WO1986005513A1 - Hybrid-gene cassette vector - Google Patents

Abstract

A cassette vector for use in constructing a hybrid Ai-B' gene where (a) Ai is a polymorphic coding region derived from an Ai-Bi gene in a family of polymorphic A-B genes; (b) B' is derived from a selected A'-B' gene; (c) Ai and A' and Bi and B' are structurally and functionally homologous gene coding regions; and (d) the A and B coding regions and the A' and B' coding regions are separated by introns I and I', respectively. The vector includes a selectable-marker gene which permits selection in a host of a cloning vector which acquires the marker gene by recombination with the cassette vector, the B' coding regions, and, located adjacent the 5' end of said B' coding region, a gene fragment derived from intron I in one of the genes in the A-B gene family. Introduction of the vector, and a library of genomic DNA fragments containing an Ai-intron gene fragment into a recombination permissive host leads to a recombination event which produces the desired Ai-B' gene. A cloning vector containing the hybrid gene is readily selectable by the presence of the cassette vector's selectable marker incorporated into the cloning vector by the recombination event. A lambda phage cloning vector useful for constructing genomic DNA libraries for use with the cassette vectors is also provided.

Description

HYBRID-GENE CASSETTE VECTOR

Field of the Invention

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.

Government Grant

The invention described herein was made in the course of grant nos. 1R43AI21606-01 and 1R43AI21604-01 from the National Institutes of Health, United States

Department of Health and Human Resources.

References

The following references are referred to herein by the corresponding number:

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2. Parham, P., et al, in Histocompatibility Antigens: Structure and Function (1982) Chapman and Hall, London-New York.

3 Pierce, C. W., et al, in IR Genes-Past, Present and Future (1983) The Humana Press, Clifton, NJ.

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6. Seed, B., Nuc Acids Res (1983) 11(8):2427.

7. Maniatis, T., et al in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1982), p. 374. 8. Hyldig-Nielsen, J. J., et al, Nuc Acids Res (1983) 11(15):5055.

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17. Maniatis, T., ibid, p. 269.

18. Maniatis, T., ibid, p. 256. 19. Ochi, A., et al, Proc Natl Acad Sci USA (1983) 80:6351.

20. Barbosa, J. A., et al, Proc Natl Acad Sci USA (1982) 79:6327.

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28. Maniatis, T., ibid, p. 353.

29. Heiter, P. A., et al, J Biol Chem (1982) 257:1516. 30. Howard Gershenfeld, unpublished results.

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10:4071-4079.

35. Ravetch, J.V., et al, Cell (1981) 27:583-591.

36. Miller, J.H., The Operon (Cold Spring Harbor Laboratory 1978). 37. Collins, F.S., et al, Proc Natl Acad Sci USA

(1984) 81:6812-6816.

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(1985) 72:3961.

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170:827-842.

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42. Zissler, et al, The Bacteriophage Lambda p. 455 (Cold Spring Harbor Laboratory 1981). 43. Maniatis, T., ibid, p. 504.

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46. Cory, S., et al, Proc Natl Acad Sci USA (1980) 77:4943-4947.

Background of the Invention

There is a growing interest in using monoclonal antibodies for human therapeutic uses. Although the original and still most successful procedure for generating monoclonal antibodies is by way of mouse-cell hybridomas, mouse monoclonal antibodies are expected to have limited prophylactic or therapeutic applications in humans, particularly where multiple injections of the antibody might have to be given to an individual, because of the likelihood that long-term administration will lead to human immunological reactions against species-specific determinants on the antibodies. Although the immune-response problem can be solved to some extent by use of human monoclonal antibodies, there are significant limitations associated with producing human monoclonal antibodies. To date technical problems have been encountered in producing stable, high-producing human hybridoma cell lines.

Another major limitation is the obvious ethical problem of immunizing human beings against many types of antigens, such as mycotoxins, snake venoms, toxic chemicals, drugs or the like, for producing the antigen-specific human lymphocytes needed in forming human hybridomas. Therefore the types of human monoclonal antibodies that can be made by current hybridoma methods are quite limited, being confined to those antigens for which highly immune individuals can be found, for example, hepatitis B antigen and some of the red blood cell surface antigens.

Functional antibody molecules are composed of two light (L) chains and two heavy (H) chains, arranged in heavy/light pairs as H₂L₂ 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. Because the immune response in humans against a. foreign (mouse) monoclonal antibody is directed largely against the antibody's light- and heavy-chain constant regions, it has been proposed that 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. At the same time, 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. For example, in cold agglutinin autoimmune hemolytic anemia, typically 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. Alternatively, non-complement fixing antibodies could be converted to complement-fixing antibodies to increase their effects in fighting infectious diseases or malignancies.

It has been shown heretofore that functional human/mouse hybrid immunological genes may be constructed by splicing a mouse variable coding region to a human constant region at the intron between the variable and constant regions in each heavy- and light-chain gene. A mouse myeloma cell cotransfected with both hybrid light-chain and heavy-chain genes was shown to produce tetrameric hybrid antibodies whose specificity matched that of the original mouse antibody (reference 1). In constructing each hybrid gene, isolated clones of both the mouse and human genes which were to be spliced together, were cut and spliced by in vitro restriction endonuclease and ligation techniques, to join the different-source variable and constant coding regions at an intermediate intron region. It can be appreciated that the cloned gene isolation and in vitro manipulation steps required for producing the hybrid gene for each antibody chain would make generating large families of hybrid antibodies, each having a different selected antigen specificity, quite laborious. For example, to construct a family of mouse-variable/human-constant genes of a given immunological class, where each member has a different antigen specificity, it would be necessary to isolate a cloned variable region gene of both light and heavy chains for each member of the family, splice the variable coding region of the cloned gene to the associated constant region of a cloned human immunoglobulin chain gene, and select transformants having the desired hybrid gene construction. 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

2-centimorgan region of human chromosome six (references 2 and 3). 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.

It would be extremely valuable, in diagnosing propensity to disease, in transplantation matching, and potentially in immunotherapy for a variety of disease processes, to be able to produce monoclonal antibodies capable of recognizing all of the polymorphisms due to the unique products of the individual class I and class II genes from within the HLA complex. Efforts to obtain monoclonal antibodies by immunization of mice has had only very limited success because the mouse reacts immunologically to species-specific determinants that are shared by all genes that have one of several human loci, rather than to the polymorphic determinants which vary among individuals in a population.

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. In theory, 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, however, 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.

There are thus a number of important applications of 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.

Summary of the Invention it is therefore a general object of the invention to provide a method which facilitates construction of a family of hybrid genes coding for highly polymorphic immunological proteins.

