Biologic Properties of a Bispecific Single-Chain Antibody Directed Against 17-1A (EpCAM) and CD3 Tumor Cell-Dependent T Cell Stimulation and Cytotoxic Activity’ Matthias Mack, Rudi Cruber, Sabine Schmidt, Cert Riethmiiller,2 and Peter Kufer ispecific Abs have been used to redirect T lymphocytes against defined Ags on tumor cells (1-4). This approach relies on binding of one arm of the bispecific Ab to a tumor-associated Ag, while the other arm, by recognizing the CD3 rnolecule on T cells, triggers their cytotoxic activity (5-6). Crosslinkage of the TCR-associated CD? molecule by Abs immobilized to solid surfaces or cell membranes has been found to be a particularly strong activation signal. First clinical trials with conventionally generated bispecific Abs (F(ab‘), fragments from hybrid-hybridomas), however, have revealed considerable toxic effects. While these Abs preincubated with lymphocytes in vitro and injected directly into or near the tumor have shown little side effects (7-9), their systemic administration resulted in activation of T cells with subsequent release of cytokines such as TNF-a and IFN-y (10, 11). The systemic release of these cytokines has been made responsible for the dose-dependent and dose-limiting toxicity (fever, chills, and dyspnea) of current bispecific Ab preparations. The rapid onset of symptoms, accompanied by high plasma levels of cytokines and negligible toxicity against normal tissue, also expressing the detected tumorassociated Ag, points to an activation of T cells independent from the target cells. The target cell-independent activation of T cells may be attributed to contaminations of bispecific F(ab’), fragInstitute of Immunology, Munich, Germany Recewed for publlcation August 8, 1996. Accepted for publicatlon January 14, 1997. The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported by grants irom Deutsche Krebshilfe, Bonn, Genzen trum Munchen (BMFT), and Curt-Bohnewand-Fond. ‘ Address correspondence and reprint requests to Dr. Gert RiethmOller, Institute of Immunology, Goethestrasse ? l , 80?36 Munchen, Germany. Copyright 0 1997 by The American Association of lrnmunologlsts ments with intact Abs present after incomplete proteolytic digestion. This gives rise to binding of CD3 Abs to the surface of FcRpositive cells, and thus entails the activation of T cells. A study investigating different preparations of OKT3-F(ab’), fragments showed a marked reduction in T cell activation and cytokine release with meticulously purified OKT3-F(abf), fragments, and therefore strongly supports the application of bispecific Abs that are free of contaminating Fc parts ( 1 2). Recently, we have described the construction and expression of bispecific single-chain Abs in eukaryotic cells (1 3). The design of this molecules is shown in Figure 1. Four variable domains of two different Abs are joined by flexible linkers to appear on one single One Ag protein chain in the order V,(A)-V,(A)-V,(B)-V,(B). binding site of this construct is directed against 17-1A; the other against the CD3 surface molecule on T cells. The construct could be expressed in Chinese hamster ovary cells as fully functional molecule in large amounts, and was easy to purify via its C-term i d histidine tail. In contrast to bispecific Abs produced with the hybrid-hybridoma technology (14, IS) or chemical cross-linkage. the bispecific single-chain Ab preparation is devoid of any Fc parts or anti-CD3 homodimers probably responsible for the severe side effects seen after i.v. application in cancer patients. In this study, we demonstrate that the bispecific single-chain Ab does not induce an activation of PBMCs in the absence of target cells, whereas addition of target cells gives rise to a strong T cell activation measured by surface activation markers and cytokine release. This observation is consistent with plenty of data showing that monovalent nonimmobilized CD3 Abs do not stimulate T cells (16-1 8). Therefore, bispecific Abs produced in the described single-chain approach should be ideal candidates for a systemic administration in cancer patients. The systemicadministration clearly is a key element of Ab-mediated tumor therapy, as a