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Construction and expression of antibody-tumor necrosis factor fusion proteins
Mo/ecu/ar Immunology, Vol. 28, No. 9, pp. 1027-1037, Printed in Great Britain.
0161~5890/91 $3.00 + 0.00 Pergamon Press plc
1991
CONSTRUCTION AND EXPRESSION OF ANTIBODY-TUMOR NECROSIS FACTOR FUSION PROTEINS* HENNIE
GUIDO VOLCKAERT~ and JEF C. M. RAust
R. HOOGENBOOM,~$
)/
tDr. L. Willems-Instituut en Departement WNIF, Limburgs Universitair Centrum, Universitaire Campus, B-3590 Diepenbeek, Belgium and §Laboratorium voor Gentechnologie, Katholieke Universiteit Leuven, De Croylaan 42, 3030 Leuven, Belgium (Received 1 October 1990; accepted 14 November 1990) Abstract-The construction, expression and secretion of two genetically engineered antibody-cytokine hybrid fusion proteins is described. To target tumor necrosis factor (TNF) to tumor cells, recombinant antibody techniques were used to generate F(ab’),-like antibody-TNF fusion proteins. At the gene level, an antitransferrin receptor antibody heavy chain gene was linked to a synthetic gene coding for human TNF. The chimeric heavy chain-TNF genes were introduced into a light chain secreting transfectoma cell line, which was producing the light chain of the same antibody. Cell lines were isolated which secreted antibody-TNF fusion proteins of expected size and composition. Culture supernatant of these cell lines contained TNF cytotoxic activity towards murine L929 cells and human MCF-7 cells, indicating that TNF is still active in the fusion protein constructs. These results illustrate the feasibility of the antibody engineering technology to create and produce chimeric mouse-human immunotoxin-like molecules. Furthermore, they demonstrate the ability of mammalian (myeloma) cells to express and secrete antibody-cytokine hybrid molecules with potential use in anticancer therapy.
INTRODUCTION Immunotoxins, bacterial to become
i.e. combinations
or plant-derived the magic
bullets
toxins,
of antibodies were
long
for treatment
and
thought
of cancers
and Lord, 1990; Vitetta et al., 1987). It has been shown that recombinant antibodies can be constructed in vitro and expressed in transfected mammalian cells (Oi et al., 1983; Morrison et al., 1984; Sharon et al., 1984). Using this technique, genetically engineered chimeric mouse-human antibodies (Sahagan et al., 1986; Liu et al., 1987; Hoogenboom et al., 1990), humanized antibodies (Riechmann et al., 1988; Verhoeyen et al., 1988) and antibody+nzyme conjugates or antibodies linked to non-immunoglobulin proteins have been produced (Neuberger et al., 1984; Schnee et al., 1987). However, the expression of recombinant proteins in mammalian cells is limited to those combinations which are not toxic to the producer cells, at least at low concns. Consequently, immunotoxins are very difficult, if not impossible, to produce in these and other
diseases
(for reviews see Spooner
*This work was supported by a fellowship from the Belgian “Nationaal Fonds voor Wetenschappelijk Onderzoek” to H. R. Hoogenboom. JPresent address: M.R.C. Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K. IlAuthor to whom correspondence should be addressed. Abbreviations: gpr, xanthine-guanine phosphoribosyl transferase: 1~. FCS. fetal calf serum: TNF. _. immunorrlobulin: tumor necrosis faitor; C; constant; V, variadle; H; heavy; L, light.
systems. The development of an E. coli expression system for Fab or Fv antibody fragments (Skerra and Pliickthun, 1988; Better et al., 1988) or single-chain antibody fragments (Huston et al., 1988) opened an alternative route to the production of antigen-binding proteins linked to toxins. Immunotoxins bearing a Pseudomonas exotoxin have already been made (Chaudary et al., 1989; Batra et al., 1989), and combinations with other toxins like diphtheria toxin and the ricin A chain are being evaluated. However, these immunotoxins comprise extremely toxic bacterial or plant derived proteins. Since neither toxins nor antibodies (most often) are human, they will evoke an immune response when used for human therapy (Schroff er al., 1985). The use of chimeric, humanized or human antibodies linked to human proteins with toxic properties would circumvent many problems encountered with the classical immunotoxins. In this study the potential use of tumor necrosis factor (TNF) for constructing immunotoxinlike molecules is evaluated. At first TNF was identified as a tumoricidal protein causing hemorrhagic necrosis of transplanted solid tumors in mice (Carswell et al., 1975). The potential of TNF as an anticancer drug was further illustrated by its cytotoxicity against various tumor target cell lines in vitro, while normal cells were minimally harmed. However, TNF appears to be involved in many other biological processes including inflammation and antiviral defense, endotoxic shock, immunoregulation, angiogenesis, cachexia and mitogenesis (Goeddel et al., 1986; Beutler and Cerami, 1027
1028
HENNIER. H~~CENB~~Met al.
1986; Fiers ef al., 1986). The mechanisms of these multiple activities remain poorly understood. A TNF receptor has recently been cloned (Loetscher et al., 1990; Schall et al., 1990), but there is no clear correlation between density or affinity for this receptor and TNF sensitivity. Furthermore, the DNA coding for human TNF has also been cloned, sequenced and expressed in E. coli, yielding large amounts of recombinant material which has been used for human cancer therapy (for review see Goeddel et al., 1986). Phase I clinical trials show that in vir;o administration of TNF results in severe side-effects, ranging from nausea and fever, vomiting, diarrhea, headache and hypotension to death (Creaven et al., 1987). Hence, there is a restriction on the use of large amounts of TNF for anticancer therapy. In an attempt to reduce the toxic effects of TNF. and to use only the anticancer properties of the molecule, we describe in this study the design and production of two genetically engineered antibody-TNF fusion proteins. By linking TNF to an antitransferrin receptor antibody, the targeting of TNF to transferrin receptor rich cells can be evaluated. Since the transferrin receptor is a growth related cellular surface antigen found on most proliferating cells (Trowbridge and Omary, 1981) most dividing cells have the capability of binding such an antibodycytokine chimeric molecule. However, the selective antitumor activity of TNF would provide the killing of tumor cells only. Since in this approach TNF targets to the surface of cancer cells, killing of cells might be achieved with smaller amounts of TNF. As a result, significant selective cytotoxicity might be obtained with diminished or possibly no side-effects. The antibody used is specific for the human transferrin receptor. It does not compete with transferrin for binding to the receptor, which might be of importance for in Go use (Heyligen et al., 1985). To minimize the immune response towards this mouse antibody when used in human cancer therapy, mouse-human chimeric genes were constructed and expressed in myeloma cells (Hoogenboom et al., 1990). The resulting chimeric antibody retained its antigen-binding capacity. These antibody genes were used in the present study to make antibody-TNF molecules. Since most of the antihybrid bodyenzymes described so far make use of a configuration in which the antibody heavy chain’s C-terminus is linked to the N-terminus of the nonimmunoglobulin protein, two analogous heavy chain-TNF molecules were constructed. Furthermore, the expression and secretion of these molecules by mammalian cells was evaluated. One attachment site was made in the 5’ region of the C, 2 domain, and one in the C,3 domain. If these heavy chain-TNF genes can be expressed in combination with the light chain of the antibody, the proteins will give rise to F(ab’),-like molecules, with targeting properties to growing cells.
MATERIALS AND METHODS Materials Xanthine and hypoxanthine were purchased from Sigma (St Louis, MO). DNA-modifying enzymes, [alfa-32P]dCTP, [gamma-32P]ATP, [“Slmethionine and biotinylated antibodies were supplied by Amersham (Buckinghamshire, U.K.). Oligonucleotides were obtained from Eurogentec (Liege, Belgium). Restriction endonucleases, mycophenohc acid and other cell culture reagents were obtained from Bethesda Research Laboratories (Gaithersburg, MD). DNAs
and DNA manipulations
The genomic human gamma-l constant region gene (Honjo et al., 1979) and the expression vector pSVgptMOV, NP ( pSVgpt) containing regulatory sequences for Ig gene expression in lymphoid cells (Neuberger, 1983) were gifts from Dr M. S. Neuberger (MRC Laboratory of Molecular Biology, Cambridge, U.K.). pSVhyg (palysl7), the selectable shuttle vector with the hygromycin resistance gene, was a gift from Dr J. Foote (MRC Laboratory of Molecular Biology, Cambridge U.K.) (Orlandi et al. 1989). The antibody genes used in this study were previously cloned, sequenced, and used to construct and express mouse-human chimeric antibody genes (Hoogenboom et al. 1990). The TNF DNA was obtained from Dr K. Ashman (EMBL, Heidelberg Germany) (Ashman et al., 1989). The recombinant DNA work was performed by standard procedures (Maniatis et al. 1982). Sequencing was done as described by Sanger et al. (1977). Ohgonucleotidedirected mutagenesis was carried out on singlestranded DNA templates derived from M 13mpl S/19 subclones of the DNA to be mutagenized, according to the method of Eckstein (Nakamaye and Eckstein, 1986) using the oligonucleotide-directed mutagenesis kit from Amersham. Cell lines and cell culture SP2/0-Ag14 (ATCC CRL 1581) is a non-Ig producing murine hybridoma. L929 (ATCC CCLl) is a murine fibroblastic cell line; MCF-7 (ATCC HTB 22) is a breast adenocarcinoma; HL-60 (ATCC CCL240) is a promyeloid leukemia; MOLT-4 (ATCC CRL 1582) is an acute lymphoblastic T-cell leukemia. All cell lines and transfectomas were grown in RPM11640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, non-essential amino acids and 10% FCS. Introduction of DNA into myelomu cells and selection of trunsfected cells DNA was introduced into mammalian cells using electroporation (Potter et al., 1984) with the Gene Pulser Apparatus of Biorad (Richmond, CA) as described previously (Hoogenboom et al., 1990).