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:

(a) the hybrid genes are constructed from DNA fragment libraries rather than from isolated cloned genes;

(b) the hybrid genes are formed without gene cutting and splicing manipulations; and

(c) formation of the hybrid genes with correct orientation and structure results in a selectable-marker event which allows easy selection of the hybrid genes. 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) A_i is a polymorphic coding region derived from an A_i-B_i gene in a family of A-B genes;

(b) B' is derived from a selected A'-B' gene;

(c) 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. In a preferred embodiment of the invention, 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. In a cassette vector designed for use in constructing hybrid immunoglobulin genes, the B' coding region includes the constant coding region of a preferably human A-B gene of a selected class, i.e., either the κ or λ light-chain class or the α, δ, ε, γ1. γ2, γ3, γ4, or μ 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.

To construct an hybrid immunoglobulin gene according to the method of the invention, there is first produced a library of genomic DNA derived from the cell known to contain the desired A_i-B_i in functional

(rearranged) form. 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. The A_i and

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. 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, some of which contain the A_i polymorphic coding regions of the gene and adjacent intron, 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.

These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.

Brief Description of the Drawings

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 λ 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 κ 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 κ gene contained in a λ 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 κ gene and whose constant coding region is derived from the human constant coding region of a human κ chain gene contained in the cassette vector; Figure 6 shows an autoradiogram pattern of the hybrid κ gene from Figure 5, following digestion with EcoRI and hybridization to a πanl3 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;

Figure 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 μ gene contained in a λ phage and in the cassette vector of Figure 7, to form a hybrid heavy-chain gene whose upstream sequences are derived from mouse μ 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 DRα/mouse I-Eα immune response gene; and

Figure 11 illustrates the recombination event between a human DRα gene contained in a λ phage and the cassette vector of Figure 9, to form a hybrid DRα/I-Eα gene.

Figure 12 illustrates the construction of the phage vector λGLla described in Example VII from λEMBL 3 and λCh4a.

Figure 13 is a restriction map of the κ light chain cassette vector πLC_κ.Notl, described in

Example I.

Figure 14 is a restriction map of the γ4 heavy chain cassette vector irHCγ₄.NotI, whose construction is described in Example III. Figure 15 illustrates the construction of the γ1 heavy chain cassette vector πHCγ₁.NotI described in Example VIII. Figure 16 illustrates the construction of the γ3 heavy chain cassette vector πHCγ₃.NotI described in Example IX.

Figure 17 illustrates the construction of the isotype switching cassette vector πSWMG₃.NotI described in Example X.

Figure 18A and B illustrate the structure of the isotype switching cassette vectors πSWMG and πSWMG₁.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_κ and -J_κ 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

J_H probe was isolated and employed in Example XII.

Figure 21 illustrates the recombination between the light chain cassette vector of Figure 13 and a murine anti-Leu3 light chain gene cloned in λGLla to form a hybrid mouse/human light chain gene in plasmid πLeu-3.LC as described in Example XII.

Detailed Description of the Invention I. Constructing a Cassette Vector Figure 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, and designated pPL, 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. Where the cloned libarary genes are carried in a λ phage, the origin of replication typically is a ColEl replicon which permits replication in a variety of λ-infectable bacterial hosts, such as E. coli. Where the cloned library genes are carried in a plasmid, 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. Advantages of a suppressor-tRNA gene are 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 λ-phage library vectors carrying amber-mutated structural genes, acquisition by the phage of the suppressor gene allows recombination selection based on λ growth in a suppressor-minus host. With plasmid library vectors containing amber-mutated antibiotic resistance genes, the acquisition of the suppressor allows plasmid selection based on growth in the presence of the antibiotic. Of course, it is noted that where 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 λ library vector is based on the increase in phage size produced by recombination between the λ and a relatively large cassette vector. In this embodiment, 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. As a first step in constructing the cassette vector, 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.

Typically, 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. Where the cassette vector is to be used in preparing hybrid histocompatibility-antigen 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

Genetic Sequence Data Bank of the National Institutes of Health, Washington, D.C., or from the EMBL Nucleotide Sequence Data Library. Both of these data banks are accessible through Intelligenetics, Inc. (1975 El Camino Real West, Mountain View, California 94040-2216). The information available through these data banks includes, in addition to updated sequence and restriction map information, a list of references from which the information was derived. Similar updated information on human and murine class I and II MHC genes is available from these two data banks. Table I below is a partial list of human and murine immunoglobulin and MHC genes for which data base information is available.

Table I

Human Immunoqlobuline Sequences

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 IG GERMLINE H-CHAIN G-E-A REGION B: GAMMA-2 CONSTANT REGION.

HUMIGCD2 : HUMAN IG GERMLINE H-CHAIN G-E-A REGION B: GAMMA-4 CONSTANT REGION.

HUMIGCD3 : HUMAN IG GERMLINE H-CHAIN G-E-A REGION B : EPSILON-1 S' FLANK.

HUMIGHAE2 : HUMAN IG ACTIVE HEAVY CHAIN EPSILONI GENE. CONSTANT REGION.

HUMIGKC1 : HUMAN IG GERMLINE KAPPA L-CHAIN. J-REGION GENES J1-J5.

HUMIGKC2 : HUMAN IG GERMLINE 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 IG GERMLINE LAMBDA L-CHAIN C-REGION 2. ISOTYPE KE- OZ-.

HUMIGLC3 :HUMAN IG GERMLINE LAMBDA L-CHAIN C-RFGION 3. ISOTYPE KE- OZ+.

Human MHC Genes

H UMMHOCI A : HUMAN MHC CLASS II HLA-DCl-ALPHA GENE (DRWG.WG). MRNA.

HUMMHORS1 : HUMAN HLA-DR ALPHA-CHAIN (CHAIN-P34) GENE. EXON 1. HUMMHDRS2 : HUMAN HLA-DR ALPHA-CHAIN (CHAIN-P34) GENE. EXONS 2. 3. 4 AND 5. HUMMHDSA : HUMAN HLA-DS ALPHA-CHAIN MRNA. HUMMHDXA1 : HUMAN MHC CLASS II HLA-DX-ALPHA GENE (DR4.WG). EXON 1.

HUMHHDXA2 :HUMAN MHC CLASS II HLA-DX-ALPHA GENE (DR4.WG). EXON 2.

Moυse Imm unoqlobul i ne S equences

MUSIGE1 :MOUSE IG GERMLINE EPSILON GENE AND SECRETED TAIL.