large primary or metastatic tumor mass is not a suitable target in contrast to 0022-1767/97/$02.00 Downloaded from www.jimmunol.org on May 10, 2010 Anti-CD3 X anti-tumor-bispecific Abs have been usedto redirect cytotoxicT cells to tumor cells in an MHC-unrestricted fashion and to induce their rejection i n vivo. We have recently described a recombinant bispecific single-chain Ab that combines four different V regions of two Abs, anti-7 7-1A and anti-CD3, on one polypeptide chain. It folds correctly to a 60-kDa globular protein and is secreted in fully functional form by a high producer Chinese hamster ovary cell line. I n this work, we report that its remarkable cytotoxicity against 17-1A+ tumor cells i s exerted via T cells without an apparent engagement of a detectable costimulatory pathway. T cells are activated only by the bispecific Ab when coincubated with 17-1A+ target cells. In a chromium release assay, CD8+ T cells reach maximal tumor cell cytotoxicity within 4 h, while CD4+ T cells need about 20 h to reach similar levels of cytotoxicity. Addition of costimulatory CD28 Abs did notlead to a further increase in cytotoxicity. Its remarkable stabilityat 37O in serum, the ease of production, and purification by affinity chromatography via polyhistidine tail make this smaller version of a bispecific Ab a promising candidate for a therapeutic trial in patients with solid tumor. Because adjuvant therapy with an intact, much less cytotoxic lgG2a Ab against the 17-1A target had already increased the 7-yr survival of colorectal cancer patients by 30%, the presented small bispecific construct lacking the immunogenic murine Fc region as well as autochthonous-T lymphocyte Stimulatory activity warrants a therapeutic trial in patients with minimal residual 17-1A+ cancer. The Journal of Immunology, 1997, 158: 3965-3970. 3966 PROPERTIES OF A 17-lA/CD3-BISPECIFIC SINGLE-CHAIN Ab Transfectants were cultured in RPMI 1640 (Sigma Chemical Co., St. Louis, MO) with 10%FCS (Life Technologies), 2 mM L-glutamine, and 1 mg/ml (3418. Several clones were screened for expression of 17-IA by FACS analysis. The clone used in the experiments expressed 17-1A to a lesser degree than Kat0 cells. Kat0 is a 17-1A-positive human gastric cancer cell line and was obtained from American Type Culture Collection (Rockville, MD). Cytotoxicity assay FIGURE 1. Scheme of the17-1NCU3-bispecificsingle-chain Ab. Two single-chain Fv fragments, one directed against 17-1A, the other against CD3, were joined by a flexible linker consisting of five amino for easy detection, and a acids. An N-terminal flag-tag was added C-terminal histidine tail (His) for protein purification. Materials and Methods Production o f the bispecific single-chain Ab The bispecific 17-1A/CD3 single-chain Ab was produced as described previously (13). Briefly, two single-chain Fv fragments were joined through a flexible Gly-Ser linker. The single-chain Fv fragment directed against 17-1A is derived from the M79 hybridoma (22); the other directed against CD3 is derived from the TR66 hybridoma (23). The construct was expressed in Chinese hamster ovary cells as fully functional molecule and was purified via its C-terminal histidine tail on a Ni-NTA (Nitrilotriacetic acid) afinity column (Qiagen, Germany). Preparation of effectorcells Human PBMCs were isolated from the fresh buffy coat of healthy donors by Ficoll density gradient centrifugation (Pharmacia, Piscataway, NJ) and a subsequent 100 X g centrifugation step to remove thrombocytes. Where indicated, CD4+ or CDSt cells were depleted with magnetic beads (Dynabeads; Dynal, Great Neck, NY), according to manufacturer's instructions. The depleted cellpopulations were analyzed by flow cytometry (FACScan; Becton Dickinson, Mountain View, CA), which showed a 99% depletion of CD8+ or CD4' cells. For the preparation of purified CD4+ or CD8+ T cells, monocytes were first depleted by adherence to plastic for 3 h, and then magnetic beads (Dynabeads) were used for positive selection ofCD4+ or CDS+ T cells,according to the procedure described by Halvorsen et al. (24). Analysis by flow cytometry showed a purity of 98% for CD4+ Tcells and 98% for CD8+ cells (91% CD8+ T