BISPECIFIC ANTIBODIES

Abstract: A bispecific F(ab')2 antibody comprises a first Fab' arm specific for a type 1 ribosome-inactivating protein and a second Fab' arm specific for a marker on the surface of a target cell. A population of cells can be depleted by administering an effective amount of such an antibody, one of the Fab' arms of the antibody being specific for a marker on the cells, and of the type 1 ribosome-inactivating protein for which the other of the Fab' arms of the antibody is specific.

Description: BISPECIFIC ANTIBODIES This invention relates to bispecific antibodies, to their preparation and to pharmaceutical compositions containing them. Current interest in using antibody (Ab) with dual specificity for the treatment of neoplastic disease has concentrated mainly on two derivatives: IgG-heterodimers (or heteroaggregates) , prepared by chemically cross-linking two monoclonal IgG Ab (1-3); and bispecific IgG, secreted when two Ab-producing hybridomas are fused to form a hybrid- hybridoma ( , 5) . In vitro, both these products have shown some promise for targeting tumor cells, either with cellular effectors (6-8) or with pharmacological agents such as drugs or toxins (9, 10). Most effort to date has been concentrated on the use of bispecific Ab to retarget activated CTL onto tumor cells via the TCR-T3 complex. However there are problems using IgG polymers (dimers or heteroaggregates) i_n vivo. They may suffer from impeded access to targets due to their size. Their multiple Fc regions may enhance uptake in the reticuloendothelial system (11). Both the polymers and monomeric bispecific IgG might be hampered by modulation, promoted by FcR-bearing cells (12), with the result that both target and effector cells become stripped of their specific antigens. Finally, both types of derivative have the potential for damaging the effector cells they recruit. By displaying FC Y regions on these cells they put them at risk of becoming the targets in ADCC (13). According to the present invention, there are provided bispecific F(ab') 2 antibodies comprising a first Fab' arm specific for a type 1 ribosome-inactivating protein (RIP) and a second Fab' arm specific for a marker on the surface of a target cell. Such antibodies can function as an efficient vector for delivering the RIP to unwanted cells in vivo. Each Fab' arm of the bispecific antibody may be derived from a monoclonal or polyclonal antibody, which in turn may be a human, urine, rat or rabbit antibody, for example. Preferably, each Fab' arm is derived from IgG. Each arm is then a Fab'γ arm and the bispecific antibody is a bispecific F(ab'γ) 2 antibody. The Fab' arms are preferably linked by a thioether linkage, for example by a -S- (o-phenylenedisuccinimidyl) -S-group. The type 1 RIP for which one Fab' arm is specific may be an agrostin, dianthin, saporin, luffin, momordin, gelonin, tritin, PAP or docecandrin RIP. Type 1 RIP's are reviewed in Stirpe and Barberi, FEBS Letters, vol. 195, no. 1/2, 1986, 1-8. Preferably the Fab' arm specific for a type 1 RIP is specific for saporin or momordin. The other Fab 1 arm may be specific for a marker on B-cells, T-helper or -suppressor cells, monocytes, cytotoxic T cells, any unwanted cells, etc. The marker antigen may be a tumour marker. The marker may be an idiotype on an autoantibody-producing B cell (i.e. in autoimmune disease) or on any other B-cell, such as a 3-cell producing undesirable or unwanted antibodies. The marker may be an idiotype of a T-cell receptor in, for example, autoimmune T-cells or a marker expressed on a cell infected witn a microorganism such as a virus, bacterium or parasite. The virus may be a retrovirus such as HIV for which the envelope coat protein serves as a cell marker following infection of the cell by the virus. The tumour marker may be any tumour-associated antigen including the idiotype of secretory Ig of B cells or the T cell receptor of T-cells, a T cell antigen receptor. Such receptors are found on many B lymphocytic and T ly phocytic malignancies. This Fab 1 arm may therefore be specific for a B cell neoplasm or for other tumours sαch as a colorectal tumor or melanoma. Therefore, the Fab' arm may be idiotype-specific. The bispecific antibodies may be prepared according to the invention by a process comprising: (i) dissociating a F(ab') 2 antibody fragment specific for the type 1 RIP into its two component Fab' arms; (ii) dissociating a F(ab') 2 antibody fragment specific for the target cell marker into its two component Fab' arms; and (iii) linking an Fab' arm specific for the type 1 RIP to an Fab' arm specific for the target cell marker. Each F(ab') antibody fragment may be obtained by treating an antibody with the correct specificity with pepsin or any other appropriate proteolytic protein or organic or inorganic cleavage agent. The antibody may be a monoclonal antibody or polyclonal antibody, for example a human, murine, rat or rabbit antibody. Preferably the antibody is IgG, so that F(ab'γ) 2 fragments are employed in steps (i ) and (ii) . Dissociation of the F(ab') 2 fragments may be achieved in any convenient manner. Typically, however, a F(ab') fragment is reduced. This may be achieved using dithiothreitol. The two sets of Fab' arms are then linked together, preferably by a thioether linkage. One of the sets of Fab' arms may then be derivitised or alkylated with a cross-linking agent. Maleimidation may be effected, for example using o-phenylenedimaleimide. This set of arms is therefore provided with free maleimide groups and is combined with the other set of arms, which have free SH groups. Under cross-linking conditions, the bispecific F(ab') 2 antibodies are formed and can be subsequently isolated. When o-phenylenedimaleimide is used to effect maleimidation, the Fab' arms are linked by a -S-(o- phenylenedisuccinimidyl)-S- group. The bispecific F(ab') 2 antibodies are used to deliver a type 1 RIP to target cells it is wished to kill in a human or animal. This may be achieved in either of two