Method for mass humanization of non-human antibodies

1. A method of producing a population of nucleic acids encoding immunoglobulin variable domains of humanized antibodies, wherein each of the nucleic acids comprises a non-human complementarity determining region 3 (CDR3) encoding sequence embedded in a scaffold-encoding nucleic acid comprising a first human framework the region (FR1) encoding sequence, a second human framework region (FR2) encoding sequence, a third human framework region (FR3) encoding sequence, a fourth human framework region (FR4) encoding sequence, a complementarity determining region 1 (CDR1) encoding sequence, and a complementarity determining region 2 (CDR2) encoding sequence, such that the FR1 encoding sequence and FR2 encoding sequence are interspaced by the CDR1 encoding sequence, the FR2 encoding sequence and FR3 encoding sequence are interspaced by the CDR2 encoding sequence, and the FR3 encoding sequence and FR4 encoding sequence are interspaced by the non-human CDR3 encoding sequence, the method comprising the following steps:

a) providing at least one non-human CDR3 encoding sequence;

b) generating, on a computer, a first positional weight matrix (PWM) of amino acid positional variability from naturally-occurring, non-human CDR1s or CDR2s by calculating a first relative frequency of each amino acid at each position of the naturally-occurring, non-human CDR1s or CDR2s;

c) generating, on the computer, a second PWM of amino acid positional variability from naturally-occurring, human CDR1s or CDR2s by calculating a second relative frequency of amino acids at each position of the naturally-occurring, human CDR1s or CDR2s;

d) blending, on a computer, the first PWM and the second PWM to produce a blended PWM that provides for amino acid variation observed in both human and non-human CDR1s or CDR2s;

e) using the blended PWM, generating a plurality of scaffold-encoding nucleic acids comprising different CDR1 encoding sequences or different CDR2 encoding sequences, wherein amino acids of the CDR1 encoding sequences or CDR2 encoding sequences, respectively, occur at about their frequencies at their respective positions within the blended PWM; and

(f) inserting the non-human CDR3 encoding sequence into the scaffold-encoding nucleic acids, thereby generating the population of encoding humanized antibodies.

2. The method of claim 1 , further comprising producing a population of polypeptides from the population of nucleic acids.

3. The method of claim 1 , wherein at least 3 nucleic acids of the population of nucleic acids exhibit different CDR1 encoding and/or CDR2 encoding sequences.

4. The method of claim 1 , wherein the CDR1 or CDR2 encoding sequences exhibit at least 30% sequence identity to a human CDR1 or human CDR2 encoding sequence, respectively.

5. The method of claim 1 , further comprising modifying the CDR1 encoding sequences or CDR2 encoding sequences to increase the immunoglobulin variable domains' similarity to that of the CDR3's native immunoglobulin variable domain with respect to a feature selected from amino acid sequence, cosmology, length, canonical structure, heavy/light interface mount angle, and combinations thereof.

6. The method of claim 1 , comprising adjusting the blended PWM to remove a cysteine, methionine or tryptophan.

7. The method of claim 1 , comprising adjusting the blended PWM towards the amino acid positional variability of the human CDR's or CDR2s or towards the amino acid positional variability of the non-human CDR's or CDR2s.

8. The method of claim 1 wherein the humanized antibodies are selected from scFv, Fv, Fab, Fab′ and F(ab′)2.

9. The method of claim 1 , wherein at least two of the nucleic acids of the population of nucleic acids encode different CDR3 amino acid sequences.

10. The method of claim 1 , wherein at least 10 or more nucleic acids of the population exhibit different CDR1 encoding sequences or different CDR2 encoding sequences.

11. The method of claim 1 , comprising

a) generating, on a computer, the first PWM of amino acid positional variability from naturally-occurring, non-human CDR1s and CDR2s by calculating the first relative frequency of each amino acid at each position of the naturally-occurring, non-human CDR's and CDR2s;

b) generating, on the computer, the second PWM of amino acid positional variability from naturally-occurring, human CDR's or CDR2s by calculating the second relative frequency of amino acids at each position of the naturally-occurring, human CDR's or CDR2s;

c) blending, on the computer, the first PWM and the second PWM to produce the blended PWM that provides for amino acid variation observed in both human and non-human CDR's and CDR2s;

d) using the blended PWM, generating the plurality of scaffold-encoding nucleic acids comprising different CDR1 encoding sequences and different CDR2 encoding sequences, wherein amino acids of the CDR1 encoding sequences and CDR2 encoding sequences, respectively, occur at about their frequencies at their respective positions within the blended PWM.