Cancer Immunol Immunother (1997) 45: 203 ± 206
Ó Springer-Verlag 1997
O R I G I N A L A RT I C L E
B.J. Kroesen ? J. Nieken ? D.T. Sleijfer ? G. Molema E.G.E. de Vries ? H.J.M. Groen ? W. Helfrich ? T.H. The N.H. Mulder ? L. de Leij
Approaches to lung cancer treatment using the CD3E ´ GP-2-directed Bispecific Monoclonal Antibody BIS-1 Accepted: 14 October 1997
AbstractmThe bispecific monoclonal antibody (bsAb) BIS1 combines a monoclonal-antibody(mAb)-defined specificity for the CD3 complex, as present on all T lymphocytes, with a mAb-defined specificity for the pancarcinoma/ epithelium associated glycoprotein EGP-2. In vitro studies indicate that BIS-1 can direct T lymphocytes to kill EGP-2positive tumour target cells. T cell pre-activation is necessary for this activity and can be obtained either via incubation of isolated peripheral blood mononuclear cells with CD3 mAb, followed by short culturing in recombinant interleukin-2-containing medium, or via costimulation with CD5- and CD28-based bsAb. Clinical application of BIS-1 was started in a pilot study in which carcinoma patients suffering from malignant ascites or intrapleural effusion were treated. In this study, ex vivo activated autologous lymphocytes were applied locally, i.e. intraperitoneally or intrapleurally, in the presence of BIS-1. Local inflammation and antitumour activity were observed, whereas no or only minor systemic toxicity was seen in these patients. Intravenous administration of BIS-1 F(ab9)2 in combination with subcutaneously given recombinant interleukin-2 (i.v. bsAb/rIL-2 treatment) induced transient but considerable toxicity including peripheral vasoconstriction, dyspnoea and fever with a maximal tolerated dose of
5±8 mg/kg. High plasma concentrations of the inflammatory cytokines tumor necrosis factor a and interferon g were observed at this dose. Whereas bsAb-dictated antitumour activity could be demonstrated to be present in blood samples of these patients in an in vitro assay, no clear clinical responses were observed. In a rat model it was found that i.v. bsAb/rIL-2 treatment of EGP-2-positive tumours was effective when a low systemic tumour burden was present, suggesting that systemic bsAb/rIL-2 treatment might be effective in situations of minimal residual disease. Key wordsmLung cancer ? CD3 ´ EGP-2 ? Bispecific monoclonal antibody
Introduction
Bispecific antibodies (bsAb) have been used for a number of purposes like immunohistochemistry, immune cell activation, antigen scavenging and drug targeting. However, most attention has been given to a phenomenon known as bsAb-redirected cellular cytotoxicity [7]. By virtue of a dual specificity for an activation molecule on effector cells and a tumour-associated antigen (TAA) on target cells, bsAb are capable of retargeting the lytic potential of various Work presented at the 5th World Conference on Bispecific Antibodies ± cytotoxic effector cells towards selected TAA-positive under the Auspices of EFIS. 25 ± 28 June 1997, Volendam, The Nethtumour cells. The possible implications of this concept erlands for the immunotherapy of cancer have been readily recognized and, as a result, various in vitro animal and clinical B.J. Kroesen ? J. Nieken ? G. Molema ? W. Helfrich ? T.H. The studies have been performed on this subject during the past L. de Leij ( ) Department of Clinical Immunology, Internal Medicine, University years [2, 3, 7]. Hospital Groningen, Hanzeplein 1, 9713 GZ Groningen, The NetherBsAb-directed antitumour activity can be executed by lands all immune effector cells with killing capacity. This inTel. +31 50 3613420; Fax +31 50 3121576; cludes the cytotoxic T lymphocyte population, where bsAb e-mail:
[email protected] can bridge activation molecules like CD3 to TAA. In this J. Nieken ? D.T. Sleijfer ? E.G.E. de Vries ? N.H. Mulder Department of Medical Oncology, Internal Medicine, University Hos- way normal T-cell-receptor/MHC interactions are bypassed pital Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands and cytotoxicity is dictated by the bsAb specificity (bsAbmediated T lymphocyte retargeting). So, cytotoxicity ocH.J.M. Groen Department of Lung Diseases, Internal Medicine, University Hospital curs irrespective of T cell receptor specificity (i.e. all cytotoxic T lymphocytes can be mobilized) and irrespective Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands
204 Table 1mPatient's characteristics. AT activated T cells, RAT redirected activated T cells Characteristics Number of patients Female/male Age (years) Median Range Tumour type Colon cancer Mammary cancer Ovarian cancer Lung cancer Gastric cancer Site of effusion Peritoneum Pleura Treatment given AT RAT
n 9 5/4 52 45 ± 73 3 1 2 2 1 7 2 7 9
of the degree of MHC expression/peptide presentation by the tumour cell. We have exploited the concept of bsAb-mediated T lymphocyte retargeting for the treatment of carcinoma patients [2, 3]. To this end, a bsAb named BIS-1 was generated by the hybrid hybridoma technology first described by Milstein et al. BIS-1 comprises a specificity for CD3 and a specificity for EGP-2, a 38-kDa epithelial/ carcinoma-associated transmembrane glycoprotein. mAb recognizing EGP-2 have been clustered as small-cell lung cancer (SCLC) cluster 2 antibodies and include CO17-1A, KS1/4, AUA-1 and MOC-31 [9]. EGP-2 is not shed from the cell membrane and is abundantly expressed on almost all carcinomas, whereas its expression on normal tissue is restricted to simple epithelia. In the latter tissues EGP-2 appears to be shielded from the circulation by a basal membrane. The use of antibodies against EGP-2 has been described in a number of clinical studies showing both good imaging of tumours [1] and therapeutic effect [10]. Here we give a short overview of our experience with BIS-1.
