T CELL RETARGETING HETERO-DIMERIC IMMUNOGLOBULINS
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 . An immunoglobulin or fragment thereof comprising an epitope binding region which binds to human CD3 and comprises a heavy chain variable domain sequence selected from the group consisting of: SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, and SEQ ID NO: 104 and a light chain variable domain sequence selected from the group consisting of: SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 401 and SEQ ID NO: 402.
2 . The immunoglobulin or fragment thereof of claim 1 , wherein the epitope binding region comprises a heavy chain variable domain sequence and a light chain variable domain sequence pair selected from the group consisting of: SEQ ID NO: 101 and SEQ ID NO: 105, SEQ ID NO: 104 and SEQ ID NO: 106, SEQ ID NO: 104 and SEQ ID NO: 401, and SEQ ID NO: 104 and SEQ ID NO: 402.
3 . An immunoglobulin or fragment thereof of comprising an epitope binding region which binds to human CD3 and comprises a heavy chain sequence and a light chain sequence pair selected from the group consisting of: SEQ ID NO: 359 and SEQ ID NO: 399 and SEQ ID NO: 359 and SEQ ID NO: 400.
4 . The immunoglobulin or fragment thereof of claim 1 , wherein said immunoglobulin or fragment thereof is a hetero-dimeric immunoglobulin or fragment thereof, wherein the hetero-dimeric immunoglobulin or fragment thereof comprises a first polypeptide and a second polypeptide.
5 . The hetero-dimeric immunoglobulin or fragment thereof of claim 4 , wherein the epitope binding region of the second polypeptide binds to a different epitope than the epitope binding region of the first polypeptide.
6 . The hetero-dimeric immunoglobulin or fragment thereof of claim 5 , 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.
7 . The hetero-dimeric immunoglobulin or fragment thereof of claim 6 , wherein the scFv binds to human CD3 and comprises a sequence selected from the group consisting of: SEQ ID NO: 361, SEQ ID NO: 311, SEQ ID NO: 394, and SEQ ID NO: 396.
8 . The hetero-dimeric immunoglobulin or fragment thereof of according to of claim 6 , wherein the scFv has at least a twofold improvement in expression in comparison to a SP34 chimera formatted as a scFv-Fc, wherein the SP34 chimera comprises the heavy chain variable domain and the light chain variable domain sequences of SEQ ID NOs: 60 and 61, respectively.
9 . The hetero-dimeric immunoglobulin or fragment thereof of claim 6 , wherein the scFv binds to human CD3 and has at least a twofold improvement in expression in comparison to a SP34 chimera formatted as an scFv, wherein the SP34 chimera comprises the heavy chain variable domain and light chain variable domain sequences of SEQ ID NOs: 60 and 61, and wherein the human CD3 binding scFv is expressed in a bispecific antibody comprising a FAB arm.
10 . The hetero-dimeric immunoglobulin or fragment thereof of claim 4 , wherein the hetero-dimeric immunoglobulin or fragment thereof has greater thermostability in comparison a hetero-dimeric immunoglobulin or fragment thereof comprising the heavy chain variable domain and light chain variable domain sequences of SEQ ID NOs: 60 and 61.
11 . An in vitro method for the production of a hetero-dimeric immunoglobulin or fragment thereof of claim 4 , comprising the following steps:
(i)(a) 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
(i)(b) 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 (i) 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 fragment thereof of interest.
12 . The method of claim 11 , wherein the hetero-dimeric immunoglobulin or fragment thereof found in the purified material from step (v) is at least 95% pure.
13 . The method of claim 12 , wherein the hetero-dimeric immunoglobulin or fragment thereof found in the purified material from step (v) is at least 96% pure.
14 . The method of claim 13 , wherein the hetero-dimeric immunoglobulin or fragment thereof found in the purified material from step (v) is at least 97% pure.
15 . The method of claim 11 , wherein purity of the hetero-dimeric immunoglobulin or fragment thereof found in the purified material can be measured by capillary electrophoresis.