US 11,685,773 B2
Method for mass humanization of rabbit antibodies
Jacob E. Gunn Glanville, San Francisco, CA (US); Vera Molkenthin, Tännesberg (DE); Remko Albert Griep, Plzen (CZ); Ahmad Trad, Plzen (CZ); Peter Milovnik, Plzen (CZ); and Volker Lang, Wolnzach (DE)
Assigned to ABCHECK s.r.o., Plzen (CZ); and CHARLES RIVER LABORATORIES, INC., Wilmington, MA (US)
Appl. No. 15/577,153
Filed by AbCheck s.r.o., Plzen (CZ); and CHARLES RIVER LABORATORIES, INC., Wilmington, MA (US)
PCT Filed Apr. 29, 2016, PCT No. PCT/EP2016/000701
§ 371(c)(1), (2) Date Nov. 27, 2017,
PCT Pub. No. WO2016/173719, PCT Pub. Date Nov. 3, 2016.
Claims priority of application No. 15001304 (EP), filed on Apr. 30, 2015.
Prior Publication US 2018/0319869 A1, Nov. 8, 2018
Int. Cl. C07K 16/06 (2006.01); C07K 16/46 (2006.01); C07K 16/00 (2006.01); C07K 16/40 (2006.01)
CPC C07K 16/005 (2013.01) [C07K 16/40 (2013.01); C07K 2317/24 (2013.01); C07K 2317/56 (2013.01); C07K 2317/565 (2013.01); C07K 2317/567 (2013.01); C07K 2317/622 (2013.01); C07K 2317/92 (2013.01)] 17 Claims
 
1. A method for producing a population of 20 or more different nucleic acids, each encoding at least one protein comprising at least one immunoglobulin variable domain comprising a rabbit CDR3 amino acid sequence embedded in human framework sequences, wherein the nucleic acid sequences encoding the rabbit CDR3 amino acid sequences are diversified among the population, wherein the method comprises the following steps:
(a) providing, simultaneously, at least 10 nucleic acids each encoding a rabbit complementarity determining region 3 (CDR3) amino acid sequence, and
(b) generating the population of 20 or more different nucleic acids, each encoding at least one protein comprising at least one immunoglobulin variable domain comprising a rabbit CDR3 amino acid sequence embedded in human framework sequences, wherein the human framework sequences consist of a first human framework region (human FR1), a second human framework region (human FR2), a third human framework region (human FR3), and a fourth human framework region (human FR4),
such that the human FR1 and human FR2 regions are interspaced by a complementarity determining region 1 (CDR1), the human FR2 and human FR3 regions are interspaced by a complementarity determining region 2 (CDR2), and the human FR3 and human FR4 regions are interspaced by a rabbit CDR3 amino acid sequence,
wherein each nucleic acid sequence encoding the CDR1 or the CDR2 amino acid sequence of the variable domain is independently based
i) on a nucleic acid sequence encoding a human CDR1 or a human CDR2, respectively, wherein at least one of the nucleic acid sequences encoding the CDR1 or the CDR2 amino acid sequence has been modified to encode at least one amino acid present in a rabbit CDR1 or a rabbit CDR2 amino acid sequence, respectively, or
ii) on a nucleic acid sequence encoding a rabbit CDR1 or a rabbit CDR2, respectively, wherein at least one of the nucleic acid sequences encoding the CDR1 or the CDR2 amino acid sequence has been modified to encode at least one amino acid present in a human CDR1 or a human CDR2 amino acid sequence, respectively,
wherein at least 80% of the nucleic acids of the population encode different CDR1 and different CDR2 amino acid sequences, and
wherein the human FR1, human FR2, human FR3 and human FR4 regions are human framework regions selected to provide a scaffold conducive for rabbit CDR3 amino acid sequences, wherein
(1) the scaffold yields a correctly folded antibody for at least 30% of grafted rabbit CDR3 amino acid sequences; and/or
(2) the scaffold exhibits at least 30% framework homology to a rabbit framework
wherein at least 10 of the nucleic acids of the population encode different CDR3 amino acid sequences,
and wherein
the two C-terminal amino acids of human FR2 are optionally non-human, and
the two C-terminal amino acids of human FR3 are optionally non-human.