WO1986005513A1 - Vecteur cassette de gene hybride - Google Patents

Vecteur cassette de gene hybride Download PDF

Info

Publication number
WO1986005513A1
WO1986005513A1 PCT/US1986/000566 US8600566W WO8605513A1 WO 1986005513 A1 WO1986005513 A1 WO 1986005513A1 US 8600566 W US8600566 W US 8600566W WO 8605513 A1 WO8605513 A1 WO 8605513A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
vector
human
derived
hybrid
Prior art date
Application number
PCT/US1986/000566
Other languages
English (en)
Inventor
Sherman M. Weissman
Kirk Fry
Jungsuh Park Kim
Francis Collins
Original Assignee
Gene Labs, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Gene Labs, Inc. filed Critical Gene Labs, Inc.
Publication of WO1986005513A1 publication Critical patent/WO1986005513A1/fr
Priority to DK551686A priority Critical patent/DK551686A/da

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a cassette vector for use in constructing hybrid genes in particular, a family of hybrid genes which are polymorphic in one gene region.
  • Functional antibody molecules are composed of two light (L) chains and two heavy (H) chains, arranged in heavy/light pairs as H 2 L 2 tetramers. Each light and heavy chain contains a variable (V) region which is responsible for the antibody's antigen specificity and a constant (C) region which contributes receptor and structural functions.
  • V variable
  • C constant
  • the immune response in humans against a. foreign (mouse) monoclonal antibody is directed largely against the antibody's light- and heavy-chain constant regions
  • a human/mouse hybrid antibody having mouse variable regions and human constant regions would largely neutralize the antibody's ability to provoke an undesired immune response when administered over long periods for therapeutic use.
  • the hybrid antibody could be constructed with specificity against any given antigen, since the antigen-specific variable regions of the antibody would be derived from antibodies generated in mice.
  • Hybrid human antibodies whose variable and constant regions are derived from different antibody classes would also have important therapeutic uses.
  • an IgM antibody is directed against red blood cell antigens, causing agglutination and destruction.
  • a hybrid human monoclonal antibody whose variable IgM region is directed against red blood cells, but which has, for example, an IgG constant region, should be able to compete with the IgM antibody for binding to red blood cell antigen sites, thus blocking IgM antibody attachment to the red blood cells, without itself producing the undesired hemolytic response.
  • non-complement fixing antibodies could be converted to complement-fixing antibodies to increase their effects in fighting infectious diseases or malignancies.
  • hybrid transplantation antigens Another type of hybrid immunological structure which would be expected to have important medical uses includes hybrid transplantation antigens.
  • the transplantation and immune response antigens are important in numerous aspects of the immune system, including transplantation rejection, and, more generally, recognition of cell-bound antigens by cytotoxic T-cells.
  • the human transplantation antigens are coded by class I and class II genes of the human major histocompatibility complex (MHC), also referred to as the human leukocyte antigens (HLA), contained in a
  • MHC human major histocompatibility complex
  • HLA human leukocyte antigens
  • each gene is composed of a series of coding regions (exons) each separated by noncoding (intron) regions of variable length.
  • the first three exons in class I genes and the first two exons in class II genes are highly polymorphic, and are primarily responsible for the variations in transplantation antigenicity seen among individuals in a population (references 2 and 3).
  • the remaining coding regions in the class I and class II genes are associated with inter-chain interactions, transmembrane and other structural or effector functions of the antigens.
  • the class I and class II genes of the HLA antigens correspond to a number of class I and class II genes in the mouse MHC. Comparative studies on the two systems has shown correspondence between a number of human and mice class I and II genes (see, for example, references 2-4).
  • the gene correspondence, and in particular the structural and functional homology between the analogous human HLA and mouse MHC genes, would allow construction of hybrid genes containing human polymorphic coding regions, such as the first three exons of the class I human genes or the first two exons of the class II gene, and complementary mouse nonpolymorphic regions.
  • hybrid class I and II HLA/MHC genes could be constructed in a manner similar to that described above for constructing hybrid antibody genes, in which isolated cloned human and mouse genes would be spliced together at suitable intron regions and successful transformants selected and confirmed as to gene construction.
  • This method would suffer from the same limitations mentioned above, and generating a family of hybrid histocompatibility complex genes, each member having a different HLA polymorphic region, would be correspondingly difficult.
  • hybrid immunological structures including hybrid antibodies and transplantation antibodies, but also including other structures, such as T-cell receptor antigens, which are also characterized by families of genes composed of highly polymorphic gene-coding regions joined to relatively non-polymorphic regions. Accordingly, it would be highly desirable, as part of a procedure for constructing families of hybrid immunological structures, to provide a method by which families of hybrid genes could be readily generated.
  • Another object of the invention is to provide a novel cassette vector for use in the hybrid gene-construction method.
  • a more specific object of the invention is to provide such method and cassette vector having the following advantages in the construction of a family of hybrid genes:
  • hybrid genes are constructed from DNA fragment libraries rather than from isolated cloned genes
  • hybrid genes are formed without gene cutting and splicing manipulations.
  • the cassette vector of the invention is intended for use in constructing one or preferably a family of hybrid A i -B' genes, where:
  • a i is a polymorphic coding region derived from an A i -B i gene in a family of A-B genes;
  • B' is derived from a selected A'-B' gene
  • a i and A' and B i and B' are structurally and functionally homologous gene regions; and (d) A and B coding regions and the A' and B' coding regions are separated by introns I and I'. respectively.
  • the vector includes: (1) a selectable-marker gene which permits selection of a genomic library clone which acquires the marker gene by recombination with the vector, (2) the B' coding region, and (3) located adjacent the coding region, a gene fragment derived from an intron having base-sequence homology with the A/B intron I of a gene from the A-B gene family.
  • the selectable-marker gene is a suppressor tRNA gene which allows expression of amber-mutation structural genes in a phage clone which acquire the cassette vector by recombinat.ion.
  • the B' coding region includes the constant coding region of a preferably human A-B gene of a selected class, i.e., either the ⁇ or ⁇ light-chain class or the ⁇ , ⁇ , ⁇ , ⁇ 1. ⁇ 2, ⁇ 3, ⁇ 4, or ⁇ heavy-chain classes.
  • the A i -B i gene used in constructing the hybrid gene is derived from a cell capable of producing antibody specific against a selected antigen, typically a mouse hybridoma cell line.
  • the A i -B i gene can also be from a human hybridoma cell line when it is desired to change the class of antibody produced by that hybridoma.
  • an hybrid immunoglobulin gene there is first produced a library of genomic DNA derived from the cell known to contain the desired A i -B i in functional
  • the DNA library fragments some of which contain the A i variable coding region of the A i -B i gene, and the adjacent downstream intron, are introduced into a recombination positive host which harbors the cassette vector of the invention.
  • a recombination event between an A i -intron genomic library fragment and the cassette vector leads to incorporation of the vector into the genomic clone, with the B' coding region in the cassette vector positioned adjacent the A i gene in the cloning vector, and remaining portions of the cassette vector located between the intron region where recombination has occurred and portions of the A i -B i gene which are downstream of the region of recombination.
  • B' coding regions in the recombined gene are separated by a "composite" intron which may contain an immunoglobulin enhancer segment acquired from the cassette vector by the recombination event.
  • a "composite" intron which may contain an immunoglobulin enhancer segment acquired from the cassette vector by the recombination event.
  • the acquisition by the cloned gene of the selectable marker in the cassette vector e.g., a suppressor-tRNA gene, allows the vector containing the desired hybrid gene to be selected when grown in a suitable selection host, e.g., a suppressor-minus bacterial host.
