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In vitro and in vivo stability and anti-tumour efficacy of an anti-EGFR/anti-CD3 F(ab')2 bispecific monoclonal antibody.

The in vitro and in vivo stability and anti-tumour efficacy of the anti-EGFR/anti-CD3 bispecific monoclonal antibody (biMAb), M26.1, were analysed. The interaction of the intact biMAb with Fc receptor I (Fc gamma RI) present on human leucocytes was not observed when the antibody was used as an F(ab')2 fragment. A CD8+ T-cell clone coated with M26.1 F(ab')2 was as effective as the intact biMAb in inducing IGROV1 target cell lysis when tested in a 51Cr-release assay. Variable levels of reduction of F(ab')2 to monovalent F(ab') were observed upon incubation with human ovarian cancer ascitic fluid (OCAF) or with human glioblastoma cavity fluid (GCF), but not with mouse or human sera. Activated lymphocytes coated with F(ab')2 and incubated in vitro with GCF or OCAF for 24 and 48 h respectively maintained their targeting. Thus, the F(ab')2, when present as a soluble molecule, but not when bound to T cells, might lose some functional activity as a consequence of partial reduction to F(ab'). In normal mice, M26.1 F(ab')2 retained full cytotoxic activity in the circulation, and clearance values were similar to those obtained with parental and other MAb F(ab')2. Treatment of IGROV1 tumour-bearing mice with activated human lymphocytes coated with the M26.1 F(ab')2 significantly prolonged survival of the animals compared with tumour-bearing untreated and control mice treated with lymphocytes or F(ab')2 alone. Together, these results suggest the clinical usefulness of bispecific M26.1 F(ab')2 as a targeting agent for local treatment of tumours such as glioma and ovarian cancers that express variable levels of epidermal growth factor receptor (EGFR).

The epidermal growth factor receptor (EGFR), a 170 kDa transmembrane glycoprotein with tyrosine kinase activity, is overexpressed in a wide range of human malignancies (Gullick, 1991). Increased EGFR expression correlates with a poor clinical outcome in patients with cancer of the lung, bladder, oesophagus, breast, cervix and ovary (Hendler et al., 1989;Fox et al., 1994). Only in glial and head and neck tumours has EGFR over-expression been frequently associated with amplification of the gene (Libermann et al., 1985;Chaffanet et al., 1992;Wong et al., 1992), and on tumour cells expression levels can be increased by several orders of magnitude, suggesting the feasibility of therapeutic strategies that exploit the differential levels of EGFR expression on tumours vs normal cells. Like other growth factor receptors on tumour cells, EGFR may represent a suitable target molecule for antibody-driven therapy. A panel of anti-EGFR monoclonal antibodies (MAbs) is now available (Gill et al., 1984;Rodeck et al., 1987;Mendelsohn 1990) and several of them have been applied in preclinical (Bender et al., 1992;Baselga et al., 1993;Fan et al., 1993a,b) and clinical immunotherapeutic settings (Kalofonos et al., 1989;Divgi et al., 1991;Brady et al., 1992).
MAbs specific for tumour-associated antigens (TAAs) have been used to construct bispecific reagents in conjunction with MAbs against T-lymphocyte surface molecules capable of cell activation, such as the CD3/TCR complex (Segal et al., 1988;Beun et al., 1994). These biMAbs can target T-cell-mediated cytotoxicity and induce lysis of target cells in an MHCindependent manner. BiMAbs have been employed to arm large numbers of effector cells in vitro (Pupa et al., 1988), which have been used to control tumour cell growth in nude mice bearing human cancer xenografts (Mezzanzanica et al., 1991a;Renner et al., 1994) and also in humans with ovarian (Bolhuis et al., 1992) and brain tumours (Nitta et al., 1990).
We previously described an anti-EGFR/anti-CD3 biMAb, termed M26.1, that specifically targets activated T lymphocytes, but not resting peripheral blood lymphocytes (PBLs), against EGFR+ tumour cells (Ferrini et al., 1993). When low concentrations of biMAb were present, only tumour cells with a moderate to high level of EGFR were lysed, indicating that induction of tumour cell lysis was strictly dependent upon the level of the expression of the EGFR molecule on target cells (Ferrini et al., 1993).
