 113,000) is expected to exceed the renal threshold for first-pass clearance. Unlike conventional antibodies, the Tandab did not accumulate in the liver or other organs, with %ID/g values at 24 h for the main organs ranging between 0.02 and 0.2 (Table 1). Our data indicate that the bispecific Tandab is a very compact molecule with excellent tumor-targeting properties. Treatment of Raji-bearing Mice with CD3 ⴛ CD19 Diabody and Tandab. To determine the in vivo antitumor activity of the tetrameric CD3 ⫻ CD19 Tandab, we established a xenotransplant
Fig. 3. Tandab stability in vitro. A, size-exclusion FPLC profiles of Tandab samples stored at 37°C during the indicated time and cleared by centrifugation. The positions of tetrameric Tandab and dimeric diabody are indicated. B, 12% SDS-PAGE analyses of soluble protein fractions (stained with Coomassie) and protein pellets (Western blot analysis using anti-c-myc MAb 9E10). kDa, Mr in thousands.
Fig. 4. 12% SDS-PAGE analysis of plasma samples containing 125I-labeled Tandab. Lanes 1 and 7, Mr markers; Lane 2, sample of labeled Tandab before injection; Lanes 3– 6, plasma samples removed from normal mice 10, 20, 40, and 90 min, respectively, after injection. kDa, Mr in thousands.
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CURE OF B-CELL LYMPHOMA BY CD3 ⫻ CD19 TANDAB
In contrast to this limited survival improvement, none of the animals receiving the Tandab plus anti-CD28 MAb had any palpable tumors after the second injection (day 10, Fig. 6). These mice remained disease-free during the whole period of the experiment (50 days) and even 100 days after the first treatment. Compared with the other treatment groups, this result was statistically significant (Fisher’s exact test: P ⬍ 0.01). Mixed-effects model analyses demonstrated similar tumor regression rates in both of the groups receiving antiCD28 MAb for costimulation, rates that were significantly higher than in groups receiving the diabody or Tandab alone (P ⫽ 0.026 and 0.011, respectively). Our data clearly indicate that the cytolytic potential in vivo of the Tandab is significantly enhanced in the presence of anti-CD28 MAb, which induces a well-known costimulatory signal (for review, see Ref. 18). Fig. 5. The in vivo tumor targeting of radioiodinated CD3 ⫻ CD19 Tandab as determined in a biodistribution study using Raji-bearing Rag2-deficient mice. The plotted values represent the mean tumor (F) and blood (‚) retention obtained from three mice per data point. Bars, SE.
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 can be ignored. In contrast to control groups, the mice receiving diabody or Tandab demonstrated significant tumor regression. Two to 3 weeks after beginning treatment, the animals in these two groups lost one-half of their tumor mass. Afterward, however, the tumors started to grow again with comparable rates (Fig. 6). The animals were killed when the tumors reached the maximum tolerated size of 15 mm in diameter. Sacrifice dates were recorded, and the median survival time in each group was calculated (Fig. 7). The median survival time was 18 days in the control group receiving PBS, 20 days in the control group receiving human PBLs alone, 44 days in the group given diabody, and 44 days in the Tandab-treated group. The animals receiving three injections of CD3 ⫻ CD19 diabody in combination with anti-CD28 MAb displayed a minimal tumor size on days 18 –20, when 4 of 5 mice were tumor-free. Afterward, the tumors began to reappear and grew progressively in three animals (Fig. 6). An analysis of the second part of the tumor growth curves (after day 14 of treatment) demonstrated fairly comparable tumor growth rates in all of the diabody- or Tandab-treated animals. In contrast, the delays in the starting point of tumor regrowth were significantly larger for animals receiving the diabody in combination with the costimulatory anti-CD28 (Tandab: P ⫽ 0.014; other: P ⬍ 0.001). Moreover, two of five animals in this group remained tumor-free until the end of monitoring (day 100 after the first treatment). The median survival time calculated for the group receiving diabody plus MAb 15E8 was 48 days (Fig. 7).