cells and 7% CD8+ NK cells). Exposition of the bsc Ab to PBS and serum The bsc Ab was stored for 6 mo in PBS at 4°Cand compared with a freshly prepared sample of the protein. To detect thestability in human serum, 20 pl of the bsc Ab and 1 0 0 pl of freshly prepared serum from a healthy donor were incubated at 37°C for different periods of time. Experiments have been repeated two times with serum of different donors. Flow cytometric analysis and cytokine ELlSA A cytotoxicity assay was performed similar to the 5'Cr release assay described above. Effector cells (150,000) were added to each well of a Ratbottom microtiter plate and incubated overnight at 37°C. Further additions were either PBS only, target cells only, bsc Ab only, OKT3 only, PHA only, or both target cells and bsc Ab. Target cells were added in an E:T ratio of 1:1 for good cell-cell contact, the bscAb was used in different concentrations, and OKT3 mAb and PHA in final concentrations of 20 ng/ml and 2 pg/ml, respectively. Three identical wells were prepared for each test. After an incubation time of 4 h and 20 h,the supernatant and the cells were collected separately. The supernatants were frozen immediately at -70°C and stored for 24 h at most. The cells were incubated for 30 min at 4°C with different Ab mixtures in the proper dilution. One Ab mixture CD25 phycoerythrin (Becton was CD3 FlTC (Coulter Corp., Hialeah, a), Dickinson), and CD4 Tricolor (Medac, Hamburg, Germany); one was CD3 FITC (Coulter Corp.), CD25 phycoerythrin (Becton Dickinson), and CD8 Tricolor (Medac); andanother the appropriate isotype controls. After washing, the samples were analyzed byflow cytometry (Becton Dickinson). Quantitative analysis of CD25 and CD3 expression was conducted by gating the lymphocytes by virtue of their light scatter properties, with careful inclusion of activated larger lymphoblasts. For analysis of the CD3 and CD25 expression in the CD4' and CD8' subset, a second gate was set on the CD4+ andCD8' cells, respectively, and the mean channel of CD3 and CD25 expression was determined. Experiments have been repeated at least four times with effector cells from different donors, and one representative experiment is shown. Cell lines Murine X63 myeloma cells were stable transfected with 17-1A cDNA using avector with the @-actinpromotor and the neomycin resistance gene. Abbreviations used in this paper: bsc Ab, 17-1NCD3-bispecific single-chain antibody; aa, amino acid. Downloaded from www.jimmunol.org on May 10, 2010 disseminated tumor cells that are lodging frequently in interstitial tissue, easily accessible to macromolecules and effector cells (19). For an in vivo application, the presence of sufficient numbers of effector cells certainly is a critical point. Therefore, we investigated whether subpopulations of PBMCs, such as purified CD4+ and CD8+ T cells, which do not contain accessory cells in significant numbers, can be redirected efficiently against different tumor cell lines with the bispecific Ab alone. The infiuence of a costimulation of T cells with CD28 Abs is also investigated. Furthermore, an in vivo application of bispecific single-chain Abs would lead to a prolonged exposure of these moIecules to human plasma and to long term interactions between target cells and redirected T cells. Therefore, we investigated the stability of the construct, when exposed to fresh human serum, and also performed long term assays with different effector cell populations and tumor cell lines. A recent clinical trial has shown that patients with a surgically completely resected colorectal carcinoma havea significant benefit concerning the overall survival and the frequency of distant metastasis, when treated i.v. with a mAb directed against 17-1A (20), now known as epithelial cell adhesion molecule (21). The use of bispecific single-chain Abs, which are severalfold more effective than mAbs (2), would be an important improvement of this approach. For the "Cr release assay, different numbers of effector cells were added in a volume of 1 0 0 4 RPMI medium (Sigma Chemical Co.) -t 10% FCS (Life Technologies) to each well of a flat-bottom microtiter plate (Costar, Cambridge, MA) and incubated