  • the cassette vector designed for constructing human/mouse transplantation antigen genes contains a B' coding region derived from the predominantly non-polymorphic exons of a selected mouse MHC gene.
  • the intron gene fragment is derived from the intron between the homologous B i coding region in a corresponding human MHC gene, and the complementary A i , predominantly polymorphic, coding region in the gene.
  • To construct a hybrid histocompatibility antigen gene there is first provided a genomic DNA library derived from the cells of an individual having particular class I or class II gene polymorphisms.
  • the cloned DNA library fragments are introduced into a recombination-positive host also harboring the cassette vector.
  • a recombination event between the homologous intron regions in the cassette vector and the intron region of the A i -containing insert leads to incorporation of the cassette vector into the cloned B' gene with the B' nonpolymorphic coding regions in the vector being positioned adjacent and downstream of the A i human polymorphic region of the phage.
  • the invention also includes hybrid antibody and cell-surface antigen genes containing the composite intron formed by the recombination event between the cloning and cassette vectors, and hybrid histocorapatibility antigens formed in accordance with the method of the invention.
  • Figure 1 illustrates a scheme for constructing a cassette vector of the invention
  • Figure 2 illustrates the recombination event between homologous intron regions in an A i -B i gene carried in a ⁇ phage vector, and the cassette vector of Figure 1, to form a hybrid A i -B' gene
  • Figure 3 illustrates the construction of a cassette vector for use in preparing a hybrid light-chain gene with a human ⁇ constant region coding sequence
  • Figure 4 shows electrophoresis patterns obtained for the cassette vector of Figure 3, following digestion with Xbal and Hindlll (A) or with EcoRI (B);
  • Figure 5 illustrates the recombination event between the IS2 regions of a mouse germ-line ⁇ gene contained in a ⁇ phage and in the cassette vector of Figure 3, to form a hybrid light-chain gene whose sequences upstream of the IS2 region are derived from a mouse ⁇ gene and whose constant coding region is derived from the human constant coding region of a human ⁇ chain gene contained in the cassette vector;
  • Figure 6 shows an autoradiogram pattern of the hybrid ⁇ gene from Figure 5, following digestion with EcoRI and hybridization to a ⁇ anl3 probe;
  • Figure 7 illustrates the steps in the construction of a cassette vector for use in preparing a hybrid heavy-chain gene containing a human IgG 4 heavy-chain coding region
  • FIG 8 shows electrophoresis patterns obtained for the cassette vector of Figure 7, following digestion with PstI;
  • Figure 9 illustrates the recombination event between the IS2 regions of a mouse germ-line ⁇ gene contained in a ⁇ phage and in the cassette vector of Figure 7, to form a hybrid heavy-chain gene whose upstream sequences are derived from mouse ⁇ gene, and whose constant coding region is derived from the human constant region of a human IgG chain gene contained in the cassette vector;
  • Figure 10 illustrates the steps in constructing a cassette vector for preparing a hybrid human DR ⁇ /mouse I-E ⁇ immune response gene
  • Figure 11 illustrates the recombination event between a human DR ⁇ gene contained in a ⁇ phage and the cassette vector of Figure 9, to form a hybrid DR ⁇ /I-E ⁇ gene.
  • Figure 12 illustrates the construction of the phage vector ⁇ GLla described in Example VII from ⁇ EMBL 3 and ⁇ Ch4a.
  • Figure 13 is a restriction map of the ⁇ light chain cassette vector ⁇ LC ⁇ .Notl, described in
  • Figure 14 is a restriction map of the ⁇ 4 heavy chain cassette vector irHC ⁇ 4 .NotI, whose construction is described in Example III.
  • Figure 15 illustrates the construction of the ⁇ 1 heavy chain cassette vector ⁇ HC ⁇ 1 .NotI described in Example VIII.
  • Figure 16 illustrates the construction of the ⁇ 3 heavy chain cassette vector ⁇ HC ⁇ 3 .NotI described in Example IX.
  • Figure 17 illustrates the construction of the isotype switching cassette vector ⁇ SWMG 3 .NotI described in Example X.
  • Figure 18A and B illustrate the structure of the isotype switching cassette vectors ⁇ SWMG and ⁇ SWMG 1 .NotI, respectively, described in Example XI.
  • Figure 19 is a restriction map of a region of the mouse unrearranged light chain gene from which the J ⁇ and -J ⁇ probes are isolated and employed in Example XII.
  • Figure 20 is a restriction map of a region of the mouse unrearranged heavy chain gene from which the
  • Figure 21 illustrates the recombination between the light chain cassette vector of Figure 13 and a murine anti-Leu3 light chain gene cloned in ⁇ GLla to form a hybrid mouse/human light chain gene in plasmid ⁇ Leu-3.LC as described in Example XII.
  • FIG. 1 illustrates a method which is generally applicable to the construction of the cassette vector of the invention.
  • a plasmid used in the vector construction contains (1) an origin of replication (ori), (2) a selectable-marker gene (sup), and (3) a polylinker (PL) containing a number of preferably unique restriction sites at which additional gene fragments can be inserted.
  • the origin of replication is one which allows plasmid replication in a host which also harbors cloned library gene fragment(s) with which the cassette vector recombines, in forming the hybrid gene, as will be described below.
  • the origin of replication typically is a ColEl replicon which permits replication in a variety of ⁇ -infectable bacterial hosts, such as E. coli.
  • the cassette and cloning vector plasmids are designed with different (non-homologous) replicons which allow the two plasmids to replicate within a common host without recombination at the replicon sites.
  • a two-plasmid system of this type is described in reference 5.
  • the selectable-marker gene is preferably a suppressor-tRNA gene (supF) which allows expression of amber-mutated genes in the cloned library vector (s) which acquires the supF gene by recombination.
  • a suppressor-tRNA gene is its relatively small size, typically 200 to 300 bp, its well-studied characteristics, and its versatility in vector systems having different types of amber mutations. For example, in ⁇ -phage library vectors carrying amber-mutated structural genes, acquisition by the phage of the suppressor gene allows recombination selection based on ⁇ growth in a suppressor-minus host.
  • the acquisition of the suppressor allows plasmid selection based on growth in the presence of the antibiotic.
  • the cassette vector selectable marker gene is to be acquired by a plasmid library vector, the marker gene itself may be an antibiotic resistance gene.
  • An alternative type of selectable marker possible for use with a ⁇ library vector is based on the increase in phage size produced by recombination between the ⁇ and a relatively large cassette vector.
  • the phage is constructed in size so that it is conditionally viable, e.g., can be killed under certain chemical medium conditions. With the recombination event, the phage acquires an additional length of the cassette vector which allows it to survive readily under such conditions.
  • a preferred plasmid for use in constructing the cassette vector of the invention is a ir plasmid of the type described in reference 6.
  • a plasmid similar to the one described in reference 6 is shown at the upper right in Figure 3, and includes a 645 bp replicon derived from pMBl, a 198 bp tyrosine tRNA amber-suppressor gene (supF), and a 49 bp polylinker segment which includes the restriction sites indicated in the expanded scale.
  • a B' coding region which will contribute the B' coding portion and the 5' I-B' junction of a hybrid A i -B' gene formed using the vector, is inserted into a suitable site in the plasmid mentioned above.
  • the B' coding region includes the constant coding region of a selected immunoglobulin gene class and source, where the cassette vector is used in preparing hybrid immunoglobulin genes.
  • the B' coding region typically includes one or more of the non-polymorphic exons from a selected histocompatibility gene.
  • the B' coding region is derived preferably from an isolated A'-B' gene, or B'-containing gene fragment, or cloned B' cDNA.
  • a suitable cloned genomic or cDNA B' coding region can be obtained by standard methods.
  • a large number of human and raurine immunoglobulin genes or gene fragments have been cloned, and complete or partial restriction maps and, in some cases, partial gene sequences have been reported for a number of these. Up-to-date restriction site and coding information on cloned immunoglobulin genes can be obtained from the
  • HUMIGCA2 HUMAN IG GERMLINE HEAVY CHAIN D-REGION GENE. 01.
  • HUMIGCB8 HUMAN IG GERMLINE H-CHAIN J-MU-DELTA REGION: MU-MEMBRANE EXON M1
  • HUMIGCC4 HUMAN IG GERMLINE G-E-A REGION A: GAMMA-1 CONSTANT REGION.
  • HUMIGCC8 HUMAN IG GERMLINE G-E-A REGION A: ALPHA-1 CONSTANT REGION.
  • HUMIGCD1 HUMAN IGGERMLINE H-CHAIN G-E-A REGION B: GAMMA-2 CONSTANT REGION.
  • HUMIGCD2 HUMAN IGGERMLINE H-CHAIN G-E-A REGION B: GAMMA-4 CONSTANT REGION.
  • HUMIGHAE2 HUMAN IG ACTIVE HEAVY CHAIN EPSILONI GENE. CONSTANT REGION.