MUSIGASANB :MOUSE IG ALPHA S REGION FROM MG03 AFTER NON-PRODUCTIVE S-S.

MUSIGDJC07 :MOUSE IG GERMLINE D-J-C REGION: D-052. J-H GENES & S-MU. (PART A

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.

MUSIGESA :MOUSE IG EPSILON S REGION AFTER CLASS-SUITC-HING.

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.

Mouse MHC Gene Sequences

MUSMH :MOUSE MHC CLASS I TRANSPLANTATION ANTIGEN (HAPLOTYPE 0). MRNA.

MUSMHD3 :MOUSE MHC CLASS I GENE. C-TERMINAL HALF <HAPLOTYPE D>. 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.

MUSMHKB2 :MOUSE MHC CLASS I H2-K GENE ( HAPLOTYPE 8). EXONS 4-8.

MUSMHKBML :MOUSE MHC CLASS I H2-K GENE (HAPLOTYPE BML). AA 92-162. CLONE

MUSMHLD1 :MOUSE MHC CLASS I H2-L GENE (HAPLOTYPE D). EXONS 1-3.

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. Where 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. Where 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.

After the selected B'-containing gene fragment has been isolated, 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. For immunoglobulin genes, the 5' cleavage site is in the IS2 intron between the gene's variable and constant regions. For class I or class II MHC genes, 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, α or β gene, in the intron between exons 2 and 3.

Example I below describes the construction of a cassette vector whose B' coding region is derived from human germ-line κ gene. The construction of a cassette vector whose B' region is derived from the constant-coding region of a human germ-line γ4 immunoglobulin chain gene is detailed in Example III. Example V below describes the construction of a cassette vector which carries, as the B' coding region, the major portion of intron 2 for the murine I-Eα 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.

To introduce the B' coding fragment into the original plasmid (pPL in Figure 1), 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. Where the selectable marker is a suppressor-tRNA gene, 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. Such a selection system is described in Examples I, III, and V. The plasmid obtained is designated pB' in Figure 1.

In a second general step in the construction of the cassette vector, an intron gene fragment is introduced into the plasmid adjacent the B' coding fragment. According to an important feature of the invention, 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. Generally, 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. Preferably, 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 λ and ic genes, where A and B are variable- and constant-coding regions, respectively. b. The family of human immunoglobulin heavy-chain genes, including the several known α, δ, ε, μ, and γ genes, where A and B are variable and constant coding regions. c. The corresponding families of murine light- and heavy-chain genes; d. The families of individual human class I and class II histocompatibility genes, where A includes one or more of the upstream exons coding for polymorphic regions of the histocompatibility antigen and B includes the remaining downstream exons of the gene. In the immunoglobulin-chain gene families, 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. In the class I and II hisocompatibility genes, 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 κ gene, and 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 μ 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-α chain obtained in accordance with reference 13. With continued reference to Figure 1, 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.

II. Preparing a Hybrid Gene The use of the cassette vector in preparing a hybrid gene is illustrated in Figure 2. In the usual method for constructing a hybrid gene, a genomic DNA library of DNA fragments cloned in a suitable vector, such as λ phage, is prepared from a source which contains the A_i-B_i gene which will contribute its

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 λ phage, such as a Charon 4A phage, containing A^am B^am structural genes necessary for λ packaging and growth. In the absence of a suppressor tRNA gene, the λ structural genes code for incomplete proteins and the phage is unable to form plaques when plated on a suppressor-minus host. When the phage incorporates the cassette vector by a recombination event, 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. Although the recombination-selection method will be described herein with particular reference to a phage-λ/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 only constraints in the system are that: (1) recombination between the cassette vector and the cloning vector occurs only in the cloned library genes containing an intron region homologous to to that in the cassette vector, and (2) such recombination event leads to incorporation into the cloning vector of a selectable marker which permits selection, in a selective medium, of only those cloning vectors which have undergone the recombination event leading to hybrid gene formation. Procedures for infecting host cells by phage λ, by preabsorbing the phage on the cells, are described, for example, in reference 18. Phage λ library vectors are initially passaged on a recombination-positive, suppressor-positive host. In this host, 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. As seen in Figure 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.

After initial passage, 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. in a particularly preferred embodiment of the present invention, the λ 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. These two identical restriction sites, for example both NotI, are inserted at either end of the λ 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). Preferably, 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. When the source of genomic DNA is digested with the frequently recognized enzyme and inserted into the phage at the corresponding restriction site(s) of the stuffer, the resulting genomic insert is flanked proximally by the common restriction sites and flanked distally by the rare restriction site. A in Figure 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 advantage of having the rare restriction site in the vector containing the library arises after the appropriate genomic clone has reσombined with a cassette vector containing the same rare restriction site. With reference to Figure 1, 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. Thus, in pIB' of Figure 1, the restriction site would be located in the segment between sup and I. (See also NotI site in Figures 13 and 14.) When pIB', containing the rare restriction site as described, recombines into the λ genomic clone (flanked by the rare restriction site) as shown in Figure 2, the resulting λ has three of the rare restriction sites, one located between sup and the genomic I, and two flanking the genomic insert (distal ends of A_i and B_i). By cutting the phage with the rare restriction site and circularizing at low concentration, one can obtain a plasmid from the -A_i-I-B'-ori-sup- segment, which can then be easily isolated from the -I-B_i-λ-and -A^am-B^am-λ- segments. Removal of the segment containing the hybrid gene prevents the desired phage from losing the inserted cassette by recombination between the two I regions. A preferred λ vector for library construction is λGLla described in Example VIII and shown in Figure 12. The use of this λ 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.

The gene-preparation method just described is generally applicable to forming any hybrid gene of the type A_i-B' where:

(a) A_i is a polymorphic coding region derived from an A_i-B_i gene in a family of A-B genes;

(b) B' is derived from a selected A'-B' gene;

(c) A_i and A and B_i and B' are structurally and functionally homologous gene regions; and

(d) 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.

By way of examples, a cassette vector whose B' coding region is a human immunoglobulin κ-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 κ- and λ-variable/human-κ hybrid genes. Similarly, a cassette vector whose B' coding egion is a human γ₁-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 α, δ, ε, γ₁, γ₂, γ₃, γ₄, and μ/human constant γ₁ hybrid genes. Where the latter cassette vector contains, instead of a murine intron gene fragment, an intron gene fragment from one of the family of human heavy-chain genes, the vector may be used in preparing human variable α, δ, ε. γ₁, γ₂, γ₃, γ₄ , and μ/human constant region hybrid genes. Examples II and IV describe the construction of mouse-variable/human-constant light- and heavy-chain hybrid genes, respectively.

