Single domain ligands, receptors comprising said ligands, methods for their production, and use of said ligands and receptors

An isolated multimer comprising two or more polypeptides, wherein each of said polypeptides independently comprises one or more of:

• a) a variable domain of an antibody heavy or light chain, said domain having specificity for an antigen;
• or
• b) a portion of a) having specificity for said antigen,
• wherein each of said variable domains or portions binds said antigen as a single antibody variable domain or antigen-binding portion thereof in said multimer, and said multimer binds said antigen with an affinity better than 50 nM.

An isolated multimer comprising two or more polypeptides, wherein each of said polypeptides independently comprises one or more of:

• a) a variable domain of an antibody heavy or light chain, said domain having specificity for an antigen;
• or
• b) a portion of a) having specificity for said antigen,
• wherein each of said variable domains or portions binds said antigen as a single antibody variable domain or antigen-binding portion thereof with an affinity better than 50 nM, and wherein each of said variable domains or portions binds said antigen as a single antibody variable domain or antigen-binding portion thereof in said multimer.

An isolated polypeptide, comprising one or more of:

• a)a variable domain of an antibody heavy or light chain, said domain having specificity for an antigen;
• or
• b)a portion of a) having specificity for said antigen,
• wherein each of said variable domains or portions i) binds said antigen as a single antibody variable domain or antigen-binding portion thereof in said polypeptide, and ii)a CDR with one to four mutations relative to a parental mammalian antibody heavy or light chain variable domain or portion thereof, and
• wherein said polypeptide can compete with said parental domain for binding to antigen.

An isolated polypeptide, comprising one or more of:

• a) a variable domain of an antibody heavy or light chain, said domain having specificity for an antigen;
• or
• b) a portion of a) having specificity for said antigen,
• wherein each of said variable domains or portions binds said antigen as a single antibody variable domain or antigen-binding portion thereof in said polypeptide, and wherein when each of said variable domains or portions is tagged with a C-terminal myc peptide tag, antigen binding is detectable in an ELISA comprising the steps:
• i)coating a well of an ELISA plate with said antigen;
• ii)washing with phosphate buffered saline (PBS), blocking for 2 hrs at 37"C with 200μl instant dried skimmed milk powder in PBS, discarding the blocking solution, and washing three times with PBS;
• iii)adding 150 μl of a test solution comprising said polypeptide tagged with a C-terminal myc peptide tag (EQKLISEEDLN) to the well, incubating at 37"C for 2 hrs, discarding the test solution, and washing three times with PBS;
• iv)adding as first antibody 100 μl of a solution of 4 μg/ml purified murine 9E10 antibody in 2% instant dried skimmed milk powder in PBS, incubating at 37"C for 2 hrs, discarding the first antibody solution, and washing three times with PBS;
• v)adding as second antibody 100 μl of a solution of a 1/500 dilution of peroxidase conjugated anti-mouse immunoglobulin, incubating at 37"C for 2 hrs, discarding the second antibody solution, and washing three times with PBS;
• vi)adding 100 μl of 2,2'azino-bis(3-ethylbenzthiazolinesulphonic acid) solution (0.55 mg/ml solution in water containing 1 μl of 20% hydrogen peroxide per 10 ml), and allowing the color to develop for 10 minutes at room temperature;
• and
• vii)adding 0.05% sodium azide in 50 mM citric acid, pH 4.3 to stop the reaction, and determining binding in a plate reader.

An isolated multimer comprising two or more polypeptides, wherein each of said polypeptides independently comprises one or more of:

• a) a variable domain of an antibody heavy or light chain, said domain having specificity for an antigen;
• or
• b) a portion of a) having specificity for said antigen,
• wherein each of said variable domains or portions binds said antigen as a single antibody variable domain or antigen-binding portion thereof in said multimer, and wherein when each of said variable domains or portions is tagged with a C-terminal myc peptide tag, antigen binding is detectable in an ELISA comprising the steps:
• i)coating a well of an ELISA plate with said antigen;
• ii)washing with phosphate buffered saline (PBS), blocking for 2 hrs at 37"C with 200μl instant dried skimmed milk powder in PBS, discarding the blocking solution, and washing three times with PBS;
• iii)adding 150 μl of a test solution comprising said polypeptide tagged with a C-terminal myc peptide tag (EQKLISEEDLN) to the well, incubating at 37"C for 2 hrs, discarding the test solution, and washing three times with PBS;
• iv)adding as first antibody 100 μl of a solution of 4 μg/ml purified murine 9E10 antibody in 2% instant dried skimmed milk powder in PBS, incubating at 37"C for 2 hrs, discarding the first antibody solution, and washing three times with PBS;
• v)adding as second antibody 100 μl of a solution of a 1/500 dilution of peroxidase conjugated anti-mouse immunoglobulin, incubating at 37"C for 2 hrs, discarding the second antibody solution, and washing three times with PBS;
• vi)adding 100 μl of 2,2'azino-bis(3-ethylbenzthiazolinesulphonic acid) solution (0.55 mg/ml solution in water containing 1 μl of 20% hydrogen peroxide per 10 ml), and allowing the color to develop for 10 minutes at room temperature;
• and
• vii)adding 0.05% sodium azide in 50 mM citric acid, pH 4.3 to stop the reaction, and determining binding in a plate reader.

