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Heterologous polypeptide of the TNF family

a tumor necrosis factor and heteromeric ligand technology, applied in the field of apbf, can solve the problems of complex ligand/receptor interaction, increase, etc., and achieve the effect of stimulating b-cells

Inactive Publication Date: 2005-06-09
RENNERT PAUL +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009] In preferred embodiments of the invention, the heterologous polypeptide comprises more than one APRIL subunit, and more preferably two APRIL subunits, linked non-covalently to a BAFF subunit. In alternative embodiments, the heterologous polypeptide comprises more than one BAFF subunit, and more preferably two BAFF subunits, linked l0 non-covalently to an APRIL subunit. Thus, in preferred embodiments, the present invention is directed to heterologous polypeptide trimers of BAFF and APRIL subunits, in which the ratio of APRIL to BAFF subunits is 2:1, or alternatively 1:2.
[0010] The present invention also relates to therapeutic methods utilizing the heteromers of the invention. One aspect of the invention relates to methods of inhibiting B-cell, T-cell or tumor cell growth in an animal by administering a therapeutically effective amount of a composition selected from the group consisting of an isolated APBF molecule or active fragment thereof, a recombinant APBF molecule or active fragment thereof, and an antibody specific for APBF or an active fragment thereof. Another aspect of the invention relates to methods of stimulating B-cell or T-cell growth in an animal by administering a therapeutically effective amount of a composition selected from the group consisting of an isolated APBF molecule or active fragment thereof, a recombinant APBF molecule or active fragment thereof, and an antibody specific for APBF or an active fragment thereof.

Problems solved by technology

In addition to the number of new members, the complexity of the ligand / receptor interactions has also increased.

Method used

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  • Heterologous polypeptide of the TNF family
  • Heterologous polypeptide of the TNF family
  • Heterologous polypeptide of the TNF family

Examples

Experimental program
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example 1

[0065] This example describes the detection of APRIL and BAFF heteromers by immunoprecipitation following co-transfection into mammalian cells.

Methods:

[0066] The plasmids encoding FLAG-tagged human soluble APRIL, beginning with residue A105 (LT033) or K110 (PL448), soluble FLAG-tagged human TWEAK beginning at A106 (PS288) or soluble FLAG-tagged human EDA beginning at A242 (PS548) or empty vector (CH269) were co-transfected with a full-length human BAFF construct (PS544) into 293T cells using lipofectamine (Life Technologies). At 48 hrs. post-transfection, conditioned media was collected and used for immunoprecipitation experiments. The immunoprecipitation samples contained 200 μl of conditioned media, 5 μg / ml of the anti-FLAG antibody M2 (Sigma) and 800 μl of DMEM containing 10% FCS, glutamine, Pen-Strep, G418 and sodium azide and were incubated at 4° C. for 1 hour, with agitation. Then, 30 μl of ProteinA-Sepharose beads (Pharmacia) was added to the samples and the mixture was in...

example 2

[0071] This example describes the detection of APBF heteromers by immunoprecipitation following co-transfection of two soluble constructs into mammalian cells.

Methods:

[0072] Plasmids encoding the following human soluble TNF family ligands were constructed with the indicated N-terminal epitope tags beginning at the ligand amino acid residue indicated in a PCR3 based mammalian cell expression vector: FLAG-APRIL, beginning with residue A105 (plasmid #LT033) or H115 (plasmid #LT038), FLAG-TWEAK A106 (plasmid #PS288), myc-APRIL A105 (plasmid #JST557), and myc-BAFF Q136 (plasmid #JST556). Various constructs encoding FLAG-tagged ligands, full length murine APRIL(plasmid #LT022), or empty vector control (plasmid #CH269) were each co-transfected with the myc-BAFF Q136 construct into 293T cells using lipofectamine (Life Technologies, Gaithersburg, Md.). At 48 hrs. post-transfection, conditioned media was collected and used for immunoprecipitation experiments. The immunoprecipitation sample...

example 3

Production and isolation of APBF by Affinity Methods

[0077] Plasmids encoding the following human soluble TNF family ligands were constructed with N-terminal FLAG or 6×His epitope tags beginning at the amino acid residue indicated in a PCR3 based mammalian cell expression vector: FLAG-APRIL, beginning with residue A87 (plasmid Lf133) and RGS(H)6-BAFF Q134. These plasmids are then co-transfected into 293T cells using lipofectamine (Life Technologies, Gaithersburg, Md.) and at 48 hrs. post-transfection, conditioned media is collected. The conditioned media is dialyzed against 50 mM NaH2PO4, pH8.0; 300 mM NaCl; 10 mM imidazole and run over a Ni—NTA Superflow column (Qiagen, Valencia, Calif.). Homomers and heteromers containing the 6×His tagged BAFF subunit bind to the Ni column; homomeric FLAG-APRIL molecules flow through. The column is washed with 50 mM NaH2PO4, pH8.0; 300 mM NaCl; 20 mM imidazole with 5-10 column volumes. The column is eluted with 5 column volumes with 50 mM NaH2PO4,...

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Abstract

A newly identified heteromeric ligand of the Tumor Necrosis Factor (TNF)-family, referred to hereinafter as “APBF” has been identified.

Description

RELATED APPLICATIONS [0001] This is a continuation of PCT / US01 / 04121, filed on Feb. 8, 2001, which claims priority from U.S. provisional application Ser. No. 60 / 181,670 filed on Feb. 11, 2000.TECHNICAL FIELD [0002] This invention relates, in part, to a newly identified heteromeric ligand of the Tumor Necrosis Factor (TNF)-family, referred to hereinafter as “APBF”, its variants, derivatives, agonists and antagonists; and uses thereof. In particular, the invention relates to an APBF having a TNF-family member APRIL subunit linked non-covalently to a TNF-family member BAFF subunit. BACKGROUND OF THE INVENTION [0003] Tumor Necrosis Factor (TNF)-family members can best be described as master switches in the immune system controlling both cell survival and differentiation. Given the current progress in manipulating members of the TNF-family for therapeutic benefit, including anti-tumor activity as well as immune regulation and inflammation, it is likely that members of this family will pr...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00A61K39/395A61K45/00C12N15/09A61P1/16A61P11/00A61P15/00A61P17/00A61P17/02A61P19/02A61P19/04A61P29/00A61P35/00A61P37/02A61P37/04A61P37/06A61P43/00C07K14/52C07K14/525C07K14/705C07K19/00C12N5/02
CPCA61K38/00C07K14/70575C07K14/52A61P1/16A61P11/00A61P15/00A61P17/00A61P17/02A61P19/02A61P19/04A61P29/00A61P35/00A61P37/02A61P37/04A61P37/06A61P43/00
Inventor RENNERT, PAULTHOMPSON, JEFFREYAMBROSE, CHRISTINECACHERO, TERESA
Owner RENNERT PAUL
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