overnight at 37°C. Target cells were labeled for 2 h with 5'Cr, and 25,000 of them were added to each well in a ) ~ were volume of 100 4 . Different concentrations of the bispecific ( b ~ c Ab added in a volume of 25 pl together with an extra solution of 25 pl, containing either medium or CD28 Ab (mAb 9.3) in different concentrations or an Ab mixture for inhibition of cytokines. This later Ab mixture consisted of TNF-a, TNF-p, and IFN-y Abs in final concentrations of 20, IO, and 2.5 pg/ml, respectively. The incubation time was 4 or 20 h at37°C. Maximal release was determined by lysis of target cells with lysis buffer (2% SDS, 0.37% EDTA, and 0.53% Na,CO,), and spontaneous 5'Cr release was determined with target cells only. The 17-IA-negative parental X63 cells were completely refactory to lysis in 5'Cr release assays (13). Unlinked sc-Fv fragments of the 17-IA and CD3 Ab were ineffective. As a further control,we used a mutated derivative of the bsc Ab, in which the 17-1A binding was abolished after site-directed mutagenesis of aa,,, in V, and aa, in V, of the anti-17-1A part. This constmct proved to be completely inactive in the lytic assays. Incubation of target cells only with bsc Ab at the highest concentration did not result in measurable lysis. Specific lysis was calculated as: (cpm, experimental release - cpm spontaneous release)/(cpm, maximal release - cpm, spontaneous release). Triplicate samples were measured in each assay, and SD was always less than 6% of the mean. Experiments were repeated at least four times with effector cells from different donors, and one representative experiment is shown. 3967 The Journal of Immunology FIGURE 3. IL-2R up-regulation and CD3 modulation induced by the bispecific Ab on T cells in contact with target cells. The same experiment as shown in Figure 2 was performed with equal lettering of the x-axis. The expression of CD25 (IL-2R) and CD3 was measured by FACS analysis after 20 h, and is shown on the y-axes in the diagrams. Results Activation of T cells by bispecific single-chain Abs F I GU R E 2. Target cellinduced release ofcytokinesfrom PBMCs. PBMCs (150,000) were incubated for4 h and 20 h with medium alone (E), with target cells (150,000 Kato) only (E+T), with the bispecific single-chain Ab at 3 yghl or 61 n g h l only (E+bsc-Ab), with target cells and the bispecific single-chain Ab at 3 pg/ml or 61 n d m l or 1.2 ng/ml (E+T+bsc-Ab), with PHA at 1 pCLg/rnl only sPHA, or with a CD3 rnAb at 20 ng/ml only (antiLCD3 mAb). ELISA for TNF-a, IFN-y, and IL-6 All ELISAs were obtained commercially (GenzymeCorp.,Cambridge, MA) and utilized according to manufacturer's instructions. The frozen supernatants from two identical wells of each experiment were analyzed separately, and mean values were calculated. ELISA experiments were performed twice with PBMCs from different donors. 3H-/abeled thymidine uptake PBMCs were added in triplicates to a 96-well U-bottom plate at a concentration of 500,000 cells/well. Bsc Ab and anti-CD3 mAb were added in different concentrations to a final volume of 250 pVwell. Addition of medium alone served as negative control. After an incubation of 48 h at 3 7 T , 'H-labeled thymidine was added at a final concentration of 10 pCi/ml for another 18-h incubation. Cells were harvested on a semiautomated cell harvester, and incorporation of 'H-labeled thymidine was determined on a scintillation counter (Beckmann, Fullerton, CA). The stimulation index was calculated as log (counts (experimenta1)kounts (negative control)). Experiments were repeated three times with PBMCs from different donors. According to the therapeutic intention, T cells should only be activated when they are attached to tumor cells via the Ab bridge, but not when they are exposed to bispecific Abs alone in the peripheral blood or lymphoid tissue. To test the activation of T cells by bispecific Abs, we performed short and long term assays (4 h, 20 h) in the absence or presence of target cells, and used PBMCs as effector cells to imitate the in vivo condition. We explored early events in T cell activation for both the CD4+ and CD8' T cell subset, and also studied the proliferation of PBMCs by a 'H-labeled