U.S. patent application number 10/412672 was filed with the patent office on 2004-05-13 for antigenic constructs of major histocompatibility complex class i antigens with specific carrier molecules, the preparation and use thereof.
This patent application is currently assigned to Dr. Klaus Bosslet. Invention is credited to Bosslet, Klaus, Sedlacek, Hans Harald, Seeman, Gerhard.
Application Number | 20040091488 10/412672 |
Document ID | / |
Family ID | 6359731 |
Filed Date | 2004-05-13 |
United States Patent
Application |
20040091488 |
Kind Code |
A1 |
Seeman, Gerhard ; et
al. |
May 13, 2004 |
Antigenic constructs of major histocompatibility complex class I
antigens with specific carrier molecules, the preparation and use
thereof
Abstract
Antigenic constructs which result from linkage of major
histocompatibility complex (MHC) class I antigens with specific
carrier molecules are described. The linkage is effected N- or
C-terminally by covalent bonding or, in the case of non-covalent
bonding, for example by an avidin/biotin bridge. The specific
carrier molecules bind selectively to target cells and are
preferably monoclonal antibodies. Processes of genetic manipulation
for the preparation of such constructs are indicated. Antigenic
constructs according to the invention are used to damage or
eliminate target cells.
Inventors: |
Seeman, Gerhard; (Marburg,
DE) ; Bosslet, Klaus; (Marburg, DE) ;
Sedlacek, Hans Harald; (Marburg, DE) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER
LLP
1300 I STREET, NW
WASHINGTON
DC
20005
US
|
Assignee: |
Dr. Klaus Bosslet
|
Family ID: |
6359731 |
Appl. No.: |
10/412672 |
Filed: |
April 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10412672 |
Apr 14, 2003 |
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08460569 |
Jun 2, 1995 |
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6548067 |
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08460569 |
Jun 2, 1995 |
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07912677 |
Jul 14, 1992 |
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07912677 |
Jul 14, 1992 |
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07385532 |
Jul 26, 1989 |
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Current U.S.
Class: |
424/178.1 ;
530/350; 530/391.1 |
Current CPC
Class: |
C07K 2319/02 20130101;
C07K 16/00 20130101; A61K 38/00 20130101; A61K 47/6898 20170801;
C07K 2319/00 20130101; C07K 14/70539 20130101; C07K 17/02 20130101;
B82Y 5/00 20130101 |
Class at
Publication: |
424/178.1 ;
530/391.1; 530/350 |
International
Class: |
A61K 039/395; C07K
014/74; C07K 016/46 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 28, 1988 |
DE |
P 3825615.0 |
Claims
1. Antigenic constructs in which major histocompatibility complex
(MHC) class I antigens are linked at the C- or N-terminal end to
specific carrier molecules.
2. Antigenic constructs in which major histocompatibility complex
(MHC) class I antigens are linked at the amino-terminal end to
specific carrier molecules.
3. Antigenic constructs in which major histocompatibility complex
(MHC) class I antigens are linked at the C-terminal end to specific
carrier molecules.
4. Antigenic constructs as claimed in claim 1, in which one MHC
class I antigen is linked to one specific carrier molecule
respectively.
5. Antigenic constructs as claimed in claim 1, in which the
specific carrier molecules are CD4 domains.
6. Antigenic constructs as claimed in claim 1, in which the
specific carrier molecules are monoclonal antibodies.
7. Antigenic constructs as claimed in claim 6, in which the
monoclonal antibodies are shortened in the constant part of the
heavy chain.
8. Antigenic constructs as claimed in claim 1, in which the MHC
class I antigen is HLA B27w or HLA B27k.
9. Antigenic constructs as claimed in claim 1, in which the MHC
class I antigen is covalently bonded to the carrier molecule.
10. Antigenic constructs as claimed in claim 1, in which the MHC
class I antigen is bonded via avidin/biotin to the carrier
molecule.
11. Antigenic constructs as claimed in claim 1, which are prepared
by genetic manipulation by fusion of the DNAs coding for them.
