An improved method for the production of monoclonal antibodies is disclosed.

DETAILED DESCRIPTION OF THE INVENTION

I. Production of Hybridoma Cells

Hybridoma cell lines producing a desired antibody may be produced by conventional methods such as the well known methods of Kohler and Milstein. Briefly, an animal, preferably a rodent such as a Balb/C mouse is immunized and later re-immunized (boosted) with the desired immunogen, with an adjuvant as desired, as is well known in the art. Assaying the serum of the animal by conventional methods such as a specific ELISA reveals whether the animal is producing an antibody of the desired affinity and avidity. An immunized animal having an appropriate titer of the desired antibody is sacrificed and its spleen removed. The spleen cells are then carefully separated and fused with a suitable myeloma cell line by conventional procedures or otherwise immortalized, as is also well known in the art. The immortalized cells producing the desired antibody are then identified by routine, conventional screening and are then subcloned as desired.

II. Cloning Heavy and Light Chain-Encoding DNAs

Methods for cloning immunoglobulin heavy and light chains is well known in the art. See e.g. Beidler et al, 1988, J. Immunol. 141:4053 (genomic) and Liu et al, 1987, Proc. Natl. Acad. Sci. USA 84:3439 (CDNA). Briefly, CDNA or genomic libraries are constructed for the RNA or genomic DNA, respectively, from hybridomas producing a specific antibody of interest, as is known in the art. The immunoglobulin clones from such libraries can be identified by hybridization to DNA or oligonucleotide probes specific for J_H or C_H sequences for the heavy chain clones, or J_L or C_L sequences for the light chain clones. The positive clones are then further characterized by conventional restriction endonuclease site mapping and nucleotide sequencing.

III. Expression Vector Construction

Any conventional eukaryotic, preferably mammalian, expression vectors designed for high expression levels, of which many are known in the art, may be used in the practice of this invention. However, in the practice of this invention the expression vector for the light chain antibody DNA contains or is cotransfected with a first selectable, amplifiable marker gene while the expression vector for the heavy chain antibody DNA contains or is cotransfected with a second selectable, amplifiable marker. The two selectable, amplifiable markers must be differentially amplifiable, i.e. must each be susceptible to amplification under conditions which do not result in amplification of the other.

The eukaryotic cell expression vectors described herein may be synthesized by techniques well known to those skilled in this art. The components of the vectors such as the bacterial replicons, selection genes, enhancers, promoters, and the like may be obtained from natural sources or synthesized by known procedures. See Kaufman et al., J. Mol. Biol., 159:601-621 (1982); Kaufman, Proc Natl. Acad. Sci. 82:689-693 (1985). Eukaryotic expression vectors useful in practicing this invention may also contain inducible promoters or comprise inducible expression systems as are known in the art.

pMT2 and pMT3SVA are exemplary expression vectors which are described below. Both vectors contain an SV40 origin of replication and enhancer, adenovirus major late promoter and tripartite leader sequence, a cloning site followed by an SV40 polyadenylation site, the adenovirus VA I gene, E coli origin of replication and an ampicillin resistance gene for bacterial selection. PMT2 further contains a DHFR gene between the cloning site and the polyadenylation signal, while pMT3SVA contains an adenosine deaminase (ADA) gene under the expression control of the SV40 early promoter. While both of these vectors contain appropriate selectable, amplifiable markers, it should be understood that separate vectors containing the markers may be cotransfected or cotransformed by conventional means with the respective heavy and light chain DNAs.

IV. Production of Transformed Cell Lines

Established cell lines, including transformed cell lines, are suitable as hosts. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants (including relatively undifferentiated cells such as hematopoietic stem cells) are also suitable. Candidate cells need not be genotypically deficient in the selection gene so long as the selection gene is dominantly acting.