In the present preclinical analysis we show that the M26.1 biMAb F(ab')2 retains its ability to trigger T-cell activity against specific target cells either, as a soluble molecule, after recirculation in normal mice or, bound to T cells, after in vitro incubation in the presence of human pathological fluids. More importantly, we demonstrate the efficacy of biMAbarmed T lymphocytes in prolonging survival of tumourbearing mice in an ovarian cancer xenograft immunotherapy Antibodies M26.1 biMAb (IgGl/IgG2a) was produced by fusion of an anti-EGFR hybridoma (IgGI) derived from mice immunised with EGFR-overexpressing A431 cells together with an anti-CD3 hybridoma (IgG2a), derived from mice immunised with human T lymphoblasts and which recognizes human but not murine CD3 (Ferrini et al., 1993). The two parental MAbs and the hybrid biMAb were purified from mouse ascitic fluid by chromatography on a Sepharose-protein A column using a three-step pH elution gradient of 0.1 M sodium citrate. Purified bispecific M26. 1 was analysed as described previously (Ferrini et al., 1993) by hydroxyapatite highperformance liquid chromatography (HPLC) using a linear gradient (10-350 mM) of potassium phosphate, pH 6.8. MAbs 197 and 3G8-FITC (Medarex) and CD32 FcR2 (Amac), which specifically detect respectively Fc'yRI, Fc-yRIII and Fc'yRII, were used to monitor expression of the relevant target molecules.
Biological fluids Human sera from healthy donors were collected and pooled; human ascitic fluids (OCAFs) were derived from advanced ovarian cancer patients; glioblastoma cavity fluids (GCFs) were recovered from the cystic cavity remaining after tumour excision from glioblastoma patients. All biological fluid samples were centrifuged and filtered through 0.22 ym filters, and stored at -20C until assayed. Protein concentrations were calculated using the Pierce BCA protein assay. A transforming growth factor beta-l (TGF-P1) enzyme-linked immunosorbent assay (ELISA) kit (Genzyme) was used to determine the presence of TGF-P in GCF and OCAF.
Immunofluorescence assay Tumour cell lines were analysed for reactivity with MAbs by FACScan analysis. Approximately 4 x 105 cells were incubated for 30 min at 0°C with 0.1 ml of reagent (anti-EGFR, anti-CD3, M26.1 or anti-FcyRs) at a concentration of 10pgml-'. The anti-Fc'R reagents were mixed with human 'y-globulins (Sigma) at 2 mg ml-' to block Fc regionspecific binding of MAb.
Preparation of bivalent MAb fragments F(ab')2 fragments of M26.1 biMAb and anti-CD3 MAb were obtained by pepsin digestion. MAb or biMAb were dialysed overnight against 20 mm sodium acetate buffer (pH 4.2), followed by digestion with 4% (w/w) pepsin for 6 h at 37°C. Digestion was terminated by addition of Tris-HCI buffer (pH 9.2), and pepsin and fragments were removed by overnight dialysis (molecular wt cut-off, 50000) against 10 mM sodium phosphate buffer (pH 8.2) containing 150 mM sodium chloride. Undigested MAbs were separated from F(ab')2 fragments on an ImmunoPure Plus-immobilised protein A gel (Pierce, Rockford, IL, USA) using the ImmunoPure IgG purification buffers. Purity of the fragment was analysed by 4-15% SDS-PAGE in a precast slab gel (Pharmacia Biotechnology, Uppsala, Sweden), using the automated microprocessor driven Phastsystem (Pharmacia) according to the manufacturer's suggestions. The F(ab')2 fragment of anti-EGFR MAb was obtained by ficin digestion as described (Mariani et al., 1991).
Pharmacokinetics studies Pathogen-free female athymic mice (nu/nu CD1 background) or normal BALB/C mice, 6-8 weeks old, were obtained from Charles River (Calco, Como, Italy). Mice were held for 1-2 weeks before initiating experiments. Animals were housed under sterile conditions and received autoclaved food and water. According to their sensitivity to labelling procedures, M26.1 and its F(ab')2 were labelled with 1251I using lodogen, whereas anti-EGFR and its F(ab')2 were labelled using the Anti-EGFR/Anti-CD3 F(ab')2 biMAb stability and efficacy DRM Negri et al 929 lactoperoxidase method (Marchalonis, 1969). Mice were injected i.p. with 1.8-3.4 ig of 251I-labelled intact MAb or F(ab')2 (sp. act. 2.7-5.2 ILCi sg-') or with 10 lAg of unlabelled F(ab')2 M26.1. Blood samples were collected at various times (from 30min to 72h) after administration (three mice for each time point) and serum was recovered. For mice injected with radiolabelled reagents, an aliquot of each serum sample was counted in a gamma-counter and protein-bound radioactivity was measured by paper chromatography in 10% trichloroacetic acid. For mice injected with unlabelled reagent, the presence of active F(ab')2 in blood serum samples were evaluated in a standard 4 h 51Cr-release assay. Pharmacokinetic parameters were calculated using the MKMODEL modelling program (Biosoft, Cambridge, UK).