ways. First, the antibody may be conjugated to the type 1 RIP for which one of its arms is specific and then administered to the host it is wished to treat. Second, the antibody and the type 1 RIP may be administered separately to the host under treatment. They may be administered at separate times and/or at different sites. Generally, the first method is preferred. The bispecific antibody and the type RIP may be mixed together to form immune complexes. These conjugates are then administered. Preferably, a molar surplus of bispecific antibody is mixed with the type 1 RIP. For example, the molar ratio of antibody: RIP may be from 3:1 to 15:1. Mixing is effected in a suitable inert diluent, typically immediately prior to administration. For this purpose, therefore, a pack may be provided comprising the bispecifc F(ab') 2 antibody and, separately therefrom, the type 1 RIP for which one arm of the antibody is specific. The bispecific F(ab') 2 antibody, either as such or complexed with the type 1 RIP for which one of its arms is specific, is used to eradicate target cells in a host. It can therefore be used to deplete B-cells, T-helper or -suppressor cells, monocytes, cytotoxic T cells, etc. It may be used to treat a patient with a tumour or infected with a microorganism. The bispecific antibody can be used to treat a tumour which displays a T cell antigen receptor on the tumour cell surface. A B cell neoplasm or other tumour such as a colorectal tumour or melanoma may be treated. Administration of the antibody or antibody complex is typically by injection or by infusion. It can be given parenterally, for example intraperitoneally, intravenously or subcutaneously; intratumourally; or into the cerebrospinal fluid if necessary. The amount of antibody administered will depend upon a variety of factors including the condition of the patient and the RIP. An amount of the bispecific antibody is administered, consistent with administering a therapeutically effective amount of the type 1 RIP. This typically means a dose of from 1 mg to 1 g of antibody and from 100 μg to 100 mg of the type 1 RIP, parenterally, for a human patient. Such a dose may be given by continous infusion or on multiple occasions during a week, for example, daily or twice a week, or less frequently, for example weekly, for as long as is necessary depending on the response to treatment and the toxicity of the bispecific F(ab*) 2 antibody and RIP conjugate. A cocktail of bispecific antibodies may be used. For example, two or more bispecific antibodies with one arm specific for the same type 1 RIP but with their respective other arms specific for different markers, or for different epitopes on the same cell marker may be given. A cocktail may also be given with two or more bispecific antibodies with one arm specific for tne same cell marker but with their respective other arms specific for different type 1 RIPs, or more than one epitope on the same type 1 RIP. A cocktail may also be given combining both of the aoove, with two or more bispecific antibodies, where the respective "first arms" may be specific for different type 1 RIPS or more than one epitope on the same type 1 RIP. Simultaneously the "second arms" may be specific for different cell markers or for different epitopes on the same cell marker. The cocktail may consist of several, for example 5 or 6, types of monoclonal antibodies, or be derived from a polyclonal serum. Tumour cells or other unwanted (eg. infected) cells can display more than one type of distinctive cell surface antigen. By employing a cocktail of the bispecific F(ab') 2 antibodies, in particular where at least the Fab' arms specific for the tumour marker are derived from a monoclonal antibody, enhanced results may be achieved. Bispecific F(ab')„ antibody: RIP conjugates can be changed or rotated during the treatment of patients as necessary, for example in cases where patients themselves raise an immune response to the first or second, etc RIP and/or bispecific F(ab') 2 antibody during their treatment or where residual unwanted cells become or are resistant to any particular combination of bispecific antibody: RIP treatments. The bispecific antibody is formulated for use, either as such or complexed with the RIP for which one of its F(ab') 2 arms is specific, as a pharmaceutical composition. Such a composition also comprises a pharmaceutically acceptable carrier or diluent. Suitable carriers and diluents include physiological saline solution and Ringers dextrose solution. The following Examples illustrate the invention. In the accompanying drawings: 3 Figure 1 shows the uptake of ( H) leucine by L~C tumor cells when cultured in medium containing a mixture of bispecific antibody and saporin. Cells (5 x 10 /ml) were cultured in supplemented Dulbecco's Minimum Essential Medium (DMEM) containing: A, saporin at a constant 100 ng/ml (3.3 x 10 —9M) and bispecific antibody at the concentrations shown; or B, bispecific antibody at a constant 1 μg/ml and saporin at the concentrations shown. After 8h at 37°C cultures 3 were pulsed with ( H) leucine for a further 12h before harvesting the incorporated radioactivity. The (F(ab'γ) 2 derivatives investigated are as indicated: DB7-12 x anti-Id-1 and DB7-18 x anti-Id-1, are the two totally monoclonal derivatives; rab a-sap x anti-Id-1, is the rabbit Fab'γ -containing derivative ' ; and DB7-18 + anti-Id-1 is a mixture of uncoupled F(ab'γ) 2 fragments from the two parent monoclonal Ab. Figure 2 shows the blood clearance rates of saporin or bispecific (Fab'γ ) 2 Ab after administration of mixtures of these reagents. Matched pairs of guinea pigs were given intravenous infusions of 200 ug 125I-sapoπn either alone or with unlabelled bispecific antibody (DB7-18 x anti-Id-1) at the ratios indicated (•) . At various intervals after the infusion plasma samples were taken and their radioactive content determined. Also included are parallel investigations showing the blood survival of the 125 I-bispecific antibody, DB7-18 x antι-Id-1 (■) . For these experiments guinea pigs were given 1 mg radioiodinated DB7-18 x anti-Id-1, either alone or with unlabelled saporin at the proportion shown. The bar at each point represents values determined for two animals (samples were taken in duplicate) . Figure 3 shows the results of immunotherapy with bispecific antibody. Groups of guinea pigs were inoculated 5 intraperitoneally with 10 2 C cells (day 0) . After 24 h they were given, via separate i.p. injections, 100 μg of saporin plus one of the following: 330 μg of control (F(ab'γ) 2 (A) ; DB7-18 F(ab' ) 2 and anti-Id-1 F(ab'γ) 2 («) or DB7-18 x anti-Id-1 (•) . An additional control group was given 330 μg of control F(ab'γ)- alone (X) . Survival was recorded daily. Figure 4 shows the optimum ratio of bispecific Ab and saporin for the treatment of 2 C leukemia. Groups of matched guinea pigs were inoculated intraperitoneally with 10 L-C cells (day 0) . After 24 h animals were injected i.p. with a mixture containing 10 μg of saporin, plus bispecific Ab (DB7-18 x anti-Id-1) at the level indicated to give bispecific Ab:saporin ratios in each treatment group of 1:15, 1:3, 1.5:1, and 15:1. The control group were given 1 mg of a non-react control IgG. These results have also been depicted in Table II. Figure 5 shows the results of immunotherapy with saporin and polyclonal- or monoclonal-derived bispecific F(ab'γ) Ab. Groups of guinea pigs were inoculated 5 intraperitoneally with 10 2 C cells (day 0) . After 24 h they were treated i.p. with a 1.5:1 mixture containing the bispecific derivatives, rab anti-sap x anti-Id-1 (A) or DB7-18 x anti-Id-1 (B) , and saporin at the doses indicated. The control group were given 1 mg of a non-reactive control IgG. The results in (B) are also depicted in Table II. Figure 6 shows the expression of surface IgM λon leukemic cells emerging after the immunotherapy described in Fig. 5. Cells recovered after therapy were exposed to the monoclonal AD shown (5 μg/ml) and, after washing, bound mouse Ab was detected by FITC-rabbit anti-mouse Ig. Three profiles of Ig expression were detected by cytofluorimetry: A, cells recovered from animal treated with control IgG, or treated with a sub-optimal mixture of bispecific Ab and saporin which failed to protect animals for more than 23 days (Fig. 5); B, cells recovered from the majority of guinea pigs surviving beyond 23 d following treatment with a mixture of bispecific Ab and saporin (Fig. 5); and C, mixed cell population emerging in the occasional leukemic animal living beyond 23 d. Figure 7 shows the uptake of ( H) leucine in Example 2. Figure 8 shows the uptake of ( H) leucine in Example 3 . Figure 9 shows the results of immunotherapy of guinea pigs in Example 4 using saporin and a mixture of bispecific F(ab') 2 antibodies. EXAMPLE 1 MATERIALS AND METHODS Materials. The RIP saporin was purified from the seeds Saponaria officinalis. (soapwort) by water extraction as described previously (16) . SDS-PAGE of the final preparation revealed >90% of the lectin was present as a single component with an Mr corresponding to 29,500. The concentration of purified saporin was estimated from its absorbance at 280 nm, taking E mg/ml to equal 0.6. Leukemia Cells. The 2 C lymphoblastic leukemia of strain 2 guinea pigs were used throughout this investigation (17) . This tumor is characterised by the expression of monomeric surface IgM X (approximately 100,000 molecules/cell (18)), and the release of free \ light chain together with small amounts of 19 S IgM during short-term culture in vitro (19) . The line is maintained by continuous passage in vivo. 5 Animals receiving an i.p. inoculum of 10 cells die 13 to 15 days later with a white blood count close to 250,000/ul. Cells are recovered by flotation on Ficoll-Hypaque (Lymphoprep; Nycomed as. Oslo, Norway) as described previously (19) . Cell viability was always >98% as judged by trypan blue exclusion. Antibodies. Two mouse monoclonal Ab, DB7-12 and DB7-18, were raised against saporin using the method of Kohler and Milstein (20) as described by Fazekas de St Groth and Scheidegger, (21) . Briefly BALB/c mice were immunized using a conventional protocol that delivered saporin (50 μg) subcutaneously (s.c.) in Complete Freunds Adjuvant (CFA) (Difco, Detroit, MI) on days 0 and 14, and intraperitoneally (i.p.) in PBS on day 24. Four days later splenic mononuclear cells were fused with the 653 (P3-X63-Ag8-653) mouse myeloma line at a ratio of 10:1 by using a standard somatic fusion protocol with polyethylene glycol 4000 (E. Merck, Darmstadt, Germany) . Hybridoma cells secreting anti-saporin Ab were identified by ELISA and cloned by limiting dilution in microculture plates. The ELISA used was a standard indirect or sandwich assay system as described by Glennie et al. (22): saporin at 0.2 μg/ml in 1% bovine serum albumin (BSA) was first captured by incubating in microculture plates which had been precoated with 7S IgG rabbit anti-saporin Ab (20 ug/ml) . Hybridoma culture supernatants were then added and any bound mouse Ab detected with an appropriate concentration of horse-radish peroxidase-labelled rabbit anti-mouse Ig (Nordic Laboratories Ltd., Maidenhead. Berks., UK) . Each stage of the assay consisted of a 90 min incubation at 37 C, followed by five washes in phosphate buffered saline (PBS) to remove unbound reagent. The other monoclonal Ab used in this work included: anti-Id-1, which is a mouse IgGl specific for the IgM Id of L_C; anti-guinea pig !