Br. J. Cancer (1993), 67, 436-440
`
`Br. J. Cancer (1993), 67, 436-440
`
`'." Macmillan Press Ltd., 1993
`
`1993
`
`Characterisation of a humanised bispecific monoclonal antibody for
`cancer therapy
`
`A. Bruynck, G. Seemann & K. Bosslet
`
`Research Laboratories of Behringwerke AG, PO Box 11 40, W-3550 Marburg/Lahn, Germany.
`
`Summary A humanised bispecific monoclonal antibody (bsMAb) with binding specificity for carcinoem-
`bryonic antigen (CEA) on one arm and a radiolabelled chelate (DTPA-9Y) on the other arm was generated by
`consecutively transfecting the humanised genes of an anti-CEA MAb and the chimerised genes of an
`anti-chelate MAb into eucaryotic BHK cells using the calcium-phosphate coprecipitation technique. The
`antibodies secreted were of IgG3 isotype with a shortened hinge region (A gamma 3) and light chains. Double
`transfectomas were screened for the secretion of bsMAbs using a double determinant enzyme-linked immuno-
`sorbent assay (ELISA) based on solid phase attached HSA-benzyl-DTPA and an anti-idiotypic MAb selective
`for the CEA-specific arm. After purification on two immunoaffinity chromatography columns, the humanised
`bsMAbs were characterised by SDS-PAGE and a quantitative binding assay in antigen excess. The purification
`procedure resulted in 95% reactive bispecific MAb. This humanised bsMAb may be employed in two phase
`radioimmunotherapy, binding to the tumour via the anti-CEA arm and localising a radiolabelled chelate with
`the other arm, without inducing a strong immune response observed sometimes with murine MAbs.
`
`Besides the important role of murine monoclonal antibodies
`(MAbs) in basic research and in vitro diagnosis, they became
`important tools for in vivo diagnosis of tumours (Murray &
`Unger, 1988), inflammatory processes (Joseph et al., 1988)
`and thrombosis (Haber et al., 1990). Furthermore it was
`clinically proven that murine MAbs inhibit transplantation
`rejection (Goldstein, 1987; Kurrle et al., 1988) and interfere
`with gram negative sepsis (Greenman et al., 1991). No clear-
`cut clinical benefit, however, was shown so far in the therapy
`of solid tumours either with cytotoxic murine MAbs or
`MAb-drug conjugates (Dykes et al., 1987). This is mainly
`due to the unfavourable tumour tissue penetration charac-
`teristics and whole body distribution of macromolecules such
`as MAbs (Thomas et al., 1989), causing severe damages in
`normal tissue as well. This can be optimised using the
`recently described two phase immunotherapy approaches
`(Bosslet et al., 1991) based on bispecific MAbs. BsMAbs
`offer a unique possibility for the two phase immunotherapy
`consisting of a long-term binding phase of the nontoxic
`bsMAbs to tumour cells and, after the elimination of un-
`bound bsMAb, a short-term binding phase of an effector
`system. In our approach, we intend to use a bsMAb with
`binding specificity for a tumour associated antigen (carcino-
`embryonic antigen, CEA) on one arm and to a radiolabelled
`chelate (DTPA-9Y) on the other arm for a two phase radio-
`immunotherapy.
`BsMAbs have been generated either by chemical linking of
`the reduced monovalent parental Fab' fragments to form a
`bispecific F(ab')2 fragment (Brennan et al., 1985; Bagshawe et
`al., 1989), using the quadroma technique (Milstein & Cuello,
`1983), by double transfection of murine Ig genes (Lenz &
`Weidle, 1990) or by double transfection of chimeric DNA
`constructs (Songsivilai et al., 1989). A murine bsMAb with
`the above mentioned specificities, generated by double fusion
`has already been described (Bosslet et al., 1991).
`Unfortunately repetitive high dose injection of murine
`monoclonal antibodies in immunotherapy often induced the
`development of human anti-mouse-Ig antibodies (HAMA) in
`patients, which prevent prolonged treatment (Miller et al.,
`1983; Kroonenburgh, van & Pauwels, 1988). Consequently,
`the target was to produce a MAb capable of escaping surveil-
`lance by the human immune system while retaining the speci-
`ficity of the murine parental antibody. In a first approach,
`
`Correspondence: K. Bosslet.
`Received 12 December 1991; and in revised form 8 October 1992.