As an example of a hybrid histocompatibility gene which can be prepared according to the invention, a cassette vector which contains exons 3-5 of a murine I-Eα gene and an intron gene fragment from the IS2 of a human DR-α gene can be used in preparing each of the family of human-polymorphic DR-α/mouse I-Eα hybrid genes. The construction of such a gene is illustrated in Example VI.

Ill. Expressing the Hybrid Gene

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. Since the enhancer element in the hybrid gene of the present invention is typically acquired from the cassette vector, and derived from a gene of the same family as the gene which contributes the variable region in the hybrid gene, 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. Thus, in expressing hybrid mouse-variable/ human-constant region genes, a mouse cell system, such as a plasmacytoma, hybridoma, or myeloma cell line will be preferred.

For the expression of hybrid MHC genes, 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. In this application, a hybrid α or β immune response gene is transfected into mouse lymphocytes. Expression of the hybrid α or β chain leads to association of that chain with the complementary mouse α or β 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). With any transfection method used, 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). 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. Similarly, 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.

The following examples illustrate various vector constructions and hybrid gene preparations, made according to the invention, but are in no way intended to limit the scope of the invention.

Examples

Applicable Methods

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 μg of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 μl of buffer solution; in the examples herein, typically, an excess of restriction enzyme is used to ensure complete digestion of the DNA substrate.

Incubation times of about one hour or two hours at about 37°C are workable, although variations can be tolerated. After each incubation, protein is removed by extractions with phenol/chloroform, and may be followed by ether extraction, and the nucleic acid recovered from aqueous fractions by precipitation with ethanol. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in reference 26.

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 MgCl₂, 6 mM DTT and 5-10 μM dNTPs. The Klenow fragment fills in at 5' sticky ends and chews back protruding 3' single strands, even though the four dNTPs are present. If desired, 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. After treatment with Klenow, 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 μl volumes under the following standard conditions and temperatures: 50 mM Tris-Cl, pH 7.5, 10 mM MgCl₂, 10 mM DTT, 100 μg/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 μg/ml total DNA concentrations (5-100 nM total end concentration). Intermolecular blunt end ligations are performed typically at about 1 μM total ends concentration. in vector construction employing "vector fragments", 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 μg of vector, depending on size, at 37°C for about one hour. In order to recover the nucleic acid fragments, 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.

In constructions set forth in the examples below, correct ligations for plasmid construction are confirmed by first transforming E. coli strain MC1061(P3) obtained from Dr. Brian Seed. Molecular Biology, Mass General Hospital, Boston. MA 02114. Successful transformants are selected by ampicillin, tetracyline, and kanamycin antibiotic resistance. Plasmids from the transformants are then prepared according to reference 6. Vector structures are confirmed by restriction analysis. Selection for λ having a sup tRNA acquired by recombination with the cassette vector is carried out in suppressor-minus E. coli strain LG-75, also obtained from Dr. Seed. Bacterial strains W3110r-m⁺(p3) and WR110r-m⁺, Su-, available from Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, may also be used (reference 28).

Example I Constructing a Mouse/Human κ-Chain Cassette Vector This example describes the construction of a plasmid designed for use in preparing mouse/human κ-chain genes.

Human germline κ 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 κ-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_κ was isolated by electrophoresis and elution from an agarose gel.

A πanl3 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. The plasmid, seen at B in

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_κ fragment from pHuC_κ and the 0.9 kb linearized π 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 πanl3 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 π plasmid.

Successful transformants were selected using standard methods on LB plates containing 50 μg/ml kanamycin, 15 μg/ml tetracycline, and 25 μg/ml ampicillin. Minilysate plasmid preparations were analyzed by digestion with Sad to select plasmids with the 2.5 kb C_κ fragment in the proper orientation. A restriction map of the selected plasmid, designated πC_κ, is shown at C in Figure 3, with the C_κ insert and adjacent polylinker segment shown in expanded scale.

An IS2 gene fragment from a mouse κ gene was derived from a pBHC cloning vector containing a Hindlll/BamHI segment of mouse κ gene contained in a pBR322 plasmid (reference 12). A map of the κ-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 κ-gene IS2 region was purified by eleσtrophoresis and elution from agarose gel.

The πC_κ 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 π-plasmid fragment was purified by electrophoresis and elution from agarose gel. This fragment was ligated to the 1 kb Hindlll/XmnI fragment from pBHC_κ with T4DNA ligase, and the ligation products were used to transform

MC1061(P3). Successful transformants were selected as above. The expected structure of the light gene cassette is illustrated at E in Figure 3.

The final structure of the light gene cassette, designated πLC_κ, was confirmed by digestion with

EcoRI and by double digestion with Xbal and Hindlll. The restriction fragments from the two digestions were each fractionated by gel electrophoresis and visualized with ethidium bromide staining, conventionally. The pattern of EcoRI fragments is shown in lanes 2-4 in Figure 4A. Lane 1 shows patterns of size markers of HindiII fragments of wild type λ DNA. The 2.5 kb and 1.9 kb fragments correspond to the correct sizes of the EcoRI fragments expected from the πLC_κ construct shown in Figure 3. The Xbal/Hindlll fragments were similarly fractionated, with the results shown in lanes

2-4 of Figure 4B. Lane 1 again represents Hindlll fragments of wild type λ DNA. The 2.4 kb and 2.0 kb fragments also correspond to the expected sizes from the πLC_κ construct shown in Figure 3.

A NotI site was then introduced into plasmid πLC_κ. First, 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 πLC_κ.NotI (Figure 13), was recovered.

Example II Constructing a Mouse/Human Hybrid κ Gene A Charon 4A phage, designated ChC_κ-1 containing an EcoRI κ-gene insert from a Balb/c mouse germline cell, was obtained (reference 30). As shown at the top in Figure 5, the vector contains amber-mutated A and B phage genes, and the EcoRI κ-gene insert extends from a position upstream from the 3' end of the gene through a portion of the constant C_κ region. MC1061 host cells harboring either the P3 plasmid alone, designated MC1061(P3), or harboring both the P3 plasmid and the π-plasmid cassette from Example I, designated MC1061(P3) (πLC_κ) were preabsorbed with 10⁶ ChC_κ-1 phage and plated on LB plates. No plaques were obtained with MC1061(P3), the negative control, demonstrating that the ChC_κ-1 phage stock contained no wild type revertants. Confluent lysate was obtained with the MC1061(P3) (πLC_κ) cells. The titer of the plate lysate on the Sup ⁺ cells was 1.5 x 10⁹ phage/ml.

