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Polyvalent protein complex

a protein complex and polypeptide technology, applied in the field of polypeptide complexes, can solve the problem that non-covalently associated molecules are not sufficiently stable under physiological conditions to have any practical us

Inactive Publication Date: 2005-01-06
IBC PHARMACEUTICALS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0031] Also provided is a method for detecting or diagnosing a B-cell malignancy, or B-cell immune or autoimmune disorder in a subject, containing administering to the subject a diagnostic composition containing a polyvalent protein complex as above and a pharmaceutically acceptable carrier, where each antigen binding site binds a distinct epitope of CD19, CD20 or CD22, and where the complex is labeled with one or more image enhancing agents for use in magnetic resonance imaging (MRI). The image enhancing agent may be as described above
[0032] Also provided is a method of diagnosing a non-neoplastic disease or disorder, by administering to a subject suffering from the disease or disorder a complex as above, where a detectable label is attached to the complex, and where one or more of the antigen binding sites is specific for a marker substance of the disease or disorder. The disease or disorder may be caused by a fungus, such as Microsporum, Trichophyton, Epidermophyton, Sporothrix schenckii, Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum, Blastomyces dermatitidis, and Candida albican, or a virus, such as human immunodeficiency virus (HIV), herpes virus, cytomegalovirus, rabies virus, influenza virus, hepatitis B virus, Sendai virus, feline leukemia virus, Reo virus, polio virus, human serum parvo-like virus, simian virus 40, respiratory syncytial virus, mouse mammary tumor virus, Varicella-Zoster virus, Dengue virus, rubella virus, measles virus, adenovirus, human T-cell leukemia-viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus and blue tongue virus. The disease or disorder may be caused by a bacterium, such as Anthrax bacillus, Streptococcus agalactiae, Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, and Mycobacterium tuberculosis, or a Mycoplasma. The disease or disorder may be caused by a parasite, such as malaria. The disease or disorder may be an autoimmune diseas...

Problems solved by technology

However, such non-covalently associated molecules are not sufficiently stable under physiological conditions to have any practical use.

Method used

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Examples

Experimental program
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example 1

BS14HP, a Bispecific Trivalent Heterodimer

[0265] Design.

[0266] BS14HP was designed for the constitutive expression of foreign genes in Pichia pastoris using the GAP promoter system. Transfection of P. pastoris cells with a linearized DNA plasmid (BS14HP-GAP+) results in the stable and site-specific integration of the two DNA segments (FIG. 1A) into the GAP locus of the host's chromosome. These two DNA segments contain open reading frames, SEQ ID NO:1 and SEQ ID NO:2, which codes for polypeptide 1 (SEQ ID NO:9) and polypeptide 2 (SEQ ID NO:10) respectively. As each of the two DNA segments also contains nucleotide sequences for the GAP promoter, two mRNA species that encode the amino acid sequences of polypeptide 1 and polypeptide 2 are synthesized in the same host cell.

[0267] Polypeptide 1.

[0268]α-Factor-h679VH-GGGGS-hMN-14VK-LEGGGS-hMN-14VH-6His (SEQ ID NO:1)

[0269] Polypeptide 2.

[0270]α-Factor-hMN-14VK-GGGQFM-hMN-14VH-GGGGS-h679VK-6His (SEQ ID NO:2)

[0271] The “α-factor,” as s...

example 2

hBS14, a Bispecific Trivalent Heterodimer Expressed in Myeloma Cells

[0300] To demonstrate that similar PPCs could be made from other types of host cell systems we developed a scheme for production of a fusion protein named hBS14 in mammalian cell culture. The hBS14 PPC produced in mammalian cell culture was designed to be structurally and functionally similar to BS14HP, which was produced in the yeast P. pastoris (example 1). A DNA plasmid vector was engineered for hBS14 expression and used to generate transgenic cell lines in SP2 / 0-Ag14 mouse myeloma cells, NS0 mouse myeloma, and YB2 / 0 rat myeloma cells. While these particular cell lines were used, it is understood that the vectors of the invention may be used in any mammalian cell lines, such as, for example, a human cell line.

