Immunoconjugates of toxins directed against malignant cells

Inactive Publication Date: 2003-07-03
IMMUNOMEDICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Some solvents which are capable of solubilizing aggregate-forming proteins, for example SDS (sodium dodecyl sulfate), 70% formic acid, are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/

Method used

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  • Immunoconjugates of toxins directed against malignant cells
  • Immunoconjugates of toxins directed against malignant cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

A. Example 1

Expression Pattern of RNAse in Rana pipiens Tissues

[0159] A DNA sequence corresponding to amino acid residues 16-98 of Onconase.RTM. was cloned by PCR amplification of Rana pipiens genomic DNA and sequenced. The sequence, consisting of 252 bp of DNA encoding the ribonuclease was designated Rana clone 9. Total cellular RNA was isolated from either male or female Rana pipiens tissues using RNA STAT-60 (TEL-TEST "B", Inc.) according to the manufacturer's protocol. Poly A+ containing MRNA was prepared using an Oligotex mRNA kit (Qiagen). Poly (A+) RNA was size fractionated on a 1% agarose gel containing 6% formaldehyde and blotted onto Nitran.RTM. nylon membranes (Schleicher & Schuell) in 10.times. SSC overnight. The membrane was rinsed in 2.times. SSC for 5 min, air dried and the RNA was cross linked to the membrane by exposure to UV light (Ultra-Lum) for 2 min. The RNA blot was hybridized at 42.degree. C. for 16-18 hours with a [.sup.32P]-labeled DNA probe prepared from 30...

example 2

B. Example 2

Expression of RNAse in Rana pipiens

[0166] To determine if RNAse is present in Rana pipiens oocytes or other tissues, protein extracts were isolated from various Rana pipiens tissues and separated on a 4-20% Tris-Glycine SDS--containing polyacrylamide gel. The protein extracts were transferred to a nitrocellulose membrane using 1.times.transfer buffer (Novagen) at 250 mA for 45 mm. The membrane was probed with primary and secondary antibodies as described in Chen, et al., Oncogene 12:241 (1996). The primary anti-Onconase.RTM. antibody was used at 1:100 dilution. The detecting antibody (horseradish peroxidase labeled donkey anti-rabbit Ig (Amersham)) was used at 1:2500 dilution. The antibodies were visualized using an ECL detection kit from Amersham.

[0167] The western blot analysis demonstrated that a protein of the correct size (12 kDa) was present in extracts from oocytes. Other tissues, including liver, did not contain a 12 kDa protein that reacted with the anti-Onconas...

example 3

C. Example 3

Isolation and Cloning of cDNA From Rana pipiens Liver mRNA

[0168] Liver poly (A+) RNA was purified twice using the poly (A+) Pure kit (Ambion). The cDNA library was constructed using a ZAP-cDNA synthesis kit and Gigapack II gold packaging extracts according to the manufacturer's protocol (Stratagene). The library contained about 1.5.times.10.sup.6 pfu from 5 .mu.g of liver poly (A+) RNA and was amplified once according to Stratagene's protocol. The library titer after amplification was 9.times.10.sup.9 pfu / mL. About 3.times.10.sup.5 plaques were screened by using a [.sup.32P]-labeled insert of Rana clone 9 following Stratagene's procedure. Positive clones (3alb, 4alb and 5alb) were excised from the lambda ZAP II vector and subcloned into pBluescript SF-vector. Plasmid DNA was prepared using the Qiagen spin plasmid miniprep kit.

[0169] Clone 5alb was digested with KpnI and HindIII to generate 3' and 5' protruding ends, and digested with exonuclease III to generate 5alb dele...

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Abstract

This invention provides for new recombinant ribonuclease proteins which are active when expressed by bacteria. This allows the recombinant ribonucleases of this invention to be fused in-frame with ligand binding moieties to form cytotoxic fusion proteins. Furthermore, these proteins are more active than ribonucleases currently available even though the proteins of this invention lack an N-terminal pyroglutamic acid, which has been found to be necessary for ribonucleolytic activity. Because these proteins are recombinant proteins, mutations which increase cytotoxicity can be engineered.

Description

[0001] This application is a divisional of U.S. Ser. No. 09 / 622,613, filed Aug. 17, 2000, which is a continuation of international application PCT / US99 / 06641, filed Mar. 26, 1999, which in turn claims benefit to provisional application U.S. Serial No. 60 / 079,751, filed Mar. 26, 1998.[0002] Ribonucleases such as ribonuclease A ("RNase A") and their cytotoxicity towards tumor cells were discovered in the 1960s (reviewed in Roth, J., Cancer Res. 23:657-666 (1963)). In the 1970s, human serum was also discovered to contain several RNAses that are expressed in a tissue specific manner (Reddi, E., Biochem. Biophys. Res. Commun. 67:110-118 (1975); and Blank, et al., HUMAN BODY FLUID RIBONUCLEASES: DETECTION, INTERRELATIONSHIPS AND SIGNIFICANCE, pp203-209 (IRL Press, London, 1981)).[0003] Further to these early studies was the discovery that an anti-tumor protein from oocytes of Rana pipiens had homology to RNAse A (Ardelt, et al., J. Biol. Chem. 256:245-251(1991)). This protein was termed O...

Claims

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Application Information

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IPC IPC(8): A61K38/00A61K47/48C12N9/22
CPCA61K38/00C12N9/22A61K47/4843A61K47/6815
Inventor RYBAK, SUSANNA M.GOLDENBERG, DAVID M.NEWTON, DIANNE L.
Owner IMMUNOMEDICS INC
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