A method of detecting or identifying a polypeptide having specificity for an antigen, wherein said polypeptide binds said antigen with an affinity better than 50the polypeptide comprising a variable domain of an antibody heavy or light chain, wherein a single antibody variable domain is present in said polypeptide, and said variable domain binds said antigen as a single antibody variable domain in said polypeptide, the method comprising:

• a)providing a nucleic acid sample comprising a repertoire of target sequences, wherein the target sequences each encode a variable domain of an antibody heavy or light chain;
• b)conducting an extension reaction using forward and back primers with the sample repertoire of step a) as template,
• wherein the forward primer binds a sequence at or adjacent to the 3" end of the sense strand of the target sequences, and the back primer binds a sequence at or adjacent to the 3" end of the antisense strand of the target sequences, and producing a repertoire of nucleic acids each encoding a polypeptide comprising a single variable domain of an antibody heavy or light chain;
• and
• c)selecting a polypeptide that binds said antigen with an affinity better than 50

A method of detecting or identifying a polypeptide having specificity for an antigen, the polypeptide comprising a variable domain of an antibody heavy or light chain, wherein a single antibody variable domain is present in said polypeptide, and wherein said variable domain binds said antigen as a single antibody variable domain in said polypeptide, the method comprising:

• a)providing a nucleic acid sample comprising a repertoire of target sequences, wherein the target sequences each encode a variable domain of an antibody heavy or light chain;
• b)conducting an extension reaction using forward and back primers with the sample repertoire of step a) as template,
• wherein the forward primer binds a sequence at or adjacent to the 3" end of the sense strand of the target sequences, and the back primer binds a sequence at or adjacent to the 3" end of the antisense strand of the target sequences, and producing a repertoire of nucleic acids each encoding a polypeptide comprising a single variable domain of a human antibody heavy or light chain;
• and
• c)selecting a polypeptide that binds said antigen, wherein when said polypeptide is tagged with a C-terminal myc peptide tag, antigen binding is detectable in an ELISA comprising the steps:
• i)coating a well of an ELISA plate with said antigen;
• ii)washing with phosphate buffered saline (PBS), blocking for 2 hrs at 37"C with 200μl instant dried skimmed milk powder in PBS, discarding the blocking solution, and washing three times with PBS;
• iii)adding 150 μl of a test solution comprising said polypeptide tagged with a C-terminal myc peptide tag (EQKLISEEDLN) to the well, incubating at 37"C for 2 hrs, discarding the test solution, and washing three times with PBS;
• iv)adding as first antibody 100 μl of a solution of 4 μg/ml purified murine 9E10 antibody in 2% instant dried skimmed milk powder in PBS, incubating at 37"C for 2 hrs, discarding the first antibody solution, and washing three times with PBS;
• v)adding as second antibody 100 μl of a solution of a 1/500 dilution of peroxidase conjugated anti-mouse immunoglobulin, incubating at 37"C for 2 hrs, discarding the second antibody solution, and washing three times with PBS;
• vi)adding 100 μl of 2,2'azino-bis(3-ethylbenzthiazolinesulphonic acid) solution (0.55 mg/ml solution in water containing 1 μl of 20% hydrogen peroxide per 10 ml), and allowing the color to develop for 10 minutes at room temperature;
• and
• vii)adding 0.05% sodium azide in 50 mM citric acid, pH 4.3 to stop the reaction, and determining binding in a plate reader.

An isolated nucleic acid encoding a polypeptide, comprising one or more of:

• a) a variable domain of an antibody heavy or light chain, said domain having specificity for an antigen;
• or
• b) a portion of a) having specificity for said antigen,
• wherein each of said variable domains or portions binds said antigen as a single antibody variable domain or antigen-binding portion thereof with an affinity better than 50 nM, and wherein each of said variable domains or portions binds said antigen as a single antibody variable domain or antigen-binding portion thereof in said polypeptide.

A method of producing a polypeptide having specificity for an antigen, comprising one or more of:

• a) a variable domain of an antibody heavy or light chain, said domain having specificity for an antigen;
• or
• b) a portion of a) having specificity for said antigen,
• wherein each of said variable domains or portions binds said antigen as a single antibody variable domain or antigen-binding portion thereof with an affinity better than 50 nM, and wherein each of said variable domains or portions binds said antigen as a single antibody variable domain or antigen-binding portion thereof in said polypeptide, comprising expressing the polypeptide in a host cell of claim 147.