Genetically engineered antibody-tumor Briefly, lo6 cells and 5-20 pg Bum HI or PuuIlinearized plasmid in 0.8 ml of PBS at 0°C in an electroporation cuvette (0.4 cm electrode gap) were subjected to a single voltage pulse at 200 V using a capacitance setting of 960pF. Selection for hygromycin resistant cells was done by adding 400 pg/ml hygromycin for a period of 3 weeks to the culture medium, starting 48 hr after the electroporation. Selection for transfected cells containing the gpt gene was carried out with 1 pg/ml mycophenolic acid, 250 pg/ml xanthine and 15 pg/ml hypoxanthine. Clones were visible after l-2 weeks. Individual clones were selected by limiting dilution in the absence of feeder cells. Screening for antibody secreting cell lines Screening for antibody production was done by ELISA, detecting human IgG or kappa chain, as described elsewhere (Hoogenboom et al., 1990). The assay measures antibody concns ranging from 1 to lOOng/ml, and is specific for detection of human gamma or kappa chain. SDS-PAGE
and immunoblot
Protein analysis by SDS-PAGE was performed according to Laemmli (1970). Samples were prepared in 0.1 M Tri-HCl, pH 7.0, containing 2% SDS, 2% mercaptoethanol and 10% sucrose and heated at 95°C for 5 min prior to electrophoresis on 12.5% cross-linked gels. After electrophoresis, samples were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, IL) by electroblot (Novablot, LKB-Pharmacia, Uppsala, Sweden) at 0.8 mA/cm’ for 1 hr, for immunoblot analysis. The filter was incubated overnight in 3% BSA at 4°C and washed afterwards in PBS&O.O5% Tween-20. Light chain was detected by using biotinylated goat antihuman kappa Ig (l/500 in PBS) (Amersham). For detection of the heavy chain, the filter was incubated for 2 hr at room temp with a l/500 dilution of goat anti-human IgG antibody (Tago Inc., Burlingame, CA) in PBS. After washing the filter was incubated with a l/250 dilution of peroxidase labeled rabbit anti-goat Ig (Dakopatts, Copenhagen, Denmark) in PBS containing 1% BSA. BSA was added to prevent extensive cross-reaction between the labeled anti-goat Ig and the mouse-human Ig on the filter. Staining was carried out with 0.05% diaminobenzidine dissolved in 0.1% H202 in PBS. The reaction was stopped by rinsing the filter with water. TNF was detected with rabbit anti-TNF antiserum (l/500 in PBS) (Genzyme, Boston, MA), and a l/500 dilution of peroxidase labeled swine anti-rabbit Ig (Dakopatts) in PBS. TNF-assay The cytotoxic activity of TNF was determined by using mouse L929 fibroblas; cells (Ruff and Gifford, 1981). The cells were incubated in the presence of actinomycin-D (1 pg/ml) with increasing amounts of the TNF containing sample. The assay was carried
necrosis factor molecules
1029
out in microtiter plates using 2 x 104-5 x lo4 cells in 200 ~1 per well. After an 18 hr incubation period, the living cells were quantified with MTT (3-(4,5dimethyl-thiazol-2-yl)-2,5-di-phenyltetrazoliumbromid) (Mosmann, 1983). MTT (20pl/well of a 5 mg/ml stock) was added to each well, and after l-4 hr incubation, the produced formazan crystals were redissolved by adding 100 PI/well 10% SDS + 0.01 N HCl and incubating for 416 hr. The O.D. was measured at 540 nm in an automatic Titertek microtiter plate reader (Flow). TNF protein was purchased from Genzyme and had a specific activity of 2 x lO’units/mg protein. The activity of TNF (in units) was defined as the reciprocal of the dilution at which a 50% reduction in O.D. was observed compared with control cultures containing no TNF. For MCF-7, HL-60 and MOLT-4 cells no actinomycin-D was used in the assay, and the incubation period was extended to 48 hr. RESULTS Design and construction TNF genes
of two chimeric
antibody-
An antibody molecule consists of compact protein domains with the hinge region serving as a spacer separating Fab from Fc regions (Burton, 1987). This domain structure is reflected at the gene level: in the human gamma-l gene, each domain, is encoded by a separate exon. To obtain two F(ab’),-like proteins, two different chimeric genes were made, in which the human TNF gene is linked to the C, 2 or C, 3 domain of the human gamma-l chain. The first attachment point for TNF was selected in the 5’ region of the C, 2-domain, leaving all amino acid residues involved in the hinge structure and dimerization of the heavy chain untouched. Only the first three amino acid residues of the CuZdomain were retained (Fig. 3) deleting Leu-235 (EU numbering), which is the major determinant in the binding of Fc to the high-affinity receptor on monocytes (Duncan et al., 1988). Attachment near a similar site in the murine gamma-2b gene has been used previously to connect Klenow polymerase (Williams and Neuberger, 1986) and aequorin (Casadei et al., 1990). By placing TNF distal to the hinge region, the antigen binding should not be influenced, and the flexibility of this region might facilitate binding of TNF to its own receptor, yielding a multifunctional protein. In a second construct the attachment site of TNF in the C, 3 domain was made at amino acid residue 422 (EU numbering) removing the last structural /I-sheet (b6-fy3-e2; Burton, 1985). Hence, binding of the antibody-TNF fusion protein to Fc receptors, which might interfere with the projected anticancer activity, is excluded in the C,,2-TNF construct only. A synthetic gene coding for the mature human TNF protein (without leader peptide) was assembled, expressed in E. coli and the recombinant TNF was checked for activity (Ashman et al., 1989). The first
1030
HENNIER. HOOGENBOOM et al.
two amino acid residues of native TNF (Val-Arg) are absent in the synthetic gene, but this deletion has no influence on the activity (Shirai et al., 1985). The synthetic gene comprises a translation initiation codon and an EcoRI restriction site at the 5’ end and two tandem stop codons and a BamHI restriction site at the 3’ end of the TNF coding region. The EcoRI site was used for linking the gene to the Cn2 or Cn3 domain. A subclone of the human gamma-l genomic gene was made by cloning a I-kb PstI-BarnHI fragment containing the C, 2 and Cn 3 domains into M 13mp 18 (Fig. 1). An EcoRI site was introduced in the Cn2 domain by oligonucleotide-directed in vitro mutagenesis by using ohgonucleotide 5’-AGCACCTGAATTCTGGGGG-3’. A clone containing the mutation was identified by Eco RI-restriction and the mutation was confirmed by sequencing. This clone was further used to generate the C,2-TNF chimeric gene, as depicted in Fig. 1. The Cn3-TNF gene fusion was made by ligating the TNF gene fragment, cut with EcoRI cut and treated with Klenow polymerase and dNTPs, to the unique XmnI site (which generates blunt ends) in the C, 3 domain of human gamma-l (Fig. 2). Since non-immunoglobulin derived untranslated 3’ regions of immunoglobulin gene constructs can decrease expression levels by destabilizing the mRNA (Weidle et al., 1987) and since the synthetic TNF gene fragment does not include a polyadenylation signal, the human gamma-l polyadenylation signal was added to the 3’ end of the chimeric construct. Therefore, the signal containing XmnI-Sac1 restriction fragment of the human gamma-l gene was ligated to the BamHI site of the TNF gene, after filling in of the latter site with Klenow ,polymerase and dNTPs. The Sac1 site fits into the unique Sac1 site of the vector (see caption of Fig. 1). Both XmnI and BamHI sites are deleted, but the series of stop codons at the 3’ end of the TNF gene are left intact. The polyadenylation signal is preceded by 78 nucleo-
tides of the (untranslated) Cn3 coding region of the gamma- 1 gene, creating an immunoglobulin-like gene and mRNA. Figures 1 and 2 show the cloning schemes used to build the heavy chain-TNF genes. The junctions between antibody heavy chains and TNF DNA are depicted in Fig. 3. Note that in the Cn2-Cn3 constructs, two to three amino acid residues are added in the junction region, including methionine, the first residue translated when expressing the synthetic DNA in E. coli (Ashman et al., 1989). Isolation of cell lines secreting chimeric proteins
the antibody-TNF
The chimeric light chain gene (Hoogenboom et al., 1990) was cloned into pSVhyg, a pSVneo derived plasmid containing the hygromycin resistance gene (see Materials and Methods; Fig. 4). The chimeric heavy chain-TNF genes were cloned into the HindIII-Bum HI sites of pSV2gptMOVn NP, which contains the gpt gene as a selection marker (see Materials and Methods). In the final construct, the expression of the chimeric gene is regulated by an immunoglobulin transcription enhancer element and promoter, both situated upstream of the gene (Fig. 4). It has been shown previously that the heavy chain enhancer is active on the promoter at this position (Neuberger, 1983). Secretion of the chimeric heavy chain-TNF proteins is directed by the presence of an immunoglobulin gene derived signal peptide sequence. Mouse SP2/0-Ag14 cells were transfected with the light gene containing expression vector. After transfection by electroporation, selection with hygromycin yielded several clones secreting human light chain, as detected by the human kappa-detecting ELISA. The best producing clone, 12B5, was selected for further use and subcloned by limiting dilution. Cell line 12B5 was transfected with one of the heavy chain-TNF gene containing vectors. After selection for the presence of the gpt gene, culture
Fig. 1. Construction scheme of the chimeric C,2 heavy chainTNF gene. Vector sequences and promoters are shown as small boxes, introns and other non-coding regions as lines, and exons as large boxes. The heavy chain variable sequence is shown in black, Ig constant regions are white boxes, and the TNF DNA box is stippled. Only restriction sites relevant to this construction scheme are shown; structural features of the vectors (antibiotic resistance genes, etc.) are not shown. Plasmids are not drawn to scale. (1) The human gamma-l gene was cloned as a 2.3-kb ApaI fragment into pGV463 (Neesen and Volckaert, 1989). (2) In vitro mutagenesis for the creation of an EcoRI site in the Cu2 domain was carried out after subcloning into M13mp18 of the 1-kb PsrI-BumHI fragment of pGV-HuIgG containing the hinge, the C, 2 and the C, 3 domain of the human gamma- I gene. In two steps, an Ig-derived polyadenylation signal was added to the TNF gene: first (3), the 170 bp PsrI-EcoRI fragment of M13mpl8CH,-Eco was cloned together with the 0.5kb EcoRI-BumHI fragment into pUC18, yielding pUC-H-TNF. Second (4). the 0.7-kb XmnI-Sac1 fragment of M13mpl8-C,2-Eco was inserted into pUCH_TNF which was cut with BumHI, treated with Klenow polymerase and dNTPs, and cut with &cl, yielding pUC-HP-TNF. (5) The 1-kb PsfILEamHI fragment of M13mpl8-C,2-Eco was subcloned into pUC18, and the complete chimeric heavy-chain gene was restored by inserting a 2.1-kb HindIIILApaI and a 0.6-kb ApaILZ’stI fragment from pUC-chimIgG (Hoogenboom et al., 1990) containing the Ig sequences, yielding pUCF31.IgGI (6). (7) The TNF-containing 1.3-kb EcoRI fragment of pUC-HP-TNF was inserted into this plasmid. In order to gain a BamHI site at the 3’ end of the chimeric gene, which was necessary for further subcloning experiments, the 4-kb Hi&III-Sac1 fragment of pUC-IgC,2-TNF was subcloned into pGV463, yielding pGVC,2-TNF (8).
Genetically
engineered
antibody-tumor
necrosis
factor
molecules
1031
pGV-Ig
Fig. 1
-CHZ
-TNF
HENNIE R. HOOGENB~OM et al.
1032 ApaI
Soci BamHI
pUC - ChlmIgG
EcoRi
PUC-Ig-Cri3m
TN;
Fig. 2. Construction scheme of the chimeric C, 33TNF gene. Legend as in Fig. 1. The 1.5kb ApaIIXmn I fragment of pUC-chimIgG (Hoogenboom Ed al., 1990) was cloned into pUC-chimIgG cut with ApaI and BumHI, together with the 1.3-kb EcoRI-EarnHI fragment of pGVC,2-TNF (Fig. I), of which the EcoRI site was treated with Klenow polymerase and dNTPs. This subcloning yielded plasmid
pUCC, supernatant of several clones reacted positive in the human IgG-detecting ELISA. Clones 8D5 and 13A4 were identified as the best producers for the C, 22TNF and C, 3-TNF proteins, respectively, and IA)
TCCTCA
(3)
(2)
(1) A
P
E
F
M
s
s
s
GCA
CCT
GAA
TTC
ATG
TCT
TCT
TCT
---@RI--intron
(1)
CAG
(3)
(2)
GGG l
E
AAC
G
****
AA
F
M
s
s
s
TTC
ATG
TCT
TCT
TCT
___EcoRI___
cm11
Cn3-domain
of the antibody-TNF
chimeric
pro-
teins
(B)
N
were used for further analysis. The amount of secreted Cn2-TNF fusion protein gave the same optical density in the human IgG-ELISA as a 10 ng/ml solution of pure human IgG protein, compared to 200 ng/ml for the C,3-TNF protein. The actual concentration of the C, 22TNF fusion protein will be higher, because the ELISA was standardized with intact human IgG-protein (which is 6 times the size of the ELISA-positive antibody region in the fusion protein). Characterization
TNF
CH2-domain
QG
3-TNF.