After initial passage, the expanded phage population was repassaged on a suppressor-minus host strain LG75. Reσombinant 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. As seen, the recombination event has produced a hybrid human κ-coding region upstream of the site of recombination and a mouse κ-coding region downstream of the recombination site. The titer on the plate lysate for the suppressor-minus strain was 4 x 10⁶ 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 ³²P-labeled πanl3 probe, with the results shown in Figure 6. Lane 1 shows the autoradiogram pattern for ChC_κ-1 DNA, and lanes 2-4 show recombinant phage DNAs selected from three different plaques. As seen, 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 λ, to give the 2.7 EcoRI fragment indicated. The 5 kb and 2.7 kb Hindlll fragments were also hybridized to the J_κ probe (Figure 19).

Example III Constructing a Mouse/Human γ4 Cassette Vector This example describes the contruction of a plasmid designed for use in preparing hybrid mouse/human γ4 genes.

Human genomic γ4 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 γ4 constant-coding region, indicated at Cγ4 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 πanl3 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 Cγ4 fragment from p24BRH and the 0.9 kb linearized π 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.

Successful transformants were selected using standard methods on LB plates containing 50 μg/ml kanamycin, 15 μg/ml tetracycline, and 25 μg/ml ampicillin (reference 6). Plasmid minipreps were analyzed by single digestion with Xbal, PstI and Smal to select plasmids with the 2.0 kb Cγ4 fragment in the proper orientation. A restriction map of the selected plasmid, designated ττCγ4, is shown at the center portion of Figure 7, with the Cγ4 insert and adjacent polylinker segment shown in expanded scale.

An IS2 gene fragment was derived from a segment of a mouse Cμ gene containing a heavy-chain enhancer, E_H. A map of the μ-gene segment is shown in Figure

7. A Ch4A phage carrying germ-line Cμ 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 πCγ4 plasmid was digested with Xbal, and the linearized π-plasmid fragment was purified by electrophoresis and elution from agarose gel. This fragment was ligated to the 1 kb Xbal μ-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. The final structure of the cassette vector, designated πHCγ4, 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 πHCγ4 by the procedure described in Example I. A plasmid, designated ττHCγ4.NotI (Figure 14), was recovered.

Example IV

Constructing a Mouse/Human Hybrid μ Gene A Charon 4A phage vector, designated ChCμ27, containing a mouse μ-gene insert from mouse cells, is obtained as described in reference 32. The phage vector, which is shown at the top in Figure 9, contains amber-mutated phage genes, and the μ-gene insert extending from a position upstream of the 3' end of the gene through the constant Cμ region. A MC1061 host cells harboring both the P3 plasmid and the π-cassette plasmid from Example III, designated

MC1061(P3) (πHCγ4), is preabsorbed with 10⁶ ChCμ27 phage and plated on LB plates.

After initial passage, 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. As seen, the recombination event produces a hybrid gene with murine J_H coding regions upstream of the IS2 recombination site and human γ4-gene coding regions downstream of the recombination site. The plaques from the LG75 plate are replated on

LG75 agarose plates, and a few plaques are picked. The

DNA from recombinant phages are analyzed by digesting with EcoRI and hybridized to a ³²P-labeled πanl3 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-Eα 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-Eα gene fragment, containing exon 2 through the 3' end of the gene is shown at A in Figure 10. Alternatively, a genomic DNA library of mouse-tissue DNA, generated by partial Sau3a digestion and cloned into λ, could be screened with synthetic oligonucleotide probes made using the published sequence for the I-Eα gene to select the same gene, and be confirmed by limited sequence analysis.

A πanl3 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-Eα-gene vector from above is digested to completion with PstI and EcoRI and the 2.5 kb fragment and the 0.9 kb linearized π-plasmid fragment are ligated with T4 DNA ligase. The ligation products are used to transform E. coli strain MC1061 (P3). Successful transformants are selected on LB plates containing 50 μg/ml kanamycin, 15 μg/ml tetracycline, and 25 μg/ml ampicillin. Minilysate plasmid preparations are analyzed by digestion with EcoRI and PstI to select plasmids with the 2.5 kb I-Eα fragment. This plasmid is designated πI-Eα at C in Figure 10.

A human DR-α 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-α 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 πl-Eα plasmid is digested with EcoRI, phosphatased and the linearized π-plasmid fragment is purified by electrophoresis and elution from agarose gel. This fragment is ligated to the 280 bp linkered fragment from the DR-α 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 πDR/I-Eα, 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.

Example VI Constructing a Human PR/Mouse

I-A Hybrid Heavy-Chain Gene 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 λ 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 πDR/I-Eα cassette vector from Example V.

After initial passage, 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-α gene upstream of the IS2 recombination site, and exons 3-5 of a mouse I-Eα 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 ³²P-labeled DR-α exonl probe, to confirm the presence of the expected DR-α exonl fragment produced by the recombinant event. Example VII Construction of Phage Vector λGLla The following example describes the construction of λ GLla, an improved λ phage vector for the construction of gene libraries for use with the cassette vectors of the present invention.

DNA from phage vector λ EMBL 3 (reference 40; Promega Biotech, Madison, WI), which has the restriction map shown in Figure 12, was digested with BamHI and the 14 kb "stuffer" fragment purified. 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 γ- ³²P-ATP. The purified stuffer is then ligated to excess adapter AB at 22°C for 1 hr. Following ligation, excess adapter is removed by passing the reaction mixture through a column containing Bio-Gel A15m (BioRad). Phage λ vector Ch4a (reference 41) was obtained and digested with EcoRI to remove its stuffer fragment. Both arms of Ch4a were purified and then ligated under standard conditions to the EMBL 3 stuffer fragment with the NotI linkers. The desired recombinant λ vector has a restriction map as shown at the bottom of Figure 12. To isolate this construction, the ligation mixture was packaged and used to transform KM392, a lac- host derived from LE392 (reference 43), in the presence of X-gal medium (reference 36). 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 λGLla.

Example VIII Constructing a Mouse/Human γ₁ Cassette Vector

This example describes the construction of a plasmid designed for use in preparing hybrid mouse/human γ₁ genes. πanl3 (Example I) was linearized with Hindlll and a NotI site inserted as described for plasmid irLC_κ in Example I to give the structure B shown in

Figure 15. πanl3.NotI was then digested with Xba and ligated to the enhancer fragment E_H recovered from πHCγ4 (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 πE_H.NotI, shown as structure C in Figure 15, gave the expected fragment sizes of 330 and 1570 bp.

