PCR amplification of rearranged genomic variable regions of...

Details PCR amplification of rearranged genomic variable regions of... PCR amplification of rearranged genomic variable regions of...

1994-12-01

2001-07-10

Park, Hankyel T. (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S069100, C536S023530

Reexamination Certificate

active

06258529

ABSTRACT:

BACKGROUND OF THE INVENTION
The development of mouse hybridoma technology has allowed the production of antibodies (Ab) specific for a wide range of antigens. Mouse monoclonal antibodies (mAb) have been used extensively for diagnosis and, in a few cases, for human therapy and in vivo diagnostics. Administration of murine antibodies to humans has been observed to induce a strong human anti-mouse antibody response (HAMA) after single or repeated treatments, thus precluding long-term treatment using these antibodies. Moreover, rodent antibodies are rapidly cleared from human serum and often do not interact effectively with the human immune system. Since human hybridomas are generally unstable and secrete low amounts of antibodies (frequently IgMs), considerable effort has been directed at rendering foreign antibodies (e.g., murine antibodies) more similar to those of the host to which they are administered (e.g., a human). Alternatives to human hybridoma-derived antibodies have been developed in which mouse immunoglobulin sequences (e.g., constant regions) are replaced with corresponding sequences derived from human immunoglobulin genes. Two examples of this type of antibody are (1) chimeric mAbs, in which murine variable regions are combined with human constant regions (Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855, 1984; Boulianne et al., Nature 312:643-30 646, 1984), and (2) humanized mAbs, in which murine CDRs (complementarity determining regions) replace the corresponding sequences in human ixmunoglobulins (Jones et al., Nature 321:522-525, 1986; Co et al., Nature 351:501-502, 1991). These engineered antibodies retain their target specificity and generally exhibit reduced HAMA responses when injected into patients. In addition, desired effector functions of antibodies for certain clinical applications can be obtained by using constant regions corresponding to the appropriate immunoglobulin isotype.
Despite these advances, cloning of variable region sequences has been a limiting step in the rapid construction of chimeric and isotype switched antibodies. Polymerase chain reaction (PCR) amplification of immunoglobulin heavy and light chain variable regions has facilitated this step. However, the high degree of DNA sequence polymorphism in leader and variable sequences of both heavy and light chain genes has required the preparation of complex sets of degenerate primers (Jones et al., Bio/Technology 9:88-89, 1991; Kettleborough et al., Eur. J. Immunol. 23:206-211, 1993; Le Boeuf et al., Gene 82:371-377, 1989; Orlandi et al., Proc. Natl. Acad. Sci. USA 86:3833-3837, 1989). In the case of 5′ primers, these primers have usually been designed to correspond to the first framework of the variable region (FR1) and, in a few cases, to the leader peptide sequence (L). The 3′ primers have usually been designed to correspond to the framework 4 (FR4) region, which displays limited polymorphism, or to the constant region, in which conserved, isotype-specific sequences are easily identified. Although complex sets of 5′ and 3′ primers have been designed, they do not always match the DNA template completely (Gavilondo-Cowley et al., Hybridoma 9:407-417, 1990; Leung et al., BioTechniques 15:286-292, 1993). Native sequences of the immunoglobulin heavy and light chain genes may therefore be altered in the FR1 and/or FR4 regions by the PCR amplification process. Modifications of the N-terminal region of an immunoglobulin, particularly the light chain variable region (VL), in which the amino acid at position two is part of the predicted canonical structure for CDR1 (Chothia et al., Nature 342:877-883, 1989), have been shown to drastically reduce the affinity of immunoglobulins for their antigens. Moreover, expression levels of the recombinant antibodies may also be altered when mutations occur in the leader peptide. In most studies involving PCR amplification of immunoglobulin H (heavy) and &kgr;/&lgr; (light) chain variable regions using these primers, cDNA templates were used, resulting in the generation of fragments containing incomplete VH and VL sequences, which may or may not be linked to part of the constant region.
SUMMARY OF THE INVENTION
We have designed a method for isolating nucleic acids encoding immunoglobulin Fv (variable) fragments from genomic DNA of hybridoma cells producing specific monoclonal antibodies. Specific primers corresponding to (1) the 5′ untranslated region (UTR) of the variable region and (2) the intron downstream of the rearranged JH/J&kgr;/&lgr; sequences are used in this method. The method can be used to amplify and clone genomic DNA corresponding to &lgr; and &kgr; light chain variable genes, as well as heavy chain variable genes. The variable genes isolated by this method can easily be inserted into expression vectors containing heterologous (e.g., human) light and heavy chain constant genes, thus facilitating isotype switching or antibody chimerization. Using this method, we have cloned for the first time genes encoding the variable regions (Fv) of the kappa light chain and heavy chain of the antibody produced by hybridoma cell line HNK-20.
Accordingly, in one aspect the invention features substantially pure DNA (genomic DNA or cDNA) encoding a variable region of the antibody produced by hybridoma cell line HNK-20. The variable region can be from the immunoglobulin heavy chain of the antibody, or from the immunoglobulin light chain of the antibody. The DNA may further encode an immunoglobulin constant region, such as a human immunoglobulin constant region. The immunoglobulin can be of any isotype, including, but not limited to an IgA (e.g., IgA1, IgA2, and sIgA), IgG, IgM, IgD, or IgE isotype. In the case of an IgA isotype, the immunoglobulin heavy chain can be an &agr; chain.
In one embodiment, the substantially pure DNA contains the sequence of
FIG. 5B
, or degenerate variants thereof, and encodes the amino acid sequence of FIG.
5
B. In another embodiment, the substantially pure DNA contains a sequence having about 50% or greater sequence identity to the DNA sequence of FIG.
5
B. In another embodiment, the substantially pure DNA a) is capable of hybridizing to the DNA sequence of
FIG. 5B
under stringent conditions; and b) encodes a polypeptide having a biological activity of a HNK-20 variable region.
In another embodiment, the substantially pure DNA contains the sequence of FIG. SC, or degenerate variants thereof, and encodes the amino acid sequence of FIG.
5
C. In another embodiment, the substantially pure DNA contains a sequence having about 50% or greater sequence identity to the DNA sequence of FIG.
5
C. In another embodiment, the substantially pure DNA a) is capable of hybridizing to the DNA sequence of
FIG. 5C
under stringent conditions; and b) encodes a polypeptide having a biological activity of a HNK-20 variable region.
In another embodiment, the substantially pure DNA contains the sequence of
FIG. 5D
, or degenerate variants thereof, and encodes the amino acid sequence-of FIG.
5
D. In another embodiment, the substantially pure DNA contains a sequence having about 50% or greater sequence identity to the DNA sequence of FIG.
5
D. In another embodiment, the substantially pure DNA a) is capable of hybridizing to the DNA sequence of
FIG. 5D
under stringent conditions; and b) encodes a polypeptide having a biological activity of a HNK-20 variable region.
In another aspect of the invention, the DNA is operably linked to regulatory sequences, such as promoter and/or enhancer sequences, for expression of the variable region. In a related aspect, the invention features a vector (e.g., a plasmid or a viral vector) containing the DNA of the invention operably linked to a promoter sequence. The invention also features a cell (e.g., a myeloma cell) containing the DNA of the invention.
In another aspect, the invention features a recombinant antibody containing a variable region from the monoclonal antibody produced by hybridoma cell line HNK-20. In one embodiment, the variable region is from the immunoglobulin

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