Results and discussion
Establishment of BIS-1 The hybrid hybridoma BIS-1 was made by fusion of hybridoma RIV-9 (anti-CD3, mouse IgG3, a gift of Dr. H. Kreeftenberg, RIVM, The Netherlands) and a hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-negative and neomycin-resistant derivative of hybridoma MOC-31 (anti-EGP2, mouse IgG1 [8, 9]) using standard polyethylene glycol (PEG) fusion procedures. The derivative of MOC-31 was made by culturing hybridoma MOC31 in 8-azaguanine-containing medium to select a HGPRTnegative subclone, followed by retroviral transfection to introduce neomycin resistance. The hybrid hybridoma BIS1 was obtained by culturing in hypoxanthine/aminopterin/
thymidine/neomycin-containing medium followed by repeated cloning. Purification of hybrid antibody from BIS1 culture supernant was performed by protein A column chromatography. The preparation was then either dialysed against 0.9% NaCl, after which human serum albumin was added (0.5%) and the preparation was sterilised and checked for contaminants (local treatment), or further digested with pepsin to obtain F(ab9)2 (intravenous treatment). BIS-1 F(ab9)2 was isolated by G150 Sephadex gel filtration, after which the preparation was adjusted to 0.9% NaCl, 0.5% human serum albumin, sterilised and checked for contaminants and toxicity according to the protocol of the Dutch Pharmacopee (1983)[1a]. Both BIS-1 IgG, BIS-1 F(ab9)2 and a recently constructed diabody with anti-CD3/anti-EGP2 specificity were highly potent in mediating T lymphocyte cytotoxicity against EGP-2-positive targets (60%±80% lysis in a 4-h chromium-release assay at an E/T ratio of 9), whereas no appreciable cytotoxicity was present against EGP-2-negative targets. Chromium release was shown to be paralleled by induction of apoptosis of target and not effector cells [6]. For the activation of T lymphocytes, isolated peripheral blood mononuclear cells can be stimulated with anti-CD3 for 2 days, followed by culturing in low-dose recombinantinterleukin-2(rIL-2)-containing medium for 3 additional days. This protocol preferentially activates and expands T cells, as shown by immunostaining against CD3 and CD8 (generally more than 95% and more than 70% positive respectively, at day 5). An alternative way of activating T lymphocytes is to incubate freshly taken blood samples with immobilized EGP-2 in the presence of bsAb directed to EGP-2, on the one hand, and to CD3, CD28 and CD5 on the other. In this way proper costimulation is obtained, which results in the effective activation of the T lymphocytes present in the blood sample [4]. Local treatment with BIS-1-redirected autologous activated T cells Nine carcinoma patients with malignant peritoneal or pleural effusion were treated locally with autologous activated T lymphocytes in the presence (retargeted activated T lymphocytes, RAT)) or absence (activated T lymphocytes, AT) of BIS-1 [2]. Patients9 characteristics and the treatment given are shown in Table 1. All patients received RAT, whereas most of these also received AT. The activated T lymphocytes used for reinfusion resulted from an ex vivo culturing protocol using CD3/rIL-2 as indicated above. Samples taken serially from the effusions, as well as blood samples, were analysed for signs of antitumour activity and immune activation. After RAT treatment, i.e. infusion of activated T cells in the presence of BIS-1, large conjugates between infused T lymphocytes and tumour cells were found to be formed in the samples within 1±4 h. A clear reduction or complete disappearance of tumour cells was apparent in the samples 24 h after the start of treatment. Along with these cytological signs of antitumour activity, local carcinoembryonic antigen levels
205 Table 2mPatient's, characteristics Characteristic
n
Number of patients Female/male Age (years) Median Range WHO performance 0 1 2 Prior therapy Nephrectomy Chemotherapy Immunotherapy Tumour localization Primary Lung Bone Other No. metastatic sites 1 2 3 or more
14 6/8 58 48 ± 70 3 7 4 6 0 14 8 9 5 4 2 8 4
started to decrease gradually 24 h after the start of the treatment. No such evidence of antitumour activity was apparent after AT treatment, i.e. without BIS-1. RAT treatment also proved to induce a marked, but transient, local inflammatory reaction, as illustrated by a steep increase in local granulocyte numbers and tumor necrosis factor a (TNFa) levels, 24 h after the start of treatment. Subsequently, 48±72 h after the start of treatment, these returned to normal and macrophages became the most prominent cells present. As another indication of an inflammatory reaction, it was noted that adhesion molecules such as ICAM-1 became rapidly up-regulated both on leucocytes and tumour cells as soon as 4 h after the start of treatment. No clear signs of any systemic inflammation were present in these patients, i.e. only a mild feverish reaction 24±48 h after the start of therapy with no systemically present cytokines (like IL-6) or acute-phase response.