thymidine uptake test. Early events in T cell activation are the expression of IL-2R (CD25), release of IFN-y, TNF-a, and IL-6, as well as down-regulation of CD3. As shown in Figures 2 and 3, we could demonstrate that incubation of PBMCs with the bispecific Ab alone did lead neither to the expression of CD25, measurable release of cytokines, nor down-regulation of CD3. Only after addition of target cells (Kato) did T cells become activated to a similar degree as when stimulated with PHA or whole anti-CD3 Abs. Activation could be induced in both CD4+ and CD8+ subsets. In the presence of the murine cell line X63-17-1A, transfected with 17-1A cDNA, activation of T cells was diminished significantly (Fig. 4). We also used immobilized recombinant 17-1A coated to the culture well bottom as target structure for PBMCs, and found that under these conditions the bispecific single-chain Ab activated CD4+ or CD8+T cells to about the same level as the murine target cells (data not shown). With purified CD4' and CD8' T cells, we found in all experiments similar results as with PBMCs concerning the expression of CD25 and CD3 (data not shown). Even after prolonged incubation, the bispecific single-chain Ab, in concentrations up to 3 pg/ml, failed to induce proliferation of PBMCs, as measured by incorporation of 3H-labeled thymidine, while an intact CD3 mAb induced a strong proliferation with a stimulation index > I O 0 (data not shown). Downloaded from www.jimmunol.org on May 10, 2010 3T PROPERTIES OF A 17-1A/CD3-BISPECIFIC SINGLE-CHAIN Ab 3968 T - 450 400 e 2 Y E g 350 40 35 CD4* 300 CD4+, a-CD28 250 0CD8+ 8 200 CD8+, a-CD28 30 'f 25 0 20 2 1:z 5 0 150 3000 500 80 14 2.3 PBS 500 80 14 2.3 PBS 90 Retargeting ofPBMCs against17-1A-transfectedmouse tumor cells (X63-17-IA).Activation of CD4+ and CD8+ T cells, measured by IL-2R up-regulation, was determined with and without addition of CD28 Abs (1 Fglml). FIGURE 4. 80 70 Y % The lytic capacity of CD4+ and CD8+ T cells As bispecific (anti-tumor/anti-CD3) Abs can target both CD4+ and CD8+ T cells to tumor cells, resulting in a strong activation ofboth T cell subsets, we investigated whether this activation also led to the lysis of target cells. A 'lCr release cytotoxicity assay was performed with CD8-depleted PBMCs (containing 100,000 CD4+ T cells), CD4-depleted PBMCs (containing 100,000 CD8+ T cells), and nondepleted PBMCs (containing 66,000 CD4+ and 33,000 CD8+ T cells). It is noteworthy that monocytes, B cells, and NK cells were still present in these negatively depleted effector cell preparations. While in the 4-h assay (Fig. 5, top) CD8-depleted PBMCs did not cause significant lysis of target cells, the CD4-depleted cell preparations or nondepleted PBMCs showed a clearly measurable specific lysis already after 4 h. However, after an incubation of 20 h (Fig. 5, bottom), the CD8-depleted PBMCs clearly induced a significant specific lysis of almost 70%. Addition of Fas Abs (clone ZB4; Immunotech, Marseille, France), inhibiting the human Fas on Kato cells, caused a reduction of lysis (Fig. 6) mediated by PBMCs, while there was no effect on lysis of murine X63-17-1A cells not recognized by the Fas Ab, precluding unspecific effects of the Fas Ab on the function of effector cells. Addition of an Ab mixture, consisting of TNF-aP and IFN-y, did not reduce the rate of specific lysis (data not shown). To assess the effect of accessory cells such as monocytes in the effector cell population, we compared the lytic activity of purified, positively selected CD4" and CD8+ T cells with the unselected PBMCs. As shown in Figure 7, the bispecific Ab alone suffices to induce cytotoxic activity of purified CD8+ and CD4+ T cells to lyse Kat0 tumor cells as well as X63-17-1A transfectants. In comparison with the human cell line Kato, the specific lysis of the murine cell line X63-17-1A was diminished significantly, especially if only CD4+ T cells were used. Costimulatory signals delivered via CD28 do not enhance the lytic capacity of PBMCs, CD4+, or CD8+ T cells redirected against different tumor cells Many authors have shown that T cells, redirected against tumor cells with bispecific anti-tumodanti-CD3 Abs, can be