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US 10,294,307 B2Granted
Methods for producing fabs and bi-specific antibodies
AssigneesEli Lilly and Company, The University of North Carolina at Chapel Hill
InventorsStephen J Demarest, Xiufeng Wu, Raheleh Toughiri, Brian Arthur Kuhlman, Steven Morgan Lewis
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Abstract
The present invention provides methods for producing Fabs and bi-specific antibodies comprising designed residues in the interfaces of the heavy chain-light chain variable (VH/VL) domain and the heavy chain-light chain constant (CH1/CL] domain, Fabs and bi-specific antibodies produced according to said processes and host cells encoding the same.
Claims13
A method for producing a fragment, antigen binding (Fab) comprising any one of (A)-(C) (all residues are numbered according to the Kabat numbering system):
- (A) (1) co-expressing in a host cell:
- (a) a first nucleic acid encoding both a heavy chain variable (VH) domain and a human IgG heavy chain constant CH1 (CH1) domain, wherein said VH domain comprises a lysine at residue 39 and a glutamic acid at residue 62, and said CH1 domain comprises an alanine at residue 172 and a glycine at residue 174;
- (b) a second nucleic acid encoding both a light chain variable (VL) domain and a light chain constant (CL) domain, wherein said VL domain comprises an arginine at residue 1 and an aspartic acid at residue 38, and said CL domain comprises a tyrosine at residue 135 and a tryptophan at residue 176, wherein each of said VH and VL domains comprise three complementarity determining regions (CDRs) which direct binding to an antigen;
- (2) cultivating said host cell under conditions such that said VH and CH1 domains and said VL and CL domains are produced;
- and
- (3) recovering from said host cell a Fab wherein said Fab comprises said VH and CH1 domains and said VL and CL domains;
- (B) (1) co-expressing in a host cell:
- (a) a first nucleic acid encoding both a VH domain and a human IgG CH1 domain, wherein said VH domain comprises a lysine at residue 39 and a glutamic acid at residue 62, and said CH1 domain comprises an arginine at residue 172 and a glycine at residue 174;
- (b) a second nucleic acid encoding both a VL domain and a CL domain, wherein said VL domain comprises an arginine at residue 1 and an aspartic acid at residue 38, and said CL domain comprises a tyrosine at residue 135 and a tryptophan at residue 176, wherein each of said VH and VL domains comprise three CDRs which direct binding to an antigen;
- (2) cultivating said host cell under conditions such that said VH and CH1 domains and said VL and CL domains are produced;
- and
- (3) recovering from said host cell a Fab wherein said Fab comprises said VH and CH1 domains and said VL and CL domains;
- or
- (C) (1) co-expressing in a host cell:
- (a) a first nucleic acid encoding both a VH domain and a human IgG CH1 domain, wherein said VH comprises a lysine at residue 39 and a glutamic acid at residue 62, and said CH1 domain comprises an alanine at residue 172 and a glycine at residue 174;
- (b) a second nucleic acid encoding both a VL domain and a CL domain, wherein said VL domain comprises an arginine at residue 1 and an aspartic acid at residue 38, and said CL domain comprises a phenylalanine at residue 135 and a tryptophan at residue 176, wherein each of said VH and VL domains comprise three CDRs which direct binding to an antigen;
- (2) cultivating said host cell under conditions such that said VH and CH1 domains and said VL and CL domains are produced;
- and
- (3) recovering from said host cell a Fab wherein said Fab comprises said VH and CH1 domains and said VL and CL domains.
16 more elements
A method for producing a fragment, antigen binding (Fab) comprising any one of (A) or (B) (all residues are numbered according to the Kabat numbering system):
- (A) (1) co-expressing in a host cell:
- (a) a first nucleic acid encoding both a VH domain and a human IgG CH1 domain, wherein said VH domain comprises a tyrosine at residue 39 and said CH1 domain comprises a WT CH1 sequence;
- and
- (b) a second nucleic acid encoding both a VL domain and a CL domain, wherein said VL domain comprises an arginine at residue 38 and said CL domain comprises a WT CL sequence, wherein each of said VH and VL domains comprise three CDRs which direct binding to an antigen;
- (2) cultivating said host cell under conditions such that said VH and CH1 domains and said VL and CL domains are produced;
- and
- (3) recovering from said host cell a Fab wherein said Fab comprises said VH and CH1 domains and said VL and CL domains;
- or
- (B)(1) co-expressing in a host cell:
- (a) a first nucleic acid encoding both a VH domain and a human IgG CH1 domain, wherein said VH domain comprises a tyrosine at residue 39 and said CH1 domain comprises an aspartic acid at residue 228;
- and
- (b) a second nucleic acid encoding both a VL domain and a CL domain, wherein said VL domain comprises an arginine at residue 38 and said CL domain comprises a lysine at residue 122, wherein each of said VH and VL domains comprise three CDRs which direct binding to an antigen;
- (2) cultivating said host cell under conditions such that said VH and CH1 domains and said VL and CL domains are produced;
- and
- (3) recovering from said host cell a Fab wherein said Fab comprises said VH and CH1 domains and said VL and CL domains.
12 more elements
An antibody comprising a heavy chain and a light chain wherein,
- (a) said heavy chain comprises the amino acid sequence of SEQ ID NO. 18 and said light chain comprises the amino acid sequence of SEQ ID NO. 15;
- (b) said heavy chain comprises the amino acid sequence of SEQ ID NO. 19 and said light chain comprises the amino acid sequence of SEQ ID NO. 20;
- (c) said heavy chain comprises the amino acid sequence of SEQ ID NO. 18 and said light chain comprises the amino acid sequence of SEQ ID NO. 20;
- (d) said heavy chain comprises the amino acid sequence of SEQ ID NO. 25 and said light chain comprises the amino acid sequence of SEQ ID NO. 26;
- (e) said heavy chain comprises the amino acid sequence of SEQ ID NO. 29 and said light chain comprises the amino acid sequence of SEQ ID NO. 30;
- (f) said heavy chain comprises the amino acid sequence of SEQ ID NO. 29 and said light chain comprises the amino acid sequence of SEQ ID NO. 41;
- (g) said heavy chain comprises the amino acid sequence of SEQ ID NO. 37 and said light chain comprises the amino acid sequence of SEQ ID NO. 38;
- (h) said heavy chain comprises the amino acid sequence of SEQ ID NO. 39 and said light chain comprises the amino acid sequence of SEQ ID NO. 40;
- or
- (i) said heavy chain comprises the amino acid sequence of SEQ ID NO. 18 and said light chain comprises the amino acid sequence of SEQ ID NO. 64.
7 more elements
Dependencies are inferred from the claim text.