Activation and coating of human PBL Freshly obtained PBLs from healthy donors were activated, expanded and coated with M26. 1 biMAb F(ab')2 as described (Bolhuis et al., 1992).
Cytotoxicity assays Activated lymphocytes, T-cell polyclonal lines and T-cell clones (Ferrini et al., 1989) were used as effector cells and IGROVI cells were used as target at effector-target cell ratios from 10:1 to 40:1 in a 4h 51Cr-release assay. For analysis of MAb-induced cytotoxicity, various concentrations of biMAb or F(ab')2 were added at the start of the assay. To test whether biMAbs maintained their ability to trigger cytolysis, coated lymphocytes were incubated in vitro for different times in the presence of OCAF or GCF (final dilution, 1:2). Unwashed lymphocytes were added at a given time to labelled target cells at an effector-to-target cell ratio of 40:1 and specific lysis was assayed in a 4 h 5'Cr-release assay. Per cent lysis and biMAb or F(ab')2 concentrations producing half-maximal cytolysis (ED50) were evaluated as described.

Stability of F(ab')2 biMAb
An aliquot of 5 IlI of 1251I-labelled F(ab')2 biMAb was incubated at 37°C with 45 pl of BALB/C mouse or human sera, OCAF and GCF samples, or saline. At various times from 30 min to 72 h, 1 IlI (2000 c.p.m.) of each sample was analysed by 4-15% SDS-PAGE, autoradiography for a week at -80°C and densitometry.
In vivo activity of F(ab')2 biMAb Five groups of athymic mice (8-12 animals per group, see Table III) were injected i.p. with 107 IGROVI cells, maintained in vivo by serial transplants, on day 0. Animals were injected i.p., twice a day, on days + 3 and + 4 with various combinations of activated PBLs and parental MAbs or biMAb. Mice were monitored for abdominal swelling and mortality. Treatment effects were compared by Wilcoxon and log-rank non-parametric tests.

BiMAb FcR interaction
We previously described a heteroisotypic (IgGl/IgG2a) anti-EGFR/anti-CD3 biMAb M26. 1, secreted by a hybrid hybridoma (Ferrini et al., 1993). This antibody was unable to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) by CD16+ (Fc-yRIII+) natural killer (NK) cells. To determine whether the Fc region of this biMAb interacted with Fc-yRI and/or II, the binding of M26.1 and the parental anti-EGFR and anti-CD3 MAbs was evaluated by immunofluorescence and FACS analysis. Figure 1 shows the binding of control anti-Fc-yR MAbs (a) and of the relevant DRM Negri et al MAbs (b) to the two FcyR-positive cell lines U937 and K562 and to the EGFR-positive line IGROVI (4 x 104 EGF binding sites per cell). As shown in Figure la, both anti-FcyRI and anti-FcyRII bound to U937 cells, only anti-FcyRII reacted with K562 cells and none of the anti-FcyR MAbs reacted with IGROVI cells. The intact anti-EGFR MAb and biMAb molecules bound to EGFR-positive, FcyRIand IInegative IGROVI cells and to EGFR-negative, Fcy?RIand II-positive U937 cells, while intact anti-CD3 interacted only with U937 cells. BiMAb failed to bind with FcyRI-negative FcyRII-positive K562 cells. Binding with U937, but not with IGROVI cells, was abolished by preincubation of cells with human IgG (data not shown) or by removal of MAb Fc portion (Figure 1). These results indicate that intact MAbs and biMAb can bind to FcyRI present on human cells. Therefore, to avoid unwanted interactions of biMAb with FcyRI in vivo, the F(ab')2 fragment of the biMAb was generated and used for further experiments. In vitro activity and stability of biMAb F(ab')2 Comparison of the ability of the F(ab')2 vs intact biMAb to target a CD8+ T-cell clone against IGROVI cells in a "Crrelease assay revealed that the F(ab')2 biMAb was as effective as the intact biMAb (ED50 = 5.3 and 4.9 ng ml-' respectively) in inducing target cell lysis. Analysis of the biological activity of the F(ab')2 fragment in the presence of normal mouse sera indicated that the concentration of reagent required to induced half-maximal cytolysis was comparable to that required in standard culture medium (data not shown). To further analyse F(ab')2 biMAb stability in different biological fluids, labelled F(ab')2 was incubated in vitro in the presence of normal mouse and human sera, four different OCAFs or four GCFs and the samples were analysed