costimulated effectively with CD28 Abs to increase the cytolytic capacity of T cells (4,25-28). We.investigated whether addition of CD28 Abs in 50 40 30 u) 20 10 0 3000 bsc-Ab concentration (nglml) FIGURE 5. The lvsis of human Kat0 tumor cells was determined with different effector ceil populations in a 5'Cr release. EitherPBMCs(Containing 66,000 CD4+ T cells and 33,000 CD8+ T cells, white bars), or PBMCs depleted of CD8+ cells (containing 100,000 CD4+ T cells, black bars), or PBMCs depleted of CD4+ cells (containing 100,000 CD8+ T cells, gray bars) were used. Incubation times:4 h in the upper panel, and 2o i n the lower panel, 90 80 70 Anti-Fas Kato, 1 S v 60 ;. 50 40 30 20 10 5 Ea, n- 3000 430 61 8.75 1.25 PBS bsc-Ab concentration (ng/ml) FIGURE 6. Partial inhibition of lysis by FasAbs. A 20-h 5'Cr release assay was performed with 500,000 PBMCs redirected against 25,000 Kat0 cells with the bispecific single-chain Ab. Fas Abs were used in a concentration of 1 pg/rnl. SD was consistently below5% of the mean. concentrations of 5 pg/ml and 30 ng/ml to the effector cell population could increase the lysis of target cells. As shown in Figure 8, CD28 Abs do not increase the lytic capacity of redirected purified CD4+ and CD8+ T cells. The same was seen with PBMCs (data not shown). In parallel, we investigated the activation of T cells by monitoring the expression of CD25. Addition of CD28 Abs to Kat0 cells increased the activation of retargeted CD4+ and CD8+ T cells only slightly (about 2%) (data not shown). Addition of CD28 Abs to the murine X63-17-1A cells, however, induced a significantincrease in the activation of the retargeted CD4+ and CD8+ T cells almost to the same level as seen with Kat0cells (Fig. 4). With immobilized 17-1A as target structure for redirected T cells, results were comparable with the murine target cells X6317-1A (data not shown). Downloaded from www.jimmunol.org on May 10, 2010 These data show that under all tested conditions, the bispecific single-chain Ab does not activate CD3+ T cells in the absence of 17-1Af target cells, even when abundant FcR-carrying accessory cells are present and high concentration of bispecific Ab constructs are used. 60 u) 2 The Journal of Immunology 3000 430 61 3969 8.75 1.25 PBS bsc-Ab concentration (nglrnl) Kat0 cells (solid lines) and murine X63-17-1A cells (broken lines), in a 20-h *'Cr release assay. The effector cell population was either 500,000 PBMCs (containing 200,000 CD4+ and 100,000 CD8+ T cells), orpurified CD4+ and CD8+ T cells (200,000 each), asindicated with circles, triangles, and squares, respectively. K FIGURE 7. Comparison of twodifferenttargets,human Flow cytometry has shown that the tumor cell line Kat0 does not express B7-1 or B7-2 (data not shown). The stability of the bispecific single-chain Abs Discussion Bispecific Abs that redirect T lymphocytes to tumor cells have been found distinctly more efficient in target cell killing or elimination than intact unmodified Abs of the same specificity (2). However, unlike intact Igs, bispecific Abs endowed with one of their both specificities for the TCR-associated CD3 molecule displayed severe clinical toxicity ascribed to CD3-triggered systemic cytokine release (10, 11). As this remarkable toxicity is most likely caused by Fc fragments contaminating the conventionally produced bispecific Ab preparations, we have resorted to rDNA-based protein engineering to generate bispecific single-chain Abs devoid of any Fc fragments. We isolated the V, and V, genes of the Ab M79 directed against the 17-1A Ag for onearm, while for theother arm the gene of a single-chain Fv fragment of a CD3 Ab was selected. The choice of a 17-1A Ab was warranted, since an unmodified murine mAb against the 17-1A epithelial Ag had been found previously to reduce the5-yr (20) andalso the 7-yrmortality (29) of patients with minimal residual disease. Furthermore, since minimal residual cancer is intended as therapeutic indication in patients that are virtually tumor free after curative regional surgery, the avoidance of toxicity appearsto be essential for any form of therapy. The data presented in this work show that the bispecific 17" CD3 single-chain Ab does not activate T lymphocytes in the absence of tumor targets. In addition, in the presence of potent costimulatory cells that are abundant in preparation of PBMCs, T cells were not activated by the addition of bispecific Abs alone. Only when 17-1A-positive tumor cells were introduced into the system were T cells induced to express the IL-2R and to secrete cytokines, such as TNF-a, IFN-y, and IL-6. In addition, the murine X63 plasmocytoma cells transfected with the 17-1A target Ag could trigger activation of T cells, albeit to a distinctly lower degree. 