`
`the V-regions of murine antibodies were recombined with
`human constant region genes to form chimeric MAbs (Bouli-
`anne et al., 1984; Morrison et al., 1984). Lately, the antigen
`binding
`loops
`complementarity-determining
`regions
`or
`(CDRs) of the mouse VH and VL domains have successfully
`been transplanted to the VH and VL domains of human
`myeloma proteins without major impairment of the antigen
`binding capacity (Jones et al., 1986; Riechmann et al., 1988).
`One of these humanised MAb has already been applied in
`patients (Hale et al., 1988), without inducing a detectable
`immune response against the molecule.
`In this report, we describe the generation of a bifunctional
`MAb consisting of one humanised anti-CEA arm and a
`chimerised anti-DTPA-Y arm by double transfection of the
`corresponding genes into BHK cells, its purification using
`immunoaffinity chromatography and its characterisation.
`
`Materials and methods
`
`Production of CEA- and DTPA-specific murine MAbs
`The generation and screening of murine MAbs directed
`against CEA (BW 431) and DTPA-Y (BW 2050), respectively,
`was described previously (Bosslet et al., 1988 and 1991).
`
`Chimerisation of an anti-DTPA- Y MAb
`The murine variable (VH and VL) region genes of the heavy
`and light chain of MAb BW 2050 were recombined with the
`constant region genes of human IgGs (Ay3) according to the
`method of Boulianne et al. (1984) (Figure 1).
`
`Humanisation of an anti-CEA MAb
`The sequences of the heavy and light chain variable genes of
`MAb BW 431 (anti-CEA) were amplified and cloned as des-
`cribed by Orlandi et al. (1989) and the CDR regions were
`subsequently built into the framework of human VH and VL
`domains according to the methodology of Jones et al. (1986)
`and Riechmann et al. (1988) (Figure 1). The exact procedure
`for the humanisation of MAb BW 431 was described by
`Gussow and Seemann (1991).
`
`Expression of humanised bsMAb
`First, the expression vectors carrying the heavy and light
`chain genes of humanised MAb BW 431 were transfected
`
`Genzyme Ex. 1038, pg 917
`
`

`
`a
`
`Hindill
`
`P-
`
`Pvull
`
`Hindlill
`
`~~~~~~~~~~~Pvull
`
`/ iv
`
`f 8W~~SV*w__iHl
`~~~~~Prametur
`OK'hu
`
`E/
`/humUVH mu./
`2050/536 V). mu/
`
`pAM
`
`CH'I.
`
`Bl
`
`Hkp
`
`Tell~~~~~~~~ul
`
`Pati
`
`Poull
`
`~
`
`sotMal
`
`ftil
`
`Schematic diagrams of the expression plasmids pAb,
`Figure 1
`used for double transfection, containing the murine (black) CDRs
`of light a, and heavy b, chain of MAb BW 431 (anti-CEA) or the
`variable region of light c, and heavy d, chain of MAb BW 2050
`(anti-DTPA-Y) respectively, joined to the human (white) A
`gamma 3 and kappa constant regions. SV40 promoter and CMV
`enhancer are shown as well as the drug resistance marker ampi-
`cillin (amp).
`
`into eucaryotic BHK cells (ATCC CCL10, American Type
`Culture Collection, Rockville, Md., USA) using the Calcium-
`phosphate precipitation technique (Graham & van der Eb,
`1973). Two plasmids carrying resistance genes for methotrex-
`ate (Subramani et al., 1981) and G418 (Hudziak et al., 1982)
`as selection markers were cotransfected. A BHK cell line
`which had been selected for the production of humanised
`anti-CEA MAb and the binding of anti-idiotypic antibody
`(Gussow & Seemann, 1991) was transfected a second time
`with the chimeric genes coding for the heavy and light chains
`of MAb BW 2050. The resistance gene for hygromycin (Gritz
`& Davies, 1983) was cotransfected for the selection of double
`transfectomas.
`Cells were seeded out in a modified Dulbecco's medium
`containing human insulin and all three resistance markers
`(Mtx, G418, hygromycin). Clones grew out 2-3 weeks after
`transfection.
