Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1993-05-06
2001-11-20
Huff, Sheela (Department: 1642)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S070200, C530S387100
Reexamination Certificate
active
06319690
ABSTRACT:
The invention relates to the preparation and use of gene banks of human antibodies (Ab). Starting from a mixture of human B-lymphocytes, their mRNA is translated into cDNA using oligo-dT primers. Subsequently, an amplification of the Ab-specific cDNA by means of polymerase chain reaction (PCR) takes place using suitable oligonucleotide primer sequences. Expression of this amplified Ab-specific cDNA in a bacterial expression vector, e.g. the vector pFMT described below, in
E. coli
thus makes available a human-antibody library with a comprehensive repertoire for screening selected antigens in vitro.
The human or mammalian immune system comprises an estimated number of between 10
6
and 10
8
different antibodies. This number of antibodies seems to be sufficient to cause an immune reaction of the body both against all naturally occurring antigens and against artificial antigens. If it is furthermore taken into account that often several antibodies react with the same antigen, the repertoire of antibodies that are really different would be rather in the region from 10
6
to 10
7
.
Up to now specific antibodies have always been obtained starting from an immunization with the particular antigen, for example injection of the antigen into the body or in vitro incubation of spleen cells with this antigen. In the case of polyclonal antibodies, the immunoglobulins can then be isolated from the serum and the specific antibodies can be isolated therefrom, e.g. by absorption methods. Monoclonal antibodies are isolated from the cell supernatants or from the cell lysate of spleen tumor cells (hybridoma cells) which have been fused with individual B lymphocytes and cloned. The abovementioned methods are unsuitable in particular for the preparation of specific human antibodies or human monoclonal antibodies.
The present invention therefore has the object of developing a generally usable process for generating specific human monoclonal antibodies (huMAbs) or parts of antibodies, which contain the antigen binding site.
It has been found that the desired huMAbs or parts thereof which contain the variable, antigen binding domain can be isolated from gene banks of human immunoglobulins. First of all, starting from a mixture of nonactivated human B-lymphocytes, their mRNA was isolated and translated into cDNA with the aid of oligo-dT primers (SEQ ID NOS 1-10). A specific amplification of the population of antibody cDNAs within the resulting cDNA pool was achieved by using PCR. For this purpose certain oligo-nucleotide primers which are homologous to conserved sequences at both ends of the antibody cDNA were used (see below and examples). The design of the primer (SEQ ID NOS 4-11) for the reverse reaction for the synthesis of the noncoding strand of the DNA of the heavy chains is based on IgM sequences (subclass III, since this comprises most of the IgM sequences). IgM molecules occur more often in non-activated B-lymphocytes than all other immunoglobulin classes. In contrast, IgG sequences predominate in activated B-lymphocytes whose repertoire of different antibodies is very much smaller. An IgG library would additionally entail the danger of one or a few particularly strongly expressed IgGs dominating the library.
Up to 30 amplification cycles were, advantageously, carried out. The oligonucleotide primers contain suitable restriction sites for inserting the amplified DNA e.g. into the antibody expression plasmid pFMT (see below).
This expression plasmid makes possible the expression of antibody cDNA and subsequent secretion of the expression products in bacteria (
E.coli
). The antibody operon of the plasmid contains the sequences (SEQ ID NOS 26-29) of the variable parts of both the heavy and light chain of an antibody. Suitable leader sequences from the amino terminal part of a bacterial protein makes secretion of the antibody parts possible. The leader sequences (SEQ ID NOS 12-15) are cleaved off by a bacterial enzyme during the secretion. During the secretion of the antibody cDNA products, the light and heavy chains of the antibody (with or without an adjacent constant domain) become associated. This results in the formation of an antibody or antibody fragment which, in either case, contains a functional antigen binding site. Similar constructs for individual antibodies have also been described by other authors (Better et al. (1988), Science 240, 1041, and Skerras & Plückthun (1988), Science 240, 1038).
It is true that the amplification of DNA coding for the variable parts of antibodies has been described by other authors (Orlandi et al. (1989), Proc. Natl. Acad. Sci. 86, 3833; Sastry et al., (1989) Proc. Natl. Acad. Sci. 86, 5728; Ward et al. (1989), Nature 341, 544); Huse et al. (1989), Science 246, 275). In this case however, the mRNA which, inter alia, also codes for antibodies was isolated from hybridoma cells or spleen lymphocytes after treatment with a certain antigen. For this reason primer sequences which are based only on IgG sequences were also used there. This is, of course, an advantage if as many antibody DNA clones as possible which are derived from activated lymphocytes are sought. With primers from IgG sequences, the chances of finding clones which contain DNA coding for antibodies against the injected antigen are much higher. It has to be added that in the foregoing papers murine and therefore nonhuman antibody DNA was synthesized and, additionally with exclusion of regions of the lambda chain, amplified.
The present invention, in contrast, uses primer sequences which are homologous to the sequences in the constant domains of IgM cDNA. This is the best way of implementing the invention, i.e. making available a very large choice of antibodies, namely the whole antibody repertoire, in the form of a library. The expression in, preferably,
E. coli
then results in the desired human-antibody library in which the desired human antibodies or antibody parts are found by means of screening bacterial clones using the selected antigen.
Oligonucleotide primers suitable for amplification are compiled in Tab. 1 (SEQ ID NOS 1-11). The positions of the abovementioned primers on the &mgr;, kappa and lambda chains are shown in the form of a diagram in Tab. 2. The molecular biological constructions of, amongst others, the expression vector, i.e. the antibody expression plasmid PFMT, are described in detail in the examples below.
The invention therefore relates to human-antibody libraries, prepared by transcription of the mRNA from nonactivated (peripheral) human B-lymphocytes by means of oligo-dT primers, subsequent amplification by PCR using primers containing sequences which are homologous to conserved regions of the IgM cDNA, and subsequent incorporation into suitable expression plasmids for the expression in microorganisms, preferably in the expression vector pFMT for the expression in
E. coli
. In a preferred embodiment an additional sequence is incorporated which codes for a marker peptide, e.g. a TAG sequence so that the expression products can be detected in a simple manner using established monoclonal antibodies against the marker peptide (Wehland et al., (1984), EMBO J. 3, 1295).
The invention also relates to the use of abovementioned human-antibody libraries for isolating desired human antibodies or parts of antibodies containing a functional antigen binding site by screening using selected antigens, and to a process for isolating the said human antibodies or their antigen-binding parts, and also to a process for preparing the said human-antibody libraries.
The invention also relates to expression vectors having the properties of the antibody expression plasmid pFMT.
REFERENCES:
patent: WO 90/14430 (1990-11-01), None
patent: WO 91/10737 (1991-07-01), None
L. Riechmann et al. “Crystallization of an Anti-2,2,6,6-Tetramethyl-1-piperidinyloxy-dinitrohpenyl Monoclonal Antibody Fab Fragment With and Without Bound Hapten,” J. Mol. Biol. 203:829-830 (1988).
D. Straus et al., “Chicken Triosephosphate Isomerase Complements anEscherichia coliDeficiency,” Proc. Natl. Acad. Sci. 82:2014-20
Breitling Frank Berthold
Dubel Stefan
Klewinghaus Iris
Little Melvyn
Seehaus Thomas
Dade Behring Marburg GmbH
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Helms Larry R.
Huff Sheela
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