Landscaped antibodies and antibody fragments for clinical use
A method of making a glycosylated antibody or antibody fragment having a reactive ketone group on the glycosylated site is provided. The method comprises expressing a cell transfected with a vector encoding an antibody having one or more glycosylation sites in a culture medium comprising a ketone derivative of a saccharide or saccharide precursor and, in the case of an antibody fragment, fragmenting the resulting antibody into an antigen-binding antibody fragment. Methods of making immunoconjugates comprising the glycosylated antibodies or antibody fragments also are provided, wherein the antibody or antibody fragment is reacted with an agent comprising a ketone-reactive group. Glycosylated antibodies and antibody fragments having a reactive ketone group on the glycosylated site, immunoconjugates comprising such antibodies and antibody fragments and in vivo targeting methods using such antibodies, antibody fragments and immunoconjugates also are provided.
1. A method of making a glycosylated antibody having a reactive ketone group on the glycosylated site, comprising:
expressing SP2/0 cells that are transfected with a vector encoding an antibody having one or more N-glycosylation sites in the CH1 or Vκ domain in a culture medium comprising a ketone derivative of a saccharide or biosynthetic saccharide precursor, where the ketone derivative of the saccharide or biosynthetic saccharide precursor is selected from the group consisting of N-levulinoyl mannosamine and N-levulinoyl fucose, so that they produce a glycosylated antibody having a reactive ketone group on the glycosylated site.
2. The method of claim 1 , wherein the antibody has more than one glycosylation site.
3. The method of claim 1 , wherein the antibody is a single-chain antibody.
4. A method of making a glycosylated antigen-binding antibody fragment having a reactive ketone group on the glycosylated site comprising:
expressing SP2/0 cells that are transfected with a vector encoding an antibody having one or more N-glycosylation sites in the CH1 or Vκ domain in a culture medium comprising a ketone derivative of a saccharide or biosynthetic saccharide precursor, wherein said ketone derivative of the saccharide or biosynthetic saccharide precursor is selected from the group consisting of N-levulinoyl mannosamine and N-levulinoyl fucose, so that they produce a glycosylated antibody having a reactive ketone group on the glycosylated site, and
fragmenting the resulting glycosylated antibody to produce a glycosylated antigen-binding antibody fragment having a reactive ketone group on the glycosylated site.
5. The method of claim 4 , wherein the fragment is an F(ab′) 2 fragment.
6. A method of making a glycosylated antibody having a reactive ketone group on the glycosylated site, comprising:
expressing SP2/0 cells that are transfected with a vector encoding an antibody having a HCN1, HCN5 or Vκ N-glycosylation site in a culture medium comprising a ketone derivative of a saccharide or biosynthetic saccharide precursor, where the ketone derivative of the saccharide or biosynthetic saccharide precursor is selected from the group consisting of N-levulinoyl mannosamine and N-levulinoyl fucose, so that they produce an N-glycosylated antibody having a reactive ketone group on the glycosylated site.
7. A method making a glycosylated antigen-binding antibody fragment having a reactive ketone group on the glycosylated site, comprising:
expressing SP2/0 cells that are transfected with a vector encoding an antibody having a HCN1, HCN5 or Vκ N-glycosylation site in a culture medium compnsing a ketone derivative of a saccharide or biosynthetic saccharide precursor, where the ketone derivative of the saccharide or biosynthetic saccharide precursor is selected from the group consisting of N-levulinoyl mannosamine and N-levulinoyl fucose, so that they produce a glycosylated antibody having a reactive ketone group on the glycosylated site, and
fragmenting the resulting glycosylated antibody into a glycosylated antigen-binding antibody fragment having a reactive ketone group on the glycosylated site.