`
`Screening of the double transfectomas
`Transfectoma supernatants were screened for the presence of
`bispecific MAb using an ELISA system in which HSA-
`benzyl-DTPA (50 ng ml-') was attached to the solid phase of
`round bottom polystyrol plates (Nunc). Bound MAb was
`detected using a murine anti-idiotypic antibody (IgG2b)
`(Bosslet et al., 1990), selective for the murine as well as the
`humanised anti-CEA MAb BW 431, and an alkaline phos-
`phatase labelled goat anti-mouse-IgG2b antibody (Southern,
`Birmingham, USA) combined with the alcohol dehydrogen-
`ase-diaphorase amplification system (Stanley et al.,
`1985;
`system III). Optical density was measured at 493 nm using a
`Titertek multiscan type 3100 (Flow Laboratories).
`
`Double affinity chromatography
`Protein A-sepharose-purified anti-idiotypic MAb specific for
`BW 431 was coupled to cyanogen bromide-activated Sepha-
`
`HUMANISED BISPECIFIC MONOCLONAL ANTIBODY
`
`437
`
`rose at a concentration of 6.5 mg MAb ml - gel according to
`van Eijk and van Noort (1976). Supernatant of double trans-
`fectoma was loaded on the column. After extensive washing
`with phosphate-buffered saline (PBS), pH 7.2,
`specifically
`bound antibodies were eluted using PBS, pH 4.0. On a
`second column, antigen (HSA-benzyl-DTPA) was covalently
`coupled to CNbr-activated Sepharose at a concentration of
`8 mg ml-' gel. The MAb eluted from the anti-id column was
`loaded on the HSA-benzyl-DTPA column. After washing
`with PBS, pH 7.2, the bispecific MAbs were eluted with
`double distilled water containing 25% glycerine, and adjusted
`to pH 2.3
`The enrichment of bispecific MAb was monitored using
`three ELISA systems which allowed the determination of the
`functional anti DTPA arm (system 1), a functional anti CEA
`arm (system 2) or both arms (system 3). Technical details of
`the systems are identical to those described in screening of
`the double transfectomas.
`System I uses HSA benzyl-DTPA on the solid phase (coat-
`ing concentration: 50 ng ml-') combined with AP-labelled
`anti human K antibody (50 ng ml-', Southern Biotechnology
`h.c., Birmingham).
`In system II CEA at a concentration of 50ngml-' was
`attached to the solid phase instead of HSA-benzyl-DTPA.
`System III was described in chapter 'Screening of the
`double transfectomas'.
`
`Characterisation of bsMAbs in SDS-Polyacrylamide-gel
`electrophoresis
`Samples were analysed either unreduced or reduced with
`50 mM DTT by SDS-PAGE (Laemmli, 1970), carried out in
`a Mini Protean II Electrophoresis Cell (Bio-Rad, Richmond,
`Ca., USA) using a 10% stacking gel combined with a separa-
`tion gel of 4-15% (Bio-Rad). The gels were stained using
`Coomassie Blue according to the manufacturers' procedure
`(PhastGel'm Blue R, Pharmacia LKB, Sweden).
`
`Quantitative binding assay in antigen excess
`Four hundred ng ml-' of purified humanised bispecific MAb
`were incubated with increasing amounts of antigen. Antigen
`consisted of CEA-expressing HT 29 colon carcinoma xeno-
`grafts which were fixed using 5% formaldehyde for 5 days
`and squeezed after cutting in 1-3 mm3 cubes through a
`stainless steel sieve. This methodology generates single dead
`round cell bodies, defined as ACM (antigen containing
`material). After a 4 h incubation at room temperature the
`antigen was spun down and the amount of unbound MAb in
`the supernatant was determined using a quantitative ELISA
`specific for human IgG (anti-human-IgG attached to the
`solid-phase). The fraction of unbound MAb detectable in the
`supernatant in antigen excess represents the inactive fraction
`of the purified bsMAb preparation. Immunoreactivity (I.R.)
`was determined according to the formula
`
`I.R. = 100% -
`
`100% x IgG-concentration at point of determination
`Input IgG-concentration
`
`The point of determination was the highest amount of anti-
`gen at which there was no unspecific binding of the chimeric
`negative control MAb BW 554, selective for ganglioside GD2.
`
`Evaluation of the anti DTPA-arm
`One fg ml-' of HSA-Benzyl-DTPA-Y was used to coat
`microtitre plates. Constant amounts (1I00 tg ml-') of bsMAb
`purified by affinity chromatography were mixed with increas-
`ing amounts of DTPA-Y starting with 0.1 ng ml-' up to
`1 ttg ml-' in 1:4 fold dilution. After incubation of the mix-
`ture in the coated wells for 30' at RT, bound bsMAb was
`detected using an AP-labelled anti human K antibody as
`combined with the ADH-diaphorase amplification system.
