Production of T cell retargeting hetero-dimeric immunoglobulins

Abstract

The present invention describes novel hetero-dimeric immunoglobulins or fragments thereof which bind to CD3 and a disease associated antigen. These hetero-dimeric immunoglobulins have been engineered to promote hetero-dimer formation during expression and can be purified to a high degree using a Protein A differential purification technique.

1 . A hetero-dimeric immunoglobulin or fragment thereof, comprising:
 (a) a first polypeptide that binds to Protein A wherein the first polypeptide comprises an epitope binding region that binds a first epitope and an immunoglobulin constant region; and   (b) a second polypeptide that does not bind to Protein A wherein the second polypeptide comprises an epitope binding region, that binds a second epitope and an immunoglobulin constant region;   wherein the first and second polypeptides comprise an engineered immunoglobulin constant region with a modified CH3 domain having a protein-protein interface, wherein the protein-protein interface of the first polypeptide comprises an amino acid substitution at a position selected from the group consisting of: 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 (IMGT® numbering), and wherein the protein-protein interface of the second polypeptide comprises an amino acid substitution at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 84.4, 85.1, 86, 88 and 90 (IMGT® numbering);   wherein the epitope binding region of the first polypeptide binds the CD3 protein complex and the epitope binding region of the second polypeptide binds a disease associated antigen or wherein the epitope binding region of the first polypeptide binds a disease associated antigen and the epitope binding region of the second polypeptide binds the CD3 protein complex; and   wherein the epitope binding region that binds the CD3 protein complex comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 194, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 195 and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 196, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 197, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 198 and a light chain CDR3 comprising the amino acid sequences of: SEQ ID NO: 199; or   wherein the epitope binding region that binds the CD3 protein complex comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 200, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 201 and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 202, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 203, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 204 and a light chain CDR3 comprising the amino acid sequences of: SEQ ID NO: 205; or   wherein the epitope binding region that binds the CD3 protein complex comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 352, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 353 and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 354, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 355, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 356 and a light chain CDR3 comprising the amino acid sequences of SEQ ID NO: 357.   
     
     
         2 . A hetero-dimeric immunoglobulin or fragment thereof of  claim 1 , wherein the epitope binding region that binds a disease associated antigen comprises heavy chain CDR1, CDR2 and CDR3 amino acid sequences and light chain CDR1, CDR2 and CDR3 amino acid sequences, respectively, selected from the group consisting of:
 i) SEQ ID NOs: 206-211;   ii) SEQ ID NOs: 212-217;   iii) SEQ ID NOs: 218-223;   iv) SEQ ID NOs: 224-229;   v) SEQ ID NOs: 230-235;   vi) SEQ ID NOs: 236-241;   vii) SEQ ID NOs: 242-247;   viii) SEQ ID NOs: 248-253;   ix) SEQ ID NOs: 254-259;   x) SEQ ID NOs: 260-265;   xi) SEQ ID NOs: 266-271; and   xii) SEQ ID NOs: 272-277.   
     
     
         3 . The hetero-dimeric immunoglobulin or fragment thereof of  claim 1 , wherein the constant region of said second polypeptide comprises an IgG3 CH3 region. 
     
     
         4 . The hetero-dimeric immunoglobulin or fragment thereof of  claim 1 , wherein said protein-protein interface of the second polypeptide comprises an amino acid substitution at a position 84.4 and at least one additional amino acid substitution selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 (IMGT® numbering). 
     
     
         5 . The hetero-dimeric immunoglobulin or fragment thereof of  claim 1 , wherein the epitope binding region that binds the CD3 protein complex comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39; or
 wherein the epitope binding region that binds the CD3 protein complex comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 64, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 69; or   wherein the epitope binding region that binds the CD3 protein complex comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 104, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 106.   
     
     
         6 . The hetero-dimeric immunoglobulin or fragment thereof of  claim 1 , wherein said epitope binding region of said second polypeptide comprises a VH3 region and wherein said VH3 region of the second polypeptide comprises a modification that reduces or abrogates the binding of said second polypeptide to Protein A. 
     