TNF
Fig. 3. Junctions between antibody heavy chain and TNF DNAs (A) in the C,2-TNF construct and (B) in the C,3-TNF construct. (1) Antibody region; (2) amino acid residues introduced while cloning, including the methionine, used for expression of the TNF DNA in E. coli; (3) mature TNF sequence, with the first two codons deleted.
Immunoprecipitation was carried out using culture supernatant of clones 8D5 and 13A4 and goat antihuman IgG-Sepharose beads. After elution of the precipitated proteins, the samples were reduced, fractionated on SDS-PAGE and transferred to a PVDF membrane for immunoblot analysis. As a positive control, culture supernatant of transfectoma 13A5, a cell line secreting the antitransferrin receptor chimeric mouse-human antibody (Hoogenboom et al., 1990) was used. Anti-human IgG or anti-TNF antisera were used to detect human immunoglobulins of TNF reactive proteins, respectively. The immunoblot analysis depicted in Fig. 5(B) shows that cell line 13A5 secretes a normal heavy chain, which can be detected with the anti-human IgG antiserum only. On the other hand, transfectomas 8D5 and 13A4 clearly
Genetically engineered antibody-tumor
necrosis factor molecules
1033
(b)
HIndI ECCRI pSVgpt-CHZ-
PSVgpt
TIJF
pSVhyg
pBR322
-CH3
- TNF
- VLCK
SJ40
Fig. 4. Structures of the plasmids containing (A) the chimeric C,2-TNF gene, (B) the chimeric C,3-TNF gene or (C) the chimeric light chain. The chimeric heavy chain-TNF genes were subcloned as HindIII-BamHI fragments into expression vector pSVgpt, yielding pSVgpt-C,2/33TNF. P, promoter; S, signal peptide sequence; E, enhancer; AMP (HYG), ampicillin (hygromycin) resistance gene; ori origin of replication. secrete an anti-human IgG (Fig. 5B) and anti-TNF (Fig. 5A) reactive protein of homogenous length. The size of the fusion proteins is as expected: 46 kD for C,2-TNF and 69 kD for C,3-TNF (in both cases 17 kD is from the TNF portion). Sample 5B4 is an immunoprecipitated culture supernatant sample of a transfection secreting an Fab-like heavy chain-TNF protein (Hoogenboom et al., submitted), which is seven amino acid residues smaller than the C, 2-TNF hybrid molecule: Figures (A) and (B) clearly show this difference in size between SB4 and 8D5. In the immunoblot analysis of culture supernatant of the 8D5 and 13A4 cells, no degradation products of the antibody region of the molecule were detected, indicating that the fusion proteins are secreted as stable and relatively protease-resistant proteins. However, extensive proteolytic degradation of the C,2/3-TNF fusion proteins was seen after culturing the 8D5 and 13A4 cells in serum-free medium for 3 days, probably caused by the massive cell death and subsequent release of intracellular proteases in those cultures.
Protein A-Sepharose failed to precipitate the fusion proteins of both 8D5 and 13A4 transfectomas. The binding site of protein A is believed to be near the junction of the Cn2 and C, 3 domains, which is deleted in the C, 2-TNF construct. In the C, 3-TNF construct on the contrary, the histidine at position 435, which is involved in the contact between human IgGl and protein A (Deisenhofer et al., 1978), was deleted, which might explain the inability of protein A to bind to the C,3-TNF hybrid protein as well. Immunoprecipitation with anti-human IgG-Sepharose of in vivo radiolabeled 8D5 and 13A4 supernatant showed that the light chain coprecipitated with the heavy chain-TNF proteins (results not shown), pointing out that the antigen-binding domains are in the expected configuration. TNF activity upon mouse and human cell lines Culture supernatants of the 8D5 and 13A4 transfectomas were assayed for cytotoxic activity. Various cell lines were incubated in 200 ~1 medium containing a variable amount of culture supernatant of
HENNIE R. HGQGENBGQMet al.
Fig. 5. Immunoblot analysis of supernatant of transfectomas 13A5 (secreting chimeric antitransferrin receptor antibody), 8D5 and 13A4 (secreting light chain and C,2-TNF and C,3-TNF fusion proteins, respectively). Transfectoma 5B4 secretes another antibody_TNF derivative (see text). Supernatant was immunoprecipitated with goat anti-human IgG-Sepharose beads, elute and fractionated on 12.5% SDS-PAGE. The proteins were transferred to a PVDF membrane and antibodies were detected using anti-TNF (A) or anti-human IgG (B), as described in Materials and Methods. The position of M, markers are indicated (M, x 10-s).
SP2/0-Ag14 cells (non-producer), 13A5 (chimeric antibody), 8D5 (C&2-TNF) or 13A4 (C,3-TNF) transfectomas. The amount of living cells was determined after 1, 2 or 3 days using the MTT-coloring method. Figure 6 depects the growth curves of the mouse L929 fibroblast cell line and the relatively TNF-resistant human MCF-7 breast carcinoma. Only 8D5 and 13A4 supernatant had a growth inhibitory effect upon both cell lines. Similar assays with cell lines HL-60 and MOLT-4 showed the same growth inhibitory effect of both 8D5 and 13A4 supernatants (results not shown). 13A4 supernatant contains more IgGantibody as measured by ELISA than 8D5 supernatant, but the growth inhibition of the latter supernatant is more pronounced. Since no protein concns are known yet, no conclusions can be drawn about the relationship between structure and activity. Since there is no competition between antibody and transferrin for binding to the receptor, the antitrans-
ferrin receptor antibody of 13A5 can not cause growth inhibition (Heyligen et al., 1985; Hoogenboom, unpublished results). Furthermore, the antibody is specific for the human transferrin receptor. The growth of the murine L929 cells can only be inhibited due to the TNF-activity of the 8D5 and 13A4 sample. According to this L929-assay, the SD5 and 13A5, supernatants contain approximately 4 and 2 unit TNFjlOO ~1, respectively. The growth of MCF-7 cells however, which could not be inhibited with 400 units TNF as tested with pure recombinant human TNF, could be inhibited with both 8D5 and 13A4 supernatant. This difference might be explained by the targeting effect of the antibody-TNF molecules to human cells due to the transferrin receptor binding, increasing the actual concn of TNF near the cellular membrane. Since the antibody does not recognize the murine transferrin receptor, there is no targeting to the murine L929 cells.
Genetically engineered antibody-tumor
1035
necrosis factor molecules
Growth curves of MCF-7 cells
Growth curves of L929 cells
0.6
* ..*-.)___
OD
SP2JoP Medium
0.4
13A52
-I 4
0.6 OD
u
6D52 6D5:4
.
6D5:6
04-
..___ ,___ 0.2 -
O.OO lime
13A4:2
+
13A4:4
Fig. 6. Growth curves of L929 (left) and MCF-7 SP2/0-Ag14 (non-producer), and transfectomas body), 8D5 and 13A4 (secreting light chain and Culture supernatant was added at the indicated an MTT-assay
13A430 *...
Medium
4 llme
(days)
Medium
--_(F
--..
“.O;
ED520
(days)
(right) cells, in the presence of culture supematant of 13A5 (secreting chimeric antitransferrin receptor antiC,2--TNF or C,3-TNF fusion proteins, respectively). dilution; (:2,:4, etc.), and living cells were quantified in after 1, 2 or 3 days.
DISCUSSION
In this study the potential targeting of TNF to cancer cells by linking it to an antitransferrin receptor antibody was evaluated. Recombinant antibody techniques were used to construct and express two antibody heavy chain-TNF genes. Cell lines were obtained secreting light chain and detectable amounts of two heavy chain-TNF fusion proteins. Several genetically engineered antibody-enzymes or other antibody-like molecules expressed by mammalian cells, have been described so far, using genes encoding Staphylococcus aureus nuclease or c-myc (Neuberger et al., 1984), Klenow polymerase (Williams and Neuberger, 1986), tissue Plasminogen Activator (Schnee et al., 1987) the photoprotein aequorin (Casadei et al., 1990), or insulin-like growth factor 1 (Shin and Morrison, 1990). In most cases nonimmunoglobulin proteins were linked to the C,2-
domain of the mouse immunoglobulin gamma2b constant region, yielding potential F(ab’),-like molecules. We used the human gamma-l constant region gene and DNA encoding human TNF for the production of chimeric mouse-human antibody-TNF fusion proteins. The murine variable regions of the antibody are the only antigenic domains when used in humans, although it can not be excluded that novel combinations of components may produce neoantigenie determinants. Linkage to the “classical” site in the Cu 2 domain as well as linkage to the Cn 3 domain proved to result in expression and secretion of fusion proteins of the expected size and composition. Alike the other described antibodyenzymes, linkage of the non-antibody region (in the present study TNF) to the antibody moiety had no deleterious effect upon the secretory pathway followed by the antibody protein. Nevertheless, the level of secretion was lower than for the described antibodyenzymes
1036
HENNIE R. HOOGENBOOMet al.
(estimated to be in the ng/ml range of l&200 ng/ml), but correct values can only be obtained after purification of the fusion proteins. To obtain higher expression levels of the hybrid molecules, amplification of the chimeric genes by the dhfr system can be envisaged (Hendricks et al., 1989). However, it must be kept in mind that the low expression levels might be due to the intrinsic toxicity of TNF itself. Although no growth retardation or cell lysis was detected in the 8D5 and 13A4 cell cultures, secreting C, 2-TNF and C, 3-TNF respectively, production of high amounts of the antibody-TNF hybrid molecules might be toxic to the transfectoma cells, and low producers might be self-selected. In the latter case, expression of the recombinant proteins as Fab or Fv-like proteins in E. co/i should be considered (Chaudary et al., 1989; Batra et al., 1989). The culture supernatant of the transfected cell line 8D5, producing light chain as well as Cn2 heavy chain-TNF protein, contained growth inhibiting activity upon murine and human cell lines. Supernatant of transfectoma 13A4 had a similar effect. Therefore, the fusion products have a TNF-activity, but the specific activity has not been determined yet. Whereas the active form of TNF is a compact trimer (Jones et al., 1989), the active native form of the F(ab’),-like antibody-TNF proteins and their possible trimerization remain to be elucidated. A difference between the TNF-activity of the culture supernatant as determined by the L929-assay and the TNF-sensitivity of human MCF-7 cells was found. Although the culture supernatant contained 24 units TNF/lOOpl according to the L929 assay, this amount was sufficient to inhibit the growth of human MCF-7 cells, which are not affected up to 400 units/100 ~1 pure recombinant human TNF. These results are highly suggestive for a targeting of the antibody-TNF fusion protein to these human cells by binding to the transferrin receptor. This TNF-activity might result not only from the concentrating effect of direct binding to the plasma membrane (and mediating toxicity through the TNF receptor) but also from the appropriate trafficking of the hybrid molecule after binding and internalization of the antibody-transferrin receptor complex. Purification of the new proteins will enable us to determine the affinity constants for the human transferrin receptor and the TNF receptor, and to elucidate the mechanism of action of these new antibody-cytokine molecules. The preliminary cytotoxicity assay results presented here suggest that chimeric antibody-TNF molecules are potential immunotherapeutics for cancer therapy. These data extend the results described in a recent paper, presenting the construction, expression and secretion of an Fab-like antibody-TNF chimeric protein (Hoogenboom et al., submitted). They demonstrate the utility and feasibility of the antibody engineering technique to develop bifunctional,
more human-like and more potent immunotoxin-like anticancer agents or other immunotherapeutics. Acknowledgements-We
thank Drs K. Ashman, M. Neuberger, G. Winter and J. Foote for their generous gifts of DNAs, and Marc Withofs for graphical work.