12. A process for the preparation of antigenic constructs as
claimed in claim 1, which comprises the required parts of genes
being fused in the form of their DNA, being provided with suitable
regulation sequences and being expressed in suitable expression
systems.
13. The use of the antigenic constructs as claimed in claim 1 for
the allogenization of target cells.
14. A pharmaceutical which contains antigenic constructs as claimed
in claim 1.
Description
[0001] The invention relates to antigenic constructs resulting from
the linkage of major histocompatibility complex (MHC) class I
antigens with specific carrier molecules.
[0002] Tissue-rejection reactions are the strongest-known immune
responses mediated by T cells. In individuals of the same species
they are caused by allogenic differences in class I and class II
MHC antigens. In organ transplants, for example, any allogenic
determinants of the MHC antigens present in the donor tissue are
recognized as foreign by allospecific T cells of the recipient, a T
cell immune response is induced, and the rejection reaction takes
place unless an immunosuppressive therapy has been initiated or
such a therapy proves insufficient.
[0003] It is furthermore known that MHC class I antigens are
glycoproteins which are expressed on the surface of all nucleated
cells. They are composed of a heavy chain, which is encoded by MHC
class I genes, and of a light chain, the .beta..sub.2-microglobulin
which is non-covalently associated with the heavy chain. The
extracellular part of the heavy chain is folded in three domains,
the first two of these domains (alpha.sub.1 and alpha.sub.2)
exhibiting a pronounced polymorphism when the amino acid sequences
of hitherto known class I MHC antigens from various individuals are
compared. They assist with antigen presentation and carry the
allogenic determinants. The third extracellular domain has a more
conserved sequence. The association with .beta..sub.2-microglobuli-
n is essential for correct folding of the heavy chain and for the
transport of the molecule to the cell surface.
[0004] Isolation and characterization of mutated MHC class I
antigens in mice showed that merely a few differences in amino
acids on the alpha.sub.1 and alpha.sub.2 domains between donor and
recipient suffice to induce a rejection reaction (Nathenson et al.,
Ann. Rev. Immunol., 1986, 4, 471-502). It has also been shown in
humans that slight differences between donor and recipient lead to
rejection of a transplant (Dausset, J., Rapaport, F. T., Legrand,
L., Colombani, J., Marcelli-Barge, A.: Skin allograft survival in
238 human subjects: Role of specific relationships at the four gene
sites of the first and the second HL-A loci., Histocompatibility
Testing (1970) pages 381-397, Terasaki P. I. (Ed.)). The task which
presented itself from that said above was to utilize the specific
inducibility and strength of the cellular immune response in the
tissue-rejection reaction to damage or destroy selected target
cells.
[0005] It has been found that target-cell-specific carriers, for
example preferably monoclonal antibodies (mAb), but also polyclonal
antibodies or molecules which bind to receptors on cells, can be
coupled to the N- or C-terminal end of an allogenic MHC class I
molecule without this altering the allogenic determinants in a
disadvantageous manner. The MHC class I molecule is brought, with
the aid of this target-cell-specific carrier, specifically to the
target cells, which leads to activation of allospecific T cells and
thus to destruction of the target cells by allospecific cytotoxic T
cells. One explanation for the success of the coupling of a
target-cell-specific carrier to the N- or C-terminal end of a MHC
class I molecule while retaining the allogenic determinants is that
the N-terminal end of the MHC class I molecule is located on the
side of the alpha.sub.1 and alpha.sub.2 domains which points
towards the cell, whereas the allogenic determinants are located on
the side of the alpha.sub.1 and alpha.sub.2 domains which faces
away from the cell (FIG. 1, FIG. 34).
[0006] Because of the great polymorphism of the MHC class I
antigens in the human population, it is possible to induce a
rejection reaction in almost 100% of the population with the aid of
only two different MHC class I molecules selected, for example HLA
B27w and HLA B27k. HLA B27w and HLA B27k are two subtypes of the
serologically defined HLA B27 specificity which are defined by
cytotoxic T lymphocytes. In the caucasoid population about 7% of
individuals express HLA B27w and about 1% express HLA B27k. The use
of, for example, both HLA B27 subtypes for the allogenization in
accordance with this invention makes it possible to treat almost
100% of the caucasoid population. However, it is possible according
to the invention to couple any desired MHC class I antigen to the
relevant specific carriers if the above-mentioned antigens do not
lead in the relevant recipient to activation of allospecific T
cells and subsequent damage to or destruction of the target cells.