Discussion The treatment of leukemias and malignant lymphoma includes multiple courses of polychemotherapy and/or radiotherapy, but, despite aggressive treatment, a fairly large number of patients relapse, and most remissions cannot be extended beyond minimal residual disease. An emerging alternative approach is the retargeting of cellular effector systems such as T cells, NK cells, or Fc␥R-positive cells (granulocytes, macrophages) by bispecific antibodies (19 –21). However, the progress of these immunotherapeutic agents into clinical applications has been slow, mainly because of the low yields of clinical grade bispecific molecules and the immunogenicity of murine bispecific antibodies. More recently, recombinant bispecific “diabodies” have been created that comprise only antibody variable domains (9, 22, 23). They are potentially less immunogenic than quadroma-derived BsAbs and can be easily produced in bacteria in relatively high yields. Previously, we demonstrated that both CD3 ⫻ CD19 and CD16 ⫻ CD30 bispecific diabodies have superior cytolytic activity in vitro against human lymphoma cells compared with quadroma-derived BsAbs of the same specificity (9, 24). In vivo, the antitumor activity of these diabodies was fairly similar to that of the parental BsAbs that have longer blood retention on account of their much larger size (24, 25). To further improve the therapeutic potential of recombinant antibody fragments, we have recently constructed a novel recombinant molecule named “tandem diabody” (Tandab) that is bispecific and tetravalent (8). The Tandab is a tetravalent homodimer formed by noncovalent association of two identical polypeptide chains containing four antibody variable domains connected by peptide linkers. To check the structural integrity of the Tandab, we performed a thorough investigation of its stability both in vitro and in vivo. Previously, we had observed a gradual decrease in the antigen-binding activity of the Tandab on incubation in human serum at 37°C (8). This suggested that the Tandab undergoes no significant dissociation into bivalent sub-
Table 1 Biodistribution of 125I-labeled Tandab in tumor-bearing immunodeficient mice Five g of 125I-labeled Tandab was administered by i.v. tail-vein injection to Rag2-deficient mice bearing s.c. Raji xenografts (n ⫽ 3 mice/time point). Tissue and blood retention were determined as described in “Materials and Methods.” SE values are presented in parentheses. Time (h) 1 Organ
%ID/g
Tumor Blood Liver Kidney Lung Spleen Bone Muscle
5.02 (1.22) 9.25 (1.25) 9.03 (1.20) 11.81 (4.19) 20.42 (9.34) 6.69 (0.16) 2.17 (0.19) 1.07 (0.18)
3 T:O ratio
%ID/g
0.5 0.6 0.4 0.2 0.8 2.3 4.7
6.77 (1.48) 4.64 (0.79) 2.73 (0.80) 3.77 (0.66) 5.56 (2.62) 2.74 (0.73) 1.45 (0.41) 0.75 (0.12)
6 T:O ratio
%ID/g
1.5 2.5 1.8 1.2 2.5 4.7 9.0
6.23 (1.80) 3.25 (0.11) 1.69 (0.15) 2.55 (0.07) 2.20 (0.12) 1.70 (0.12) 1.10 (0.23) 0.61 (0.01)
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18 T:O ratio
%ID/g
1.9 3.7 2.4 2.8 3.7 5.6 10.3
2.71 (1.19) 0.33 (0.04) 0.18 (0.03) 0.22 (0.01) 0.44 (0.04) 0.18 (0.02) 0.05 (0.03) 0.03 (0.02)
24 T:O ratio
%ID/g
T:O ratio
8.1 14.7 12.5 6.2 15.3 60.7 98.0
1.72 (0.24) 0.13 (0.01) 0.11 (0.04) 0.17 (0.05) 0.17 (0.13) 0.11 (0.01) 0.04 (0.01) 0.02 (0.01)
13.3 16.4 10.1 9.9 15.6 39.1 104.2
CURE OF B-CELL LYMPHOMA BY CD3 ⫻ CD19 TANDAB
Fig. 6. Treatment of SCID mice bearing human Burkitt’s lymphoma xenografts. The mice received PBS, preactivated human PBLs alone, or preactivated human PBLs followed 4 – 6 h later by the administration of CD3 ⫻ CD19 diabody, Tandab, CD3 ⫻ CD19 diabody plus MAb 15E8 or Tandab in combination with MAb 15E8. Tumor growth curves of individual animals are presented. Tumor size was measured every 2nd day (E). F, the final day of measurement; ⫻, eradicated tumors.
units, because this would cause sharp drop in activity attributable to lower avidity (8). In the present study, we demonstrated that the loss of Tandab activity in vitro was caused by aggregation and not by dissociation. In vivo, however, blood proteins seem to stabilize the Tandabs and prevent aggregation. This conclusion was made from the observation that the Tandabs had a significantly longer half-life in human serum than in phosphate buffer. Supporting evidence was obtained from biodistribution studies. We demonstrated that the labeled Tandab was cleared mainly through the kidneys, whereas ag-
Fig. 7. Survival of SCID mice bearing human Burkitt’s lymphoma xenografts. The mice received PBS (䡺); or preactivated human PBLs alone (f); or preactivated human PBLs followed 4 – 6 h later by the administration of CD3 ⫻ CD19 diabody (‚), Tandab (E), CD3 ⫻ CD19 diabody plus MAb 15E8 (Œ), or Tandab in combination with MAb 15E8 (F).