Systemic treatment of carcinoma patients with BIS-1 F(ab9)2 A phase I study for the systemic treatment of carcinoma patients with BIS-1 was started. Since the implementation of a protocol for ex vivo activation of enough autologous lymphocytes for such a purpose was deemed unrealistic, it was argued that in vivo activation of lymphocytes by the application of rIL-2 could be an easy and already standard treatment procedure, at least for renal cell carcinoma (RCC) patients. Therefore we started a phase I study [increasing amounts of intravenously applied BIS-1 F(ab9)2 in combination with standard s.c. rIL-2] in RCC patients who had failed to respond to previous treatment (i.e. standard s.c. rIL-2 treatment) [3]. The patients9 characteristics are given in Table 2. To avoid aspecific immune activation through
interaction with FcR-positive cells, F(ab9)2 fragments of BIS-1 were prepared. In vitro, BIS-1 F(ab9)2 induced efficient lysis of EGP-2-positive tumour cells, whereas FcR-positive target cells (P815) were not killed, indicating the absence of an intact BIS-1-associated Fc domain. Patients were treated with 1, 3 or 5 mg/kg BIS-1 F(ab9)2 during concomitant s.c. IL-2 therapy. Despite the use of F(ab9)2 fragments, transient but severe treatment-associated toxicity was observed at the 3-mg/kg and 5-mg/kg dose levels, with the latter dose considered to be the maximum tolerated. No significant clinical tumour responses were seen. Toxic side-effects were accompanied by high systemic concentrations of TNFa and interferon g (IFN-g) and a transient drop, in the number of circulating lymphocytes, monocytes and eosinophils. Neutrophil numbers did not drop, suggesting a VLA-4-mediated redistribution of leucocytes. This study is now being extended to a novel phase I trial in which i.v. BIS-1 in combination with s.c. rIL-2 is given repeatedly. Preliminary data indicate that BIS-1 F(ab9)2 can be given in doses of 5±8 mg/kg when applied on 5 consecutive days. However, in this trial also, no objective antitumour responses were observed. The degree to which CD3 is occupied in the blood of the above patients was determined flow-cytometrically and proved to increase dose-dependently. It is known from in vitro data that maximal cytolytic activity of retargeted T lymphocytes is obtained when about 20%±30% of CD3 is occupied with BIS-1 F(ab9)2. A dose of 5±8 mg BIS-1/kg appears enough to reach such a level in the blood of treated patients. Systemic treatment of EGP-2-positive lung carcinoma metastases in a rat model To investigate the possibilities of systemic treatment of metastatic tumour with bsAb in more detail we have developed a rat tumour model [5]. In this model a rat squamous cell carcinoma cell line (L37) has been transfected with the gene encoding human EGP-2. These transfected cells can be injected i.v. into immunocompetent rats and give rise to tumour outgrowth in the lungs. Treatment of these rats, 7 days after tumour inoculation, with a bsAb combining anti-EGP-2 specificity with anti-rat T cell receptor specificity in the presence of rIL-2 during 5 consecutive days, resulted in an almost complete disappearance of tumour. Apparently, if a treatment schedule comparable to the one given above in the clinical study is used, the treament of rat tumour is effective. One important difference between the rat model and the patient treatment setting was the stage of disease being treated. Whereas rats with only a minimal tumour load were treated, patients received treatment at a late stage of disease. To study the events in the rat model, we have followed the redistribution of leucocytes into tissue during bsAbbased treatment. No significant accumulation of leucocytes was observed in tumour nodules as compared to normal tissues. Rather a general transmigration of leucocytes, including bsAb-loaded T lymphocytes, through all endothelia was observed. These data can be taken as an indication
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that the present i.v. bsAb/rIL-2 treatment may be effective only in situations of minimal residual disease, since only with low tumour burden can a sufficiently high E/T ratio be obtained from the randomly transmigrating bsAb-loaded lymphocytes.
Conclusion We have shown that EGP-2 is a good target antigen to which an effective cytolytic activity of bsAb-retargeted T lymphocytes can be directed both in vitro and in vivo. The latter included local treatment in patients and systemic treatment in a rat tumour model. However, in the systemic treatment of patients in a phase I setting, the toxicity proved to be rather high (maximum tolerated dose: 5±8 mg/kg), whereas no significant tumour response was obtained. We therefore conclude that the current CD3-targeted bsAb treatment of cancer is likely to be effective only in situations of minimal residual disease, e.g. after initial tumour reduction by conventional treatment modalities. In lung cancer it is known that small-cell lung cancer patients often show initially an apparently complete response to chemotherapy, but subsequently almost inevitably die of recurrent disease with a chemotherapy- and/or radiotherapy-resistant phenotype. These patients might benefit from the bsAbbased treatment protocols described here.
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