at various time intervals by SDS-PAGE followed by densitometry (Table I). In OCAFs, but not in mouse and human sera, there was a slight reduction of intact F(ab')2 biMAb, indicated by the appearance of a band corresponding to F(ab') (50 000 kDa) detected at 72 h. In GCFs, variable levels of reduction to F(ab') were evident at 24 h (5-23%), increasing up to 53% by 48 h after incubation with GCFs 1 and 4. The difference in reducing capacity did not depend on protein concentration. The large variability might be due to differences in reducing agents in the different samples. Indeed despite a wide variability among patients, reproducible results were obtained when individual samples were analysed in repeated tests (data not shown). However the biMAb F(ab')2, when bound to T cells, was remarkably stable during prolonged in vitro incubation with different pathological fluids. Human activated T lymphocytes coated with M26.1 F(ab')2 and incubated in vitro in the presence of GCFs or OCAFs for 24 h and 48 h respectively, maintained their ability to lyse IGROVI target cells in a 5"Cr-release assay (Figure 2). The presence of the immunosuppressive cytokine TGF-01, which was detected in variable amounts in GCF and OCAF, did not seem to influence the cytotoxic ability of activated lymphocytes.

Pharmacokinetics
To test whether the biMAb F(ab')2 fragment retained its targeting activity in vivo BALB/C mice were injected with the reagent and serum samples obtained at different time intervals were tested for their ability to trigger cytolytic T lymphocytes in a 51Cr-release assay against IGROVI target cells (Figure 3). At 2 and 6 h after injection, the dilutions of mouse sera able to induce lysis were superimposable. F(ab')2 biMAb activity progressively decreased after 24 h but was still detectable at 72 h. The pharmacokinetic and the half-life values (t,/2p) calculated on the basis of this functional assay were similar to those of radiolabelled F(ab')2 evaluated in conventional pharmacokinetics assay (Table II). The same labelled reagent had a shorter t1/2P (approximately 7 h) in athymic mice, suggesting a different pharmacokinetic behaviour in the two mouse strains, as already reported by others (Sharkey et al., 1991). The shorter t1/2P of M26.1 F(ab')2 as compared to parental anti-EGFR F(ab')2 might reflect a more rapid degradation of the heteroisotypic form. When biMAb and parental anti-EGFR MAb were compared, similar values were obtained.

In vivo anti-tumour activity of human T cells targeted by biMAb F(ab')2
The in vivo anti-tumour activity of PBLs targeted by biMAb F(ab')2 was evaluated in a preclinical model of human tumour xenografts in nude mice. Intraperitoneal injection of 107 IGROVI cells into the mice caused death rapidly, with a mean survival time of 10.75 days (Table III) Anti-EGFR/Anti-CD3 F(ab)2 biMAb stability and efficacy DRM Negri et al 931 the diaphragm and liver. Nude mice bearing 3 day i.p. IGROVI tumours and treated i.p. on day 3 and 4 with PBLs alone, PBLs plus parental MAbs or M26. 1 F(ab')2 alone showed no increase in mean survival time (10.91 * . 11.83 days) as compared to untreated mice (10.75 ± 0.41 days), whereas mice treated with activated PBLs coated with M26.1 F(ab')2 showed a significant increase in mean survival time (23.17 ± 2.67 days, P = 0.003) (Table III, Figure 4).

Discussion
In this report, we show that the anti-EGFR/anti-CD3 biMAb M26. 1, following removal of the Fc region, retains its ability to target human T lymphocytes against EGFR+ tumours in vitro in the presence of different mouse and human biological fluids, and in vivo in a xenotransplanted mouse model.
Other biMAbs have been shown to interact with the three types of IgG FcRs involved in ADCC (Fanger et al., 1989;Mezzanzanica et al., 1991b), and with anti-TAA/anti-CD3 biMAbs, the binding with human cells via FcyR might induce unwanted phenomena such as damage of biMAbcoated T cells by FcyR' effectors or killing of FcyR+ targets    Mice were injected i.p. with tumour cells on day 0 and were treated on days +3 and +4 twice a day with: physiological solution; (0), PBLs alone (A), biMAb F(ab')2 alone (+), PBL-+ parental F(ab')2 (0) or PBLs coated with biMAb F(ab')2 (X).
