Production of T cell retargeting hetero-dimeric immunoglobulins

• US 9,493,563 B2
• Filed: 11/04/2014
• Issued: 11/15/2016
• 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, Ollier, Romain, Skegro, Darko, Hou, Samuel
• Primary Examiner(s): BRISTOL, LYNN ANNE
• Application Number:
  US14/532,923
• Publication Number:
  US 20150133640A1
• Time in Patent Office: 742 Days
• Field of Search: 435/69.6, 530/387.3
• US Class Current: 1/1
• 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 first polypeptide and a second polypeptide, 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 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 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 amino acid sequence of SEQ ID NO;

47 and hinds 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 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 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 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 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 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 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 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 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 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.

2. 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.

3. A method according to claim 2, 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.