  • HUMIGKC1 HUMAN IG GERMLINE KAPPA L-CHAIN. J-REGION GENES J1-J5.
  • HUMIGKC2 HUMAN IGGERMLINE KAPPA L-CHAIN PARTIAL J-C INTRON.
  • HUMIGKC3 HUMAN IG GERMLINE KAPPA L-CHAIN. CONSTANT REGION ( INV3 ALLELE).
  • HUMIGLAA HUMAN IG ACTIVE LAMBDA L-CHAIN CONSTANT REGION GENE. KE- OZ+.
  • HUMIGLC1 HUMAN IG GEFMLINF LAMBDA L-CHAIN C-REGION ISOTYPE MCG.
  • HUMIGLC2 HUMAN IGGERMLINE LAMBDA L-CHAIN C-REGION 2.
  • HUMIGLC3 HUMAN IGGERMLINE LAMBDA L-CHAIN C-RFGION 3.
  • H UMMHOCI A HUMAN MHC CLASS II HLA-DCl-ALPHA GENE (DRWG.WG). MRNA.
  • HUMMHORS1 HUMAN HLA-DR ALPHA-CHAIN (CHAIN-P34) GENE.
  • HUMMHDRS2 HUMAN HLA-DR ALPHA-CHAIN (CHAIN-P34) GENE.
  • HUMMHDSA HUMAN HLA-DS ALPHA-CHAIN MRNA.
  • HUMMHDXA1 HUMAN MHC CLASS II HLA-DX-ALPHA GENE (DR4.WG).
  • HUMHHDXA2 HUMAN MHC CLASS II HLA-DX-ALPHA GENE (DR4.WG). EXON 2.
  • MUSIGE1 MOUSE IG GERMLINE EPSILON GENE AND SECRETED TAIL.
  • MUSIGASANB MOUSE IG ALPHA S REGION FROM MG03 AFTER NON-PRODUCTIVE S-S.
  • MUSIGDJC31 MOUSE IG GERMLINE D-J-C REGION: S-GAMMAZA REGION. PART B.
  • MUSIGDJC38 MOUSE IG GERMLINE D-J-C REGION: GAMMAZA MEMBRANE EXON REGION.
  • MUSJGL1A1 MOUSE IG LAMBDA1 ACTIVE GENE VARIABLE/JOINING REGION.
  • MUSIGL1A2 MOUSE IG LAMBDA1 ACTIVE GENE CONSTANT REGION.
  • MUSIGL2A1 MOUSE IG LAMBDA2 ACTIVE GENE VARIABLE/JOINING REGION.
  • MUSIGL2A2 MOUSE IG LAMBDA2 ACTIVE GENE: CONSTANT REGION.
  • MUSMH MOUSE MHC CLASS I TRANSPLANTATION ANTIGEN (HAPLOTYPE 0). MRNA.
  • MUSMHD3 MOUSE MHC CLASS I GENE.
  • CLONE MUSMHIEAO MOUSE MHC CLASS II H2-IE-ALPHA GENE (HAPLOTYPE D).
  • MUSMHKB MOUSE MHC CLASS I H2-K GENE ( HAPLOTYPE B). MRNA. CLONE PH202.
  • MUSMHLD2 MOUSE MHC CLASS I H2-L GENE ( HAPLOTYPE D ). EXONS 4-8. Methods for isolating cloned A'-B' genes or B' -containing gene fragments or obtaining cDNA clones have been described exhaustively, and appropriate references can be obtained for each gene of interest from the data base information noted above. In many cases, cloned fragments and/or probes suitable for isolating the cloned fragments are available.
  • probes or cloned gene fragments are not readily available, the latter may be obtained, conventionally, by first constructing a synthetic oligonucleotide corresponding in sequence to a sequenced portion of the gene, then using the probe to identify suitable cloned gene fragments.
  • restriction maps of regions of interest in the cloned genes are not available, these may be constructed by conventional restriction mapping methods, such as described in reference 7.
  • the B'-coding region is excised, conventionally, by treating the B'-region cloning vector with one or more selected restriction endonucleases.
  • Suitable restriction sites can be determined from restriction maps of the A'-B' gene, obtained from published sources, data base information, or by conventional restriction analysis of the cloned B' fragment.
  • the B' gene is preferably excised near its 5' end, in a region of the 5'-adjoining intron, and at its 3' end, at a location downstream of post-transcriptional and post-translational signals, i.e., downstream of the gene's polyadenylation site.
  • the 5' cleavage site is in the IS2 intron between the gene's variable and constant regions.
  • the 5' cleavage site is in the intron between the gene's polymorphic and non-polymorphic exons, e.g., in the murine MHC I-A or I-E, ⁇ or ⁇ gene, in the intron between exons 2 and 3.
  • Example I describes the construction of a cassette vector whose B' coding region is derived from human germ-line ⁇ gene.
  • the construction of a cassette vector whose B' region is derived from the constant-coding region of a human germ-line ⁇ 4 immunoglobulin chain gene is detailed in Example III.
  • Example V describes the construction of a cassette vector which carries, as the B' coding region, the major portion of intron 2 for the murine I-E ⁇ chain through to the downstream region encoding beyond the polyadenylation site for the gene.
  • the gene can be cloned from many murine genomic libraries, and can be isolated following the procedure of reference 8.
  • the cassette plasmid is digested with one or more selected restriction endonucleases, and preferably at unique restriction sites contained in a polylinker region of the plasmid, to linearize the plasmid at a site suitable for insertion of the isolated B' fragment.
  • the sites at which the B' fragment and/or the plasmid may be cleaved are preferably sites which will provide proper orientation of the B' fragment in the plasmid by sticky-end ligation at one or both fragment ends.
  • the linearized plasmid and isolated B' fragment are ligated conventionally under sticky-end and/or blunt-end conditions, and successful transformants are selected using standard procedures.
  • the host preferably includes a second plasmid having one or more amber-mutated antibiotic genes which are expressed only in the presence of the plasmid suppressor-tRNA gene.
  • a selection system is described in Examples I, III, and V.
  • the plasmid obtained is designated pB' in Figure 1.
  • an intron gene fragment is introduced into the plasmid adjacent the B' coding fragment.
  • the intron gene fragment is homologous to an intron region in a gene which contains the complementary A i coding region of the hybrid A i -B' gene which is to be prepared using the cassette vector. More specifically, the gene fragment has sufficient homology with an intron region of the A i -containing gene to allow a recombination between the two intron regions in a recombination-competent host system.
  • the extent of homology between the cassette vector's gene fragment and that of the A i -containing gene intron must be at least about 90% in order for such recombination to occur efficiently, although the extent of homology required becomes greater for relatively short homologous regions.
  • the intron gene fragment is derived from intron between the A and B coding regions of a gene which is itself a member of the family of A-B genes which contains the A i -B i gene (which contributes the A i coding region to the hybrid A i -B' gene).
  • A-B gene families used in the invention include, but are not limited to: a.
  • the family of human immunoglobulin light-chain genes including the several known ⁇ and ic genes, where A and B are variable- and constant-coding regions, respectively.
  • the intron gene fragment is derived from the IS2 region between the variable and constant coding regions of the gene, and may include the enhancer element contained in the IS2 region, as described below.
  • the intron is also preferably derived from the IS2 region which, in an unrearranged immunoglobulin gene, is upstream of the switch signal at which class switching occurs.
  • the intron gene fragment is, of course, derived from the intron between the adjacent exons in the selected A and B coding regions.
  • the sources and methods for obtaining suitable isolated cloned genes or gene fragments containing the IS2 portion of the immunoglobulin genes, or the desired intron in a histocompatibility gene are similar to those described above with respect to gene fragments containing a B' coding gene fragment, as are sources for and methods of obtaining restriction maps of the gene regions.
  • the cloned intron-containing gene or gene fragment is treated with one or more selected restriction endonucleases to excise a desired intron gene fragment.
  • the size and location of the fragment which is excised will depend on a variety of considerations, including the availability of suitable restriction sites in the intron region.
  • the intron fragment derived from an immunoglobulin-chain A-B family preferably includes the enhancer element in the IS2 gene region. This element has been shown in both human and mouse immunoglobulin genes to contribute to efficient transcription of the immunoglobulin gene in same-species B-lineage cells (reference 9-12).
  • the total length of the intron fragment is preferably between about 60 and 1000 bp, and the fragment must be free of repetitive sequences if unwanted recombination events are to be avoided. Methods for screening gene fragments for the presence of repetitive sequences are well known. Finally, the gene fragment may be cut at sites which facilitate insertion into the B'-containing (pB') plasmid with proper orientation.
  • Example I illustrates the construction of a cassette vector having a 1 kb IS2 region derived from a Hindlll/BamHI segment of mouse ⁇ gene
  • Example III describes the construction of a heavy-chain cassette vector in which the gene fragment is a 1 kb Xbal segment of a mouse ⁇ gene. Both of the IS2 regions include enhancer regions from the respective genes.
  • Example V below describes construction of a hybrid MHC-class II heavy-chain cassette vector containing a 272 bp gene fragment from the 5' portion of the second intervening sequence from a human DR- ⁇ chain obtained in accordance with reference 13.
  • the pB' plasmid constructed as above, is linearized by digestion with one or more restriction endonucleases which preferably cleave the plasmid only in the polylinker and/or intron portion of the B' segment upstream of the B' coding region.
  • the linearized plasmid is isolated, conventionally, and ligated to the isolated intron gene fragment from above using conventional sticky-end and/or blunt-end ligation conditions, to insert the intron gene fragment adjacent the 5' end of the B' coding fragment.
