Soluble heavy-chain only antibodies
The present invention provides a high affinity, antigen-specific, soluble heavy chain-only antibody which: lacks hallmark camelid-related amino acid substitutions and has FR2 substitutions which are not found in antibodies which comprise heavy and light chain; shows increased net hydrophobicity within CDR1 and an increased number of charged amino acids present in CDR3; and comprises one or more amino acid substitutions within the framework β-pleated sheet leading to increased net hydrophobicity within FR1 and increased number of charged amino acids present in FR3. Also provided are VH domains having the same properties, gene segments for their production, methods for their production, transgenic animals and uses of the antibody of the VH domains in therapy.
1. A transgenic non-human mammal comprising a transgene, the transgene comprising at least one heterologous immunoglobulin heavy chain locus comprising human VH gene segments, one or more human D gene segments, human J gene segments, and at least one mouse constant region gene segment wherein the human VH gene segments comprise up to 20 VH gene segments and all of said human VH gene segments are naturally occurring, the human D gene segments comprise at least 5 human D gene segments, the human J gene segments comprise all six human J gene segments, and the mouse constant region gene segment comprises a Cγ lacking CH1.
2. The transgenic non-human mammal according to claim 1 , wherein said locus further comprises a selectable marker.
3. The transgenic non-human mammal according to claim 1 , wherein the mammal is a rodent.
4. The transgenic non-human mammal according to claim 3 , wherein the rodent is a mouse.
5. A method of producing a heavy chain-only antibody which binds specifically to an antigen comprising:
(a) immunising the non-human transgenic mammal of claim 1 with the antigen, wherein the mammal expresses heavy chain-only antibodies which lack CH1 functionality in the transcribed and processed heavy chain mRNA;
(b) isolating long-lived plasma cells or memory B-cells from the immunised mammal;
(c) isolating an mRNA population from cells derived from step (b);
(d) cloning a cDNA population derived from the mRNA isolated in step (c) into an expression vector and expressing the cDNA in a cell-line of choice; and
(e) selecting at least one cell-line which produces a heavy chain-only antibody which binds specifically to the antigen.
6. A method of producing a heavy chain-only antibody which binds specifically to an antigen comprising:
(a) immunising the non-human transgenic mammal of claim 1 with the antigen, wherein the mammal expresses heavy chain-only antibodies which lack CH1 functionality in the transcribed and processed heavy chain mRNA;
(b) isolating long-lived plasma cells or memory B-cells from the immunised mammal;
(c) isolating an mRNA population from cells derived from step (b);
(d) cloning a cDNA population comprising VH domains derived from the mRNA isolated in step (c) into expression vector of choice such that a VH fusion protein, which may comprise a heavy chain effector region, is expressed in a cell-line of choice; and
(e) selecting at least one cell-line which produces a VH fusion protein which binds specifically to the antigen.
7. A method of deriving a human, antibody from a hybrid antibody comprising:
(a) carrying out the steps of claim 5 or 6 ;
(b) cloning and sequencing the V H domain from the cell line;
(c) recloning selected sequences comprising the V H binding domain coding sequences with human constant effector domains of choice; and
(e) expressing the recloned sequences using an expression vector in a cell type of choice.
8. A vector comprising an immunoglobulin heavy chain locus comprising human VH gene segments, one or more human D gene segments, human J gene segments, and at least one mouse constant region gene segment wherein the human VH gene segments comprise up to 20 VH gene segments and all of said human VH gene segments are naturally occurring, the human D gene segments comprise at least 5 human D gene segments, the human J gene segments comprise all six human J gene segments, and the mouse constant region gene segment comprises a Cγ lacking CH1.