_- chain; anti-guinea pig λ chain; and a control IgGl without reactivity for the L 2 C All these reagents have been fully characterised and described previously (18) . Polyclonal antisera reacting with saporin and mouse Ig were raised in rabbits using our standard immunization protocol (14) . Antibody titers were monitored by precipitation reactions with antigen in agar, using the double-diffusion method of Ouchterlony (23) . Those animals showing the best responses bled at weekly intervals. The 7S IgG fractions of monoclonal ascites and polyclonal antisera were isolated as described (18) by precipitation in 2 M ammonium sulfate, followed by ion exchange chromatography on Trisacryl-M-DEAE (LKB-Produkter AB, Bromma, Sweden) . Rabbit IgG Ab specific for mouse IgG was adsorbed by passage through a Sepharose 4B-linked guinea pig globulin column and then coupled to FITC iso er 1 (BDH Chemicals Ltd, Poole, United Kingdom) (18). The resulting FITC-IgG Ab showed no detectable reactivity with L 2 C before they had been coated with mouse Ig. F(ab'γ) 2 fragments from both monoclonal and polyclonal IgG were prepared by limited proteolysis with pepsin at pH 4.2 in 0.1 M sodium acetate (18, 24). The reaction was monitored at regular intervals by rapid fractionation of lOO g samples on a Zorbax GF250 HPLC column (Du Pont, Wilmington, Delaware, USA); when less than 10% of the IgG remained chromatography on Ultrogel AcA44 (LKB), in 0.1 M NaCl, 0.02 M T-ris-HCl, pH 8, was used to stop the digestion and fractionate the products. Immunofluorescence Studies. Binding of mouse Ab to the Id IgM of L 2 C and its variants was monitored as described (22) using indirect immmunofluorescence and the FACS III flow cytofluorimeter (Becton Dickinson, Mountain View, CA). Radioiodination of Proteins. Bispecific Ab derivatives and saporin were trace radioiodinated for blood clearance studies using carrier-free 125 l (Radiochemical Centre, Amersham, United Kingdom) with IODO-GEN (Pierce Chemicals Co., Rockford, IL) as the oxidizing agent (25). Radiation damage to proteins was minimized by maintaining their specific activity below 30 Ci/mg. Radioactivities were measured in a Rackgamma spectrometer (LKB). Preparation of bispecific antibody. Heterodimeric F(ab'γ) 2 molecules containing two different mouse Fab'y fragments, or a mixture of mouse and polyclonal rabbit Fab'γ fragments, were constructed according to the method described by Glennie et al. 1987 (14). Briefly, F(ab'γ) 2 from the two chosen Ab were first reduced to provide Fab' with hinge-region SH groups (Fab'γ SH ). The SH groups on one of the Fab'γ SH partners (the mouse component in the case of a mouse x rabbit hybridization ) were then fully alkylated with excess o-phenylenedimaleimide to provide free maleimide groups. Finally the two preparations, Fab'γ ]nal and Fab'γ SH , were combined under conditions which allow cross-linking of the maleimide and the SH groups and avoided reoxidation of SH groups. The final products were reduced and alkylated to remove any untoward minor product, including F(ab'γ) 2 homodimers, which may have formed by oxidation or disulfide exchange, before fractionating on Ultrogel AcA44 (LKB). The F(ab'γ) 2 heterodi er normally constituted between 50 and 70% of the recovered material. Uptake of [ 3 H]Leucine by L^C Cells in Culture. The assay for estimating the incorporation of [ 3 H]leucine into L 2 C cells was essentially the same as described in (14). However particular attention was paid to the humidity of the cell cultures at 37°C. Allowing the relative humidity to drop significantly may result in evaporation from the outer microculture wells, effectively increasing the concentration of bispecific Ab and saporin to which cells in these wells are exposed. All experimental points on graphs were determined in triplicate. The concentration of saporin or bispecific Ab at which [ 3 H]leucine uptake by cells was inhibited by 50% were taken as the IC 50 value. Blood clearance of Saporin and Bispecific Antibody. The technique for following the survival of radioiodinated saporin and bispecific Ab in the circulation of guinea pigs has been described previously (22). Animals were first given an i.v. injection of 125 I-labelled saporin (200 μg ) , either with or without unlabelled bispecific Ab at the ratio specified, and then at various intervals duplicate blood samples (50 μl ) were taken from an ear vein for radioactive counting in a Rackgamma spectrometer (LKB). Similarly, in separate experiments animals received 1 mg of radiolabelled bispecific Ab, with or without unlabelled saporin, and again blood samples were taken to determine the survival of radioactivity. Immunotherapy Studies. Groups of five or ten age-matched strain 2 guinea pigs (450 to 550 g) were inoculated i.p. with 10 5 fresh L 2 C cells (day 0) in 0.5 ml of DMEM. Then at 24 h (day 1) treatment was administered as described in the individual experiments, usually entailing a single i.p. injection of control or bispecific Ab which had been premixed with saporin at the stated ratio. Survival was monitored daily and the results expressed as the average increase in survival (number of days + SD) for each group over an appropriate control group. For the purposes of this calculation, the survival limit of animals that did not develop tumor within a 50 day period was taken as the last day on which a death occurred within that group. RESULTS Specific delivery of saporin to cultured L,C cells by bispecific Ab. Three bispecific F(ab'γ)- Ab have been constructed for the current work to link saporin to the surface IgM expressed by L 2 C tumor cells. All three derivatives received one of their Fab'γ arms from an anti-Id Ab, anti-ld-1 (18). This reagent was chosen from a panel of monoclonal anti-L 2 C Id Ab (18) due to its unusually high association constant (1.1 x 10 9 /M), and its readiness to yield F(ab'γ) 2 when digested with pepsin. The second Fab'γ Ab arm in each bispecific derivative bound saporin, and was selected from either one or two mouse monoclonal Ab, DB7-12 and DB7-18, or from an affinity-purified rabbit polyclonal Ab. Initially the effectiveness of these bispecific derivatives at delivering a lethal dose of saporin to L 2 C cells was investigated n vitro. Cells were cultured in supplemented medium containing a sub-toxic concentration of saporin (100 ng/ml; see saporin titration curve in Figure IB), in the presence or absence of bispecific Ab. As shown in Figure 1A all three derivatives transformed such medium into a highly toxic mixture for the L 2 C cells, in which they