`
`Genzyme Ex. 1038, pg 918
`
`

`
`Table I ELISA for the evalution of double immunoaffinity chromato-
`graphy
`
`III
`0.240
`
`I
`1.220
`
`II
`0.990
`
`Experimental procedure
`Concentrate before chromatography
`(1:20)
`Flow through a-id-column
`0.340
`1.200
`0.100
`Eluate (1:10)
`0.330
`0.340
`1.100
`Flow through antigen-column
`0.250
`0.170
`0.350
`Eluate (1: 10)
`0.440
`0.920
`0.350
`Optical density at 492 nm in detection system: (I) HSA-benzyl-DTPA
`solid phase + anti-K-AP; (II) CEA solid phase + anti-K-AP; (III) HSA-
`benzyl-DTPA solid phase + anti-id (IgG2b) + anti-IgG2b-AP.
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`150 _
`
`110 _
`
`70-
`
`44-
`
`28-
`
`18-
`
`Figure 2
`SDS-Polyacrylamide gel electrophoresis (10% stacking
`gel) with unreduced (lanes 1-3) and reduced (lanes 4-6) samples
`of protein marker (lanes 1 and 4); monospecific humanised MAb
`BW 431 (lanes 2 and 5); bsMAb A 10 (lanes 3 and 6). Molecular
`weights in kDa.
`
`*
`
`-
`
`.+.
`
`| l X W | t t .......
`
`1500
`
`1000 _
`_
`
`-
`
`500
`
`w E
`
`C 0 C
`
`.)cJ
`
`xw
`
`33.3 +.+
`33.3
`100
`300
`
`0
`
`0
`
`0.4
`
`1.2
`
`3.6
`11.1
`mg ACM
`Figure 3 ELISA for the quantitative evaluation of unbound
`bispecific humanised MAb after double affinity chromatography.
`Individual
`wells contained
`the
`antigen
`containing
`material
`(ACM = CEA) (indicated on the abscissa) mixed with 400 ng of
`humanised bsMAb A 10 (
`), with 400 ng of humanised
`MAb BW 431 (. ), or with 400 ng of irrelevant chimeric MAb
`BW 554 (------). Optical density expressed in mE (492 nm) at the
`ordinate represents the amount of unbound MAb in the super-
`natant of each antigen concentration. The arrow indicates the
`point of determination.
`
`Table II
`
`hum. BW 431
`hum. bispecific
`A 10
`
`Immunoreactivity (I.R.), expressed as percentage, of human-
`ised bsMAb A 10 and humanised MAb BW 431
`IgG-concentration
`IgG-concentration
`(input)
`(p. of determination) % IR.
`400 ng ml-'
`15 ng ml-'
`96
`468 ng ml-'
`22 ng ml-'
`95
`
`438
`
`A. BRUYNCK et al.
`
`Results
`
`Generation of humanised anti-CEA x anti-DTPA- Y bsMAbs
`By double transfection of BHK cells we produced clones
`secreting bispecific IgG3 with one humanised anti-CEA arm
`and a chimerised anti-DTPA-Y arm. Arising clones were
`screened using a double determinant ELISA based on solid
`phase attached HSA-benzyl-DTPA and anti-idiotypic anti-
`body, selective for the CEA-specific arm (see Material and
`methods; system III). Out of 730 clones that were isolated
`2-3 weeks after transfection, three clones remained stable
`producers of bsMAbs after two rounds of cloning. The clone
`with the highest production rate was A 10/32/255, further
`named A 10 or humanised bsMAb. A 10 produced 4-8 pg
`MAb ml-' and was cultured in roller bottles in a modified
`Dulbecco's medium supplemented with human insulin and
`the resistance markers (G418, Mtx, hygromycin).
`
`Purification of humanised bsMAb
`Ten litres supernatant of clone A 10 were concentrated and
`purified using two consecutive affinity chromatography col-
`umns, first an anti-id column and second an antigen column.
`The effect of the individual purification steps was investigated
`using three different ELISAs, detecting the anti-DTPA-Y
`arm (system I), the anti CEA arm (system II), and both arms
`(system III). BsMAb could be separated from the non bi-
`specific molecules which appear by false recombinations of
`heavy and light chains. The data, presented in Table I, show
`a) the increase of activity in system II (specific for CEA) after
`anti-idiotypic affinity chromatography, and b) the increase of
`activity in system I (specific for DTPA-Y) after antigen
`affinity chromatography in the eluates in question. Coinci-
`dental there is a decrease of activity in the flow throughs of
`both columns.