Target cells may be regarded as cells which are undesired and/or
pathogenic in the body, such as, for example, tumor cells. The
antigenic constructs according to the invention are accordingly
suitable for tumor therapy. However, it is also possible with the
MHC class I antigenic constructs according to the invention to
treat other diseases which are caused by cells or the products
thereof and are favorably affected by elimination of these cells.
The mode of action of the hybrid molecules described in example
groups I and II derives from the fact that they are able, because
of the specifity of the antibody portion, to bind to an antigen on
the cell. The HLA B27 portion of the fused molecule results in
masking of the surface of the target cell with an allogenic MHC
class I molecule. These allogenic class I molecules can then be
recognized by syngeneic, allospecific, cytotoxic T cells, which
leads to destruction of the target cells by the allospecific
cytotoxic T cells. Accordingly, the invention relates to
[0007] a) MHC class I antigens which are linked N- or C-terminally
to specific carriers, the linkage preferably being brought about
covalently but also possibly being non-covalent, for example by a
biotin-avidin bridge, and the specific carriers binding selectively
to target cells and denoting preferably monoclonal, but also
polyclonal, antibodies, but being very generally receptor-binding
molecules which bind to the particular cell receptors,
[0008] b) a process for the preparation of the MHC class I
antigenic constructs, and
[0009] c) the use of the MHC class I antigenic constructs mentioned
in a) and b) for damaging or eliminating target cells.
[0010] The invention is furthermore described in the examples which
follow and in the patent claims, but it is not to be regarded as
restricted thereto.
[0011] Examples 1-17 detailed hereinafter describe a construct
according to the invention composed of the nitrophenol
(NP)-specific mouse mAb B/1-8 V.sub.H gene (1), of a human IgG C
F(ab').sub.2 gene (2) and of an HLA B27w gene (3). (1) and (2) are
to be regarded in this context as examples of the specific carrier
portion--in this case an mAb against NP--whereas (3) represents an
HLA class I antigen.
[0012] The abovementioned construct is, after appropriate
transformation, expressed and secreted by those myeloma cells which
contain a human .beta..sub.2-microglobulin and a light chain of an
immunoglobulin and whose V gene forms with V.sub.H B/1-8 a NP
binding-site, such as, for example, the mice myeloma cell J 558 L
(Oi, V. T., Morrison, S. L., Herzenberg, L. A., Berg, P.:
Immunoglobulin gene expression in transformed lymphoid cells. Proc.
Natl. Acad. Sci. USA 80, 825, 1983). It is possible, by exchanging
the V.sub.H gene of the heavy chain and using an appropriate light
chain, to provide the mAb/HLA B27w fusion product with any desired
specificity for which a specific or selective mAb exists.
EXAMPLES
[0013] I Examples 1 to 13 Show the Construction of an HLA B27/mAb
Fusion Gene with the HLA B27 Portion at the 3' End of the
Monoclonal Antibody
[0014] A) Preparation of the mAb C Gene Portion (IgG.sub.3 C
Gene)
Example 1
[0015] A human IgG.sub.3 C gene was isolated from a human gene bank
in EMBL3 phages (Frischauf, A.-M., Lehrach, H., Proustka, A.,
Murray, N.: Lambda replacement vectors carrying polylinker
sequences. J. Mol. Biol. 170, 827-842 (1983) and Seemann, G. H. A.,
Rein, R. S., Brown, C. S., Ploegh, H. L.: Gene conversion-like
mechanisms may generate polymorphism in human class I genes. The
EMBO Journal 5, 547-552 (1986)) and subcloned as a HindIII/Sph I
fragment 3.1 kb in size into the plasmid vector pUC 19 (clone
54.1.24) (FIG. 2).