could no longer incorporate [ H]leucine into their proteins. The rabbit Fab'γ Ab-containing derivative (rab anti-sap x anti-Id-1) proved the most effective at enhancing saporin toxicity in this assay. Of the two monoclonal anti-saporin Ab-containing reagents, DB7-18 was the more effective with an IC 50 close to 4 ng/ml. Despite differences in IC 50 , all the bispecific Ab were most effective when added to cultures at approximately 1 g/ml, i.e. when the molar ratio of bispecific Ab:saporin was around 3:1. Figure 1A shows that any significant increase above a 3:1 ratio was accompanied by a reduction in saporin toxicity. This prozone-like effect may have stemmed from bispecific Ab failing to cross-link saporin and L 2 C cells due to saturation of both antigens by separate Ab molecules. The efficiency with which bispecific Ab could deliver saporin to cultured L 2 C cells and prevent the uptake of [ 3 H]leucine became most evident when we reversed the assay-maintaining cells in a constant level of bispecific Ab (1 g/ml) while diluting out the saporin. Figure IB shows that without any Ab, or with a mixture of uncoupled F(ab'γ) fragments from anti-saporin and anti-ld-1, the IC 50 for free saporin was approximately 6 x 10 ~ M (18.000 ng/ml). Following the addition of a bispecific Ab, however, the saporin toxicity curve shifted to a point where, in the case of the rabbit Fab'γ-containing derivative, its IC 50 was below 7 x 10 "12 M (0.21 ng/ml): i.e. close to a 90,000-fold increase in toxicity. Smaller but still striking shifts in toxicity were also obtained with the DB7-12 and -18 Ab-containing derivatives. The position of the three curves show that the relative performance of each of the derivatives (rab anti-sap x anti-Id-1 > DB7-18 x anti-Id-1 > DB7-12 x anti-Id-1) remained the same as in the first in vitro assay (Figure 1A) . Throughout this work, the addition of anti-Id-1 alone to L 2 C cells did not influence their ability to incorporate [ 3 H]leucine into protein. Furthermore a mixture of anti-Id-1 and anti-saporin F(ab'γ) 2 Ab was not seen to increase or decrease the toxicity of saporin to cultured L 2 C cells. Blood Clearance of Saporin and Saporin-Bispecific Antibody Complexes. A small dose (200 μg ) of radioiodinated saporin disappeared rapidly from the plasma of guinea pigs, so that 15 minutes after intravenous infusion only 10 percent of the injected material remained .in the circulation (Fig. 2). This extremely rapid disappearance is followed by a somewhat slower decline in the second phase of the survival curve, but the relevance of this region of the curve is questionable as only 2-3 percent of the radioactivity remains. By contrast, almost all the radiolabelled bispecific Ab (DB7-18 x anti-Id-1) was detected at 15 minutes after its infusion, and from there on it disappeared with a half-life of approximately 17 hours. The unmodified F(ab'γ) 2 fragments from DB7-18 and anti-Id-1 showed survival curves and half-lives identical with those of (DB7-18 x anti-ld-1) (data not shown), strong evidence that the manipulations carried out during the chemical synthesis of this bispecific Ab do not damage its structure in any untoward fashion. To investigate the influence of bispecific Ab on the survival of circulating saporin, matched pairs of guinea pigs were injected intravenously with radioiodinated saporin (200 μg ) which had been premixed with bispecific Ab at various ratios. Figure 2 shows that the effect of complexing with the bispecific Ab was to extend the time saporin persisted in the circulation, and also that the magnitude of any increase depended on the level of Ab in the mixture. Thus by injecting a molar 7:1 of bispecific Ab: saporin, it was possible to increase the half-life of the detected saporin to around 5 hours. Unfortunately, even when using bispecific Ab in such excess, almost 50 percent of the injected material was still lost before the first blood sample was taken. Such a result might have been predicted if the monoclonal Ab, DB7-18, used in thse experiments had failed to recognize 50 percent of the injected saporin, perhaps due to protein denaturation or to a major undetected contaminant. The latter explanation seems unlikely: first because no major contaminant can be detected on SDS-PAGE gel; and second because a comparable initial drop was also seen when this experiment was repeated using the polyclonal rabbit Fab'γ-containing bispecific Ab. As this polyclonal Ab was raised against the same saporin preparation it would presumably also contain activity to any radiolabelled contaminants, and therefore should capture and extend the circulation time of all the injected material. This leaves the possibility of damage to the saporin during iodination. Therapeutic Performance of Bispecific Antibody and Saporin. Initial attempts to treat tumor-bearing guinea pigs demonstrated at once that bispecific Ab had considerable potential for delivering saporin to unwanted cells _in vivo. Figure 3 shows that single separate i.p. injections of saporin (100 μg ) and DB7-18 x anti-Id-1 (330 μg ) given 24 hours after an i.p. inoculum of 10 5 L 2 C cells increased the mean survival of animals from 14 to over 26 days. Saporin given alone, or together with a control F(ab'γ). fragment which had no Ab activity for the cells or the saporin, did not show any significant protection of leukemic animals. Slightly extended survival was seen in animals given saporin plus a mixture of F(ab r ) 2 fragments from DB7-18 anti anti-Id-1. To investigate further the efficiency with which a bispecific Ab could capture and deliver saporin to tumor cells residing __n vivo, groups of five guinea pigs were treated in the way described above, but using a combination of separate i.p. and ε.c. .routes to administer the saporin and bispecific Ab. The results show (Table 1) that the maximum potency of the treatment was retained only while the saporin was being given via the peritoneal cavity. Giving saporin at a s.c. site reduced the increase in mean survival of animals