Plasmid 13AHP, containing human genomic Cγ₁ in pBR322 (reference 34) was obtained from Dr. Jay Ellison. After digesting 13AHP with Hindlll and PvuII, the 3.0 kb fragment containing Cγ₁ 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. πE_H.NotI was digested with Sst-1 and ligated to the Sst-1-digested Cγ1 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 Cγ1 in the proper orientation. The orientation was tested by digestion with PstI alone, and with a combination of PstI and EcoRI. A plasmid designated πHCγ1.NotI, shown as structure D in Figure 15, gave fragments of the expected size after digestion: PstI - 400, 1500, 1850, and 1050 bp; PstI + EcoRI - 400, 300, 1200, 1850, 150, and 900 bp.

Example IX

Constructing a Mouse/Human γ3 Cassette Vector This example describes a construction of a plasmid designed for use in preparing mouse/human γ3 genes. λ phage Ch4a.H.Igγ122 (reference 39) was obtained from Dr. Tasuku Honjo of Kyoto University, Japan. The phage, containing the human Cγ3 region, is shown as structure A in Figure 16. Phage DNA was digested with Hindlll and EcoRI and the 3.2 kb fragment containing Cγ3 was eluted. This fragment was hybridized with a human Cγ4 probe to confirm that the 3.2 kb fragment contains the Cγ3 coding sequence. The sticky ends of the fragment were then trimmed back. This fragment is then ligated into πanl3 (Example I), which had previously been linearized with SacI, sticky ends trimmed and the 5' ends dephosphorylated. ττanl3 containing the Cγ3 coding sequences was used to transform MC1061(P3) cells and transformants selected to determine the orientation of the insert.

A plasmid designated πCγ3-14 was obtained with the Cγ3 insert in the wrong orientation. ττCγ3-14 aquired an unexpected additional EcoRI site. The orientation of the Cγ3 insert was determined by double digestion with Hindlll + Bglll, which gave the following size fragments: Hindlll + Bglll - 1800 and 2300 bp. τrCγ3-14 then was digested with EcoRI and religated to reorient the Cγ3 segment.

Plasmids from transformants were then tested again for correct orientation. A plasmid designated ττCγ3, structure C in Figure 16, gave fragments of the correct size: Hindlll + Bglll - 900 and 3200 bp. ττCγ3 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 πHCγ3, 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 πHCγ3 as described in Example I. This gave a plasmid designated ττHCγ3.NotI, shown as structure E in Figure 16.

Example X Constructing a Human Isotype

Switching Cassette Vector for IgM to IgG₃ This example describes the construction of a plasmid designed for use in changing the isotype of a human immunoglogulin heavy chain gene. In this example, the cassette vector is useful for changing the isotype of an IgM antibody to IgG₃.

A clone of the human germ line immunoglobulin heavy chain gene (structure A in Figure 17) was obtained in plasmid PJ shown as structure B in Figure 17. (References 33 and 35.) Plasmid PJ is digested with Bglll and Hindlll, and the 0.75 kb fragment, which contains the intron IS, is purified. Plasmid πan7, obtained from Dr. Brian Seed (reference 6) and shown as structure C in Figure 17, was digested with Bglll and Hindlll. The larger of the linear fragments was circularized by ligation with the IS fragment after dephosphorylating the 5' ends. The ligation mixture was used to transform MC1061(P3) cells and a plasmid designated pHC-1 was recovered (structure D in Figure 17). The Cγ3-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 plasmid designated πSWMG3, structure E in Figure 17, gave fragments of the correct size: 1600 and 3200 bp. A NotI site (adapter AB, Example I) was ligated between the EcoRI and BamHI sites of ττSWMG3 to give a plasmid designated ττSWMG3.NotI, depicted as structure F in Figure 17.

Example XI Constructing a Human Isotype Switching

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 IgG₁. 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 Cγ3 fragment into pHC-1, the 3.0 kb Cγ1 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 πSWMGl.NotI (structure B in Figure 18). Example XII

Constructing an Anti-Leu3

Mouse/Human Light-Chain Immunoglobulin

Several probes were used in the procedures described below. Figure 19 shows the restriction map of the J_κ-E_κ-C_κ-region of an unrearranged mouse gene. (Reference 45.) J_κ probe and -J_κ probes are the 2.7 kb and 0.7 kb Hindlll fragments, respectively. The J_κ probe is present in both the unrearranged immunoglobulin gene, as well as normally rearranged immunoglobulin genes. The -J_κ 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_μ- region of the mouse unrearranged heavy chain immunoglobulin gene.

(Reference 46.) The 1.7 kb BamH-EcoRI fragment was used as the J_H probe.

This example shows the use of the πLC_κ.NotI cassette vector (Example I, Figure 13) and a library from an anti-Leu3 murine hybridoma in λGLla (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_κ 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_κ probe (Figure 19). The

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 λGLla DNA and packaged. The number of recombinant phage was estimated by Spi selection (reference 42). 1.2 x 10⁶ 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

TTLC_κ.NotI, where, presumably, there occurred a recombination between the phage containing the rearranged active murine light chain gene and the light chain cassette (Structures A and B in Figure 21.) The resulting phage was selected by being titered on both E. coli LG-75 (SupF-) and KM392. The ratio of titer on

LG-75 to KM392 was 10^-5 to 10^-7.

Phage were then screened by hybridization to the J_κ probe. Eleven sublibraries of the twelve were positive. J⁺ _kSupF phage was then plaque purified, and then hybridized to the J_κ, human C_κ (Figure 3A), and -J_κ probes to eliminate unrearranged clones. Three of the eleven sublibraries belonged to unrearranged light chain genes as indicated by hybridization to probe -J_κ. All of the remaining eight phage clones hybridized to J_κ and human _Cκ 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

J_κ, human C_κ and SupF. About 0.5 μg of phage DNA was digested with NotI and ligated at low DNA concentration (reference 37; structures B and C in

Figure 21). Competent MC1061(P3) cells were then transformed with each circular π plasmid DNA.

Transformants were picked on duplicate nitrocellulose filters, which were hybridized to J_κ and human

C_κ probes. Over 95% of the colonies contained both probe sequences. A plasmid designated πLeu-3.LC is selected, and by restriction enzyme analysis and hybridization with the appropriate probes is demonstrated to have structure D shown in Figure 21.

Deposit of Biological Materials

The following materials were deposited on 14 March 1986 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland

20852, U.S.A. in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure:

ATCC

Material Accession No.