(For doses see Table III). (Segal et al., 1988;Fanger et al., 1992). We have previously shown that intact IgGl/IgG2a heteroisotypic biMAb does not interact with FcyRIII present on human NK cells, being unable to mediate ADCC by CD16+ NK cells (Ferrini et al., 1993). In our present report, we evaluated M26.1 Fc binding with FcyRI and II, which are expressed on different cells such as monocytes, polymorphonuclear cells, B lymphocytes and also microglial (murine) cells (Bender et al., 1992) and observed that it interacted with FcTRI+ cells. Use of F(ab')2 fragments prevents this interaction, which assumes importance since one possible clinical application of this biMAb is local treatment of human glioma.
In view of its heteroisotypic nature (IgGl/IgG2a), we investigated the in vitro and in vivo stability of M26.1 F(ab')2 fragments. As previously reported, the pepsin cleavage site for IgGl and IgG2a is localised in different regions, i.e. the reaction produces a smaller segment in IgGl than in IgG2a (Parham, 1983). Consequently, F(ab')2 of the heteroisotypic biMAb is formed by two fragments of different length which might lead to an increased susceptibility to proteolytic or reducing activities. Indeed, our analysis of the stability of the biMAb F(ab')2 vs the parental anti-EGFR fragment in the presence of mouse and human sera or pathological human fluids revealed a variable level of reduction of intact F(ab')2 to monovalent F(ab') after incubation with pathological fluids, particularly in the case of GCFs. As expected, the phenomenon was more evident with biMAb F(ab')2. However F(ab')2 biMAb-coated lymphocytes maintained the ability to lyse EGFR+ targets even after 24 h and 48 h of incubation with GCFs and OCAFs respectively. Thus, it appears that biMAb F(ab')2, when present as a soluble molecule in some biological fluids, but not when bound to T cells, can lose functional activity as a consequence of partial reduction.
GCF and ascitic fluids have been shown to contain immunosuppressive factors, such as TGF-P (Hirte and Clark, 1991), which can inhibit in vitro activation and proliferation of lytic effector cells (Wahl et al., 1989) as well as antitumour cytotoxicity of cultured lymphokine activated killer (LAK) cells (Ruffini et al., 1993). Variable levels of TGF-P were found in all of our tested fluids, but its effect on the biMAb-directed lytic activity of activated lymphocytes appeared to be minimal, although the same fluids were able to inhibit the lymphocyte activation phase (data not shown). Further confirmation of the stability of the reagent in vivo came from analysis in normal mice injected with M26. 1 F(ab')2, where it retained full T-cell targeting activity in the circulation, and clearance values were comparable to those obtained with parental and other MAb F(ab')2 fragments (Fan et al., 1993b;Van Dijk et al., 1991).
To verify the in vivo anti-tumour activity of our reagent, we chose a preclinical survival model of nude mice using the EGFR+ human ovarian carcinoma cell line IGROVI. This model was selected because the tumour remains localised to the peritoneum and can therefore be treated with local immunotherapy. Specific in vivo targeting therapy using in vitro-activated lymphocytes coupled with biMAb has been described (Nitta et al., 1990;Bolhuis et al., 1992), and other clinical trials with different biMAbs are currently underway. Consistent with data from preclinical and clinical studies, we find that mice treated with activated human PBLs coated with M26.1 F(ab')2 survive significantly longer, although the treatment did not completely eradicate the implanted tumour. This failure probably reflects the high growth rate of the xenograft model which causes death more rapidly (mean survival time = 10.75 days) than in other preclinical models (Mezzanzanica et al., 1991a;Renner et al., 1994). More aggressive treatment might further improve survival rates and eventually lead to tumour cure.
This biMAb retargeting approach offers several advantages over others that utilise anti-EGFR MAbs therapeutically. Targeting of T cells by biMAb should allow a local release of inhibitory cytokines at the tumour site (Qian et al., 1991) which might act on all cells within a tumour, including cells that were sterically inaccessible to targeted PBLs or that had lost antigen expression. In our experimental model, adverse effects on normal tissues would not be expected in mice because the biMAb does not bind to the mouse EGFR (Valota et al., submitted) and to murine CD3 (Ferrini et al., 1993). Since human epithelia express moderate/low EGFR levels (Rodriguez et al., 1991;Banks-Schlegel et al., 1986) the systemic use of anti-EGFR MAbs in humans must be considered with caution. However, bispecific M26. 1 F(ab')2 might be used as a targeting agent for local treatment of tumours such as gliomas or ovarian cancers that express variable levels of EGFR molecules, but whose surrounding accessible normal tissues are EGFR negative.