Genentech, Inc.

City of Hope

Beckman Research Institute of the City of Hope

Arthur D Riggs

Herbert L Heyneker

William E Holmes

John A Zaia

Keiichi Itakura

Hema Pande

Ronald B Wetzel

Shmuel Cabilly

John J Rossi

Qiang Liu

John E Shively

Brian R Clark

Steve S Sommer

Michaela Scherr

Michael Neumaier

Kenneth C Reed

John M Corbett

The invention relates to processes for producing an immunoglobulin or an immunologically functional immunoglobulin fragment containing at least the variable domains of the immunoglobulin heavy and light chains. The processes can use one or more vectors which produce both the heavy and light chains or fragments thereof in a single cell. The invention also relates to the vectors used to produce the immunoglobulin or fragment, and to cells transformed with the vectors.

Methods of making antibody heavy and light chains having specificity for a desired antigen

A novel method of pyrophosphorolysis activated polymerization (PAP) has been developed. In PAP, pyrophosphorolysis and polymerization by DNA polymerase are coupled serially for each amplification by using an activatable oligonucleotide P* that has a non-extendible 3'-deoxynucleotide at its 3&#x2032; terminus. PAP can be applied for exponential amplification or for linear amplification. PAP can be applied to amplification of a rare allele in admixture with one or more wild-type alleles by using an activatable oligonucleotide P* that is an exact match at its 3&#x2032; end for the rare allele but has a mismatch at or near its 3&#x2032; terminus for the wild-type allele. PAP is inhibited by a mismatch in the 3&#x2032; specific sequence as far as 16 nucleotides away from the 3&#x2032; terminus. PAP can greatly increase the specificity of detection of an extremely rare mutant allele in the presence of the wild-type allele. Specificity results from both pyrophosphorolysis and polymerization since significant nonspecific amplification requires the combination of mismatch pyrophosphorolysis and misincorporation by the DNA polymerase, an extremely rare event. Using genetically engineered DNA polymerases greatly improves the efficiency of PAP.

Pyrophosphorolysis activated polymerization (PAP)

A method for identifying sites on a target RNA which are accessible to pairing by antisense DNA, ribozymes or DNAzymes. Native or in vitro-synthesized target RNA is incubated with defined ODNs and RNase H, or with a random or semi-random ODN library and RNase H, or with defined ribozymes or DNAzymes, or with a semi-random ribozyme or DNAzyme library, in a cell extract containing endogenous RNA-binding proteins, or in a medium which mimics a cell extract due to presence of one or more RNA-binding proteins. Any antisense ODN, ribozyme or DNAzyme which is complementary to an accessisble site in the target RNA hybridizes to that site and the RNA is cleaved at that site. Reverse transcription can be used to generate a first strand DNA from the RNA cleavage product, and terminal deoxynucleotidyl transferase-dependent polymerase chain reaction (TDPCR) can be used to identify sites on target RNA to which antisense ODNs, ribozymes or DNAzymes have bound and cleavage has occurred.

Method for identifying accessible binding sites on RNA

A novel method of pyrophosphorolysis activated polymerization (PAP) has been developed. In PAP, pyrophosphorolysis and polymerization by DNA polymerase are coupled serially for each amplification by using an activatable oligonucleotide P* that has a non-extendible 3'-deoxynucleotide at its 3&#x2032; terminus. PAP can be applied for exponential amplification or for linear amplification. PAP can be applied to amplification of a rare allele in admixture with one or more wild-type alleles by using an activatable oligonucleotide P* that is an exact match at its 3&#x2032; end for the rare allele but has a mismatch at or rear its 3&#x2032; terminus for the wild-type allele. PAP is inhibited by a mismatch in the 3&#x2032; specific sequence as far as 16 nucleotides away from the 3&#x2032; terminus. PAP can greatly increase the specificity of detection of an extremely rare mutant allele in the presence of the wild-type allele. Specificity results from both pyrophosphorolysis and polymerization since significant nonspecific amplification requires the combination of mismatch pyrophosphorolysis and misincorporation by the DNA polymerase, an extremely rare event. Using genetically engineered DNA polymerases greatly improves the efficiency of PAP.