λGLla

E. coli MC1061(P3)(πLC_κ.NotI) 67034

E. coli MC1061(P3) (πHCγ1.NotI) 67035

E. coli MC1061(P3) (πHCγ3.NotI) 67036

E. coli MC1061(P3)(πHCγ4.NotI) 67037

E. coli MC1061(P3)(πSWMGl.NotI) 67038

E. coli MC1061(P3)(πSWMG3.NotI) 67039 While particular applications and embodiments of the invention have been described herein, it will be readily apparent to those in the art how the general method used for preparing hybrid immunoglobulin chain and histocompatibility antigen genes can be applied to other hybrid-gene applications where families of polymorphic genes are involved. Since modifications of the above embodiments will be apparent to those of skill in the art, it is intended that the scope of the present invention be defined solely by the appended claims.

Claims

IT IS CLAIMED:

1. A cassette vector for use in constructing a hybrid A_i-B' gene where: (a) A_i is a polymorphic coding region derived from an A_i-B_i gene in a family of polymorphic A-B genes;

(b) B' is derived from a selected A' -B' gene;

(c) A_i and A' and B_i and B' are structurally and functionally homologous gene coding regions; and

(d) The A and B coding regions and the A' and B' coding regions are separated by introns I and I'. respectively; said vector comprising a selectable-marker gene which permits selection in a host of a cloning vector which acquires the marker gene by recombination with the cassette vector, the B' coding region, and a gene fragment derived from intron I in one of the genes in the A-B gene family, the 3' end of said gene fragment being adjacent to the 5' end of said B' coding region.

2. The vector of claim 1, for use with a λ phage cloning vector containing two amber-mutated genes whose expression allows phage selection in a suppressor-minus host, wherein said selectable-marker gene is a suppressor-tRNA gene.

3. The vector of claim 1, for use in constructing a hybrid immunoglobulin chain gene composed of mouse-variable and human-constant coding regions, wherein B' includes the constant coding region from a human immunoglobulin gene, and said gene fragment is derived from the IS2 intron of a mouse immunoglobulin gene.

4. The vector of claim 3, wherein the intron gene fragment includes a transcription enhancer element.

5. The vector of claim 3, wherein the intron gene fragment is derived from the IS2 region upstream of the switching signal in an unrearranged immunoglobulin gene.

6. The vector of claim 1, for use in constructing a hybrid light-chain immunoglobulin chain, wherein B' includes the constant-coding region of a human κ or λ. gene, and said gene fragment is derived from the IS2 region of a mouse κ or λ gene.

7. The vector of claim 6 that is plasmid πLC_κ.NotI.

8. The vector of claim 1, for use in constructing a hybrid heavy-chain immunoglobulin chain, wherein B' includes the constant-coding region of a human α, δ, ε, γ ₁-γ₄, or μ gene, and said gene fragment is derived from the IS2 region of a mouse heavy-chain gene.

9. The vector of claim 8 that is a plasmid selected from the group consisting of πHCγ1.NotI, πHCγ3.NotI and πHCγ4.NotI.

10. The vector of claim 1, for use in constructing a hybrid heavy-chain immunoglobulin gene composed of human-variable and constant-coding regions from different heavy-chain classes, wherein B' includes the constant-coding region of a human heavy-chain immunoglobulin gene of a selected class, and said gene fragment is derived from the IS2 region of a human heavy-chain gene, in the region upstream of the switch signal of the unrearranged gene.

11. The vector of claim 10, for use in constructing a hybrid gene containing a μ variable region and a γ constant region gene, wherein B' includes the constant-coding region from a human γ gene.

12. The vector of claim 11 that is a plasmid selected from the group consisting of πSWMG1.NotI and πSWMG3.NotI.

13. The vector of claim 1, for use in constructing a hybrid class I or II MHC gene composed of human polymorphic and mouse non-polymorphic coding regions, where B' includes one or more of the non-polymorphic exons from a murine MHC gene and said intron gene fragment is derived from the intron just upstream of the analogous non-polymorphic exon(s) from the corresponding human MHC gene.

14. The vector of claim 13, for use in constructing a human/mouse class II MHC hybrid gene, where B' includes exons 3-5 of a murine I-Aα. I-Aβ, I-Eα, or I-Eβ gene, and said intron gene fragment is derived from the IS2 region of a homologous DR, DC, or SB human α or β gene.

15. The vector of claim 1 that is a plasmid which further comprises a rare restriction site that is cut by an enzyme that rarely cuts genomic DNA from the cell source of the A_i-B_i gene wherein said rare restriction site is located adjacent the 5' end of said gene fragment derived from the intron I and neither said plasmid's origin of replication nor said selectable marker gene being located between said rare restriction site and said gene fragment derived from intron I.

16. The vector of claim 15 wherein said rare restriction site is selected from the group consisting of NotI, Sfil and Xhol.

17. The vector of claim 15 wherein said rare restriction site is NotI.

18. A method of producing a hybrid A_i-B', where

(a) A_i is a polymorphic coding region derived from an A_i-B_i gene in a family of A-B genes; (b) B' is a substantially non-polymorphic coding region derived from a selected A'-B' gene;

(c) A_i and A' and B_i and B' are structurally and functionally homologous gene coding regions; and (d) the A and B coding regions and the A' and

B' coding regions are separated by introns I and I', respectively; said method comprising providing a cassette vector containing a selectable-marker gene which permits selection in a host of a cloning vector which acquires the marker gene by recombination with the cassette vector, the B' coding region, and a gene fragment derived from intron I in one of the genes in the A-B gene family, the 3' end of said gene fragment being adjacent to the 5' end of said B' coding region, preparing a genomic PNA library of cloned gene fragments from a cell source containing such A_i-B_i gene, introducing the cassette vector and cloned library fragments into a recombination-permissive host, and selecting library fragments containing said selectable marker gene.

19. The method of claim 18, wherein the cassette vector is provided with a suppressor-tRNA selectable-marker gene, the library gene fragments are cloned in a λ phage cloning vector having an amber-mutated structural protein, and said selecting is based on phage plaque formation in a suppressor-minus host.

20. The method of claim 19 wherein: the cassette vector contains a selected restriction site that is rare in the PNA from said cell source of A_i-B_i and is located adjacent to the 5' end of said intron I gene fragment with neither said selectable-marker gene nor the cassette vector's origin of replication located between said rare restriction site and the 5' end of said intron I gene fragment; and said λ phage cloning vector contains (i) a pair of identical restriction sites that occur more frequently in the DNA from said cell source of A_i-B_i than said rare restriction site and that flank proximally said library gene fragments, and (ii) a pair of said rare restriction sites that flank distally said library gene fragments.