  • Successful transformants are selected on a suitable host under growth conditions which require the presence of the cassette vector's selectable-marker gene, as discussed above.
  • the desired construct, indicated at pIB' at the bottom in Figure 1, can be confirmed by restriction analysis.
  • A. coding region to the hybrid gene For example, in the construction of a human/mouse hybrid immunoglobulin gene whose variable coding region is derived from mouse gene, the gene source is typically a mouse hybridoma cell line prepared to produce antibodies against a selected antigen. In constructing a hybrid histocompatibility antigen gene, a common source of genomic DNA material is any EBV-transformed lymphoblast line obtained from the subject of interest by standard means (references 14-16).
  • the cells which provide the source of A i -B i gene are cultured under standard conditions, and genomic DNA is extracted from the cells and partially digested with a suitable restriction endonuclease, such as Sau3a, according to known procedures.
  • the restriction digest pieces are ligated into the vector, followed by plating on an appropriate bacterial host. Methods for forming such genomic libraries are well known and are detailed, for example, in reference 17.
  • a preferred cloning vector for the genomic DNA fragments is a ⁇ phage, such as a Charon 4A phage, containing A am B am structural genes necessary for ⁇ packaging and growth.
  • the ⁇ structural genes code for incomplete proteins and the phage is unable to form plaques when plated on a suppressor-minus host.
  • the suppressor tRNA gene in the cassette vector is able to repress the phage amber mutation, allowing selection, on the basis of plaque formation, of those phage which have acquired the cassette vector.
  • recombination-selection method will be described herein with particular reference to a phage- ⁇ /suppressor tRNA system of this type, it is understood that other recombination selection systems, such as one in which an antibiotic resistance gene in a cassette vector is acquired by a cloning plasmid, allowing growth of the cloning plasmid in an antibiotic selection medium could be carried out readily by those skilled in the field.
  • the desired recombination event between the cassette vector and cloning vector with a homologous intron region will occur, typically at a frequency of between about 10 -4 and 10 -2 .
  • the recombination event places the B' coding region in the cassette vector adjacent the A i coding region in the cloning vector to form the desired hybrid A i -B' gene, and places the entire cassette vector fragment between the original A i and B i coding regions in the coding vector.
  • the expanded phage population is plated on a suppressor-minus host which allows plaque formation only in those phage which have acquired the cassette vector by the recombination event.
  • the plaques may be repassaged on a suitable host one or more times to increase the total amount of hybrid-gene phage, preferably on a suppressor-minus host, to prevent back-recombination events.
  • the construction of the hybrid-gene phage may be confirmed by restriction analysis to eliminate hybrid genes resulting from cassette vector recombination with inactive or incomplete A i -containing gene fragments.
  • the ⁇ phage vector used to construct the genomic library is modified to contain two restriction sites recognized by an enzyme, such as Notl, Sfil or Xhol, that rarely recognizes a restriction site in the source of the A i -B i or A'-B' genes.
  • an enzyme such as Notl, Sfil or Xhol
  • These two identical restriction sites for example both NotI, are inserted at either end of the ⁇ phage's "stuffer"fragment and flanking one or more pairs of more frequently recognized restriction sites, such as BamHI, Hindlll and EcoRI (see Figure 12).
  • it is an enzyme that cuts the genomic DNA at enough sites to be suitable for use in making a genomic library. Even more preferably, it is the enzyme used to make the library.
  • FIG. 21 shows such a construction where the common EcoRI sites and rare NotI sites flank a genomic clone of a murine light-chain immunoglobulin gene.
  • the restriction site should be located in the cassette vector adjacent to the 5' end of segment I and with neither the selection marker (e.g., SupF), nor the cassette's origin of replication located between the rare restriction site and I.
  • the restriction site would be located in the segment between sup and I.
  • a preferred ⁇ vector for library construction is ⁇ GLla described in Example VIII and shown in Figure 12.
  • the use of this ⁇ with a genomic insert in recombination with a cassette vector containing the same rare restriction site (NotI) is described in Example XII and shown in Figure 21.
  • a i is a polymorphic coding region derived from an A i -B i gene in a family of A-B genes;
  • B' is derived from a selected A'-B' gene
  • a i and A and B i and B' are structurally and functionally homologous gene regions
  • a and B coding regions and A' and B' coding regions are separated by introns I and I', respectively.
  • the I and I' introns may be, but are not necessarily, homologous.
  • a cassette vector whose B' coding region is a human immunoglobulin ⁇ -chain coding region, and whose intron gene fragment is from one of the family of murine light-chain genes, can be used to form both mouse ⁇ - and ⁇ -variable/human- ⁇ hybrid genes.
  • a cassette vector whose B' coding egion is a human ⁇ 1 -gene coding region and whose intron gene is derived from a gene portion 5' with respect to the switch region from one of the family of murine heavy-chain genes, may be used in forming mouse variable genes from ⁇ , ⁇ , ⁇ , ⁇ 1 , ⁇ 2 , ⁇ 3 , ⁇ 4 , and ⁇ /human constant ⁇ 1 hybrid genes.
  • the vector may be used in preparing human variable ⁇ , ⁇ , ⁇ . ⁇ 1 , ⁇ 2 , ⁇ 3 , ⁇ 4 , and ⁇ /human constant region hybrid genes.
  • Examples II and IV describe the construction of mouse-variable/human-constant light- and heavy-chain hybrid genes, respectively.
  • a cassette vector which contains exons 3-5 of a murine I-E ⁇ gene and an intron gene fragment from the IS2 of a human DR- ⁇ gene can be used in preparing each of the family of human-polymorphic DR- ⁇ /mouse I-E ⁇ hybrid genes.
  • the construction of such a gene is illustrated in Example VI.
  • Hybrid genes formed in accordance with the foregoing methods can be expressed in a variety of cell systems.
  • For the expression of hybrid immunoglobulin genes production of functional immunoglobulin genes by mouse myeloma cell lines (reference 1) and plasmacytoma and raouse-hybridoma cell lines (reference 19) have been shown in cells transfected with cloned immunoglobulin chain genes.
  • a cell used in expressing hybrid immunoglobulin genes is preferably one which is compatible with the enhancer element contained in the hybrid gene's intron. Recent studies, referenced above, indicate that the enhancer element functions best when the expression cells are B-lineage cells of the same species from which the enhancer element was derived.
  • the expression system will preferably be a B-lineage cell of the same species as that from which the variable region in the gene is derived.
  • a mouse cell system such as a plasmacytoma, hybridoma, or myeloma cell line will be preferred.
  • a preferred system is murine lymphocyte cells, although other cell lines, such as those suitable for expressing immunoglobulin-chain hybrid genes, may also be used (references 20, 21).
  • Murine lymphocyte cells are especially advantageous in expressing hybrid human/mouse immune response genes which are to be used to generate murine monoclonal antibodies directed against each of a variety of human polymorphic regions of cell-surface antigens.
  • a hybrid ⁇ or ⁇ immune response gene is transfected into mouse lymphocytes. Expression of the hybrid ⁇ or ⁇ chain leads to association of that chain with the complementary mouse ⁇ or ⁇ chain produced normally by the cell, leading to expression of a hybrid cell-surface antigen.
  • Lymphocytes expressing the composite gene are then used to immunize mice of the donor strain.
  • the principal foreign immune response antigens in these cells are those coded by the human polymorphic coding region of the hybrid gene.
  • Transfection of the selected expression system cells with the hybrid-gene phage, or other suitable transfection vector is carried out by known methods.
  • One standard technique involves calcium phosphate-precipitated, DNA-mediated gene transfer (reference 22).
  • Protoplast fusion is an alternative procedure that may give greater transfection efficiency in some cells (reference 23).
  • a relatively recent procedure relies on strong-voltage fluctuations to increase the permeability of the cells to the transfecting vector (reference 24).
  • the efficiency of transfection can be monitored by cotransfection with a selectable-marker plasmid carrying a bacterial antibiotic-resistance gene, such as a neomycin-resistance gene (reference 25).
  • a selectable-marker plasmid carrying a bacterial antibiotic-resistance gene such as a neomycin-resistance gene (reference 25).