Methods of producing immunoglobulins, vectors and transformed host cells for use therein

Disclosed are amino acid sequences of the late 64 kilodalton protein of human cytomegalovirus (HCMVgp64), useful in diagnosing and preventing HCMV infections.

Method for detection and prevention of human cytomegalovirus infection

A DNA probe has been isolated which is capable of hybridizing to an oligonucleotide sequence coding for a polypeptide from a major 64 Kilodalton protein of human cytomegalovirus (HCMVgp64). The probe has a sequence of at least seventeen (17) to as many as seven hundred twenty-one (721) nucleotides. The DNA fragments coding for the major late protein of human cytomegalovirus (HCMVgp64) may be hybridized to DNA fragments of HCMV DNA from an individual having human cytomegalovirus infection. The major late protein of human cytomegalovirus (HCMVgp64) also reacts with T-lymphocytes of an individual after natural infection of that individual with human cytomegalovirus. Thus, the HCMVgp64 protein may be used as a vaccine to prevent HCMV infection.

Method and means for microbial polypeptide expression

1. Recombinant microbial cloning vehicles comprising heterologous DNA coding for the expression of mammalian hormone (e.g., somatostatin) and other polypeptides, including plasmids suited for the transformation of bacterial hosts. The latter incorporate a regulon homologous to the host in its untransformed state, in reading phase with the structural gene for the heterologous DNA; 2. Cloning vehicles coding for the microbial expression of a protein variously comprising (a) a polypeptide hapten and additional protein sufficient in size to confer immunogenicity on the product of expression, which may find use in raising antibodies to the hapten for assay use or in the manufacture of vaccines; and (b) a desired polypeptide product and additional protein from which the desired product may be cleaved; and 3. Methods of preparing synthetic structural genes coding for the expression of mammalian polypeptides in microbial cloning systems.

The present invention provides an apparatus and method for the amplification of a particular sequence(s) of DNA in a sample using polymerased chain reaction. The method of the present invention involves the injection of a reaction mixture into a stream of carrier fluid. The carrier fluid then passes through a plurality of temperature zones in which the polymerase chain reactions take place. The method of the present invention allows the sequential processing of a number of samples.

Device and method for the automated cycling of solutions between two or

A chimeric anti CEA antibody comparable to ATCC Accession No. BH 8747 is described.

Chimeric anti-CEA antibody

A DNA probe has been isolated which is capable of hybridizing to an oligonucleotide sequence coding for a polypeptide from a major 64 Kilodalton protein of human cytomegalovirus (HCMVgp64). The probe has a sequence of at least seventeen (17) to as many as seven hundred twenty-one 721) nucleotides. The probe may be labelled as by radioactivity. The probe has been used to screen DNA fragments constituting a subgenomic library of human cytomegalovirus DNA to obtain DNA fragments coding for the major late protein of human cytomegalovirus. The DNA fragments coding for the major late protein of human cytomegalovirus (HCMVgp64) may be hybridized to DNA fragments of HCMV DNA from an individual having human cytomegalovirus infection. The viral DNA can be used as whole HCMV DNA or as fragments formed by digesting the human cytomegalovirus DNA with a restriction endonuclease such as one of the restriction endonucleases EcoRI, BamHI, XbaI, HindIII and PrtI. During the screening of clinical specimens, the DNA fragment coding for HCMVgp64 of human cytomegalovirus hybridizes to whole HCMV DNA or to a particular one of the DNA fragments produced by digesting the human cytomegalovirus DNA with the restriction endonucleases. As a resuslt of such hybridization, the identity of human cytomegalovirus may be established. The particular DNA fragment may have a map location of approximately 0.50 to 0.51 units in the human cytomegalovirus (strain Towne) genome. It may have a sequence of about 721 nucleotides. The major late protein of human cytomegalovirus (HCMVgp64) also reacts with T-lymphocytes of an individual after natural infection of that individual with human cytomegalovirus. Thus, the HCMVgp64 protein may be used as a vaccine to prevent HCMV infection.

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La Verne, CA, United States of America

Arthur D Riggs