H. R. Hoogenboom “Nationaal Fonds (N.F.W.O.).
was a research assistant voor Wetenschappelijk
of the Belgian Onderzoek”
REFERENCES Ashman K., Matthews N. and Frank R. W. (1989) Chemical synthesis, expression, and product assessment of a gene coding for biologically active human TNF-alfa. Prof. Engng 2 387-391. Batra J. K., Jinno Y., Chaudary V. K., Kondo T., Willingham M. C., FitzGerald D. J. and Pastan I. (1989) Antitumor activity in mice of an immunotoxin made with anti-transferrin receptor and a recombinant form of Pseudomonas exotoxin. Proc. natn. Acad. Sri. U.S.A. 86, 8545-8549. Better M., Chang C. P., Robinson R. R. and Horwitz A. H. (1988) Escherichiu coli secretion of an active chimeric antibody fragment. Science 240, 1041-1044. Beutler B. and Cerami A. (1986) Cachectin and Tumour Necrosis Factor as two sides of the same biological coin. Nafure, Land. 320, 584588. Burton D. R. (1985) Immunoglobulin G: Functional sites. Molec. Immun. 22, 161-206. Burton D. R. (1987) In Molecular Genetics of Immunoglobulin. New Comprehensive Biochemistry, Vol. 17 (Edited bv Calabi F. and Neuberaer M. S.). London. Carswell E.-A., Old J., Kassel R., Green S., Fiore N. and Williamson B. (1975) An endotoxin-induced serum factor that causes necrosis of tumours. Proc. natn. Acad. Sci. U.S.A. 72, 36663670. Casadei J., Powell M. J. and Kenten J. H. (1990) Expression and secretion of aequorin as a chimeric antibody by means of a mammalian expression vector. Proc. natn. Acad. Sci. U.S.A. 81 2047-2051. Chaudary V. K., Queen C., Junghans R. P., Waldmann T. A., Fitzgerald D. J. and Pastan I. (1989) A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin. Nature, Land. 339, 394-397. Creaven P. J., Plager J. E., Dupere S., Huben R. P., Takita H., Mittelman A. and Proefrock A. (1987) Phase-I clinical trial of recombinant human Tumor Necrosis Factor. Cancer Chemother. Pharmacol. 20, 1377144. Deisenhofer J., Jones T. A. and Huber R. (1978) Crystallization, crystal structure analysis and atomic model of the complex formed by a human Fc fragment an Fragment B of Protein A from Slaphylococcus aureus. Hoppe-Seyler’s Z. physiol. Chem. 359, 975-985. Duncan A., Woof J., Partridge L., Burton D. and Winter G. (1988) Localization of the binding site for the human high affinity Fc-receptor on IgG. Nature Lond. 332 563-566. Fiers W., Brouckaert P., Devos R., Franssen L., LerouxRoels G., Remaut E., Suffys P., Tavernier J., Van der Heyden J. and Van Roy F. (1986) Lymphokines and monokines in anticancer therapy. Cold Spring Harb. Symp. quant. Biol. 51, 587-595. Goeddel D. V., Aggarwal B. B., Gray P. W., Leung D. W., Nedwin G. E., Palladino M. A., Patton J. S., Pennica D., Shepard H. M., Sugarman B. J. and WongG. H. W. (1986) Tumor Necrosis Factors: gene structure and biological activity. Cold Spring Harb. Symp. quant. Biol. 51,597-609. Hendricks M. B., Luchette C. A. and Banker M. J. (1989) Enhanced expression of an immunoglobulin-based vector in myeloma cells mediated by coamplification with a mutant dihydrofolate reductase gene. Biotechnology 7, 1271-1274.
Genetically
engineered
antibody-tumor
Heyligen H., Thijs C., Weber W., Bosmans E. and Raus J. (1985) Monoclonal antibodies detecting human T-cell activation antigens: development of monoclonal antibodies and expression of antigens on activated T-cells and leukemic cell lines. Fedn Proc. 44, 787. Honjo T., Obata M., Yamawaki-Kataoka Y., Kataoka T., Kawakami T. Takahashi N. and Mano Y. (1979) Cloning and complete nucleotide sequence of mouse immunoglobulin gamma-l chain gene. Cell 18, 559-568. Hoogenboom H. R., Raus J. C. M. and Volckaert G. (1990) Cloning and expression of a chimeric antibody directed against the human transferrin receptor. J. Immun. 144, 321 l-3217. Huston J. S., Levinson D., Mudgett-Hunter M., Tai M.-S., Novotny J., Margolies M. N., Ridge R. J., Bruccoleri R. E., Haber E., Crea R. and Oppermann H. (1988) Protein engineering of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in E. coli. Proc. natn. Acad. Sci. U.S.A. 85, 5879-5883. Jones E. Y., Stuart D. I. and Walker N. P. C. (1989) Structure of tumour necrosis factor. Nature Lond. 338, 225-228. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680-684. Liu A. Y., Robinson R. R., Murray E. D., Ledbetter J. A., Hellstriim I. and Hellstrijm K. E. (1987) Production of a mouse-human chimeric monocloial antibody to CD20 with potent Fc-dependent biologic activity. J. Immun. 139, 3521-3526. Loetscher H., Pan Y.-C. E., Lahm H.-W., Gentz R., Brockhaus M., Tabuchi H. and Lesslauer W. (1990) Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor. Cell 61, 351-359. Maniatis T., Fritsch E. F. and Sambrook J. (1982) In Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Morrison S. L., Johnson M. J., Herzenberg L. A. and Oi. V. T. (1984) Chimeric human antibody molecules: Mouse antigen-binding domains with human constant region domains. Proc. nafn. Acad. Sci. U.S.A. 81, 6851-6855. Mosmann T. (1983) Rapid calorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immun. Meth. 65, 55-63. Nakamaye K. and Eckstein F. (1986) Inhibition of restriction endonuclease NciI cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis. Nucleic Acids Res. 14, 9679-9698. Neesen K. and Volckaert G. (1989) Construction and shuttling of novel bifunctional vectors for Streptomyces spp. and Escherichia co/i. J. Bacterial. 171, 1569-1573. Neuberger M. S. (1983) Expression and regulation of imminoglobulin heavy &ain gene transiected into lvmphoid cells. EMBO J. 2. 1373-1378. Ne;b&ger M. S., Williams 6. T. and Fox R. 0. (1984) Recombinant antibodies possessing novel effector functions. Nature, Lond. 312, 604-608. Oi V. T., Morrison S. L., Herzenberg L. A. and Berg P. (1983) Immunoglobulin gene expression in transformed lymphoid cells. Proc. natn. Acad. Sci. U.S.A. 80, 825-829. Orlandi R., Giissow D. H., Jones P. T. and Winter G. (1989) Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc. nafn. Acad. Sci. ti.S.A. 86; 3833-3837. Potter H., Weir L. and Leder P. (1984) Enhancer-dependent expression of human K immunoglobulin genes introduced
necrosis
factor
molecilles
1037
into mouse pre-B lymphocytes by electroporation. Proc. natn. Acad. Sri. U.S.A. 81, 7161-7165. Riechmann L., Clark M., Waldmann H. and Winter G. (1988) Reshaping human antibodies for therapy. Nature, Lond. 332, 323-327. Ruff M. and Gifford G. (1981) Tumor necrosis factor. In Lymphokines, Vol. 2 (Edited by Pick E.). Academic Press, New York. Sahagan B. G., Dorai H., Saltzgaber-Muller J., Toneguzzo F., Guindon C. A., Lilly S. P., McDonald K. W., Morrissey D. V., Stone B. A., Davis G. L., McIntosh P. K. and Moore G. P. (1986) A genetically engineered murine/human chimeric antibody retains specificity for human tumor-associated antigen. J. Immun. 137, 10661074. Sanger F., Nicklen S. and Coulson A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. natn. Acad. Sci. U.S.A. 74, 5463-5467. Schall T. J., Lewis M., Keller K. J., Lee A., Rice G. C., Wong G. H. W., Gatanaga T., Granger G. A., Lentz R., Raab H., Kohr W. J. and Goeddel D. V. (1990) Molecular cloning and expression of a receptor for human tumor necrosis factor. Cell 61, 361-370. Schnee J. M., Runge M. S., Matsueda G. R., Hudson N. W., Seidman J. G., Haber E. and Quertermous T. (1987) Construction and expression of a recombinant antibodytargeted plasminogei activator. Proc. nam. Acad. S&. U.S.A. 84. 6904-6908. Schroff R. w., Foon A. F., Beatty S. M., Oldham R. K. and Walton C. M. (1985) Human anti-murine immunoglobulin responses in patients receiving monoclonal antibody therapy. Cancer Res. 45, 879-885. Sharon J., Gefter M. L., Manser T., Morrison S. L., Oi V. T. and Ptashne M. (1984) Expression of a VHCK chimaeric protein in mouse myeloma cells. Nature, Lond. 309, 364-367. Shin S.-U. and Morrison S. L. (1990) Expression and characterization of an antibody binding specificity joined to insulin-like growth factor 1: potential applications for cellular targeting. Proc. natn. Acad. Sci. U.S.A. 87, 5322-5326. Shirai T., Yamaguchi H., Ito H., Todd C. W. and Wallace R. B. (1985) Cloning and expression in E. coli of the gene for human Tumor necrosis factor. Nafure, Lond. 313, 803-806. Skerra A. and Pliickthun, A. (1988) Assembly of a functional Immunoglobulin Fv fragment in Escherichia coli. Science 240, 1038-1041. Spooner R. A. and Lord J. M. (1990) Immunotoxins: status and prospects. TIBTECH 8, 189-193. Trowbridge I. S. and Omary M. B. (1981) Human cell surface glycoprotein related to cell proliferation is the receptor for transferrin. Proc. natn. Acad. Sci. U.S.A. 78, 3039-3043. Verhoeyen M., Milstein C. and Winter G. (1988) Reshaping human antibodies: grafting an anti-lysozyme activity. Science 239, 1534-1537. Vitetta E. S., F&on R. J., May R. D., Till M. and Uhr J. W. (1987) Redesigning Nature’s poisons to create antitumor reagents. Science 238, 1099-l 104. Weidle U. H., Koch S. and Buckel P. (1987) Expression of antibody cDNA in murine myeloma cells: possible involvement of additional regulatory elements in transcription of immunoglobulin gene. Gene 60, 205-216. Williams G. T. and Neuberger M. S. (1986) Production of antibody-tagged enzymes-by myeloma cells: application to DNA polymerase I Klenow fragment. Gene 43, 319-324.