  • Coinfection with a plasmid containing an antibiotic resistance-selectable marker also allows for selection for cells which have been transfected by the hybrid gene vector, since cotransfection of the cell by the phage DNA and selectable-marker plasmid occurs with a high probability.
  • the level of hybrid gene transcription and expression can be determined by known hybridization and antibody precipitation techniques, respectively, under conditions in which radiolabeled transcripts and polypeptides are produced.
  • the production of functional hybrid antibodies can be monitored, of course, by immunospecific reaction with selected antigens.
  • the production of hybrid histocompatibility genes containing polymorphic human class I or class II regions can be confirmed by the ability of the antigens, typically cell bound, to induce human-specific antibodies. From the foregoing, it can be appreciated how various objects of the invention are met.
  • the method of the invention allows for the construction, using a single cassette vector, of families of hybrid genes, in which the members of the families are polymorphic in the region of hybrid gene complementary to a coding region contained in the cassette vector.
  • Each member of the family can be prepared from library genomic DNA, rather than from isolated cloned gene fragments, and gene construction avoids in vitro restriction cutting and splicing steps. The effort required to obtain hybrid genes, and particular large families of hybrid genes, is thus greatly reduced. Further, the formation of a hybrid gene according to the method of the invention is readily detectable, and the hybrid gene easily isolated, e.g., from plaque-forming phage.
  • Site specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog. In general, about 1 ⁇ g of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 ⁇ l of buffer solution; in the examples herein, typically, an excess of restriction enzyme is used to ensure complete digestion of the DNA substrate.
  • Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using incubation times of about 15 to 25 minutes at 20 to 25°C. in 50 mM Tris-HCl, pH 7.6. 50 mM NaCl, 6 mM MgCl 2 , 6 mM DTT and 5-10 ⁇ M dNTPs. The Klenow fragment fills in at 5' sticky ends and chews back protruding 3' single strands, even though the four dNTPs are present.
  • E. coli DNA polymerase I Klenow
  • dNTPs deoxynucleotide triphosphates
  • selective repair can be performed by supplying only a selected one or more dNTPs within the limitations dictated by the nature of the sticky ends.
  • the mixture is extracted with phenol/chloroform and ethanol precipitated.
  • Treatment under appropriate conditions with S1 nuclease results in hydrolysis of any single-stranded portion.
  • Synthetic oligonucleotides may be prepared by the triester method of Matteucci, et al (reference 27), or the diethylphosphoramidite method of Caruthers, described in U.S. Patent No. 4,415,732, issued 15 November 1983.
  • Ligations are performed typically in 15-30 ⁇ l volumes under the following standard conditions and temperatures: 50 mM Tris-Cl, pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 100 ⁇ g/ml BSA, 1 mM ATP, and either 0.01-0.02 (Weiss) units T4 DNA ligase at 0°C (for "sticky end” ligation) or 0.3-0.6 (Weiss) units T4 DNA ligase at 14°C (for "blunt end” ligation). Intermolecular "sticky end” ligations are usually performed at 33-100 ⁇ g/ml total DNA concentrations (5-100 nM total end concentration).
  • Intermolecular blunt end ligations are performed typically at about 1 ⁇ M total ends concentration.
  • the vector fragment is commonly treated with calf intestine phosphatase (CIP) in order to remove the 5' phosphate and prevent religation of the vector.
  • CIP digestions are conducted typically at about pH 9, in approximately 50 mM Tris, in the presence of Zn +2 and Mg +2 , using about 0.01 unit of CIP per ⁇ g of vector, depending on size, at 37°C for about one hour.
  • the preparation is extracted with phehol/chloroform and ethanol precipitation. Alternatively, religation can be prevented in vectors which have been double digested by additional restriction enzyme digestion of the unwanted fragments.
  • Example I Constructing a Mouse/Human ⁇ -Chain Cassette Vector This example describes the construction of a plasmid designed for use in preparing mouse/human ⁇ -chain genes.
  • Human germline ⁇ gene was originally obtained from a BamHI phage library of human DNA, as described in reference 29, and subcloned into the EcoRI site of PBR322, to form a pHuC vector, whose ⁇ -gene insert is shown at A in Figure 3.
  • the pHuC plasmid was digested to completion with EcoRI, and the 2.5 kb fragment containing the constant coding region C ⁇ was isolated by electrophoresis and elution from an agarose gel.
  • a ⁇ anl3 plasmid used in constructing the cassette vector was constructed according to procedures similar to those outlined in reference 6 and can be obtained from Dr. Seed.
  • Figure 3 includes a 645 base-pair origin of replication from pMBl, a 198 base-pair synthetic tyrosine tRNA suppressor gene, and the polylinker region shown in expanded scale in the figure.
  • the plasmid was linearized by treatment with EcoRI, treated with calf intestinal phosphatase to remove 5'-phosphate, and the 0.9 kb plasmid fragment was purified by electrophoresis and elution from agarose gel.
  • the 2.5 kb EcoRI C ⁇ fragment from pHuC ⁇ and the 0.9 kb linearized ⁇ plasmid fragment were ligated with T4 DNA ligase, and the ligation products were used to transform E. coli strain MC1061 (P3).
  • the bacterium harbors a P3 plasmid having amber mutations in both ampicillin- and tetracycline-resistance genes. Transformation of cells already carrying P3 with the ⁇ anl3 plasmid thus confers simultaneous resistance to ampicillin and tetracyline to these cells.
  • a kanamycin-resistance gene in the P3 plasmid is used in maintaining the plasmid in the host in the absence of the ⁇ plasmid.
  • An IS2 gene fragment from a mouse ⁇ gene was derived from a pBHC cloning vector containing a Hindlll/BamHI segment of mouse ⁇ gene contained in a pBR322 plasmid (reference 12).
  • a map of the ⁇ -gene fragment insert is shown at D in Figure 3.
  • the pBHC plasmid was digested to completion with HindiII and XmnI, and the 1 kb fragment which contains the enhancer element in the ⁇ -gene IS2 region was purified by ele ⁇ trophoresis and elution from agarose gel.
  • the ⁇ C ⁇ plasmid was prepared by alkaline lysis and digested with Hindlll/Smal, releasing a small Hindlll/Smal fragment from the polylinker region of the plasmid.
  • the larger linearized ⁇ -plasmid fragment was purified by electrophoresis and elution from agarose gel. This fragment was ligated to the 1 kb Hindlll/XmnI fragment from pBHC ⁇ with T4DNA ligase, and the ligation products were used to transform
  • Lane 1 again represents Hindlll fragments of wild type ⁇ DNA.
  • the 2.4 kb and 2.0 kb fragments also correspond to the expected sizes from the ⁇ LC ⁇ construct shown in Figure 3.
  • a NotI site was then introduced into plasmid ⁇ LC ⁇ .
  • the plasmid was linearized by digestion at the single Hindlll site shown at E in Figure 3.
  • the 4.4 kb linear fragment was purified and blunt ended by filling in with Poll-Klenow fragments.
  • Kinased NotI linkers (Adapter AB, Figure 12 and Example VII) were then ligated to the blunt ends, followed by digestion with NotI.
  • the resulting linear fragment with NotI sticky ends was recircularized and used to transform MC1061(P3) cells. Transformants were selected for the presence of Sup, and plasmid structure verified by cutting with NotI.
  • a plasmid, designated ⁇ LC ⁇ .NotI ( Figure 13) was recovered.
  • Example II Constructing a Mouse/Human Hybrid ⁇ Gene
  • ChC ⁇ -1 containing an EcoRI ⁇ -gene insert from a Balb/c mouse germline cell, was obtained (reference 30).
  • the vector contains amber-mutated A and B phage genes, and the EcoRI ⁇ -gene insert extends from a position upstream from the 3' end of the gene through a portion of the constant C ⁇ region.
  • MC1061 host cells harboring either the P3 plasmid alone, designated MC1061(P3), or harboring both the P3 plasmid and the ⁇ -plasmid cassette from Example I, designated MC1061(P3) ( ⁇ LC ⁇ ) were preabsorbed with 10 6 ChC ⁇ -1 phage and plated on LB plates. No plaques were obtained with MC1061(P3), the negative control, demonstrating that the ChC ⁇ -1 phage stock contained no wild type revertants. Confluent lysate was obtained with the MC1061(P3) ( ⁇ LC ⁇ ) cells. The titer of the plate lysate on the Sup + cells was 1.5 x 10 9 phage/ml.
  • the expanded phage population was repassaged on a suppressor-minus host strain LG75.
  • Re ⁇ ombinant phage which acquired the suppressor-tRNA gene by recombination with the cassette vector were selected by growth.
  • the structure of the recombination region of the phage is shown at the bottom in Figure 5.
  • the recombination event has produced a hybrid human ⁇ -coding region upstream of the site of recombination and a mouse ⁇ -coding region downstream of the recombination site.
  • the titer on the plate lysate for the suppressor-minus strain was 4 x 10 6 phage/ml. Therefore, the recombination frequency between the homologous two regions of the cassette plasmid and the phage was about 3 x 10 -3 .