4h 20 h I FIGURE 8. Effectof a costimulation with CD28 Abs on the lytic activity of T cells redirected with the bispecific single-chain Ab. A 5'Cr release assay was performed with purifiedCD4+ or CD8+T cells (200,000each), of 4 or 20 h, and different Kat0 orX63-I 7-1 A target cells, incubation times concentrations of CD28 Abs. In each experiment, specific lysis was determined with five different concentrations of the bsc Ab (as in Fig. 5), and mean values werecalculated. Relative lytic activityin percentageof mean values obtained in experiments without CD28 Abs. As T lymphocytes require a costimulatory signal for activation, the question was whether the activation observed in this study via bispecific Abs bound to tumor cells depended on the main costimulatory pathway CD28&37. On human Kat0 cells, we could neither detect B7-1/2 by sensitive FACS analysis, nor did the addition of stimulatory CD28 Ab result in a measurable increased activation by bispecific Abs. In the presence of 17-1A-transfected murine target cells (X63-17-1A), however, CD28 Abs costimulated T cells significantly. This difference between the human and murine target cell may be partially due to a lower concentration of 17-1A onX63-17-1A cells, as well as to other as yet unrecognized costimulatory molecules on the human cell not present or fully active on the xenogenic murine target. As both major subsets of T cells, Le., CD4+ and CD8+ cells, were equally well stimulated when bound to the targets via the bispecific bridge, it was of interest to see whether activation correlated with cytotoxicity. While purified CD4+ and CD8+ T cells induced a similar degree of cytotoxicity of Kat0 targets in a 20-h 51Cr release assay, in a 4-h assay only CD8+ cells proved to be cytotoxic. This difference may be explained by the preformed lytic molecules of CD8+ cells that are readily released from storage granules by an activation signal, whereas CD4+ T cells acquire the cytotoxic phenotype only after prolonged activation (30). The significantly lower T cell activation induced by the murine target cells correlated well to the much smaller lysis of these cells. Consistently, murine X63-17-1A cells hardly induced lytic activity of CD4' cells, also not after prolonged incubation. Although costimulatory CD28 Abs increased the activation of redirected T cells, their cytotoxic capability was not increased (Fig. 8). Thus, we suggest that activation parameters (CD25) do not necessarily predict the cytotoxic activity of T cells. As the Fas ligand can be induced by prolonged activation in both CD8+ and CD4+ T cells (31), Fas-mediated apoptosis, the cytotoxic pathway used predominantly by CD4 T cells (32, 33), may contribute to the cytotoxicity observed after 20 h of incubation. The partial inhibition of tumor cell lysis (Fig. 6) by anti-Fas Abs indicates that also this form of lytic mechanism can be mobilized by bispecific Abs. Interestingly, the most pronounced inhibition is seen at lower concentrations of the bsc Ab. As killing by T cells is mediated by more than one mechanism, the inhibitory effect of anti-Fas Abs may be seen only with moderately activated T cells, Le., at lower bsc Ab concentrations (see also Fig. 3), while Downloaded from www.jimmunol.org on May 10, 2010 In general, bispecific single-chain Abs appeared rather labile, as the four variable domains, although joined by flexible linkers, are kept in the right quaternary structure only by noncovalent interactions between the light and heavy variable domains. We investigated the stability of the 17-1NCD3-bispecific Ab, when stored in PBS at 4°C or in freshhuman