0161~5890/91 $3.00 + 0.00 Pergamon Press plc
1991
CONSTRUCTION AND EXPRESSION OF ANTIBODY-TUMOR NECROSIS FACTOR FUSION PROTEINS* HENNIE
GUIDO VOLCKAERT~ and JEF C. M. RAust
R. HOOGENBOOM,~$
)/
tDr. L. Willems-Instituut en Departement WNIF, Limburgs Universitair Centrum, Universitaire Campus, B-3590 Diepenbeek, Belgium and §Laboratorium voor Gentechnologie, Katholieke Universiteit Leuven, De Croylaan 42, 3030 Leuven, Belgium (Received 1 October 1990; accepted 14 November 1990) Abstract-The construction, expression and secretion of two genetically engineered antibody-cytokine hybrid fusion proteins is described. To target tumor necrosis factor (TNF) to tumor cells, recombinant antibody techniques were used to generate F(ab’),-like antibody-TNF fusion proteins. At the gene level, an antitransferrin receptor antibody heavy chain gene was linked to a synthetic gene coding for human TNF. The chimeric heavy chain-TNF genes were introduced into a light chain secreting transfectoma cell line, which was producing the light chain of the same antibody. Cell lines were isolated which secreted antibody-TNF fusion proteins of expected size and composition. Culture supernatant of these cell lines contained TNF cytotoxic activity towards murine L929 cells and human MCF-7 cells, indicating that TNF is still active in the fusion protein constructs. These results illustrate the feasibility of the antibody engineering technology to create and produce chimeric mouse-human immunotoxin-like molecules. Furthermore, they demonstrate the ability of mammalian (myeloma) cells to express and secrete antibody-cytokine hybrid molecules with potential use in anticancer therapy.
INTRODUCTION Immunotoxins, bacterial to become
i.e. combinations
or plant-derived the magic
bullets
toxins,
of antibodies were
long
for treatment
and
thought
of cancers
and Lord, 1990; Vitetta et al., 1987). It has been shown that recombinant antibodies can be constructed in vitro and expressed in transfected mammalian cells (Oi et al., 1983; Morrison et al., 1984; Sharon et al., 1984). Using this technique, genetically engineered chimeric mouse-human antibodies (Sahagan et al., 1986; Liu et al., 1987; Hoogenboom et al., 1990), humanized antibodies (Riechmann et al., 1988; Verhoeyen et al., 1988) and antibody+nzyme conjugates or antibodies linked to non-immunoglobulin proteins have been produced (Neuberger et al., 1984; Schnee et al., 1987). However, the expression of recombinant proteins in mammalian cells is limited to those combinations which are not toxic to the producer cells, at least at low concns. Consequently, immunotoxins are very difficult, if not impossible, to produce in these and other
diseases
(for reviews see Spooner
*This work was supported by a fellowship from the Belgian “Nationaal Fonds voor Wetenschappelijk Onderzoek” to H. R. Hoogenboom. JPresent address: M.R.C. Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K. IlAuthor to whom correspondence should be addressed. Abbreviations: gpr, xanthine-guanine phosphoribosyl transferase: 1~. FCS. fetal calf serum: TNF. _. immunorrlobulin: tumor necrosis faitor; C; constant; V, variadle; H; heavy; L, light.
systems. The development of an E. coli expression system for Fab or Fv antibody fragments (Skerra and Pliickthun, 1988; Better et al., 1988) or single-chain antibody fragments (Huston et al., 1988) opened an alternative route to the production of antigen-binding proteins linked to toxins. Immunotoxins bearing a Pseudomonas exotoxin have already been made (Chaudary et al., 1989; Batra et al., 1989), and combinations with other toxins like diphtheria toxin and the ricin A chain are being evaluated. However, these immunotoxins comprise extremely toxic bacterial or plant derived proteins. Since neither toxins nor antibodies (most often) are human, they will evoke an immune response when used for human therapy (Schroff er al., 1985). The use of chimeric, humanized or human antibodies linked to human proteins with toxic properties would circumvent many problems encountered with the classical immunotoxins. In this study the potential use of tumor necrosis factor (TNF) for constructing immunotoxinlike molecules is evaluated. At first TNF was identified as a tumoricidal protein causing hemorrhagic necrosis of transplanted solid tumors in mice (Carswell et al., 1975). The potential of TNF as an anticancer drug was further illustrated by its cytotoxicity against various tumor target cell lines in vitro, while normal cells were minimally harmed. However, TNF appears to be involved in many other biological processes including inflammation and antiviral defense, endotoxic shock, immunoregulation, angiogenesis, cachexia and mitogenesis (Goeddel et al., 1986; Beutler and Cerami, 1027
1028
HENNIER. H~~CENB~~Met al.
1986; Fiers ef al., 1986). The mechanisms of these multiple activities remain poorly understood. A TNF receptor has recently been cloned (Loetscher et al., 1990; Schall et al., 1990), but there is no clear correlation between density or affinity for this receptor and TNF sensitivity. Furthermore, the DNA coding for human TNF has also been cloned, sequenced and expressed in E. coli, yielding large amounts of recombinant material which has been used for human cancer therapy (for review see Goeddel et al., 1986). Phase I clinical trials show that in vir;o administration of TNF results in severe side-effects, ranging from nausea and fever, vomiting, diarrhea, headache and hypotension to death (Creaven et al., 1987). Hence, there is a restriction on the use of large amounts of TNF for anticancer therapy. In an attempt to reduce the toxic effects of TNF. and to use only the anticancer properties of the molecule, we describe in this study the design and production of two genetically engineered antibody-TNF fusion proteins. By linking TNF to an antitransferrin receptor antibody, the targeting of TNF to transferrin receptor rich cells can be evaluated. Since the transferrin receptor is a growth related cellular surface antigen found on most proliferating cells (Trowbridge and Omary, 1981) most dividing cells have the capability of binding such an antibodycytokine chimeric molecule. However, the selective antitumor activity of TNF would provide the killing of tumor cells only. Since in this approach TNF targets to the surface of cancer cells, killing of cells might be achieved with smaller amounts of TNF. As a result, significant selective cytotoxicity might be obtained with diminished or possibly no side-effects. The antibody used is specific for the human transferrin receptor. It does not compete with transferrin for binding to the receptor, which might be of importance for in Go use (Heyligen et al., 1985). To minimize the immune response towards this mouse antibody when used in human cancer therapy, mouse-human chimeric genes were constructed and expressed in myeloma cells (Hoogenboom et al., 1990). The resulting chimeric antibody retained its antigen-binding capacity. These antibody genes were used in the present study to make antibody-TNF molecules. Since most of the antihybrid bodyenzymes described so far make use of a configuration in which the antibody heavy chain’s C-terminus is linked to the N-terminus of the nonimmunoglobulin protein, two analogous heavy chain-TNF molecules were constructed. Furthermore, the expression and secretion of these molecules by mammalian cells was evaluated. One attachment site was made in the 5’ region of the C, 2 domain, and one in the C,3 domain. If these heavy chain-TNF genes can be expressed in combination with the light chain of the antibody, the proteins will give rise to F(ab’),-like molecules, with targeting properties to growing cells.
MATERIALS AND METHODS Materials Xanthine and hypoxanthine were purchased from Sigma (St Louis, MO). DNA-modifying enzymes, [alfa-32P]dCTP, [gamma-32P]ATP, [“Slmethionine and biotinylated antibodies were supplied by Amersham (Buckinghamshire, U.K.). Oligonucleotides were obtained from Eurogentec (Liege, Belgium). Restriction endonucleases, mycophenohc acid and other cell culture reagents were obtained from Bethesda Research Laboratories (Gaithersburg, MD). DNAs
and DNA manipulations
The genomic human gamma-l constant region gene (Honjo et al., 1979) and the expression vector pSVgptMOV, NP ( pSVgpt) containing regulatory sequences for Ig gene expression in lymphoid cells (Neuberger, 1983) were gifts from Dr M. S. Neuberger (MRC Laboratory of Molecular Biology, Cambridge, U.K.). pSVhyg (palysl7), the selectable shuttle vector with the hygromycin resistance gene, was a gift from Dr J. Foote (MRC Laboratory of Molecular Biology, Cambridge U.K.) (Orlandi et al. 1989). The antibody genes used in this study were previously cloned, sequenced, and used to construct and express mouse-human chimeric antibody genes (Hoogenboom et al. 1990). The TNF DNA was obtained from Dr K. Ashman (EMBL, Heidelberg Germany) (Ashman et al., 1989). The recombinant DNA work was performed by standard procedures (Maniatis et al. 1982). Sequencing was done as described by Sanger et al. (1977). Ohgonucleotidedirected mutagenesis was carried out on singlestranded DNA templates derived from M 13mpl S/19 subclones of the DNA to be mutagenized, according to the method of Eckstein (Nakamaye and Eckstein, 1986) using the oligonucleotide-directed mutagenesis kit from Amersham. Cell lines and cell culture SP2/0-Ag14 (ATCC CRL 1581) is a non-Ig producing murine hybridoma. L929 (ATCC CCLl) is a murine fibroblastic cell line; MCF-7 (ATCC HTB 22) is a breast adenocarcinoma; HL-60 (ATCC CCL240) is a promyeloid leukemia; MOLT-4 (ATCC CRL 1582) is an acute lymphoblastic T-cell leukemia. All cell lines and transfectomas were grown in RPM11640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, non-essential amino acids and 10% FCS. Introduction of DNA into myelomu cells and selection of trunsfected cells DNA was introduced into mammalian cells using electroporation (Potter et al., 1984) with the Gene Pulser Apparatus of Biorad (Richmond, CA) as described previously (Hoogenboom et al., 1990).