from 10 days to between 4.6 and 7 days. In contrast, the route by which the bispecific Ab was given did not appear to influence therapeutic performance to any significant extent, and the Ab was perfectly capable of escaping from a s.c. site to recruit saporin which had been located in the peritoneal cavity (Table I). The results in Table ' also show that premixing the bispecific Ab and saporin to allow complex formation before injection, while giving good therapeutic performance after i.p. treatment, was disadvantageous when using the s.c. route. TABLE I Optimal route for treatment with saporin and bispecific Ab Route of Treatment Days (SD)increase in Ab(330 μg) + SapdOO μg)' survival over controls' i.p. i.p. 10.0(3.3) s.c. s.c. 4.6(3.1) s.c. i.p. 10.0(1.2) i.p. s.c. 7.0(4.5) s.c. b 1.6(0.5) 1.p. b 14.2(1.9) * groups of guinea pigs (five/group) were given an i.p. inoculum of 10 5 L 2 C cells on day 0, followed 24 hours later by separate injections of saporin and bispecific Ab (Ab in Table) at the dose and site indicated. in these experiments the saporin and bispecific Ab were premixed before injection at the site shown. c results show the average days increase in survival of treated animal over a control group receiving the saporin only. Survival was recorded daily. The SD for the individual groups are shown in parenthesis. The treatment regimens described above all employed bispecific Ab and saporin at a molar ratio of one. In our initial attempts to optimise these proportions, groups of leukemic guinea pigs were given a low i.p. dose of saporin (10 μg ) which had been premixed with bispecific Ab at various weight ratios between 1:15 and 15:1 (bispecific Ab:saporin). Figure 4 shows that, at this concentration of saporin, progressive increases in the level of bispecific Ab resulted in a concomitant prolongation of animal survival. Thus while animals receiving bispecific Ab and saporin at a ratio of 1.5:1 lived on average 2.4 days longer than controls, those given 15:1 survived for an extra 10.2 days. To investigate further the optimum balance between bispecific Ab and saporin, and also the effective minimum dose of saporin, this experimental approach of premixing the bispecific Ab and saporin before i.p. injection was extended to cover a range of saporin concentrations. The data generated in this work and that shown in Figure 4 have been summarised and expressed in Table II. Here a number of separate therapeutic experiments have been summarized, each with its own control group of untreated animals. TABLE II Mean increase in survival of leukemic guinea pigs treated with different ratios of bispecific Ab and saporin* j-atio ug of Eaporin animal (Ab:Sap) 0.1 10 25 50 100 0:1 n.d. b n.d. n.d. n.«3. 0.6(2.6) -0.8(0.6) 0.0(0.6) 1:15 1.0(0.6) 1.4(0.7) ι..d. . 3.4(0.7) 4.0(5.9) 8.4(4.5) 7.4(6.6) 1:3 1.2(0.6) 3.4(1.2) n.d. 8.2(1.1) 10.8(0.6) 10.9(3.-9) 11.0(3.9) 1.5:1 0.4(0.7) 3.4(2.0) n.d. 8.2(1.8) 12.8(3.3) 13.9(3.9) 13.8(3.2) 3:1 0.8(0.6) 1.6(0.8) n.d. 10.6(0.8) 12.1(1.5) 14.8(4.7) 14.8(1.1) 7:1 1.6(2.0) 7.0(6.4) 11.4(6.2) 10.0(1.6) 8.8(1.6) n.d. n.d. 15:1 0.6(0.6) 4.4(2.8) 10.2(3.5) 10.2(2.7) 8.8(2.1) n.d. n.d. groups of guinea pigs (five/group) were inoculated i.p. with 10 5 L 2 C cells on day 0 and then with a mixture of bispecific Ab (Ab in Table) and saporin given as a single i.p. injection at 24 h. Survival was recorded daily. The concentration of saporin and the ratio of bispecific Ab : sap are as indicated. b results are expressed as the mean increase in days survival over a control group of ten leukemic animals treated with a non-reactive IgG. The SD for each group is shown in parenthesis. Table II shows that the maximum therapeutic protection which we could achieve for this particular combination of bispecific Ab and saporin was close to 15 days. It is also clear that such protection could be attained, or very nearly attained, using various proportions of bispecific Ab and saporin. For example, just 5 μg of saporin when given together with a large excess (>125 μg ) of bispecific Ab could prolong the survival of leukemic guinea pigs by more than 11 days, while comparable survival was achieved with more saporin (25 μg ) plus less Ab (25 μg ) . Results from earlier ii vitro experiments (Fig. 1), had suggested that a large excess of bispecific Ab could perhaps block delivery of saporin to tumor by way of a prozone effect. No such effect was apparent i_n vivo. The slight loss of protection seen when animals were treated with 24 μg of saporin together with 625 μg (7:1) of 1250 μg (15:1) of bispecific Ab, did suggest the possibility of blocking by excess Ab. However this observation was not confirmed at lower concentrations of saporin and cannot be considered significant. Finally the results in line one (0:1) of Table II verify and extend the finding shown in Figure 3 that saporin given in the absence of bispecific Ab is not selectively cytotoxic to L 2 C cells, and consequently confers no protection on leukemic animals. Polyclonal Bispecific Antibody. The polyclonal rabbit Fab'γ-containing derivative (rab anti-sap x anti-Id-1) which had proven so efficient at targeting saporin iτ_ vitro (Fig. 1), was next investigated in the treatment of L 2 C i_n vivo. The design of this experiment was similar to that described in Table II: animals were first given 10 5 L 2 C cells (i.p.) followed 24 hours later by treament, consisting of a single i.p. injection containing bispecific Ab and saporin premixed at a ratio of 1.5:1. The results indicate (Fig. 5A) that the rabbit Fabγ-containing derivative was a highly efficient vector for saporin in vivo. For the three highest levels of treatment investigated (100, 25 and 10 μg of saporin/animal) , the average guinea pig survival was extended by between 13 and 16 days. Furthermore, even when the dose of saporin in the treatment was reduced to as little as 1.0 μg it still sufficed, in combination with bispecific Ab, to prolong the lives of 80% of the animals by at least 10 days. Comparing these results with