serum at37°C for prolonged periods of time. Storage inPBS at a concentration of 200 pg/ml over 6 mo did not reduce the amount of active molecules nor the affinity of the Ab significantly. Incubation of the bsc Ab in serum at 37°C causes a slow decrease in biologic activity to 87% over 56 h, as measured in a 51Cr release assay (data not shown). I X 3970 Acknowledgments We thank E. Kopp for protein purification and S . Dittrich for excellent technical assistance. This study includes work performed by M. Mack in partial fulfillment of requirements for a medical thesis of the Faculty of Medicine, University of Munich. References I. Titus, J. A,, M. A. Garrido, T. T. Hecht, D. F. WinMer, J. R. Wunderlich, and D. M. Segal. 1987. Human T-cells targeted with antiGT3 cross-linked to antitumor antibody prevent tumor growth in nude mice. J. Immunol. 138:4018. 2. Weiner, G. J., and J. R. Hillstrom. 1991. Bispecific anti-idiotype/anti-CD3 antibody therapy of murine B cell lymphoma. J. Immunol. 147:4035. 3. Demanet, C.. J. Brlssinck, 0. 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Immunol. 152:1127. 33 Hanabuchi, S., M. Koyanagi, A. Kawasaki. N. Shinohara, A. Matsuzawa, Y . Nishimura, Y. Kobayashi, S. Yonehara. H. Yagita, and K. Okumura. 1994. Fas and Its ligand in a general mechanism of T-cell-mediated cytotoxicity. Proc. N a f l . Acali. Sci. USA YI:4Y30. 34 Yokota, T., D. E. Milenic. M. Whitlow, and J. Schlom. 1992. Rapid tumor penetration of single-chain Fvand comparison with other immunoglobulin forms. Cunrer Res. 52:3402. 35 Shen, J., B. Atkinson. H. Koprowski, and H. F. Sears. 1984. Binding of human immunoglobulin to human tissues after immunotherapy with anticolorectal carcinoma monoclonal antibody. Inr. J. Cancer 33:465. Downloaded from www.jimmunol.org on May 10, 2010 at maximum T cell activation other lytic mechanisms may override the Fas blockade. Taken together, the described bispecific Ab appears to be a promising candidate for a therapeutic trial in minimal residual disease. Its secretion from theproducer cell line in a fully functional form and the ease of its purification via aftinity chromatography on Ni-NTA columns will greatlyfacilitate the procurement of clinical grade material. Another major advantage is clearly its small molecular mass of 60 kDa, since size is important for tumor and tissue penetration(34). The remarkable stability of the construct in protease-rich human serum at 37°C is a further asset. Moreover, as the bispecific single-chain Ab does not modulate the CD3 molecule on nonengaged T cells, and as the 17-1A Ag is not endocytosed by tumor cells (35), the Ab should be resident for sufficient periods of time on both effectors and targets. The data with purifiedCD4+ and CD8+ T cells show that large numbers of accessory cells within the tumor may not be necessary for efficient activation of retargeted T cells and lysis of tumor targets. However, the strongest argument for its clinical testing may be derived from its high lytic activity and the lack of autochthonous stimulatory activity on T cells in the absence of tumor targets. The peculiar characteristics of the single-chain construct should allow the administration of higher doses required for tissue penetration, as well as for optimal tumor cell cytotoxicity. A caveat needs to be added because of the wide distribution of the 17-1A target Ag on normal simple epithelia. However, since the unmodified murine 17-1A Ab (20) and a conventionally produced bispecific 17- lA/CD3 Ab derived from hybrid-hybridoma (1 1) did not exhibit major tissue-specific toxicity, one can therefore expect that the presented bispecific single-chain Ab is also well tolerated. The immunogenicity of the construct could be reduced further by humanization to prevent the formation of human anti-mouse Abs, which could lead to tumor-independent T cell activation via their Fc parts. Particularly for patients with minimal residual disease, who are essentially asymptomatic afterresection of theirprimary tumor, a nomutagenic therapywith no orlittle side effectsoffers a great benefit. PROPERTIES OF A 17-1A/CD3-BISPECIFIC SINGLE-CHAIN Ab
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