  • the plaques from the LG75 plate were replated on LG75 agarose plates, and a few plaques were picked.
  • the DNA from recombinant phages was digested with EcoRI, fractionated by agarose gel electrophoresis, and hybridized to a 32 P-labeled ⁇ anl3 probe, with the results shown in Figure 6.
  • Lane 1 shows the autoradiogram pattern for ChC ⁇ -1 DNA
  • lanes 2-4 show recombinant phage DNAs selected from three different plaques.
  • the probe hybridized to a 2.7 kb EcoRI fragment from the recombinant phage DNAs, confirming the structure shown at the bottom in Figure 5, where the recombination event has introduced an EcoRI site and adjacent suppressor-tRNA gene into the phage ⁇ , to give the 2.7 EcoRI fragment indicated.
  • the 5 kb and 2.7 kb Hindlll fragments were also hybridized to the J ⁇ probe ( Figure 19).
  • Example III Constructing a Mouse/Human ⁇ 4 Cassette Vector This example describes the contruction of a plasmid designed for use in preparing hybrid mouse/human ⁇ 4 genes.
  • Human genomic ⁇ 4 was obtained as described in reference 31, and subcloned into pBR322 to form a p24BRH vector, whose insert construction is shown at the top in Figure 7.
  • the insert region contains the ⁇ 4 constant-coding region, indicated at C ⁇ 4 in a 2.0 kb EcoRI/Hindlll fragment.
  • the p24BRH plasmid is digested to completion with Hindlll, blunt ended, and then digested to completion with EcoRI.
  • the 2.0 kb fragment containing the constant region is isolated by electrophoresis and elution from an agarose gel.
  • the ⁇ anl3 plasmid from Example I was linearized by digestion with SacI, blunt-ended with T4 DNA polymerase, then further digested to completion with EcoRI.
  • the 0.9 kb plasmid fragment was purified by electrophoresis and elution from agarose gel.
  • the 2.0 kb Hindlll/EcoRI C ⁇ 4 fragment from p24BRH and the 0.9 kb linearized ⁇ plasmid fragment are ligated with T4 DNA ligase and the ligation products were used to transform E. coli strain MC106KP3), as described in Example I.
  • Plasmid minipreps were analyzed by single digestion with Xbal, PstI and Smal to select plasmids with the 2.0 kb C ⁇ 4 fragment in the proper orientation.
  • a restriction map of the selected plasmid, designated ⁇ C ⁇ 4, is shown at the center portion of Figure 7, with the C ⁇ 4 insert and adjacent polylinker segment shown in expanded scale.
  • An IS2 gene fragment was derived from a segment of a mouse C ⁇ gene containing a heavy-chain enhancer, E H .
  • a map of the ⁇ -gene segment is shown in Figure
  • a Ch4A phage carrying germ-line C ⁇ was digested to completion with Xbal, and the 1 kb fragment containing the E H element was purified by electrophoresis and elution from agarose gel.
  • the ⁇ C ⁇ 4 plasmid was digested with Xbal, and the linearized ⁇ -plasmid fragment was purified by electrophoresis and elution from agarose gel. This fragment was ligated to the 1 kb Xbal ⁇ -gene fragment with T4 DNA ligase, and the ligation products were used to transform MC1061(P3). Successful transformants were selected as above.
  • the expected structure of the heavy gene cassette is illustrated at the bottom in Figure 7.
  • cassette vector ⁇ HC ⁇ 4 The final structure of the cassette vector, designated ⁇ HC ⁇ 4, was confirmed by digestion with PstI, which gave expected 2.0 kb, 1.5 kb, and 0.4 kb fragments ( Figure 8) and by double digestion with Smal and EcoRI. which yields expected 1.8, 1.77, and 0.3 kb fragments.
  • a NotI site was introduced into cassette vector ⁇ HC ⁇ 4 by the procedure described in Example I.
  • the phage vector which is shown at the top in Figure 9, contains amber-mutated phage genes, and the ⁇ -gene insert extending from a position upstream of the 3' end of the gene through the constant C ⁇ region.
  • MC1061(P3) ( ⁇ HC ⁇ 4), is preabsorbed with 10 6 ChC ⁇ 27 phage and plated on LB plates.
  • the expanded phage population is repassaged on a suppressor-minus host strain LG75, and recombinant phage which have acquired the suppressor-tRNA gene by recombination with the cassette vector are selected by growth.
  • the structure of the recombinant region of the phage is shown at the bottom in Figure 9.
  • the recombination event produces a hybrid gene with murine J H coding regions upstream of the IS2 recombination site and human ⁇ 4-gene coding regions downstream of the recombination site.
  • the plaques from the LG75 plate are replated on
  • DNA from recombinant phages are analyzed by digesting with EcoRI and hybridized to a 32 P-labeled ⁇ anl3 probe, to confirm the presence of the expected 3.9 kb fragment produced by the recombination event.
  • Example V Constructing a Human PR/Mouse I-E Heavy-Chain Cassette Vector
  • the murine I-E ⁇ chain gene is isolated following the procedure in reference 8. Briefly, the identified gene clone is grown up in a phage vector, then cloned into pBR322. The I-E ⁇ gene fragment, containing exon 2 through the 3' end of the gene is shown at A in Figure 10.
  • a genomic DNA library of mouse-tissue DNA generated by partial Sau3a digestion and cloned into ⁇ , could be screened with synthetic oligonucleotide probes made using the published sequence for the I-E ⁇ gene to select the same gene, and be confirmed by limited sequence analysis.
  • a ⁇ anl3 plasmid (Example I) is linearized by digestion with EcoRI and PstI, and the 0.9 kb plasmid fragment is purified by electrophoresis and elution from agarose gel.
  • the I-E ⁇ -gene vector from above is digested to completion with PstI and EcoRI and the 2.5 kb fragment and the 0.9 kb linearized ⁇ -plasmid fragment are ligated with T4 DNA ligase.
  • the ligation products are used to transform E. coli strain MC1061 (P3).
  • a human DR- ⁇ chain gene shown at D in Figure 10, is isolated according to the procedure of reference 13.
  • the gene includes a 491 bp intron 2 having the restriction sites indicated in expanded scale in the figure.
  • the plasmid carrying the DR- ⁇ gene fragment is digested to completion with Rsal and EcoRV, yielding a 272 bp intron 2 fragment which is purified by electrophoresis and elution from agarose gel. EcoRI linkers are then ligated to the 272 base pair Rsal-EcoRV fragment and then the fragment is digested with EcoRI.
  • the ⁇ l-E ⁇ plasmid is digested with EcoRI, phosphatased and the linearized ⁇ -plasmid fragment is purified by electrophoresis and elution from agarose gel. This fragment is ligated to the 280 bp linkered fragment from the DR- ⁇ gene with T4 DNA ligase, and the ligation products are used to transform MC1061 (P3). Successful transformants are selected as above and the orientation of the 280 base pair fragment is confirmed using Ncol.
  • the final structure of the MHC heavy-chain gene cassette, seen at E in Figure 10 and referred to as ⁇ DR/I-E ⁇ , is confirmed by digestion with EcoRI, giving expected 280 and 3400 fragments, and by double digestion with PstI and Ncol, giving expected 1110 and 2570 bp fragments.
  • a human EBV transformed lymphoblast line is obtained from the subject of interest by standard means.
  • the cells are cultured under standard conditions.
  • Genomic DNA is extracted from the cells and partially digested with Sau3a.
  • the digest fragments are ligated into ⁇ EMBL 3a under standard conditions followed by in vitro packaging and plating on the appropriate bacterial host.
  • the library phage vectors are passed initially in MC1061 cells harboring both the P3 plasmid (Example I) and the ⁇ DR/I-E ⁇ cassette vector from Example V.
  • the expanded phage population is repassaged on a suppressor-minus host, strain LG75, and recombinant phage which have acquired the suppressor-tRNA gene by recombination with the cassette vector are selected by growth.
  • the structure of the recombinant region of the phage is shown at the bottom in Figure 11. As seen, the recombination event produces a hybrid gene with exons 1 and 2 of the human DR- ⁇ gene upstream of the IS2 recombination site, and exons 3-5 of a mouse I-E ⁇ gene downstream of the recombination site.
  • a plaque from the LG75 plate is expanded on LG75 agarose plates. DNA from these recombinant phages are analyzed by hybridization to a 32 P-labeled DR- ⁇ exonl probe, to confirm the presence of the expected DR- ⁇ exonl fragment produced by the recombinant event.
  • Example VII Construction of Phage Vector ⁇ GLla The following example describes the construction of ⁇ GLla, an improved ⁇ phage vector for the construction of gene libraries for use with the cassette vectors of the present invention.
  • Adapter AB oligonucleotide, shown in Figure 12, was synthesized by Applied Biosystems, Foster City, CA. It is a NotI linker molecule also containing an inactivated EcoRI site, and active Xhol and BamHI sites. Adaptor AB is kinased in the presence of ⁇ - 32 P-ATP. The purified stuffer is then ligated to excess adapter AB at 22°C for 1 hr.
  • Recombinant phages which lack the stuffer fragment from Ch4a form colorless plaques. Phage DNA from colorless plaques were isolated, and subjected to enzyme restriction analysis by digestion with NotI. A recombinant phage that gave the expected 20 kb, 14 kb, and 11kb fragments was designated ⁇ GLla.
  • Example II This example describes the construction of a plasmid designed for use in preparing hybrid mouse/human ⁇ 1 genes.