[0301] Generation of an hBS14 DNA Expression Vector

[0302] The nucleic acid encoding the hBS14 polypeptides were recombinantly inserted into the mammalian expression vector pdHL2, which permits the amplifi...

example 3

Affinity Purification of hBS14

[0317] hBS14 was purified to homogeneity using a novel affinity resin that was prepared and used as described below.

[0318] Activation and Coupling of IMP291-affigel

[0319] IMP291 peptide (see structure in FIG. 18) was coupled to Affigel 102 (BIO-RAD Laboratories, Hercules Calif.) using chloroacetic anhydride (CAA). CCA (1.5 g, 8.8 mmol) was dissolved in acetonitrile and added to 30 ml of Affigel 102 slurry. The pH was adjusted to 9.0 with triethylamine and reacted for 1 hour at room temperature to allow coupling of CAA to amine groups on the Affigel. The CAA-Affigel was washed and exchanged into 0.2M NaBorate, pH 8.0. A total of 166 mg of IMP291 was dissolved in 10 ml of 0.2M NaBorate, pH 8.0 and then added to the slurry, which was then rocked overnight at room temperature to allow coupling of the peptide to the CAA-Affigel via thioether bond formation. The resin was quenched by adding cysteine in 0.2M NaBorate, pH 8.0 to a final concentration of 20 m...

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Abstract

The invention provides for a polyvalent protein complex (PPC) comprising two polypeptide chains generally arranged laterally to one another. Each polypeptide chain typically comprises 3 or 4 “v-regions”, which comprise amino acid sequences capable of forming an antigen binding site when matched with a corresponding v-region on the opposite polypeptide chain. Up to about 6 “v-regions” can be used on each polypeptide chain. The v-regions of each polypeptide chain are connected linearly to one another and may be connected by interspersed linking regions. When arranged in the form of the PPC, the v-regions on each polypeptide chain form individual antigen binding sites.

Description

[0001] This application claims priority to U.S. Provisional Application No. 60 / 464,532, filed Apr. 22, 2003, and 60 / 525,391, filed Nov. 24, 2003, the contents of which are hereby incorporated by reference in their entirety.FIELD OF THE INVENTION [0002] The present invention relates to polyvalent protein complexes, including trivalent bispecific proteins, useful for the treatment and diagnosis of diseases, and to methods of producing such proteins. BACKGROUND OF THE INVENTION [0003] Throughout this specification, various patents, published applications and scientific references are cited to describe the state and content of the art. Those disclosures, in their entireties, are hereby incorporated into the present specification by reference. [0004] The present invention is directed to a novel protein structures, termed a “polyvalent protein complex” or PPC, that comprise three or four antigen binding sites (ABS). These PPC comprise novel properties, such as trivalence and tetravalence,...

Claims

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Application Information

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IPC IPC(8): A61K39/395C07H21/04C07K14/475C07K14/52C07K16/00C07K16/30C07K16/44C07K16/46C12NC12N15/00C12P21/04C12Q1/68
CPCA61K2039/505C07K16/3007C07K16/44C07K2319/00C07K2317/24C07K2317/626C07K2318/20C07K16/468A61P1/04A61P1/16A61P11/00A61P13/12A61P17/00A61P17/06A61P19/00A61P21/00A61P21/04A61P25/00A61P25/14A61P25/28A61P29/00A61P31/00A61P31/04A61P31/10A61P31/12A61P33/06A61P33/10A61P35/00A61P35/02A61P37/02A61P37/06A61P37/08A61P43/00A61P5/14A61P5/40A61P7/04A61P7/06A61P9/00A61P9/10A61P3/10
Inventor ROSSI, EDMUNDCHANG, CHIENMCBRIDE, WILLIAM
Owner IBC PHARMACEUTICALS INC
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