Genetically engineered antibody-tumor Briefly, lo6 cells and 5-20 pg Bum HI or PuuIlinearized plasmid in 0.8 ml of PBS at 0°C in an electroporation cuvette (0.4 cm electrode gap) were subjected to a single voltage pulse at 200 V using a capacitance setting of 960pF. Selection for hygromycin resistant cells was done by adding 400 pg/ml hygromycin for a period of 3 weeks to the culture medium, starting 48 hr after the electroporation. Selection for transfected cells containing the gpt gene was carried out with 1 pg/ml mycophenolic acid, 250 pg/ml xanthine and 15 pg/ml hypoxanthine. Clones were visible after l-2 weeks. Individual clones were selected by limiting dilution in the absence of feeder cells. Screening for antibody secreting cell lines Screening for antibody production was done by ELISA, detecting human IgG or kappa chain, as described elsewhere (Hoogenboom et al., 1990). The assay measures antibody concns ranging from 1 to lOOng/ml, and is specific for detection of human gamma or kappa chain. SDS-PAGE
and immunoblot
Protein analysis by SDS-PAGE was performed according to Laemmli (1970). Samples were prepared in 0.1 M Tri-HCl, pH 7.0, containing 2% SDS, 2% mercaptoethanol and 10% sucrose and heated at 95°C for 5 min prior to electrophoresis on 12.5% cross-linked gels. After electrophoresis, samples were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, IL) by electroblot (Novablot, LKB-Pharmacia, Uppsala, Sweden) at 0.8 mA/cm’ for 1 hr, for immunoblot analysis. The filter was incubated overnight in 3% BSA at 4°C and washed afterwards in PBS&O.O5% Tween-20. Light chain was detected by using biotinylated goat antihuman kappa Ig (l/500 in PBS) (Amersham). For detection of the heavy chain, the filter was incubated for 2 hr at room temp with a l/500 dilution of goat anti-human IgG antibody (Tago Inc., Burlingame, CA) in PBS. After washing the filter was incubated with a l/250 dilution of peroxidase labeled rabbit anti-goat Ig (Dakopatts, Copenhagen, Denmark) in PBS containing 1% BSA. BSA was added to prevent extensive cross-reaction between the labeled anti-goat Ig and the mouse-human Ig on the filter. Staining was carried out with 0.05% diaminobenzidine dissolved in 0.1% H202 in PBS. The reaction was stopped by rinsing the filter with water. TNF was detected with rabbit anti-TNF antiserum (l/500 in PBS) (Genzyme, Boston, MA), and a l/500 dilution of peroxidase labeled swine anti-rabbit Ig (Dakopatts) in PBS. TNF-assay The cytotoxic activity of TNF was determined by using mouse L929 fibroblas; cells (Ruff and Gifford, 1981). The cells were incubated in the presence of actinomycin-D (1 pg/ml) with increasing amounts of the TNF containing sample. The assay was carried
necrosis factor molecules
1029
out in microtiter plates using 2 x 104-5 x lo4 cells in 200 ~1 per well. After an 18 hr incubation period, the living cells were quantified with MTT (3-(4,5dimethyl-thiazol-2-yl)-2,5-di-phenyltetrazoliumbromid) (Mosmann, 1983). MTT (20pl/well of a 5 mg/ml stock) was added to each well, and after l-4 hr incubation, the produced formazan crystals were redissolved by adding 100 PI/well 10% SDS + 0.01 N HCl and incubating for 416 hr. The O.D. was measured at 540 nm in an automatic Titertek microtiter plate reader (Flow). TNF protein was purchased from Genzyme and had a specific activity of 2 x lO’units/mg protein. The activity of TNF (in units) was defined as the reciprocal of the dilution at which a 50% reduction in O.D. was observed compared with control cultures containing no TNF. For MCF-7, HL-60 and MOLT-4 cells no actinomycin-D was used in the assay, and the incubation period was extended to 48 hr. RESULTS Design and construction TNF genes
of two chimeric
antibody-
An antibody molecule consists of compact protein domains with the hinge region serving as a spacer separating Fab from Fc regions (Burton, 1987). This domain structure is reflected at the gene level: in the human gamma-l gene, each domain, is encoded by a separate exon. To obtain two F(ab’),-like proteins, two different chimeric genes were made, in which the human TNF gene is linked to the C, 2 or C, 3 domain of the human gamma-l chain. The first attachment point for TNF was selected in the 5’ region of the C, 2-domain, leaving all amino acid residues involved in the hinge structure and dimerization of the heavy chain untouched. Only the first three amino acid residues of the CuZdomain were retained (Fig. 3) deleting Leu-235 (EU numbering), which is the major determinant in the binding of Fc to the high-affinity receptor on monocytes (Duncan et al., 1988). Attachment near a similar site in the murine gamma-2b gene has been used previously to connect Klenow polymerase (Williams and Neuberger, 1986) and aequorin (Casadei et al., 1990). By placing TNF distal to the hinge region, the antigen binding should not be influenced, and the flexibility of this region might facilitate binding of TNF to its own receptor, yielding a multifunctional protein. In a second construct the attachment site of TNF in the C, 3 domain was made at amino acid residue 422 (EU numbering) removing the last structural /I-sheet (b6-fy3-e2; Burton, 1985). Hence, binding of the antibody-TNF fusion protein to Fc receptors, which might interfere with the projected anticancer activity, is excluded in the C,,2-TNF construct only. A synthetic gene coding for the mature human TNF protein (without leader peptide) was assembled, expressed in E. coli and the recombinant TNF was checked for activity (Ashman et al., 1989). The first
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HENNIER. HOOGENBOOM et al.
two amino acid residues of native TNF (Val-Arg) are absent in the synthetic gene, but this deletion has no influence on the activity (Shirai et al., 1985). The synthetic gene comprises a translation initiation codon and an EcoRI restriction site at the 5’ end and two tandem stop codons and a BamHI restriction site at the 3’ end of the TNF coding region. The EcoRI site was used for linking the gene to the Cn2 or Cn3 domain. A subclone of the human gamma-l genomic gene was made by cloning a I-kb PstI-BarnHI fragment containing the C, 2 and Cn 3 domains into M 13mp 18 (Fig. 1). An EcoRI site was introduced in the Cn2 domain by oligonucleotide-directed in vitro mutagenesis by using ohgonucleotide 5’-AGCACCTGAATTCTGGGGG-3’. A clone containing the mutation was identified by Eco RI-restriction and the mutation was confirmed by sequencing. This clone was further used to generate the C,2-TNF chimeric gene, as depicted in Fig. 1. The Cn3-TNF gene fusion was made by ligating the TNF gene fragment, cut with EcoRI cut and treated with Klenow polymerase and dNTPs, to the unique XmnI site (which generates blunt ends) in the C, 3 domain of human gamma-l (Fig. 2). Since non-immunoglobulin derived untranslated 3’ regions of immunoglobulin gene constructs can decrease expression levels by destabilizing the mRNA (Weidle et al., 1987) and since the synthetic TNF gene fragment does not include a polyadenylation signal, the human gamma-l polyadenylation signal was added to the 3’ end of the chimeric construct. Therefore, the signal containing XmnI-Sac1 restriction fragment of the human gamma-l gene was ligated to the BamHI site of the TNF gene, after filling in of the latter site with Klenow ,polymerase and dNTPs. The Sac1 site fits into the unique Sac1 site of the vector (see caption of Fig. 1). Both XmnI and BamHI sites are deleted, but the series of stop codons at the 3’ end of the TNF gene are left intact. The polyadenylation signal is preceded by 78 nucleo-
tides of the (untranslated) Cn3 coding region of the gamma- 1 gene, creating an immunoglobulin-like gene and mRNA. Figures 1 and 2 show the cloning schemes used to build the heavy chain-TNF genes. The junctions between antibody heavy chains and TNF DNA are depicted in Fig. 3. Note that in the Cn2-Cn3 constructs, two to three amino acid residues are added in the junction region, including methionine, the first residue translated when expressing the synthetic DNA in E. coli (Ashman et al., 1989). Isolation of cell lines secreting chimeric proteins
the antibody-TNF
The chimeric light chain gene (Hoogenboom et al., 1990) was cloned into pSVhyg, a pSVneo derived plasmid containing the hygromycin resistance gene (see Materials and Methods; Fig. 4). The chimeric heavy chain-TNF genes were cloned into the HindIII-Bum HI sites of pSV2gptMOVn NP, which contains the gpt gene as a selection marker (see Materials and Methods). In the final construct, the expression of the chimeric gene is regulated by an immunoglobulin transcription enhancer element and promoter, both situated upstream of the gene (Fig. 4). It has been shown previously that the heavy chain enhancer is active on the promoter at this position (Neuberger, 1983). Secretion of the chimeric heavy chain-TNF proteins is directed by the presence of an immunoglobulin gene derived signal peptide sequence. Mouse SP2/0-Ag14 cells were transfected with the light gene containing expression vector. After transfection by electroporation, selection with hygromycin yielded several clones secreting human light chain, as detected by the human kappa-detecting ELISA. The best producing clone, 12B5, was selected for further use and subcloned by limiting dilution. Cell line 12B5 was transfected with one of the heavy chain-TNF gene containing vectors. After selection for the presence of the gpt gene, culture
Fig. 1. Construction scheme of the chimeric C,2 heavy chainTNF gene. Vector sequences and promoters are shown as small boxes, introns and other non-coding regions as lines, and exons as large boxes. The heavy chain variable sequence is shown in black, Ig constant regions are white boxes, and the TNF DNA box is stippled. Only restriction sites relevant to this construction scheme are shown; structural features of the vectors (antibiotic resistance genes, etc.) are not shown. Plasmids are not drawn to scale. (1) The human gamma-l gene was cloned as a 2.3-kb ApaI fragment into pGV463 (Neesen and Volckaert, 1989). (2) In vitro mutagenesis for the creation of an EcoRI site in the Cu2 domain was carried out after subcloning into M13mp18 of the 1-kb PsrI-BumHI fragment of pGV-HuIgG containing the hinge, the C, 2 and the C, 3 domain of the human gamma- I gene. In two steps, an Ig-derived polyadenylation signal was added to the TNF gene: first (3), the 170 bp PsrI-EcoRI fragment of M13mpl8CH,-Eco was cloned together with the 0.5kb EcoRI-BumHI fragment into pUC18, yielding pUC-H-TNF. Second (4). the 0.7-kb XmnI-Sac1 fragment of M13mpl8-C,2-Eco was inserted into pUCH_TNF which was cut with BumHI, treated with Klenow polymerase and dNTPs, and cut with &cl, yielding pUC-HP-TNF. (5) The 1-kb PsfILEamHI fragment of M13mpl8-C,2-Eco was subcloned into pUC18, and the complete chimeric heavy-chain gene was restored by inserting a 2.1-kb HindIIILApaI and a 0.6-kb ApaILZ’stI fragment from pUC-chimIgG (Hoogenboom et al., 1990) containing the Ig sequences, yielding pUCF31.IgGI (6). (7) The TNF-containing 1.3-kb EcoRI fragment of pUC-HP-TNF was inserted into this plasmid. In order to gain a BamHI site at the 3’ end of the chimeric gene, which was necessary for further subcloning experiments, the 4-kb Hi&III-Sac1 fragment of pUC-IgC,2-TNF was subcloned into pGV463, yielding pGVC,2-TNF (8).
Genetically
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antibody-tumor
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pGV-Ig
Fig. 1
-CHZ
-TNF
HENNIE R. HOOGENB~OM et al.
1032 ApaI
Soci BamHI
pUC - ChlmIgG
EcoRi
PUC-Ig-Cri3m
TN;
Fig. 2. Construction scheme of the chimeric C, 33TNF gene. Legend as in Fig. 1. The 1.5kb ApaIIXmn I fragment of pUC-chimIgG (Hoogenboom Ed al., 1990) was cloned into pUC-chimIgG cut with ApaI and BumHI, together with the 1.3-kb EcoRI-EarnHI fragment of pGVC,2-TNF (Fig. I), of which the EcoRI site was treated with Klenow polymerase and dNTPs. This subcloning yielded plasmid
pUCC, supernatant of several clones reacted positive in the human IgG-detecting ELISA. Clones 8D5 and 13A4 were identified as the best producers for the C, 22TNF and C, 3-TNF proteins, respectively, and IA)
TCCTCA
(3)
(2)
(1) A
P
E
F
M
s
s
s
GCA
CCT
GAA
TTC
ATG
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TCT
TCT
---@RI--intron
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(2)
GGG l
E
AAC
G
****
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M
s
s
s
TTC
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cm11
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of the antibody-TNF
chimeric
pro-
teins
(B)
N
were used for further analysis. The amount of secreted Cn2-TNF fusion protein gave the same optical density in the human IgG-ELISA as a 10 ng/ml solution of pure human IgG protein, compared to 200 ng/ml for the C,3-TNF protein. The actual concentration of the C, 22TNF fusion protein will be higher, because the ELISA was standardized with intact human IgG-protein (which is 6 times the size of the ELISA-positive antibody region in the fusion protein). Characterization
TNF
CH2-domain
QG
3-TNF.