[0016] All the techniques used in these and in the following
examples were taken, unless otherwise indicated, from Lehrach, H.
and Frischauf, A.-M. Laboratory Manual EMBL (1982), Heidelberg;
Maniatis, T., Fritsch, E. F., Sambrook, J.: Molecular Cloning: A
laboratory manual (1982), Cold Spring Harbor Laboratory.
Example 2
[0017] The 54.1.24 clone was subjected to complete HindIII and
partial PstI restriction digestion. This results, inter alia, in
restriction fragments which contain the C.sub.H1 exon and one, two
or three hinge exons. These fragments were cut out of an agarose
gel and cloned into a pUC 19 vector cut with HindIII and PstI (FIG.
3).
[0018] The plasmid clone with the C.sub.H1 and three hinge exons
(F(ab').sub.2 3H) was then cleaved with BamHI and Asp 718, the
cleavage sites were filled in and religated with T.sub.4 ligase
(FIG. 4). This deletes the pUC 19 polylinker between the Xba I and
the SstI cleavage site.
[0019] I B) Preparation of the HLA B27 Gene
Example 3
[0020] An HLA B27w gene was isolated from a genomic gene bank
cloned in EMBL3 bacteriophages (Frischauf, A.-M., loc. cit., and
Seemann, G. H. A., loc. cit.) and characterized by restriction
mapping and nucleotide sequence analysis (Maxam, A., Gilbert, W.:
Sequencing end-labeled DNA with base specific chemical cleavage.
Meth. Enzymol. 65, 499560 (1980) and Sanger, F., Nicklen, S.,
Coulson, A. R.: DNA sequencing with chain terminating inhibitors.
Proc. Natl. Acad. Sci. USA 74, 5463-5471i (1977)) (FIG. 5).
[0021] The HLA B27w gene was then digested with the restriction
enzymes SstI and BglII and subcloned into the SstI and BamHI
cleavage sites of pUC 19. Plasmid clones with the subfragments A, B
and C (FIG. 5) were isolated.
Example 4
[0022] The plasmid with subfragment A was cleaved completely with
SstI and partially with SmaI and, after fractionation on an agarose
gel, the fragment A' (FIG. 6) was cloned in a pUC 19 plasmid
cleaved with HincII and SstI.
Example 5
[0023] The plasmid with the subfragment B was digested with XbaI,
and the resulting XbaI insert (B') was cloned in a XbaI-cleaved pUC
19 plasmid (FIG. 7).
Example 6
[0024] The plasmid with the subfragment C was cleaved completely
with HindIII and partially with SstI, and the fragment which, in
the HLA B27w gene, is attached to fragment A (C') was, after
fractionation on an agarose gel, isolated and cloned into the
Bluescript KS+ phasmid vector (Stratagene, LaJolla, Calif., USA)
cleaved with HindIII and SstI (FIG. 8).
Example 7
[0025] Single-stranded phages were prepared from the KS+ phasmid
vector C' by infection with VCS-M13 helper phages (Stratagene, Cat
# 200251) and were purified (Stratagene: Bluescript Exo/Mung DNA
sequencing system: Instruction Manual). A synthetic oligonucleotide
(I=5'CCTTACCTCATCTCAGG3'- ) was hybridized onto these single
strands, and the remainder of the second strand was synthesized
using Klenow polymerase. The double-stranded phasmids generated in
this way were transformed into XL Blue bacteria and then
single-stranded phages were again generated from the resulting
plasmid clones by infection with helper phages, and the nucleotide
sequence was determined with the aid of an oligonucleotide primer
II (5'TGAGGGCTCCTGCTT3') (Sanger, F. et al., loc. cit.). A clone in
which the codon TGG (amino acid 274) at the 3' end of the alpha3
exon had been mutated to a stop codon (TGA) was identified (C")
(FIG. 9).
Example 8
[0026] The plasmid with the fragment A' was cleaved with SstI and
ligated with the C" fragment which had been generated by a complete
HindIII and partial SstI cleavage of the phasmid clone C" and had
been isolated after fractionation on an agarose gel. After ligation
at 14.degree. C. for 30 minutes, the unligated ends were filled in
with T.sub.4 polymerase and subsequently ligated once again.