those produced by the monoclonal bispecific derivative, DB7-18 x anti-Id-1 (Fig. 5B) , shows that as in the _ n vitro situation the rabbit Fab'γ-containing species has out-performed the monoclonal derivative. Although it is not possible to quantify precisely this increase in performance, a direct comparison of the survival curves for a tumor with a doubling time of 19h would suggest that inserting a single polyclonal Fab 1 arm into our monoclonal bispecific Ab has resulted in at least a 10-fold increase in targeting efficiency i_τ_ vivo. Emergence of Ig-Tu or Variants during Therapy. We have shown previously (22) that one of the consequences of treating the L„C with a potent anti-tumor agent working via the surface Id, is the emergence of stable Ig-negative variant tumors which have lost the ability to produce Ig neavy chain. Although these surface Ig-negative cells appear to prevent complete eradication of tumor using anti-Id directed therapy, their emergence in the absence of wild-type L„C can be used as an indication of the selective presure imposed by the treatment. In the current work the Ig expression on cells emerging after therapy with saporin and bispecific Ab was examined by flow cytofluorimetry. As expected tumor cells emerging in animals which had not been treated, or had failed to suvive for more than 22 days following treatment, all showed the normal Ig phenotype for the L 2 C (Fig. 6A) : strong reactivity with anti-λ chain, anti-μ chain and anti-Id-1 Ab. In contrast, tumour emerging later than 23 days following treatment often, but not always, showed the Ig-negative phenotype described above (Fig. 6B) . For example, out of the 30 animals receiving the three highest doses of saporin (100, 25 or 10 μg/animal) and rab anti-sap x anti-Id-1 (500, 125, or 10 μg/animal) in Figure 5A, 25 were checked for surface I . Of these 25, 24 were completely negative (Fig. 6B) and one showed a mixture of surface Ig-negative and -positive cells (Fig. 6C) . Tumors emerging in animals treated with the next level of saporin (1 μg) and rab anti-sap x anti-Id-1 (5 μg) showed a range of all three Ig phenotypes (Fig. 6) , with cells from at least 4 animals showing the mixed Ig phenotype (Fig. 6C) . A similar selection Ig expression was found in tumors emerging after treatment with saporin and the fully monoclonal derivative, DB7-18 x anti-Id-1. Again only in those animals where survival was increased by more than 10 days over control animal did emerging L 2 C start to show the Ig-negative phenotype. EXAMPLE 2: Targeting different RIPs with bispecific antibodies The suitability of saporin, a type 1 RIP, and the A chain of ricin, a type 2 RIP, for targeting by bispecific 3 antibody was compared. The uptake of ( H) leucine by L„C tumor cells when cultured in medium containing a mixture of bispecific antibody and either saporin or ricin A chain was examined. Cells (5 x 10 /ml) were cultured in supplemented DMEM containing saporin or ricin A chain in the presence or absence of bispecific Fab'γ ) 2 at 1 μg/ml throughout. After 8h at 37 C, cultures were pulsed with ( H) leucine for a furtner 12 h before harvesting the incorporated radioactivity. The F(ab'**f-) 2 derivatives investigated were DB7-18 x anti-Id-1 and 207 x anti-Id-1. 207 is an anti-ricin antibody. The results are shown in Figure 7. Figure 7 shows the ability of saporin and ricin A chain, in the presence and absence of the appropriate bispecific antibody, to 3 inhibit the incorporation of [ H]-leucine into cultured L 2 C cells. The results show that while both RIPs display similar levels of toxicity in the absence of bispecific F(abY ) antibody (50% inhibition: IC50 of about 10 μg/ml) , when a bispecific antibody is included in the assay at 1 μg/ml the saporin oecomes approximately 10 times more toxic than the ricin. EXAMPLE 3: Cocktails of bispecific F(ab' Investigations in Example 1 with bispecific F(ab' ) 2 and saporin had indicated that derivatives that bound saporin via a polyclonal rabbit antibody were at least 10 times more efficient than equivalent reagents which relied on single monoclonal antibodies. A panel of five monoclonal anti-saporin antibodies was therefore produced as described in Example 1. Each of these has been converted into a bispecific F(ab'γ ) 2 derivative and its performance as a targeting agent for saporin compared with that of the polyclonal reagent and with that of a mixture (cocktail) of equal proportions of all five monoclonal bispecific antioodies. 3 The uptake of ( H) leucine by L~C tumor cells when cultured in medium containing a mixture of bispecific antibody and saporin was investigated. Cells (5 x 10 /ml) were cultured in supplemented DMEM containing saporin in the presence or absence of bispecific F(ab'γ ) 2 antibody (1 ug/ml) . The bispecific F(ab* γ) - antibodies were all constructed with the anti-Id-1 monoclonal antibody together with one of the five monoclonal anti-saporin antibodies (1-5) or an affinity purified polyclonal rabbit anti-saporin reagent (Example 1) . After 8h at 37°C cultures were pulsed 3 with ( H) leucine for a further 12h before harvesting the incorporated radioactivity. The affinity constants for each of the monoclonal antibodies, measured using competition enzyme-linked immunosorbent assay and radioimmunoassay, were as follows: Antibody Affinity 1 6 x 10 "8 M 2 5.9 x 10 -8 M 3 5 x 10 "8 M 4 4 x 10~ 7 M 5 1 x 10~ 6 M The results are shown in Figure 8. Figure 8 shows tnat the hierarchy of affinity of the monoclonal antibodies does not correlate exactly with their performance as bispecific derivatives. Figure 8 also demonstrates that a cocktail containing a mixture of equal amounts of all five monoclonal bispecific antibodies (1-5) is as good if not better than tne polyclonal rabbit bispecific antibody. Thus it appears that the advantage of the polyclonal reagent lies not in an increased affinity for saporin, but in its multivalency for different epitopes which has been mimicked by mixing monoclonal reagents. EXAMPLE 4: Immunotherapy of