     
         7 . The hetero-dimeric immunoglobulin or fragment thereof of  claim 6 , wherein the modified VH3 region comprises an amino acid substitution selected from the group consisting of: 57, 65, 81, 82a and combination 19/57/59 (Kabat numbering). 
     
     
         8 . The hetero-dimeric immunoglobulin or fragment thereof of  claim 6 , wherein the modified VH3 region comprises an amino acid substitution selected from the group consisting of: 57A, 57E, 65S, 81E, 82aS and combination 19G/57A/59A (Kabat numbering). 
     
     
         9 . The hetero-dimeric immunoglobulin or fragment thereof of  claim 1 , wherein the heavy chain variable framework region comprises an amino acid substitution selected from the group consisting of: I34M, V48I, A49G, R58N/Y, I69L, A71T and T73K (Kabat numbering) and the light chain variable framework region comprises an amino acid substitution selected from the group consisting of: M4L, V33M, A34N, L46R, L47W, R66G, F71Y and P96F (Kabat numbering); or wherein the heavy chain variable framework region comprises the amino acid substitutions I34M, A49G and A71T (Kabat numbering) and the light chain variable framework region comprises the amino acid substitutions M4L, L46R, L47W and F71Y (Kabat numbering). 
     
     
         10 . The hetero-dimeric immunoglobulin or fragment thereof of  claim 1 , wherein the heavy chain variable region comprises an amino acid substitution selected from the group consisting of: W100eF and W100eY (Kabat numbering) and the light chain variable region comprises an amino acid substitution selected from the group consisting of: A2I, S25A, T27A, G27aA, V27cA, T28A, T29A, S30A, N31A, Y32A, E38Q, F44P, G46L, T51A N52A, K53A, R54A, P56A, L66G, D69T, F87Y, Q89A, W91F, Y92A, S93A, N94A, and Q100G (Kabat numbering); or wherein the heavy chain variable region comprises the amino acid substitutions W100eY (Kabat numbering) and the light chain variable region comprises the amino acid substitutions A2I, T29A, S30A, T51A, F87Y, Q89A, and W91F (Kabat numbering) or light chain variable region comprises the amino acid substitutions A21, E38Q, F87Y, and Q89A. 
     
     
         11 . The hetero-dimeric immunoglobulin or fragment thereof of  claim 1 , wherein the epitope binding region of the first polypeptide is a FAB and the epitope binding region of the second polypeptide is a scFv or wherein the epitope binding region of the first polypeptide is a scFv and the epitope binding region of the second polypeptide is a FAB. 
     
     
         12 . (canceled) 
     
     
         13 . An in vitro method for the production of a hetero-dimeric immunoglobulin or fragment thereof of  claim 1  comprising the following steps:
 ia) preparing a DNA vector encoding a heavy chain of the first polypeptide and a DNA vector encoding a heavy chain of the second polypeptide wherein one or both DNA vectors or a third DNA vector optionally encode a common light chain or a light chain that assembles with a heavy chain of the first or second polypeptide; or 
 ib) preparing one DNA vector encoding heavy chains of the first and second polypeptides wherein the DNA vector optionally encodes a common light chain or a light chain that assembles with a heavy chain of the first or second polypeptide; and 
 
       wherein said DNA vectors are suitable for transient or stable expression in a mammalian host cell;
 ii) transfecting or co-transfecting the DNA vector(s) from (ia) or (ib) in a mammalian host cell line; 
 iii) culturing the transfected cell line or stably selected clone therefrom and harvesting the cell culture supernatant; 
 iv) contacting the cell culture supernatant on a Protein A affinity chromatography resin; and 
 v) eluting and collecting the hetero-dimeric immunoglobulin of interest. 
 
     
     
         14 . A method according to  claim 13 , wherein the hetero-dimeric immunoglobulin or fragment thereof found in the purified material from step (v) is at least 95% pure as determined by capillary electrophoresis.