Restriction mapping was used to identify the plasmid D (FIG. 10) in
which the fragment A' is connected to the fragment C" via the SstI
cleavage site in the alpha2 exon.
Example 9
[0027] The plasmid with the fragment D was cleaved with XbaI and
ligated with the fragment B' which had been cut out of the plasmid
B' with XbaI and had been purified after fractionation on an
agarose gel (FIG. 11). Nucleotide sequence analyses (17) were used
to identify a plasmid (E) in which the B' fragment is ligated in
the correct 5'-3' orientation to the fragment D.
[0028] C) Fusion of the Modified HLA B27w Gene with the IgG3 C
F(ab').sub.2 3H Gene Fragment
Example 10
[0029] The fragment E was cut out of the plasmid E by cleavage with
EcoRI and HindIII, the ends were filled in with T.sub.4 polymerase
and purified after fractionation on an agarose gel. This purified
fragment E was then ligated with the plasmid which contains the
IgG3 F(ab').sub.2 3H fragment after the latter had been cleaved and
the XbaI ends had been filled in with T.sub.4 polymerase (FIG. 12).
Restriction mapping was used to identify the clone which contained
the plasmid F, in which the modified HLA B27w gene is fused in the
correct 5'-3'orientation to the F(ab').sub.2 3H gene.
Example 11
[0030] The fragment F was cut out with HindIII and EcoRI in order
to place a polylinker in front of the 5' end of the fragment F. The
HindIII and XbaI ends were filled in with T.sub.4 polymerase and
cloned into a pUC 19 which had been cleaved with SstI and whose
SstI ends had been filled in with T.sub.4 polymerase. Restriction
analyses were used to identify the clone with the plasmid G which
has the pUC 19 polylinker 5' from the fragment F (FIG. 13).
Example 12
[0031] The plasmid G was cleaved with HindIII and EcoRI, and the
insert with the IgG F(ab').sub.2 HLA B27w fusion gene was isolated
and cloned into a Bluescript KS+ phasmid vector (Stratagene:
Bluescript Exo/Mung DNA sequencing system: Instruction Manual)
cleaved with HindIII and EcoRI (FIG. 14).
Example 13
[0032] The plasmid H resulting from this cloning was then cleaved
with BamHI, and the insert was cloned into the eukaryotic
expression vector pEV.sub.H (Simon, T., Rajewsky, K., Nucl. Acids
Res. 16, 354, (1988), which contains the IgG H promoter/enhancer
sequences and the V.sub.H gene originating from the NP-specific
mouse mAb B/1-8 which had been cleaved with BamHI (FIG. 15)
(Neuberger, M. N.: EMBO Journal 2, 1375-1378 (1983)). Restriction
analysis was used to identify the plasmid I in which the IgG 3
F(ab').sub.2 HLA B27w fusion gene is cloned in the correct 5'-3'
orientation behind the V.sub.H gene.
[0033] The mAb/HLA B27w fusion gene now possesses intact 5' and 3'
ends having all the signals required for expression in eukaryotic
cells. The construct is, as stated in the introduction, expressed
and secreted in every myeloma cell which contains a human
.beta..sub.2-microglobulin and a light chain of an immunoglobulin
and whose V gene forms with V.sub.H B/1-8 a NP binding site, such
as, for example, the mouse myeloma cell J 558L (Oi, V. T.,
Morrison, S. L., Herzenberg, L. A., Berg, P., Proc. Natl. Acad.
Sci. USA 80, 825 (1983)).
[0034] II Examples 14 to 17 Show the Construction of an HLA B27/mAb
Fusion Gene with the HLA B27 Portion at the 5' End of the
Monoclonal Antibody
[0035] A) Preparation of the HLA B27 Gene
Example 14
[0036] An HLA B27w gene was isolated from a genomic gene bank
cloned in EMBL3 bacteriophages (Frischauf et al., loc. cit. and
Seemann, G. H. A., loc. cit.) and characterized by restriction
mapping and nucleotide sequence analysis (Maxam et al., loc. cit.
and Sanger et al., loc. cit.) (FIG. 5).