leukemic animals with saporin and a mixture of bispecific F(ab' ) 2 antibodies Treatment of leukaemia-bearing animals with a combination of one bispecific F(ab'γ)_ antibody and saporin is effective at prolonging survival but is often thwarted by the escape of antigen (IgM idiotype)-negative variants which could not be destroyed by this therapy (see Figure 6) . These variant cells exist at low frequency (<1%) in the L«C tumor and are completely stable over multiple generations in vivo. Furthermore, similar cells have also been identified in other animal tumors and in human lymphomas and leukemias following selective pressure from immunotherapy. To overcome this escape route by tumours the L-C tumor was attacked via two surface antigens. The chances of a variant cell pre-existing or emerging during treatment which is negative for two surface antigens is clearly very remote. For the second anti-L 2 C antibody (in addition to anti-idiotype) a monoclonal antibody was raised which binds to guinea pig B cells, including L 2 C tumor and its Ig-negative variant. Immunotherapy with saporin and a mixture of bispecific antibodies was thus investigated. Guinea pigs were inoculated intraperitoneally with 10 5 L 2 C cells (day 0). After 24 h they were treated i.p. with a mixture containing saporin (100 μg/animal) and the bispecific derivatives (500 μg/animal) ; rab anti-sap x anti-Id-1 (a-Id), rab anti-sap x a-guinea pig B cell (a-B-cell) or an equal mixture of rab anti-sap x anti-Id-1 and rab anti-sap x anti-B cell (a-B cell + a-Id). The control group received saporin only. The results are shown in Figure 9. Figure 9 shows that by using a combination of saporin and a mixture of the two bispecific antibodies, one containing targeting via anti-idiotype and the other via the anti-B cell antibody, 80% of tumor-bearing animals have managed to be cured (healthy beyond 100 days) with a single treatment. Thus this strategy has blocked one of the major escape routes of tumors from immunotherapy. REFERENCES 1. Karpovεky, B., J.A. Titus, D.A. Stephany, and D.M. Segal. 1984. Production of target-specific effector cells using hetero-cross-linked aggregates containing anti-target cell and anti-Fct receptor antibodies. J. Exp. Med. 160:1686. 2 . Staerz, U.D., 0. Kanagawa, and M.J. Bevan. 1985. Hybrid antibodies can target sites for attack by T cells. Nature 319:628. 3. Liu, M. A., D.M. Kranz, J.T. Kurnick, L.A. Boyle, R. Levy, and H.N. Eisen. 1985. Heteroantibody duplexes target cells for lysis by cytotoxic T lymphocytes. Proc. Natl. Acad. Sci. USA 82:8648. 4. Milstein, C, and A.C. Cuello. 1983. 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Claims: CLAIMS 1. A bispecific F(ab') 2 antibody comprising a - first Fab' arm specific for a type 1 ribosome-inactivating protein and a second Fab' arm specific for a marker on the surface of a target cell. 2. An antibody according to claim 1, which is a bispecific F(ab , y) 2 antibody. 3. An antibody according to claim 1, wherein the first Fab' arm is specific for saporin or momordin. 4. An antibody according to claim 1, wherein the second Fab' arm is specific " for a tumour marker. 5. An antibody according to claim 4, wherein the marker is a T cell antigen receptor. 6. An antibody according to claim 1, wherein the second Fab' arm is specific for the envelope coat protein of HIV. 7. An antibody according to claim 1, wherein the Fab 1 arms are linked by a -S-(o-phenylenedisuccinimidyl)-S- group. 8. A process for the preparation of a bispecific F(ab') 2 antibody as claimed in claim 1, which process comprises: (i) dissociating a F(ab') antibody fragment specific for the type 1 ribosome-inactivating protein into its two component Fab' arms; (ii) dissociating a F(ab')_ antibody fragment specific for the target cell marker into its two component Fab' arms; and (iii) linking a Fab' arm specific for the type 1 ribosome-inactiving protein to a Fab' arm specific for the target cell marker. 9. A process according to claim 8, wherein the F(ab') 2 antibody fragments have been obtained by treating antibodies with the said specificities with pepsin. 10. A process according to claim 8, wherein the Fab' arms are linked by treating the Fab', arms produced in one of step (i) or step (ii) with o-phenylenedimaleimide and combining the thus-treated Fab' arms with the Fab' arms produced in the other of step (i) or step (ii) under cross- linking conditions. 11. A conjugate comprising a bispecific F(ab')_ antibody as defined in claim 1 complexed with the type 1 ribosome-inactivating protein for which one of the Fab' arms of the antibody is specific. 12. A process for the preparation of a conjugate as claimed in claim 10, which process comprises mixing the bispecific F(ab') 2 antibody with the type 1 ribosome-inactivating protein. 13. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and as active ingredient: (a) at least one bispecific F(ab') antibody as defined in claim 1, or (b) at least one conjugate as claimed in claim 10. 14. A composition according to claim 13 which comprises as active ingredient: (a) two or more bispecific F(ab') 2 antibodies with different specifities as defined in claim 1, or (b) two or more conjugates with different specifities as claimed in claim 10. 15. A pack comprising a bispecific F(ab')_ antibody as defined in claim 1 and, seperately therefrom, the type 1 ribosome-inactivating protein for which one of the Fab' arms of the antibody is specific. 16. A method of depleting a population of cells in a host, which method comprises administering to the host an effective amount of a bispecific F(ab') 2 antibody as defined in claim 1, one of the Fab' arms of the antibody being specific for a marker on the said cells, and of the type 1 ribosome-inactivating protein for which the other of the Fab 1 arms of the antibody is specific.

Inventors: GLENNIE MARTIN JOHN, GLENNIE, MARTIN, JOHN
Applicants: GLENNIE MARTIN JOHN, GLENNIE, MARTIN, JOHN
Application year: 1989
Main classification: A61K39
IPC codes: A61K38, A61K47, C07K16