`Out of 32 mg of IgG which could be detected via ELISA
`in 10 1 of supernatant of A 10, 3.1 mg were left over after
`double immunoaffinity purification. The yield of 10% was in
`the range suggested according to the theoretical considera-
`tions from Milstein and Cuello (1983).
`
`Characterisation of the humanised bsMAbs
`In order to characterise the immunoaffinity purified human-
`ised bsMAb and to investigate whether the bispecific mole-
`cule retains the characteristics of the humanised monospecific
`anti-CEA MAb 431, both molecules were compared using
`SDS-PAGE.
`In SDS-PAGE (Figure 2) both immunoglobulins showed a
`major band with an apparent molecular weight of approxi-
`mately 150 kDa and a minor band of approximately 125
`kDa. Under reducing conditions, the humanised anti CEA
`MAb showed a 50 kDa heavy chain and a 25 kDa light
`chain, whereas in the bsMAb preparation two light chains
`with an approximate molecular weight of 25 and 26.5 kDa
`were detected in addition to a 50 kDa heavy chain band.
`
`Quantitative binding capacity of humanised bsMAbs in antigen
`excess
`The immunoreactivity of the immunopurified bsMAb A 10
`was determined in comparison to the humanised monospecific
`anti-CEA MAb BW431 and an irrelevant chimeric antibody
`(BW 554) in an ELISA after incubation of constant amounts
`of antibody with increasing amounts of particle attached
`CEA as described in Materials and methods (Figure 3).
`Immunoreactivity was calculated, based on the IgG con-
`centrations determined by ELISA, according to the formula
`given in Materials and methods. Calculations indicated that
`95% of the preparation of bispecific MAb consisted of
`immunoreactive material, compared to 96% as determined
`for the monospecific humanised MAb BW 431 (see Table II).
`
`Genzyme Ex. 1038, pg 919
`
`

`
`Evaluation of the anti DTPA- Y arm
`The binding potential of the bsMAb to HSA-benzyl DTPA-
`Y was determined in a competitive ELISA system as des-
`cribed in Materials and methods. Increasing amounts of
`DTPA-Y as a competitor resulted in a decrease of the bind-
`ing signal in the ELISA down to background level (data not
`shown). These data indicate that free DTPA-Y is able to
`block the binding of the bsMAb to solid phase attached
`HSA-benzyl DTPA-Y, arguing for the functional integrity of
`the anti DTPA-Y arm.
`
`Discussion
`
`We have established a humanised bispecific monoclonal anti-
`body consisting of a humanised anti-CEA arm and a chime-
`rised anti-DTPA-Y arm. The bispecific molecule is of IgG3
`isotype and has a shortened hinge region: instead of normally
`four, the AIgG3 gene has only one exon, reducing the possi-
`ble disulfide bonds from 11 to 3. We were able to generate
`this bsMAb by two successive, calcium-phosphate mediated
`transfections of an eucaryotic BHK cell line. Out of several
`hundred clones, only three stably produced bsMAbs detect-
`able in a double determinant ELISA. For purification pur-
`poses we had to load the culture supernatant on two
`consecutive immunoaffinity chromatography columns, first
`presenting an anti-idiotypic antibody, selective for the CEA
`arm, and second the antigen for the anti-DTPA arm. Puri-
`fication was controlled by three ELISA systems (Table I),
`which proved that there was an increase of activity in the
`eluates and a coincidental decrease of activity in the flow
`through of the two columns. We were able to quantitatively
`separate 10% of bispecific monovalent antibodies from false-
`ly recombined molecules, a yield which correlates with the
`theoretical considerations of Milstein and Cuello (1983).
`Ninety-five per cent of the bispecific molecules were immuno-
`reactive as revealed by a quantitative binding assay in anti-
`gen excess (Table II). Compared to other methods (Doussal
`et al., 1989; Lenz & Weidle, 1990) this purification method
`leads to exceptionally high yields. Disadvantageous was the
`yield of the BHK cells secreting the immunoglobulins: from a
`cell line which had produced 4-8 g ml-' in the beginning,
`we only harvested about 30 mg immunoglobulin from 101
`supernatant instead of 40-80 mg expected. It has to be
`accepted that to some extent the cells lost the specificity of
`antibody production. In another case of antibody production
`in BHK cells in our laboratory it was revealed that the cells
`produced much more light chains than heavy chains (Bosslet,
`unpublished data), a fact which leads to reduced yields of
`intact molecules. Combined with the fact that two affinity
`chromatography columns are necessary for purification, these
`conditions are unfavourable for larger production scales.
