Heterologous polypeptide of the TNF family

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...

Examples

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,...

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...

Examples

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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