  • ⁇ anl3 (Example I) was linearized with Hindlll and a NotI site inserted as described for plasmid irLC ⁇ in Example I to give the structure B shown in
  • Figure 15 ⁇ anl3.NotI was then digested with Xba and ligated to the enhancer fragment E H recovered from ⁇ HC ⁇ 4 ( Figure 7). Recircularized plasmid DNA was then used to transform MC1061(P3) cells and plasmids from transformants analyzed for proper orientation of the E H fragment by digestion with EcoRI. A plasmid designated ⁇ E H .NotI, shown as structure C in Figure 15, gave the expected fragment sizes of 330 and 1570 bp.
  • Plasmid 13AHP containing human genomic C ⁇ 1 in pBR322 (reference 34) was obtained from Dr. Jay Ellison. After digesting 13AHP with Hindlll and PvuII, the 3.0 kb fragment containing C ⁇ 1 was purified and the ends trimmed back. Kinased Sst-1 linkers were ligated to the blunt ends of the 3.0 kb fragment, followed by digestion with Sst-1. ⁇ E H .NotI was digested with Sst-1 and ligated to the Sst-1-digested C ⁇ 1 fragment to recircularize as shown in Figure 15.
  • the ligated DNA was used to transform MC1061(P3) cells and transformants were analyzed for plasmids containing C ⁇ 1 in the proper orientation.
  • the orientation was tested by digestion with PstI alone, and with a combination of PstI and EcoRI.
  • ⁇ anl3 (Example I), which had previously been linearized with SacI, sticky ends trimmed and the 5' ends dephosphorylated.
  • ⁇ anl3 containing the C ⁇ 3 coding sequences was used to transform MC1061(P3) cells and transformants selected to determine the orientation of the insert.
  • a plasmid designated ⁇ C ⁇ 3-14 was obtained with the C ⁇ 3 insert in the wrong orientation.
  • ⁇ C ⁇ 3-14 aquired an unexpected additional EcoRI site.
  • the orientation of the C ⁇ 3 insert was determined by double digestion with Hindlll + Bglll, which gave the following size fragments: Hindlll + Bglll - 1800 and 2300 bp.
  • ⁇ rC ⁇ 3-14 then was digested with EcoRI and religated to reorient the C ⁇ 3 segment.
  • Plasmids from transformants were then tested again for correct orientation.
  • a plasmid designated ⁇ C ⁇ 3, structure C in Figure 16, gave fragments of the correct size: Hindlll + Bglll - 900 and 3200 bp.
  • ⁇ C ⁇ 3 was digested with Xba and the 1 kb -
  • Xba fragment of enhancer E H was inserted as described in Example VIII. These recombinant plasmids were used to transform MC1061(P3) cells. Transformants were then tested for the presence of plasmid containing enhancer region in the proper orientation.
  • a plasmid designated ⁇ HC ⁇ 3, structure D in Figure 16 was digested with EcoRI and gave fragments of the correct size: 3200, 1600, and 340 bp.
  • a NotI site was inserted in the Hindlll site of ⁇ HC ⁇ 3 as described in Example I. This gave a plasmid designated ⁇ HC ⁇ 3.NotI, shown as structure E in Figure 16.
  • Switching Cassette Vector for IgM to IgG 3 This example describes the construction of a plasmid designed for use in changing the isotype of a human immunoglogulin heavy chain gene.
  • the cassette vector is useful for changing the isotype of an IgM antibody to IgG 3 .
  • Plasmid PJ is digested with Bglll and Hindlll, and the 0.75 kb fragment, which contains the intron IS, is purified.
  • the ligation mixture was used to transform MC1061(P3) cells and a plasmid designated pHC-1 was recovered (structure D in Figure 17).
  • the C ⁇ 3-containing 3.2 kb fragment from Ch4a (Example IX) with added Hindlll linkers was then inserted into the Hindlll site of pHC-1.
  • MC1061(P3) cells were transformed by the ligation mixture and transformants tested for the presence of plasmids with the proper orientation by digestion with Bglll.
  • a NotI site (adapter AB, Example I) was ligated between the EcoRI and BamHI sites of ⁇ SWMG3 to give a plasmid designated ⁇ SWMG3.NotI, depicted as structure F in Figure 17.
  • Cassette Vector for IgM to IgGl This example describes the construction of a cassette vector similar to the one constructed in the previous example, however, it switches IgM to IgG 1 .
  • the procedure used to construct this cassette vector is substantially the same as that described in Example X, except that rather than inserting the 3.2 kb C ⁇ 3 fragment into pHC-1, the 3.0 kb C ⁇ 1 fragment described in Example VIII ( Figure 15) with added Hindlll linkers was inserted to give a plasmid designated ITSWMGI (structure A in Figure 18).
  • a NotI site was inserted as described in Example X to give a plasmid designated ⁇ SWMGl.NotI (structure B in Figure 18).
  • FIG 19 shows the restriction map of the J ⁇ -E ⁇ -C ⁇ -region of an unrearranged mouse gene.
  • J ⁇ probe and -J ⁇ probes are the 2.7 kb and 0.7 kb Hindlll fragments, respectively.
  • the J ⁇ probe is present in both the unrearranged immunoglobulin gene, as well as normally rearranged immunoglobulin genes.
  • the -J ⁇ probe is not present in the normally rearranged gene, but is present in the unrearranged gene.
  • Figure 20 shows the restriction map of the -J H -E H -C ⁇ - region of the mouse unrearranged heavy chain immunoglobulin gene.
  • This example shows the use of the ⁇ LC ⁇ .NotI cassette vector (Example I, Figure 13) and a library from an anti-Leu3 murine hybridoma in ⁇ GLla (Example VII, Figure 12) to construct a hybrid immunoglobulin gene consisting of the murine variable region of the light chain produced by the hybridoma, and the human C ⁇ region from the cassette vector.
  • Hybridoma cell line SBC3.5 producing anti-Leu3 antibodies, was obtained from Dr. Edward Engleman, Stanford University.
  • the cellular DNA from the hybridoma was extracted and digested with Hindlll. Restriction patterns were analyzed by Southern blot analysis employing the J ⁇ probe ( Figure 19).
  • Southern blot analysis showed a 4.2 kb band that was unique to the anti-Leu3 hybridoma when compared to SP2 (fusion partner of the hybridoma) and Balb/c liver DNAs. This suggests that the 4.2 kb band contains the rearranged anti-Leu3 light chain gene.
  • Hybridization employing the J H probe indicated that, judging from the intensities of the autoradiograras relative to liver DNA, the copy number of the anti-Leu3 heavy chain gene is much lower than that of the light chain gene.
  • Hybridoma DNA was then extracted and partially digested with EcoRI* activity (reference 44) under conditions to give an average fragment size of 15 kb. Size-selected DNA was then ligated into the EcoRI site of ⁇ GLla DNA and packaged. The number of recombinant phage was estimated by Spi selection (reference 42). 1.2 x 10 6 recombinant phage was divided into twelve sublibraries and then amplified on E. coli KM392 (SupF + ). The plate lysates were replated onto MC1061(P3) cells containing cassette vector
  • LG-75 to KM392 was 10 -5 to 10 -7 .
  • Phage were then screened by hybridization to the J ⁇ probe. Eleven sublibraries of the twelve were positive. J + k SupF phage was then plaque purified, and then hybridized to the J ⁇ , human C ⁇ ( Figure 3A), and -J ⁇ probes to eliminate unrearranged clones. Three of the eleven sublibraries belonged to unrearranged light chain genes as indicated by hybridization to probe -J ⁇ . All of the remaining eight phage clones hybridized to J ⁇ and human C ⁇ probes, suggesting that they contain the right sequences. Primary lysate was prepared from a single plaque. Extracted DNA was confirmed by digestion with the appropriate enzymes, and by hybridization to probes
  • Competent MC1061(P3) cells were then transformed with each circular ⁇ plasmid DNA.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Un vecteur cassette est utilisé pour construire un gène hybride Ai-B' dans lequel (a) Ai est une région polymorphe de codage dérivée d'un gène Ai-Bi appartenant à une famille de gènes polymorphes A-B; (b) B' est dérivé d'un gène A'-B' sélectionné; (c) Ai et A' et Bi et B' sont des régions structurellement et fonctionnellement homologues de codage de gènes; et (d) les régions de codage A et B et les régions de codage A' et B' sont séparées par les introns I et I', respectivement. Le vecteur comprend un gène sélectionnable de marquage qui permet de sélectionner dans un hôte un vecteur de clonage qui acquiert le gène de marquage par recombinaison avec le vecteur cassette, la région B' de codage, et, adjacent à l'extrémité 5' de ladite région B' de codage, un fragment de gène dérivé de l'intron I de l'un des gènes appartenant à la famille de gènes A-B. L'introduction du vecteur et d'une génothèque de fragments d'ADN génomique contenant un fragment de gène Ai-intron dans un hôte recombinable entraîne la recombinaison qui produit le gène Ai-B' voulu. Un vecteur de clonage contenant le gène hybride peut être aisément sélectionné grâce à la présence du gène de marquage sélectionnable du vecteur cassette, incorporé dans le vecteur de clonage lors de la recombinaison. Un vecteur de clonage de phages lambda est utilisé pour construire des génothèques d'ADN génomique utilisables avec les vecteurs cassettes.
PCT/US1986/000566 1985-03-18 1986-03-18 Vecteur cassette de gene hybride WO1986005513A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
DK551686A DK551686A (da) 1985-03-18 1986-11-18 Hybridgenkassettevektor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US71305285A 1985-03-18 1985-03-18
US713,052 1985-03-18