`
`HUMANISED BISPECIFIC MONOCLONAL ANTIBODY
`
`439
`
`Comparison of the humanised bsMAb and the humanised
`monospecific anti-CEA antibody in SDS-PAGE proved that
`both molecules resemble each other to a large extent. The
`most obvious differences appear in the behaviour of the light
`chains of both antibodies which point to the presence of two
`different K-chains in the bsMAb (Figure 2, lanes 5 and 6).
`This may be due to the fact that one of the light chains still
`has a whole murine variable region. The integrity of the
`bispecific MAb was further proven by our immunoreactivity
`studies indicating that >95% of the bispecific humanised
`MAb were functionally active. This immunoreactivity is only
`marginally inferior to the value generated using the human-
`ised monospecific MAb (>96%-.
`The anti DTPA-Y could only be evaluated qualitatively.
`Data from the competitive ELISA system in which the bind-
`ing of the bsMAb to solid phase attached HSA-benzyl
`DTPA-Y was completely blocked by a > 100 fold excess of
`free DTPA-Y, argue for the functional integrity and speci-
`ficity of those bsMAb molecules which bound to the solid
`phase attached HSA-benzyl DTPA-Y. This type of analysis is
`less quantitative than the immunoreactivity assay performed
`for the anti CEA arm, but is nevertheless suited to support
`the usefulness of the hu bsMAb.
`The bispecific MAb generated is intended to be employed
`in two phase radioimmunotherapy, having dual specificity for
`carcino-embryonic antigen (CEA) and a radiolabelled chelate
`(DTPA-90Y). Concerning the specific tumour localisation,
`Bosslet et al. (1991) showed that it is possible to obtain a
`significant tissue penetration of solid human carcinoma xeno-
`grafts after long time application of the murine anti-CEA
`MAb BW 431. Since the two phase therapy concept is built
`up on repetitive long-term and high dose injections of
`bispecific antibodies, immunisation of patients is most prob-
`able. The humanised bsMAb is hoped to overcome the prob-
`lem of immunogenicity currently seen with murine and
`chimeric antibodies used in human therapy (Briiggemann et
`al., 1989).
`Considering the yields and the cumbersome purification
`procedure, attempts have been made to improve the yield of
`bispecific monovalent MAb using recombinant DNA techno-
`logy, e.g. the construction of an Ig heavy chain, consisting of
`VH of MAB 1 and a CHl domain, which is linked by a
`polypeptide spacer to the other heavy chain, consisting of VH
`of MAb 2 and a CH3 domain, to form a 'tandem heavy
`chain'. The association of such a 'tandem heavy chain' with
`the two corresponding light chains (VK of MAb 1 and a Cx
`domain or VK of MAb 2 and a CH3 domain) should lead
`predominantly to the formation of the desired bispecific
`molecule.
`
`The technical assistance of N. Doring, E. Herz, H. Lind and M.
`Matthai as well as the secretarial assistance of S. Lehnert are greatly
`appreciated.
`
`References
`
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`
`BOULAINNE, G.L., HOZUMI, N. & SHULMAN, M.J. (1984). Produc-
`tion of functional chimeric mouse/human antibody. Nature, 312,
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`BRENNAN, M., DAVISON, P.F. & PAULUS, H. (1985). Preparation of
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`immunoglobulin GI fragments. Science, 229, 81-83.
`BRJGGEMANN, M., WINTER, G., WALDMANN, H. & NEUBERGER,
`M.S. (1989). The immunogenicity of chimeric antibodies. J. Exp.
`Med., 170, 2153-2157.
`DYKES, P.W., BRADWELL, A.R., CHAPMAN, C.E. & VAUGHAN,
`A.T.M. (1987). Radioimmunotherapy of cancer: clinical studies
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`DOUSSAL, J.M., LE, MARTIN, M., GAUTHERAT, E., DELAAGE, M. &
`BARBET, M. (1989). In vitro and in vivo targeting of radiolabeled
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