[0037] The HLA B27w gene was then digested with the restriction
enzymes SstI and BglII and subcloned into the SstI and BamHI
cleavage sites of pUC 19. Plasmid clones with the subfragments A, B
and C (FIG. 5) were isolated.
[0038] The plasmid with the HLA B27 subclone C was partially
cleaved with PstI. The protruding 3' ends of the PstI cleavage
sites were removed with T.sub.4 polymerase, with addition of dGTP,
and religated with T.sub.4 ligase. Restriction analysis was used to
identify the plasmid clone Cl which contains no PstI cleavage site
in the intron between the alpha2 and alpha3 exon (FIG. 16).
[0039] The plasmid clone Cl was cleaved partially with SstI and
completely with HindIII, and the Cl' fragment was isolated and
cloned into a double-stranded M13 mp18 vector cleaved with SstI and
HindIII. The M13 clone Cl' with the Cl' fragment was identified by
determining the nucleic acid sequence of the insert (FIG. 17).
[0040] Using the protocol of the Bio-Rad Muta-Gene M13 mutagenesis
kit there were isolated from the Cl' M13 mp18 phages in the
bacterial strain CJ236 single-stranded phages which contained
uracils. An oligonucleotide (oligonucleotide III) with the sequence
.sup.5' GCGCGCTGGAGCGTCTC.sup.3' was hybridized onto these
single-stranded phages and the second strand was synthesized with
addition of T.sub.4 polymerase, DNTP and T.sub.4 ligase.
[0041] After infection of the bacterial strain MV 1190 the mutated
clone C2 was identified by restriction analysis of the M13 mp18
double-stranded DNAs and confirmed by nucleic acid sequence
analysis (FIG. 18). The mutagenesis resulted in destruction of the
PstI restriction cleavage site in the alpha2 exon without altering
the reading frame or the encoded amino acid sequence.
[0042] Single-stranded phages were in turn produced from the M13
clone C2 in the bacterial strain CJ236 and were hybridized with the
oligonucleotide IV (oligonucleotide
IV=.sup.5'GGGGACGGTGGAATTCGAAGACGGCTC- .sup.3'). The second strand
was then synthesized with T.sub.4 polymerase, T.sub.4 ligase and
dNTP. After transformation into MV 1190 bacteria, the M13 mp18
clone C2' was identified by restriction analysis, and the mutation
was verified as correct by nucleotide sequence analysis (FIG. 19).
This mutagenesis resulted in an EcoRI and an AsuII cleavage site
being introduced into the TM exon of the HLA B27 gene, and in amino
acid 279 being converted from glutamine into asparagine (FIG.
20).
Example 15
[0043] The plasmid clone with the subfragment B was digested with
XbaI, and the resulting XbaI insert (B') was cloned in an
XbaI-cleaved pUC 19 plasmid (FIG. 7).
[0044] The plasmid clone with the fragment A was cleaved completely
with HindII and partially with SmaI, and religated. Restriction
analysis was used to identify the clone A' in which part of the pUC
19 polylinker is deleted (FIG. 21).
[0045] The plasmid clone A' was cleaved partially with SstI and
ligated with the C2 fragment generated by an SstI cleavage of the
plasmid clone C2 and isolated after fractionation on an agarose
gel. Restriction mapping was used to identify the plasmid D.sub.1
(FIG. 22) in which the fragment A is connected to the fragment C2
via the SstI cleavage site in the alpha2 exon. The 5' end of the
HLA B27w gene is thus complete.
