Production of T cell retargeting hetero-dimeric immunoglobulins

1. A hetero-dimeric immunoglobulin or fragment thereof, comprising:

(a) a first polypeptide that binds to Protein A comprising an epitope binding region that binds a first epitope and an immunoglobulin constant region; and

(b) a second polypeptide comprising a VH3 based epitope binding region that binds a second epitope and an immunoglobulin constant region;

wherein the VH3 based epitope binding region of the second polypeptide has a modified VH3 domain with reduced binding to Protein A compared to an unmodified VH3 domain and wherein said modified VH3 domain has a substitution at one or more of residues 57, 65, 81 or 82a according to Kabat numbering;

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 according to 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 according to IMGT numbering;

wherein the epitope binding region of the first polypeptide binds a CD3 protein complex and the epitope binding region of the second polypeptide binds a cancer antigen or wherein the epitope binding region of the first polypeptide binds a cancer 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. The hetero-dimeric immunoglobulin or fragment thereof of claim 1 , wherein the epitope binding region that binds a cancer antigen binds to:

i) HER2 and comprises heavy chain CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NOs: 206-208 and light chain CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NOs: 209-211;

ii) CD38 and comprises heavy chain CDR1, CDR2 and CDR3 amino acid sequences of SEQ D NOs: 212-214 and light chain CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NOs: 215-217;

iii) CD38 and comprises heavy chain CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NOs: 218-220 and light chain CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NOs: 221-223;

iv) CD38 and comprises heavy chain CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NOs: 230-232 and light chain CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NOs: 233-235;

v) EGFR and comprises heavy chain CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NOs: 254-256 and light chain CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NOs: 257-259;

vi) CD19 and comprises heavy chain CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NOs: 260-262 and light chain CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NOs: 263-265;

vii) IgE and comprises heavy chain CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NOs; 266-268 and light chain CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NOs: 269-271; or

viii) IgE and comprises heavy chain CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NOs: 272-274 and light chain CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NOs: 275-277.

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 according to 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 the modified VH3-based epitope binding region comprises an amino acid substitution selected from the group consisting of: 57, 65, 81, 82a and a combination of 19/57/59 according to Kabat numbering.

7. The hetero-dimeric immunoglobulin or fragment thereof of claim 6 , wherein the modified VH3-based epitope binding region comprises an amino acid substitution selected from the group consisting of: 57A, 57E, 81E, and a combination of 19G/57A/59A according to Kabat numbering.

8. The hetero-dimeric immunoglobulin or fragment thereof of claim 1 , comprising a heavy chain variable framework region and a light chain variable framework region, 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 according to 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 according to Kabat numbering; or wherein the heavy chain variable framework region comprises the amino acid substitutions I34M, A49G and A71T according to Kabat numbering and the light chain variable framework region comprises the amino acid substitutions M4L, L46R, L47W and F71Y according to Kabat numbering.

9. The hetero-dimeric immunoglobulin or fragment thereof of claim 1 , comprising a heavy chain variable framework region and a light chain variable framework region, wherein the heavy chain variable region comprises an amino acid substitution selected from the group consisting of: W100eF and W100eY according to 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 according to Kabat numbering; or wherein the heavy chain variable region comprises the amino acid substitutions W100eY according to Kabat numbering and the light chain variable region comprises the amino acid substitutions A2I, T29A, S30A, T51A, F87Y, Q89A, and W91F according to Kabat numbering or light chain variable region comprises the amino acid substitutions A2I, E38Q, F87Y, and Q89A.

10. 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.

11. A hetero-dimeric immunoglobulin or fragment thereof, wherein the hetero-dimeric immunoglobulin or fragment thereof comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a FAB and the second polypeptide comprises a scFv or wherein the first polypeptide comprises a scFv and the second polypeptide comprises a FAB, and wherein the hetero-dimeric immunoglobulin or fragment thereof binds to:

i) the CD3 protein complex and HER2, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 159 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 47 and binds CD3 epsilon, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 160 and binds HER2;

ii) the CD3 protein complex and HER2, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 161 and is assembled with a cognate light chain of amino acid sequence of SEQ ID NO: 3 and binds HER2, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 162 and binds CD3 epsilon;

iii) the CD3 protein complex and HER2, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 163 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 47 and binds CD3 epsilon, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 164 and binds HER2;

iv) the CD3 protein complex and HER2, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 165 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 166 and binds CD3 epsilon, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 167 and binds HER2;

v) the CD3 protein complex and HER2, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 168 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 89 and binds CD3 epsilon, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 167 and binds HER2;

vi) the CD3 protein complex and CD38, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 169 and is assembled with a cognate light chain of amino acid sequence of SEQ ID NO: 119 and binds CD38, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 162 and binds CD3 epsilon;

vii) the CD3 protein complex and CD38, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 170 and is assembled with a cognate light chain of amino acid sequence of SEQ ID NO: 138 and binds CD38, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 171 and binds CD3 epsilon;

viii) the CD3 protein complex and CD38, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 176 and is assembled with a cognate light chain of amino acid sequence of SEQ ID NO: 119 and binds CD38, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 177 and binds CD3 epsilon;

ix) the CD3 protein complex and CD38, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 178 and is assembled with a cognate light chain of amino acid sequence of SEQ ID NO: 128 and binds CD38, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 179 and binds CD3 epsilon;

x) the CD3 protein complex and OX40 wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 172 and is assembled with a cognate light chain of amino acid sequence of SEQ ID NO: 173 and binds OX40, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 162 and binds CD3 epsilon;

xi) the CD3 protein complex and EGFR wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 174 and is assembled with a cognate light chain of amino acid sequence of SEQ ID NO: 175 and binds EGFR, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 171 and binds CD3 epsilon; or

xii) the CD3 protein complex and CD20, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 180 and is assembled with a cognate light chain of amino acid sequence of SEQ ID NO: 181 and binds CD20, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 177 and binds CD3 epsilon.

12. An in vitro method for the production of a hetero-dimeric immunoglobulin or fragment thereof of any one of the preceding claims 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 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 encodes a common light chain or a light chain that assembles with a heavy chain of the first or second polypeptide; or

(ic) preparing a DNA vector encoding a heavy chain of the first polypeptide, a DNA vector encoding a heavy chain of the second polypeptide and a third DNA vector encoding 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 comprise a promoter sequence, a polyadenylation sequence and a leader peptide sequence and are suitable for transient or stable expression in a mammalian host cell;

ii) transfecting or co-transfecting equal quantities of, 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.

13. The method according to claim 12 , 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.