The host cells preferably will be established mammalian cell lines. For stable integration of the vector DNA into chromosomal DNA, and for subsequent amplification of the integrated vector DNA, both by conventional methods, CHO (Chinese Hamster Ovary) cells are currently preferred. Other usable mammalian cell lines include HeLa, human 293 cells, COS-1 monkey cells, melanoma cell lines such as Bowes cells, mouse L-929 cells, 3T3 lines derived from Swiss, Balb/c or NIH mice, BHK or HaK hamster cell lines and the like, as well as lymphocyte derived cell lines such as the murine hybridoma SP2/0-Ag14 or murine myeloma cells such as P3.653 and J558L or Abelson murine leukemia virus transformed pre-B lymphocytes.

The expression vectors may be introduced into the host cells by purely conventional methods, of which several are known in the art. Electroporation has been found to be particularly useful.

Stable transformants may then be screened for the presence and relative amount of incorporated antibody DNA and corresponding mRNA and polypeptide synthesis by standard methods. For example, the presence of the DNA encoding the desired antibody chain may be detected by standard procedures such as Southern blotting, the corresponding mRNA by Northern blotting and the protein thereby encoded by Western blotting.

It should be appreciated that the two antibody genes may be introduced serially into the same host cells, or may be introduced in parallel into separate host cells. In the former case, the antibody genes would be transfected separately, and the transfectants after the first of the two transfections, may or may not be selected in iteratively increasing amounts of the appropriate selective agent, prior to the second transfection. In the latter case, the two transfectants may be fused by conventional means to produce a cell containing and capable of expressing both antibody chains, as well as both selectable markers to facilitate isolation of hybrid cells, as exemplified in the Examples which follow. One of the parental cells of a fusion may be exposed to ionizing radiation before the fusion event. In addition, both heavy and light chain DNAs may be co-transfected with a single selectable, amplifiable marker, and the transfectants then passaged in iteratively increasing amounts of the selective agent. Once the relative levels of the heavy and light chains expressed in such a transfectant has been determined, a DNA encoding the chain found in limiting amounts can then be transfected into the cell, linked to a different selectable, amplifiable marker. The expression level for that chain can then be increased by iterative amplification as previously described.

V. Specific Amplification

Specific and independent amplification of the two DNAs may be readily accomplished using conventional amplification procedures appropriate for each of the respective markers. See e.g. published International Application WO 88/08035 for an exemplary description of independently amplifying a first gene linked to a DHFR gene and a second gene linked to an ADA gene. Other selectable, amplifiable markers can also be used, and examples are reviewed in Kaufman, R. J., Genetic Engineering, 9:155, J. K. Setlow, ed. (Plenum Publishing Corp.) 1987.

VI. Characterization of MAbs

The MAbs so produced by the amplified cell lines can be characterized by standard immunochemical techniques, including SDS-PAGE, Western blotting and immunoprecipitation of intrinsically ³⁵S-methionine-labeled proteins. The levels of heavy and light chains produced can be quantitated by ELISAs, and binding to solid-phase antigens can be demonstrated by ELISA. The binding characteristics of the antibodies can also be studied in similar antigen-binding ELISAs in the presence of varying concentrations of free antigen. The effector functions of the antibodies can be characterized by standard techniques, e.g. for complement fixation and antibody-dependent cellular cytotoxicity.
 

Claim 1 of 31 Claims

1. A method of optimizing the expression level of an antibody or a fragment thereof, which comprises:

(a) producing a eukaryotic host cell containing and capable of expressing a first DNA sequence encoding an antibody heavy chain, said first DNA sequence being associated with a first heterologous selectable amplifiable marker gene, and a second DNA sequence encoding an antibody light chain, said second DNA sequence being associated with a second heterologous selectable amplifiable marker gene;

(b) culturing said host cell in a suitable culture medium;

(c) measuring the relative amounts of said first and second DNA sequences expressed; and

(d) differentially amplifying said amounts of said first and second DNA sequences with appropriate selective agents to allow maximized production of said antibody or fragment thereof.
 

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