gregated material would accumulate in the liver. Furthermore, no proteolytic degradation of the Tandab was observed either in vitro (human serum at 37°C; Ref. 8) or in vivo (mice, present study). Pharmacokinetic and biodistribution studies demonstrated that the Tandab had a somewhat faster clearance from the blood stream than would be expected for its size. However, the size of the molecule is not the only factor determining its susceptibility to filtration by the kidneys. Other factors are the conformation, electrical charge, and binding characteristics of the molecule. For example, weak ionic interactions can increase the propensity of antibodies to be phagocytosed by the reticuloendothelial cells of the liver, or to be bound to the highly charged glomerular basement membranes of the kidneys. Recently, it was demonstrated that lowering the isoelectric point (pI) of a single-chain Fv antibody fragment can significantly decrease its renal uptake (26). A comparison of the pharmacokinetic parameters determined for different antibody fragments also demonstrated that molecules containing constant immunoglobulin domains are retained in the blood stream significantly longer than molecules of the same size but composed only of variable domains (27). The Tandab seems to be a fairly compact molecule that is positively charged under physiological conditions (pI ⬃ 8) and can, therefore, be attracted by negatively charged cell surfaces of the proximal tubular cells. It is possible, therefore, that lowering the isoelectric point or the fusion with an antibody CH3 domain could further improve the pharmacokinetics and therapeutic effect of recombinant bispecific molecules. The CD3 ⫻ CD19 diabody and Tandab were designed for treating minimal residual disease in patients with B-cell malignancies. They can retarget T-cell mediated lysis in a MHC-independent fashion and prevent tumor growth in an animal model. The antitumor activity of both constructs in vivo was significantly enhanced in the presence of
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CURE OF B-CELL LYMPHOMA BY CD3 ⫻ CD19 TANDAB
anti-CD28 MAb, which induces a costimulatory signal (18). Costimulation is thought to be required not only to activate T cells but also to prevent deletion of the activated T cells, by rendering them apoptosisresistant (28), and to reactivate exhausted T cells (29). However, it is still somewhat surprising that the anti-CD28 MAb improves the efficacy of B-cell lysis in our system, inasmuch as the Raji B cells carry both costimulatory molecules CD80 and CD86. One explanation would be that the circulating anti-CD28 MAbs saturate the CD28 molecules on the effector cells faster and more efficiently than the B7 that is present only at the tumor site. Here, we demonstrate that the tetravalent Tandab has a significantly higher antitumor activity than the bivalent diabody when applied together with the anti-CD28 MAb. There are two explanations for the observed effect: (a) stronger binding of the Tandab to target cells attributable to its bivalency for the CD19 surface antigen; and (b) a longer blood circulation of the Tandab (8). The CD3 ⫻ CD19 Tandab bound to target B cells more strongly than to effector T cells, because of the almost 5-fold higher off-rate from CD3-positive cells (8). Such relatively strong binding to a target tumor cell and weaker binding to an effector cell may have certain advantages for tumor therapy. Artificial signaling via the CD3 antigen mimics the physiological antigen-specific activation of T lymphocytes by MHC-bound antigen. Accordingly, in a model of TCR serial triggering (30), a high off-rate of the TCR is essential because it allows a single peptide-MHC complex to engage many TCRs in successive rounds of ligation, triggering, and dissociation. High affinities attributable to a low off-rate may, therefore, inhibit this process. The toxicity associated with anti-CD3 ⫻ antitumor immunotherapy is usually caused by nonspecific T-cell activation attributable to the BsAb-mediated cross-linking of CD3 (31). Having two CD3-binding sites, the Tandab could theoretically activate T cells distant from the tumor site. However, probably because of steric reasons, the Tandab appears to bind bivalently only a small population of CD3-positive cells with densely clustered CD3 (8). In vitro experiments demonstrated that the CD3 ⫻ CD19 Tandab was consistently more effective than the diabody in inducing T-cell proliferation (8), but this stimulation was observed only in the presence of CD19-positive cells (data not shown). Similarly, the in vivo experiments with the SCID mouse model did not reveal any toxic effects associated with human-antimouse T-cell reactions. In view of the limitations associated with tumor therapy using CTLs (31), we have also constructed bispecific molecules to recruit NK cells via CD16 to a tumor site (24). Which type of response would be most beneficial for tumor immunotherapy remains to be investigated and will probably depend on the type and location of the tumor. One of the main goals of this study was to compare the therapeutic efficacy of two different recombinant bispecific molecules using the same tumor model. We demonstrated that the Tandab format had superior in vivo properties compared with the diabody format that is currently widely used for making bispecific molecules. To our knowledge, this is the first report on the successful use of recombinant BsAbs to cure a xenografted human B-cell lymphoma. References 1. Fanger, M. W., Morganelli, P. M., and Guyre, P. M. Bispecific antibodies. Crit. Rev. Immunol., 12: 101–124, 1992. 2. Canevari, S., Stoter, G., Arienti, F., Bolis, G., Colnaghi, M. I., Re, E. M. D., Eggermont, A. M. M., Goey, S. H., Grazama, J. W., Lamers, C. H. J., Nooy, M. A., Parmiani, G., Raspagliesi, F., Ravagnani, F., Scarfone, G., Trimbos, J. B., Warnaar, S. O., and Bolhuis, R. L. H. Regression of advanced ovarian carcinoma by intraperitoneal treatment with autologous T lymphocytes retargeted by a bispecific monoclonal antibody. J. Natl. Cancer Inst., 87: 1463–1469, 1995.
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