Publications (1)

Publication Number Publication Date
WO1986005513A1 true WO1986005513A1 (fr) 1986-09-25

Family

ID=24864557

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1986/000566 WO1986005513A1 (fr) 1985-03-18 1986-03-18 Vecteur cassette de gene hybride

Country Status (4)

Country Link
EP (1) EP0217916A4 (fr)
JP (1) JPS62502586A (fr)
AU (1) AU596585B2 (fr)
WO (1) WO1986005513A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0247091A4 (fr) * 1985-11-01 1987-10-27 Internat Genetic Engineering I Assemblage modulaire de genes d'anticorps, anticorps ainsi prepares et utilisation.
US5530101A (en) * 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5576195A (en) * 1985-11-01 1996-11-19 Xoma Corporation Vectors with pectate lyase signal sequence
US5595898A (en) * 1985-11-01 1997-01-21 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
EP0469089B1 (fr) * 1989-04-18 1997-11-19 Applied Biotechnology, Inc. Generation de genes hybrides et de proteines par recombinaison a mediation virale
US6358740B1 (en) 1999-03-05 2002-03-19 Maxygen, Inc. Recombination of insertion modified nucleic acids

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3526995A1 (de) * 1985-07-27 1987-02-05 Hoechst Ag Fusionsproteine, verfahren zu ihrer herstellung und ihre verwendung

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60224492A (ja) * 1984-04-24 1985-11-08 Yuu Honshiyo 異種遺伝子の結合法
JPS6147500A (ja) * 1984-08-15 1986-03-07 Res Dev Corp Of Japan キメラモノクロ−ナル抗体及びその製造法
EP0173494A3 (fr) * 1984-08-27 1987-11-25 The Board Of Trustees Of The Leland Stanford Junior University Récepteurs chimériques par liaison et expression de l'ADN
GB8422238D0 (en) * 1984-09-03 1984-10-10 Neuberger M S Chimeric proteins

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CEll, Volume 33, issued July 1983, (Cambridge, Massachussetts, USA) (BANERJI, et al), "A Lymphocyte-Specific Cellular Enhancer is Located Down Stream of the Joining Region in Immunoglobulin Heavy Chain Genes", see page 729 and 737 *
Nature, Volume 312, issued 13 December 1984, (London, GB) (BOULIANNE, et al), "Production of Functional Chimaeric Mouse/Human Antibody", see pages 643-646 *
Nature, Volume 314, issued 21 March 1985. (London, GB), (NEUBERGER, et al.) "A Hapten-Specific Chimaeric IgE Antibody with Human Physiological Effector Function" see pages 268-270. *
Nucleic Acids Research, Volume 11, Number 8, issued 25 April 1983, (Amsterdam, NL), (Seed, B.), "Purification of Genomic Sequences from Bacteriophage Libraries by Recombination and Selection in Vivo" see pages 2427-2431 *
Proceedings National Academy of Sciences (USA), (Washington. D.C., USA) Volume 81. issued November, 1984, (MORRISON, et al), "Chimeric Human Antibody Molecules: Mouse Antigen-Binding Domains with Human Constant Region Domains", see pages 6851-6855 *
See also references of EP0217916A4 *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6204023B1 (en) 1985-11-01 2001-03-20 Xoma Ltd. Modular assembly of antibody genes, antibodies prepared thereby and use
EP0247091A1 (fr) * 1985-11-01 1987-12-02 Int Genetic Eng Assemblage modulaire de genes d'anticorps, anticorps ainsi prepares et utilisation.
EP0247091A4 (fr) * 1985-11-01 1987-10-27 Internat Genetic Engineering I Assemblage modulaire de genes d'anticorps, anticorps ainsi prepares et utilisation.
US5698417A (en) * 1985-11-01 1997-12-16 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US5698435A (en) * 1985-11-01 1997-12-16 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US5595898A (en) * 1985-11-01 1997-01-21 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US5618920A (en) * 1985-11-01 1997-04-08 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US5846818A (en) * 1985-11-01 1998-12-08 Xoma Corporation Pectate lyase signal sequence
US5693493A (en) * 1985-11-01 1997-12-02 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US5576195A (en) * 1985-11-01 1996-11-19 Xoma Corporation Vectors with pectate lyase signal sequence
US5585089A (en) * 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5530101A (en) * 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5693761A (en) * 1988-12-28 1997-12-02 Protein Design Labs, Inc. Polynucleotides encoding improved humanized immunoglobulins
US6180370B1 (en) 1988-12-28 2001-01-30 Protein Design Labs, Inc. Humanized immunoglobulins and methods of making the same
US5693762A (en) * 1988-12-28 1997-12-02 Protein Design Labs, Inc. Humanized immunoglobulins
US7022500B1 (en) 1988-12-28 2006-04-04 Protein Design Labs, Inc. Humanized immunoglobulins
EP0469089B1 (fr) * 1989-04-18 1997-11-19 Applied Biotechnology, Inc. Generation de genes hybrides et de proteines par recombinaison a mediation virale
US6358740B1 (en) 1999-03-05 2002-03-19 Maxygen, Inc. Recombination of insertion modified nucleic acids
US6365377B1 (en) 1999-03-05 2002-04-02 Maxygen, Inc. Recombination of insertion modified nucleic acids
US6406910B1 (en) 1999-03-05 2002-06-18 Maxygen, Inc. Recombination of insertion modified nucleic acids
US6413745B1 (en) 1999-03-05 2002-07-02 Maxygen, Inc Recombination of insertion modified nucleic acids

Also Published As

Publication number Publication date
EP0217916A4 (fr) 1989-02-23
AU5668086A (en) 1986-10-13
EP0217916A1 (fr) 1987-04-15
JPS62502586A (ja) 1987-10-08
AU596585B2 (en) 1990-05-10

Similar Documents

Publication Publication Date Title
DE69133476T2 (de) Transgene Mäuse fähig zur Produktion heterologer Antikörper
US5633425A (en) Transgenic non-human animals capable of producing heterologous antibodies
US5874299A (en) Transgenic non-human animals capable of producing heterologous antibodies
CA2124967C (fr) Animaux transgeniques non humains capables de produire des anticorps heterologues
KR100855998B1 (ko) 비동족형 스위치 영역에 의해 인간 항체의 특정 이소타입을 생산하는 트랜스제닉 동물
Siebenkotten et al. The murine IgG1/IgE class switch program
Eckhardt et al. Independent immunoglobulin class-switch events occurring in a single myeloma cell line
FI121339B (fi) Geneettisesti muunnetut hiiret ihmisen vasta-aineiden tuottamiseen, ja menetelmät niiden valmistamiseksi
US5338669A (en) Method of producing fusion proteins
EP0765937B1 (fr) Méthode de production de protéines de fusion
WO1991000906A1 (fr) Animaux chimeriques et transgeniques pouvant produire des anticorps humains
WO1991016427A1 (fr) Procedes de creation de phenotype a partir de populations de genes multiples
KR20120068757A (ko) 가용성 “항체만의” 결합분자
GB2216126A (en) Antibodies to Campath-1
JP2619232B2 (ja) ヒト免疫グロブリンｅ結合因子活性ポリペプチドをコードする遺伝子系
AU596585B2 (en) Hybrid genes (eg. ig's & mhc proteins) via lambda library andplasmid casette vector
JPH11206387A (ja) 異種免疫グロブリンを作る方法及びそのためのトランスジェニックマウス
WO1991010722A2 (fr) Immunoglobuline chimerique pour recepteurs de cd4
JPS62269697A (ja) エリスロポエチンの製造方法
Morrison et al. Transfectomas Provide Antibodies with Novel Structures and Functions
Morrison et al. SECTION V IMMUNOLOGIC INTERVENTION WITH MONOCLONAL ANTIBODIES
Smith et al. Detection of gene targeting by co-conversion of a single nucleotide change during replacement recombination at the immunoglobulin μ hesvy chain locus
Teillaud et al. 22. Monoclonal Antibodies

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BR DK FI HU JP KP NO

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LU NL SE

WWE Wipo information: entry into national phase

Ref document number: 1986902598

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1986902598

Country of ref document: EP

WWR Wipo information: refused in national office

Ref document number: 1986902598

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1986902598

Country of ref document: EP