21. The method of claim 20 wherein said frequently occurring restriction sites are cut by the enzyme used to digest the genomic DNA from said cell source in said library preparation step.

22. The method of claim 20 wherein said frequently occurring restriction sites are cut by an enzyme selected from the group consisting of EcoRI, Hindlll and BamHI.

23. The method of claim 20 wherein said rare restriction site is cut by an enzyme selected from the group consisting of NotI, Sfil and Xhol.

24. The method of claim 20 wherein said rare restriction site is cut by the enzyme NotI.

25. The method of claim 22 wherein said rare restriction site is cut by the enzyme NotI.

26. The method of claim 20 wherein said λ phage cloning vector is λGLla.

27. The method of claim 18, for use in constructing a hybrid immunoglobulin gene having selected variable and constant-coding regions, wherein the cassette vector is provided with a B' coding region containing such selected constant-coding region, and an intron gene fragment derived from an immunoglobulin gene in the family of immunoglobulin genes containing such selected variable coding region, and the DNA library provided is derived from a source containing the selected variable coding region.

28. The method of claim 27, for use in constructing a gene having mouse variable and human hybrid immunoglobulin gene coding regions and adapted to code for a chain in an antibody which is specific against a selected antigen, wherein the B' coding region is derived from a selected human immunoglobulin constant region, and the DNA library provided is derived from a mouse hybrid cell line capable of producing monoclonal antibodies specific against the selected antigen.

29. The method of claim 28 wherein said cassette vector is a plasmid selected from the group consisting of πHCγ1.NotI, πHCγ3.NotI, πHCγ4.NotI and πLC_κ.NotI.

30. The method of claim 27, for use in constructing a hybrid heavy-chain immunoglobulin gene having a variable coding region from a first heavy-chain class and a constant coding region from a second, different heavy-chain class, wherein the cassette vector is provided with a B' coding region containing a human immunoglobulin heavy-chain gene of such second class and an intron gene fragment derived from the IS2 region of a human heavy-chain gene, in the region upstream of the switch signal of an unrearranged gene, and the DNA library provided is derived from a cell source containing a variable coding region from the gene of such first class.

31. The method of claim 30 wherein said cassette vector is a plasmid selected from the group consisting of πSWMG1.NotI and πSWMG3.NotI.

32. The method of claim 27, wherein the cassette vector is provided with an intron gene fragment containing an enhancer element.

33. The method of claim 18, for use in constructing a hybrid human polymorphic/mouse non-polymorphic MHC gene, wherein the cassette vector is provided with a B' coding region containing one or more of the substantially non-polymorphic exons from a selected mouse MHC gene, the intron gene fragment is derived from the intron just upstream of the analogous exon(s) in the corresponding human MHC gene, and the DNA library is derived from a human cell containing human polymorphic gene regions from the corresponding human MHC gene.

34. The method of claim 33, for use in constructing a human/mouse class II MHC hybrid gene, wherein the cassette vector is provided with a B' coding region. containing exons 3-5 of a murine I-Aα, I-Aβ, I-Eα, or I-Eβ gene, and the intron gene fragment is derived from the IS2 region of a homologous DR, DC. or SB human α or β gene.

35. A hybrid A_i-B' gene comprising a polymorphic A_i coding region derived from an A_i-B_i gene in a family of polymorphic A-B genes, a substantially non-polymorphic B' coding region derived from a selected A'-B' gene, where A_i and A' and B_i and B' are structurally and functionally homologous gene coding regions and the A and B coding regions and the A' and B' coding regions are separated by introns I and I', respectively, and separating the A_i and B' coding regions, a composite intron composed of an upstteam segment derived from the A_i-B_i gene immediately 3' of the A_i coding region, an intermediate segment derived from another gene in the A-B gene family, and a downstream segment derived from A'-B' gene immediately 5' of the B' coding region.

36. The hybrid gene of claim 35, wherein A_i and B' are the variable and constant coding regions, respectively, and are from difference heavy-chain or different light-chain immunoglobulin genes.

37. The gene of claim 36, wherein the intermediate segment contains a transcription enhancer element.

38. The gene of claim 36, wherein the intermediate segment is derived from an immunoglobulin gene IS2, upstream of the switch signal in the unrearranged gene.

39. The gene of claim 35, wherein A_i contains one or more of the polymorphic exons of a human HLA gene, and B' includes the complementary, substantially non-polymorphic exons of a corresponding mouse MHC gene.

40. The gene of claim 39, wherein A_i includes the exons 1 and 2 from a human DR, DC, of SB human α or β gene, and B' includes exons 3-5 of a murine I-Aα. I-Aβ, I-Eα, or I-Eβ gene.

41. The gene of claim 35 wherein A_i is from a murine gene and B' is from a human gene.

42. The gene of claim 36 wherein A_i is from a murine gene and B' is from a human gene.

43. The gene of claim 37 wherein A_i is from a murine gene and B' is from a human gene.

44. The gene of claim 38 wherein A_i is from a murine gene and B' is from a human gene.

45 The gene of claim 42 wherein A_i and B' are coding regions from light-chain immunoglobulin genes.

46. The gene of claim 42 wherein A_i and B' are coding regions from heavy-chain immunoglobulin genes.

47. The gene of claim 45 wherein said light-chain gene in the κ chain gene.

48. The gene of claim 40 wherein A_i includes exons 1 and 2 froma human DR-α coding region and B' includes exons 3-5 of a murine I-Eα.

49. A λ phage cloning vector comprising two phage DNA arms flanking a stuffer DNA fragment, said stuffer DNA fragment flanked (i) proximally by a first pair of restriction sites that is cut by a single enzyme which is suitable to digest murine or human genomic DNA to form a gene library, and (ii) distally by a second pair of restriction sites that are cut by a single enzyme which rarely cuts murine or human genomic DNA, both said first and second pair of restriction sites being flanked by said phage DNA arms.

50. The λ phage cloning vector of claim 49 wherein said first pair of restriction sites is cut by an enzyme selected from the group consisting of EcoRI, Hindlll and BamHI.

51. The λ phage cloning vector of claim 49 wherein said second pair of restriction sites is cut by an enzyme selected from the group consisting of NotI, Sfil and Xhol.

52. The λ phage cloning vector of claim 50 wherein said second pair of restriction sites is cut by an enzyme selected from the group consisting of NotI, Sfil and Xhol.

53. The λ phage cloning vector of claim 49 wherein the enzyme that cuts said first pair of restriction sites is EcoRI and the enzyme that cuts said second pair of restriction sites NotI.

54. The λ phage cloning vector of claim 49 that is λGLla.