TNF
Fig. 3. Junctions between antibody heavy chain and TNF DNAs (A) in the C,2-TNF construct and (B) in the C,3-TNF construct. (1) Antibody region; (2) amino acid residues introduced while cloning, including the methionine, used for expression of the TNF DNA in E. coli; (3) mature TNF sequence, with the first two codons deleted.
Immunoprecipitation was carried out using culture supernatant of clones 8D5 and 13A4 and goat antihuman IgG-Sepharose beads. After elution of the precipitated proteins, the samples were reduced, fractionated on SDS-PAGE and transferred to a PVDF membrane for immunoblot analysis. As a positive control, culture supernatant of transfectoma 13A5, a cell line secreting the antitransferrin receptor chimeric mouse-human antibody (Hoogenboom et al., 1990) was used. Anti-human IgG or anti-TNF antisera were used to detect human immunoglobulins of TNF reactive proteins, respectively. The immunoblot analysis depicted in Fig. 5(B) shows that cell line 13A5 secretes a normal heavy chain, which can be detected with the anti-human IgG antiserum only. On the other hand, transfectomas 8D5 and 13A4 clearly
Genetically engineered antibody-tumor
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(b)
HIndI ECCRI pSVgpt-CHZ-
PSVgpt
TIJF
pSVhyg
pBR322
-CH3
- TNF
- VLCK
SJ40
Fig. 4. Structures of the plasmids containing (A) the chimeric C,2-TNF gene, (B) the chimeric C,3-TNF gene or (C) the chimeric light chain. The chimeric heavy chain-TNF genes were subcloned as HindIII-BamHI fragments into expression vector pSVgpt, yielding pSVgpt-C,2/33TNF. P, promoter; S, signal peptide sequence; E, enhancer; AMP (HYG), ampicillin (hygromycin) resistance gene; ori origin of replication. secrete an anti-human IgG (Fig. 5B) and anti-TNF (Fig. 5A) reactive protein of homogenous length. The size of the fusion proteins is as expected: 46 kD for C,2-TNF and 69 kD for C,3-TNF (in both cases 17 kD is from the TNF portion). Sample 5B4 is an immunoprecipitated culture supernatant sample of a transfection secreting an Fab-like heavy chain-TNF protein (Hoogenboom et al., submitted), which is seven amino acid residues smaller than the C, 2-TNF hybrid molecule: Figures (A) and (B) clearly show this difference in size between SB4 and 8D5. In the immunoblot analysis of culture supernatant of the 8D5 and 13A4 cells, no degradation products of the antibody region of the molecule were detected, indicating that the fusion proteins are secreted as stable and relatively protease-resistant proteins. However, extensive proteolytic degradation of the C,2/3-TNF fusion proteins was seen after culturing the 8D5 and 13A4 cells in serum-free medium for 3 days, probably caused by the massive cell death and subsequent release of intracellular proteases in those cultures.
Protein A-Sepharose failed to precipitate the fusion proteins of both 8D5 and 13A4 transfectomas. The binding site of protein A is believed to be near the junction of the Cn2 and C, 3 domains, which is deleted in the C, 2-TNF construct. In the C, 3-TNF construct on the contrary, the histidine at position 435, which is involved in the contact between human IgGl and protein A (Deisenhofer et al., 1978), was deleted, which might explain the inability of protein A to bind to the C,3-TNF hybrid protein as well. Immunoprecipitation with anti-human IgG-Sepharose of in vivo radiolabeled 8D5 and 13A4 supernatant showed that the light chain coprecipitated with the heavy chain-TNF proteins (results not shown), pointing out that the antigen-binding domains are in the expected configuration. TNF activity upon mouse and human cell lines Culture supernatants of the 8D5 and 13A4 transfectomas were assayed for cytotoxic activity. Various cell lines were incubated in 200 ~1 medium containing a variable amount of culture supernatant of
HENNIE R. HGQGENBGQMet al.
Fig. 5. Immunoblot analysis of supernatant of transfectomas 13A5 (secreting chimeric antitransferrin receptor antibody), 8D5 and 13A4 (secreting light chain and C,2-TNF and C,3-TNF fusion proteins, respectively). Transfectoma 5B4 secretes another antibody_TNF derivative (see text). Supernatant was immunoprecipitated with goat anti-human IgG-Sepharose beads, elute and fractionated on 12.5% SDS-PAGE. The proteins were transferred to a PVDF membrane and antibodies were detected using anti-TNF (A) or anti-human IgG (B), as described in Materials and Methods. The position of M, markers are indicated (M, x 10-s).
SP2/0-Ag14 cells (non-producer), 13A5 (chimeric antibody), 8D5 (C&2-TNF) or 13A4 (C,3-TNF) transfectomas. The amount of living cells was determined after 1, 2 or 3 days using the MTT-coloring method. Figure 6 depects the growth curves of the mouse L929 fibroblast cell line and the relatively TNF-resistant human MCF-7 breast carcinoma. Only 8D5 and 13A4 supernatant had a growth inhibitory effect upon both cell lines. Similar assays with cell lines HL-60 and MOLT-4 showed the same growth inhibitory effect of both 8D5 and 13A4 supernatants (results not shown). 13A4 supernatant contains more IgGantibody as measured by ELISA than 8D5 supernatant, but the growth inhibition of the latter supernatant is more pronounced. Since no protein concns are known yet, no conclusions can be drawn about the relationship between structure and activity. Since there is no competition between antibody and transferrin for binding to the receptor, the antitrans-
ferrin receptor antibody of 13A5 can not cause growth inhibition (Heyligen et al., 1985; Hoogenboom, unpublished results). Furthermore, the antibody is specific for the human transferrin receptor. The growth of the murine L929 cells can only be inhibited due to the TNF-activity of the 8D5 and 13A4 sample. According to this L929-assay, the SD5 and 13A5, supernatants contain approximately 4 and 2 unit TNFjlOO ~1, respectively. The growth of MCF-7 cells however, which could not be inhibited with 400 units TNF as tested with pure recombinant human TNF, could be inhibited with both 8D5 and 13A4 supernatant. This difference might be explained by the targeting effect of the antibody-TNF molecules to human cells due to the transferrin receptor binding, increasing the actual concn of TNF near the cellular membrane. Since the antibody does not recognize the murine transferrin receptor, there is no targeting to the murine L929 cells.
Genetically engineered antibody-tumor
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necrosis factor molecules
Growth curves of MCF-7 cells
Growth curves of L929 cells
0.6
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.
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Fig. 6. Growth curves of L929 (left) and MCF-7 SP2/0-Ag14 (non-producer), and transfectomas body), 8D5 and 13A4 (secreting light chain and Culture supernatant was added at the indicated an MTT-assay
13A430 *...
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Medium
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(right) cells, in the presence of culture supematant of 13A5 (secreting chimeric antitransferrin receptor antiC,2--TNF or C,3-TNF fusion proteins, respectively). dilution; (:2,:4, etc.), and living cells were quantified in after 1, 2 or 3 days.
DISCUSSION
In this study the potential targeting of TNF to cancer cells by linking it to an antitransferrin receptor antibody was evaluated. Recombinant antibody techniques were used to construct and express two antibody heavy chain-TNF genes. Cell lines were obtained secreting light chain and detectable amounts of two heavy chain-TNF fusion proteins. Several genetically engineered antibody-enzymes or other antibody-like molecules expressed by mammalian cells, have been described so far, using genes encoding Staphylococcus aureus nuclease or c-myc (Neuberger et al., 1984), Klenow polymerase (Williams and Neuberger, 1986), tissue Plasminogen Activator (Schnee et al., 1987) the photoprotein aequorin (Casadei et al., 1990), or insulin-like growth factor 1 (Shin and Morrison, 1990). In most cases nonimmunoglobulin proteins were linked to the C,2-
domain of the mouse immunoglobulin gamma2b constant region, yielding potential F(ab’),-like molecules. We used the human gamma-l constant region gene and DNA encoding human TNF for the production of chimeric mouse-human antibody-TNF fusion proteins. The murine variable regions of the antibody are the only antigenic domains when used in humans, although it can not be excluded that novel combinations of components may produce neoantigenie determinants. Linkage to the “classical” site in the Cu 2 domain as well as linkage to the Cn 3 domain proved to result in expression and secretion of fusion proteins of the expected size and composition. Alike the other described antibodyenzymes, linkage of the non-antibody region (in the present study TNF) to the antibody moiety had no deleterious effect upon the secretory pathway followed by the antibody protein. Nevertheless, the level of secretion was lower than for the described antibodyenzymes
1036
HENNIE R. HOOGENBOOMet al.
(estimated to be in the ng/ml range of l&200 ng/ml), but correct values can only be obtained after purification of the fusion proteins. To obtain higher expression levels of the hybrid molecules, amplification of the chimeric genes by the dhfr system can be envisaged (Hendricks et al., 1989). However, it must be kept in mind that the low expression levels might be due to the intrinsic toxicity of TNF itself. Although no growth retardation or cell lysis was detected in the 8D5 and 13A4 cell cultures, secreting C, 2-TNF and C, 3-TNF respectively, production of high amounts of the antibody-TNF hybrid molecules might be toxic to the transfectoma cells, and low producers might be self-selected. In the latter case, expression of the recombinant proteins as Fab or Fv-like proteins in E. co/i should be considered (Chaudary et al., 1989; Batra et al., 1989). The culture supernatant of the transfected cell line 8D5, producing light chain as well as Cn2 heavy chain-TNF protein, contained growth inhibiting activity upon murine and human cell lines. Supernatant of transfectoma 13A4 had a similar effect. Therefore, the fusion products have a TNF-activity, but the specific activity has not been determined yet. Whereas the active form of TNF is a compact trimer (Jones et al., 1989), the active native form of the F(ab’),-like antibody-TNF proteins and their possible trimerization remain to be elucidated. A difference between the TNF-activity of the culture supernatant as determined by the L929-assay and the TNF-sensitivity of human MCF-7 cells was found. Although the culture supernatant contained 24 units TNF/lOOpl according to the L929 assay, this amount was sufficient to inhibit the growth of human MCF-7 cells, which are not affected up to 400 units/100 ~1 pure recombinant human TNF. These results are highly suggestive for a targeting of the antibody-TNF fusion protein to these human cells by binding to the transferrin receptor. This TNF-activity might result not only from the concentrating effect of direct binding to the plasma membrane (and mediating toxicity through the TNF receptor) but also from the appropriate trafficking of the hybrid molecule after binding and internalization of the antibody-transferrin receptor complex. Purification of the new proteins will enable us to determine the affinity constants for the human transferrin receptor and the TNF receptor, and to elucidate the mechanism of action of these new antibody-cytokine molecules. The preliminary cytotoxicity assay results presented here suggest that chimeric antibody-TNF molecules are potential immunotherapeutics for cancer therapy. These data extend the results described in a recent paper, presenting the construction, expression and secretion of an Fab-like antibody-TNF chimeric protein (Hoogenboom et al., submitted). They demonstrate the utility and feasibility of the antibody engineering technique to develop bifunctional,
more human-like and more potent immunotoxin-like anticancer agents or other immunotherapeutics. Acknowledgements-We
thank Drs K. Ashman, M. Neuberger, G. Winter and J. Foote for their generous gifts of DNAs, and Marc Withofs for graphical work.
