Production of T cell retargeting hetero-dimeric immunoglobulins

• US 20150133640A1
• Filed: 11/04/2014
• Published: 05/14/2015
• Est. Priority Date: 11/04/2013
• Status: Active Grant

• Current Assignee: Ichnos Sciences SA
• Original Assignee: Glenmark Pharmaceuticals SA (Glenmark Pharmaceuticals Limited)
• Inventors: BLEIN, Stanislas, Skegro, Darko, Hou, Samuel, Ollier, Romain
• Granted Patent: US 9,493,563 B2
• Time in Patent Office: Days
• US Class Current: 530/387.3
• CPC Class Codes: C07K 16/2803 against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/2878 against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 16/2887 against CD20
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 16/4291 against IgE
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/33 Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/524 CH2 domain
• CPC Class Codes: C07K 2317/526 CH3 domain
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/567 Framework region [FR]
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/71 : Decreased effector function due to an Fc-modification
• CPC Class Codes: C07K 2317/73 : Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2317/94 : Stability, e.g. half-life, pH, temperature or enzyme-resistance

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.

Claims

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 that does not bind to Protein A comprising 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 A2I, 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.

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.

12. A hetero-dimeric immunoglobulin or fragment thereof that 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;

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.