[0046] Construction of the Linker:
[0047] Two oligonucleotides were synthesized:
1 oligonucleotide Va: .sup.5'TCGAATTCCG GCGAGGCAGC TCCCCCAGCT
GCACCCGCAG CAGCCGCAGC AGGCCGGCAG GTCCAACTGC AGGA .sup.3'
oligonucleotide Vb: .sup.5'TCCTGCAGTT GGACCTGCCC GCCTGCTGCG
GCTGCTGCGG GTGCAGCTGC GGGAGCTGCC TCGCCGGAAT TCGA .sup.3'
[0048] The two oligonucleotides were hybridized together. This
resulted in double-stranded DNA fragments with an EcoRI restriction
cleavage site at one end and a PstI restriction cleavage site at
the other end. These fragments were cleaved with EcoRI and PstI and
cloned into an EcoRI- and PstI-cleaved pUC 19 plasmid vector (FIG.
23). The plasmid clone L was identified by restriction analysis and
verified by nucleotide sequence analysis.
[0049] The immunoglobulin V gene was synthesized by P. T. Jones et
al. (Jones, P. T., Dear, P. H., Foote, J., Neuberger, M. S. Winter,
G., Nature 321: 522, (1986)) using oligonucleotides. It contains a
PstI restriction cleavage site in the 5' region of the clone and is
cloned as HindIII/BamHI fragment in an M13 mp8 vector whose PstI
cleavage site had been destroyed by cleavage, removal of the
protruding ends and religation (FIG. 24).
[0050] II B) Preparation of the mAb C Gene Portion:
Example 16
[0051] A human IgG3 C gene was isolated from a human gene bank in
EMBL3 phages (Frischauf et al., loc. cit. and Seeman et al., loc.
cit.) and subcloned into the plasmid vector pUC 19 as a
HindIII/SphI fragment 3.1 kb in size (clone 54.1.24) (FIG. 2).
[0052] The plasmid clone 54.1.24 was cleaved with HindII and
Asp718, the protruding ends of the Asp718 cleavage site were
removed with T.sub.4 polymerase and religated with T.sub.4 ligase.
Restriction analysis and nucleic acid sequence determination were
used to identify the clone 54.1.24 Delta Pol which, apart from
SphI, PstI, SstI and EcoRI, no longer contains any restriction
cleavage sites 3' of the human IgG3 C gene (FIG. 25).
[0053] The plasmid clone 54.1.24 Delta Pol was digested with BglII
and SphI. The protruding ends were removed with T.sub.4 polymerase
and religated with T.sub.4 ligase. Restriction analysis and nucleic
acid sequence determination were used to identify the clone I which
now contains only the CH.sub.1 exon of the human IgG3 C gene (FIG.
26).
[0054] The plasmid clone I was cleaved with PstI, and the
protruding ends were removed with T.sub.4 polymerase. The B' insert
which had been cut with XbaI and filled in with T.sub.4 polymerase
to give blunt ends was ligated into the resulting blunt ends.
Restriction analysis and nucleic acid sequence determination were
used to identify the clone K (FIG. 27) which contains a human
IgG.sub.3C.sub.H1 exon and a 3' end of a HLA class I gene.
[0055] The plasmid clone K was cleaved with HindIII and EcoRI, the
protruding ends were removed, and the insert was ligated in an
SstI-cleaved pUC 19 plasmid whose ends had likewise been made
blunt. The clone L which harbors the polylinker of the pUC 19
vector 51 from the C.sub.H1 exon was identified (FIG. 28).
[0056] The plasmid clone L was cleaved with EcoRI and HindIII, and
the insert was purified and ligated into a HindIII- and
EcoRI-cleaved KS.sup.+ vector (Stratagene; Bluescript Exo/Mung DNA
Sequencing System) whose PstI cleavage site had previously been
destroyed by cleavage with PstI, T.sub.4 polymerase treatment and
religation. The clone M, from which it is possible to cut out the
human C.sub.H1 exon with the HLA class 13' end by a Bam HI
cleavage, was identified (FIG. 29).
Example 17
[0057] Double-stranded DNA was prepared from the M13 mp8 clone V
and cleaved with BamHI. The KS.sup.+ clone M was cleaved with
BamHI, and the insert M was purified. The M fragment was ligated
into the BamHI-cleaved clone V, and nucleic acid sequence
determination was used to identify the M13 clone N which contains
an intact IgG3 gene (FIG. 30).
