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Immunoglobulin DNA cassette molecules, monobody constructs, methods of production, and methods of use therefor

a technology of immunoglobulin and cassette molecules, which is applied in the field of immunoglobulin dna cassette molecules, can solve the problems of time-consuming processes, inability to generate therapeutically useful antibodies, and inability to generate effective antigens in animals/humans, and achieves the effect of simple generation of desired immunoglobulin molecules and rapidity

Inactive Publication Date: 2006-07-06
MILLENNIUM PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a system for quickly and easily generating desired immunoglobulin molecules. The invention features DNA cassette constructs that allow for the rapid and simple production of immunoglobulin molecules. The DNA cassette constructs include an immunoglobulin DNA cassette that can be a light chain or heavy chain immunoglobulin cassette. The DNA cassette constructs also include a poly-adenylation region and a transcriptional promoter region. The invention also includes expression vectors that contain the DNA cassette molecules and host cells that can be used for production of antibodies. The invention provides a more efficient and flexible way to produce antibodies, which can be useful in various applications.

Problems solved by technology

However, such methods are limited in that they may require large amounts of antigen to produce response, and they are time-consuming processes.
Furthermore, antigen may not generate effective response in the animal, and generation of therapeutically useful antibodies is questionable.
Therefore the time and effort expended to produce antibodies using such present methods may not culminate in sufficient antibody production.
Currently utilized methods of production of immunoglobulin expression systems, however, require multiple cloning steps resulting in production of only a single desired molecule.

Method used

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  • Immunoglobulin DNA cassette molecules, monobody constructs, methods of production, and methods of use therefor
  • Immunoglobulin DNA cassette molecules, monobody constructs, methods of production, and methods of use therefor
  • Immunoglobulin DNA cassette molecules, monobody constructs, methods of production, and methods of use therefor

Examples

Experimental program
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Effect test

example 1

Creation of Leader Sequences

[0164] The VH heavy chain leader (nVHL) was created by reverse translation of human and mouse VH signal sequence available in the public database to determine those with the potential to have useful restriction enzyme cloning sites added within the signal sequence. The sequence chosen for the VH leader comes from the mouse gene U60820 which has been described previously (See Mus musculus anti-digoxin immunoglobulin heavy chain variable region precursor mRNA, partial cds. Genbank Submission (13-JUN.-1996) Mironova, R. S., et al.; Gene activity regulation, Inst. Molecular Biology, Bulgarian Academy of Sciences, Acad G. Bonchev bl 21, Sofia 1113, Bulgaria.) [0165] U60820 (SEQ ID NO: 101): MAVLGLLFCLVTLPNCVLS

[0166] DNA sequences were designed that would encode both the original protein sequence and the desired unique restriction cloning site, MfeI, flanked by an EcoRI restriction enzyme site and a Kozak sequence on the 5′ end. At its 3′ end a stuffer sequen...

example 2

Creation of Human Cassettes

[0174] The human heavy chain constant region, human IgG1-WT, was constructed by a standard PCR reaction of human splenic cDNA (Invitrogen) with the primers pCHhum1 and pCHhum2 (Table 2). The primer pCHhum1 contains the cloning site BlpI within its 5′ sequence and the primer pCHhum2 contains a stop codon followed by the cloning site XbaI. Utilization of these primers resulted in generation of the human constant region sequences shown in SEQ ID NO:7 & 8.

[0175] The human IgG1-WT cassette, named PLKTOK55, was then created by cloning the nVHL fragment together with the IgG1 fragment into the expression vector pLKTOK10 which contains an EF-1a promoter and a BGH polyadenylation region flanking the cloning site (FIG. 3). This method was used to create all the described heavy chain cassettes.

[0176] The entire region of promoter through polyadenylation region is flanked with BglII and BamHI restriction enzyme sites. This allows the entire cassette to be transferr...

example 3

Creation of Macaque Cassettes

[0185] Published sequences for the cynomologous and rhesus macaques IgG1 cDNA's were different at 3 amino acids so cDNA for IgG1 isolated from PBLs from both monkey species (See, e.g., Calvas P, et al. Characterization of the three immunoglobulin G subclasses of macaques. 1999 Scand J Immunol 49(6):595-610; and Lewis A P, et al. Cloning and sequence analysis of kappa and gamma cynomolgus monkey immunoglobulin cDNAs. 1993. Dev Comp Immunol 17(6):549-60). Sequencing analysis determined that the reported differences did not truly exist so a single macaque IgG1 cassette could be created for use in both monkey species.

[0186] The macaque heavy chain constant region, macaque IgG1-WT, was constructed by a standard PCR reaction of rhesus PBL cDNA with the primers pIgGrh1 and pIgGrh4 (Table 2). The primer pIgGrh1 contains the cloning site BlpI within its 5′ sequence and the primer pIgGrh4 contains a stop codon followed by the cloning site XbaI. Resultant sequenc...

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Abstract

Featured are DNA molecules, expression vectors, and host cells useful for creation of immunoglobulins, as well as novel immunoglobulin molecules termed monobodies. Additionally provided are methods of production of immunoglobulins, including monobodies, as well as methods of using the disclosed immunoglobulin and monobody constructs, expression vectors and host cells containing DNA encoding molecules for production of immunoglobulin and monobody proteins.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 60 / 350,166, filed Oct. 19, 2001, and U.S. Provisional Application No. 60 / 392,364, filed Jun. 26, 2002, the contents of which are incorporated herein by this reference.FIELD OF THE INVENTION [0002] The present invention relates to immunoglobulin DNA cassette molecules, including novel constructs termed monobody constructs, as well as methods for producing expression vectors and host cells containing DNA cassettes and capable of producing immunoglobulin and monobody molecules. More particularly, the invention relates to DNA cassette molecules useful for creating a variety of immunoglobulin therapeutic molecules, including novel monobodies, as well as methods for producing and using such molecules. BACKGROUND OF THE INVENTION [0003] Immunoglobulins (Igs), or antibodies, as the main effectors of humoral immunity and endogenous modulators of the immune response, have pro...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395C07H21/04C12P21/06C12N1/21C12N5/06C12N1/18C07K16/00C07K16/28
CPCC07H21/04C07K16/00C07K16/2866C07K2317/24C07K2319/00
Inventor O'KEEFE, THERESAPONATH, PAUL
Owner MILLENNIUM PHARMA INC
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