H. R. Hoogenboom “Nationaal Fonds (N.F.W.O.).
was a research assistant voor Wetenschappelijk
of the Belgian Onderzoek”
REFERENCES Ashman K., Matthews N. and Frank R. W. (1989) Chemical synthesis, expression, and product assessment of a gene coding for biologically active human TNF-alfa. Prof. Engng 2 387-391. Batra J. K., Jinno Y., Chaudary V. K., Kondo T., Willingham M. C., FitzGerald D. J. and Pastan I. (1989) Antitumor activity in mice of an immunotoxin made with anti-transferrin receptor and a recombinant form of Pseudomonas exotoxin. Proc. natn. Acad. Sri. U.S.A. 86, 8545-8549. Better M., Chang C. P., Robinson R. R. and Horwitz A. H. (1988) Escherichiu coli secretion of an active chimeric antibody fragment. Science 240, 1041-1044. Beutler B. and Cerami A. (1986) Cachectin and Tumour Necrosis Factor as two sides of the same biological coin. Nafure, Land. 320, 584588. Burton D. R. (1985) Immunoglobulin G: Functional sites. Molec. Immun. 22, 161-206. Burton D. R. (1987) In Molecular Genetics of Immunoglobulin. New Comprehensive Biochemistry, Vol. 17 (Edited bv Calabi F. and Neuberaer M. S.). London. Carswell E.-A., Old J., Kassel R., Green S., Fiore N. and Williamson B. (1975) An endotoxin-induced serum factor that causes necrosis of tumours. Proc. natn. Acad. Sci. U.S.A. 72, 36663670. Casadei J., Powell M. J. and Kenten J. H. (1990) Expression and secretion of aequorin as a chimeric antibody by means of a mammalian expression vector. Proc. natn. Acad. Sci. U.S.A. 81 2047-2051. Chaudary V. K., Queen C., Junghans R. P., Waldmann T. A., Fitzgerald D. J. and Pastan I. (1989) A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin. Nature, Land. 339, 394-397. Creaven P. J., Plager J. E., Dupere S., Huben R. P., Takita H., Mittelman A. and Proefrock A. (1987) Phase-I clinical trial of recombinant human Tumor Necrosis Factor. Cancer Chemother. Pharmacol. 20, 1377144. Deisenhofer J., Jones T. A. and Huber R. (1978) Crystallization, crystal structure analysis and atomic model of the complex formed by a human Fc fragment an Fragment B of Protein A from Slaphylococcus aureus. Hoppe-Seyler’s Z. physiol. Chem. 359, 975-985. Duncan A., Woof J., Partridge L., Burton D. and Winter G. (1988) Localization of the binding site for the human high affinity Fc-receptor on IgG. Nature Lond. 332 563-566. Fiers W., Brouckaert P., Devos R., Franssen L., LerouxRoels G., Remaut E., Suffys P., Tavernier J., Van der Heyden J. and Van Roy F. (1986) Lymphokines and monokines in anticancer therapy. Cold Spring Harb. Symp. quant. Biol. 51, 587-595. Goeddel D. V., Aggarwal B. B., Gray P. W., Leung D. W., Nedwin G. E., Palladino M. A., Patton J. S., Pennica D., Shepard H. M., Sugarman B. J. and WongG. H. W. (1986) Tumor Necrosis Factors: gene structure and biological activity. Cold Spring Harb. Symp. quant. Biol. 51,597-609. Hendricks M. B., Luchette C. A. and Banker M. J. (1989) Enhanced expression of an immunoglobulin-based vector in myeloma cells mediated by coamplification with a mutant dihydrofolate reductase gene. Biotechnology 7, 1271-1274.
Genetically
engineered
antibody-tumor
Heyligen H., Thijs C., Weber W., Bosmans E. and Raus J. (1985) Monoclonal antibodies detecting human T-cell activation antigens: development of monoclonal antibodies and expression of antigens on activated T-cells and leukemic cell lines. Fedn Proc. 44, 787. Honjo T., Obata M., Yamawaki-Kataoka Y., Kataoka T., Kawakami T. Takahashi N. and Mano Y. (1979) Cloning and complete nucleotide sequence of mouse immunoglobulin gamma-l chain gene. Cell 18, 559-568. Hoogenboom H. R., Raus J. C. M. and Volckaert G. (1990) Cloning and expression of a chimeric antibody directed against the human transferrin receptor. J. Immun. 144, 321 l-3217. Huston J. S., Levinson D., Mudgett-Hunter M., Tai M.-S., Novotny J., Margolies M. N., Ridge R. J., Bruccoleri R. E., Haber E., Crea R. and Oppermann H. (1988) Protein engineering of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in E. coli. Proc. natn. Acad. Sci. U.S.A. 85, 5879-5883. Jones E. Y., Stuart D. I. and Walker N. P. C. (1989) Structure of tumour necrosis factor. Nature Lond. 338, 225-228. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680-684. Liu A. Y., Robinson R. R., Murray E. D., Ledbetter J. A., Hellstriim I. and Hellstrijm K. E. (1987) Production of a mouse-human chimeric monocloial antibody to CD20 with potent Fc-dependent biologic activity. J. Immun. 139, 3521-3526. Loetscher H., Pan Y.-C. E., Lahm H.-W., Gentz R., Brockhaus M., Tabuchi H. and Lesslauer W. (1990) Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor. Cell 61, 351-359. Maniatis T., Fritsch E. F. and Sambrook J. (1982) In Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Morrison S. L., Johnson M. J., Herzenberg L. A. and Oi. V. T. (1984) Chimeric human antibody molecules: Mouse antigen-binding domains with human constant region domains. Proc. nafn. Acad. Sci. U.S.A. 81, 6851-6855. Mosmann T. (1983) Rapid calorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immun. Meth. 65, 55-63. Nakamaye K. and Eckstein F. (1986) Inhibition of restriction endonuclease NciI cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis. Nucleic Acids Res. 14, 9679-9698. Neesen K. and Volckaert G. (1989) Construction and shuttling of novel bifunctional vectors for Streptomyces spp. and Escherichia co/i. J. Bacterial. 171, 1569-1573. Neuberger M. S. (1983) Expression and regulation of imminoglobulin heavy &ain gene transiected into lvmphoid cells. EMBO J. 2. 1373-1378. Ne;b&ger M. S., Williams 6. T. and Fox R. 0. (1984) Recombinant antibodies possessing novel effector functions. Nature, Lond. 312, 604-608. Oi V. T., Morrison S. L., Herzenberg L. A. and Berg P. (1983) Immunoglobulin gene expression in transformed lymphoid cells. Proc. natn. Acad. Sci. U.S.A. 80, 825-829. Orlandi R., Giissow D. H., Jones P. T. and Winter G. (1989) Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc. nafn. Acad. Sci. ti.S.A. 86; 3833-3837. Potter H., Weir L. and Leder P. (1984) Enhancer-dependent expression of human K immunoglobulin genes introduced
necrosis
factor
molecilles
1037
into mouse pre-B lymphocytes by electroporation. Proc. natn. Acad. Sri. U.S.A. 81, 7161-7165. Riechmann L., Clark M., Waldmann H. and Winter G. (1988) Reshaping human antibodies for therapy. Nature, Lond. 332, 323-327. Ruff M. and Gifford G. (1981) Tumor necrosis factor. In Lymphokines, Vol. 2 (Edited by Pick E.). Academic Press, New York. Sahagan B. G., Dorai H., Saltzgaber-Muller J., Toneguzzo F., Guindon C. A., Lilly S. P., McDonald K. W., Morrissey D. V., Stone B. A., Davis G. L., McIntosh P. K. and Moore G. P. (1986) A genetically engineered murine/human chimeric antibody retains specificity for human tumor-associated antigen. J. Immun. 137, 10661074. Sanger F., Nicklen S. and Coulson A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. natn. Acad. Sci. U.S.A. 74, 5463-5467. Schall T. J., Lewis M., Keller K. J., Lee A., Rice G. C., Wong G. H. W., Gatanaga T., Granger G. A., Lentz R., Raab H., Kohr W. J. and Goeddel D. V. (1990) Molecular cloning and expression of a receptor for human tumor necrosis factor. Cell 61, 361-370. Schnee J. M., Runge M. S., Matsueda G. R., Hudson N. W., Seidman J. G., Haber E. and Quertermous T. (1987) Construction and expression of a recombinant antibodytargeted plasminogei activator. Proc. nam. Acad. S&. U.S.A. 84. 6904-6908. Schroff R. w., Foon A. F., Beatty S. M., Oldham R. K. and Walton C. M. (1985) Human anti-murine immunoglobulin responses in patients receiving monoclonal antibody therapy. Cancer Res. 45, 879-885. Sharon J., Gefter M. L., Manser T., Morrison S. L., Oi V. T. and Ptashne M. (1984) Expression of a VHCK chimaeric protein in mouse myeloma cells. Nature, Lond. 309, 364-367. Shin S.-U. and Morrison S. L. (1990) Expression and characterization of an antibody binding specificity joined to insulin-like growth factor 1: potential applications for cellular targeting. Proc. natn. Acad. Sci. U.S.A. 87, 5322-5326. Shirai T., Yamaguchi H., Ito H., Todd C. W. and Wallace R. B. (1985) Cloning and expression in E. coli of the gene for human Tumor necrosis factor. Nafure, Lond. 313, 803-806. Skerra A. and Pliickthun, A. (1988) Assembly of a functional Immunoglobulin Fv fragment in Escherichia coli. Science 240, 1038-1041. Spooner R. A. and Lord J. M. (1990) Immunotoxins: status and prospects. TIBTECH 8, 189-193. Trowbridge I. S. and Omary M. B. (1981) Human cell surface glycoprotein related to cell proliferation is the receptor for transferrin. Proc. natn. Acad. Sci. U.S.A. 78, 3039-3043. Verhoeyen M., Milstein C. and Winter G. (1988) Reshaping human antibodies: grafting an anti-lysozyme activity. Science 239, 1534-1537. Vitetta E. S., F&on R. J., May R. D., Till M. and Uhr J. W. (1987) Redesigning Nature’s poisons to create antitumor reagents. Science 238, 1099-l 104. Weidle U. H., Koch S. and Buckel P. (1987) Expression of antibody cDNA in murine myeloma cells: possible involvement of additional regulatory elements in transcription of immunoglobulin gene. Gene 60, 205-216. Williams G. T. and Neuberger M. S. (1986) Production of antibody-tagged enzymes-by myeloma cells: application to DNA polymerase I Klenow fragment. Gene 43, 319-324.