[0058] Double-stranded DNA was prepared from the M13 clone N and
was cleaved with EcoRI, and the insert was purified. The plasmid
clone D.sub.1 was cleaved with EcoRI and ligated with the fragment
N. The phage clone 0 in which the fragment N is cloned in the
correct orientation into the clone D.sub.1 was isolated (FIG.
31).
[0059] The plasmid clone O was subjected to a complete PstI
cleavage and partial EcoRI cleavage and ligated with the linker
fragment cut out of the plasmid vector L with EcoRI and PstI. The
plasmid clone P contains the complete HLA B27w mAb fusion gene
(FIG. 32, FIG. 33). This fusion gene can be expressed and secreted
in human cells alone or in mouse cells together with the human
beta.sub.2 microglobulin gene if the expressing cells also contain
an immunoglobulin light chain.
[0060] Key to FIG. 1
[0061] alpha 1, alpha 2 and alpha 3 denote the domains of the class
I MHC antigen chain. The arrows point to the alpha helices which
carry the allodeterminants. CM is meant to represent the cell
membrane, and C the cell.
[0062] Key to FIG. 2 et Seq.
[0063] EcoRI etc. represents the cleavage with the particular
restriction end nuclease or represents the corresponding cleavage
site denotes a restriction cleavage site destroyed by religation
after filling-in.
[0064] TM denotes the transmembrane region.
[0065] 3'NT denotes 3' non-translated IgH p/E denotes the
immunoglobulin heavy chain promoter/enhancer
[0066] * denotes: incomplete digestion
[0067] DS-DNA denotes: double-stranded DNA
[0068] SS-DNA denotes: single-stranded DNA
[0069] Key to FIG. 34:
[0070] s.CTL denotes: syngeneic cytotoxic T lymphocyte
[0071] TcR denotes: T-cell receptor
[0072] a.MHC class I denotes: allogenic MHC class I antigen
[0073] t.a.a. denotes: tumor-associated antigen
[0074] s.t.c. denotes: syngeneic tumor cell
Sequence CWU 1
1
13 1 17 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 1 ccttacctca tctcagg 17 2 15 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 2 tgagggctcc tgctt 15 3 17 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 3
gcgcgctgga gcgtctc 17 4 27 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 4 ggggacggtg
gaattcgaag acggctc 27 5 74 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 5 tcgaattccg
gcgaggcagc tcccgcagct gcacccgcag cagccgcagc aggcgggcag 60
gtccaactgc agga 74 6 74 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 6 tcctgcagtt
ggacctgccc gcctgctgcg gctgctgcgg gtgcagctgc gggagctgcc 60
tcgccggaat tcga 74 7 28 DNA Artificial Sequence CDS (2)..(28)
Description of Artificial Sequence Synthetic oligonucleotide 7 a
gag ccg tct tcs mak tcc acc gtc ccc 28 Glu Pro Ser Ser Xaa Ser Thr
Val Pro 1 5 8 9 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 8 Glu Pro Ser Ser Xaa Ser Thr Val Pro 1
5 9 28 DNA Artificial Sequence CDS (2)..(28) Description of
Artificial Sequence Synthetic oligonucleotide 9 a gag ccg tct tcg
aat tcc acc gtc ccc 28 Glu Pro Ser Ser Asn Ser Thr Val Pro 1 5 10 9
PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 10 Glu Pro Ser Ser Asn Ser Thr Val Pro 1 5 11 27
PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 11 Glu Pro Ser Ser Asn Ser Gly Glu Ala Ala Pro
Ala Ala Ala Pro Ala 1 5 10 15 Ala Ala Ala Ala Gly Gly Gln Val Gln
Leu Gln 20 25 12 12 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 12 Gly Val His Asp Gln Val
Gln Leu Gln Glu Ser Gly 1 5 10 13 30 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 13 Glu Pro Ser
Ser Asn Ser Gly Glu Ala Ala Pro Ala Ala Ala Pro Ala 1 5 10 15 Ala
Ala Ala Ala Gly Gly Gln Val Gln Leu Gln Glu Ser Gly 20 25 30
* * * * *