CD3 binding polypeptides
The present invention relates to mono-specific and multi-specific polypeptides that specifically bind or interact with CD3. These polypeptides can be, but are not limited to, antibodies, fragments thereof, scFvs, Fabs, di-scFvs single domain antibodies, diabodies, dual variable domain binding proteins and polypeptides containing an antibody or antibody fragments. In one embodiment, a multi-specific polypeptide binds both a T-cell receptor complex on T-cells and a tumor antigen to induce target-dependent T-cell cytotoxicity, activation and proliferation.
1. A CD3 binding polypeptide comprising a CD3 binding domain, wherein the CD3 binding domain has a reduced isoelectric point as compared to the isoelectric point of a binding domain with the amino acid sequence of SEQ ID NO:41;
wherein the CD3 binding domain comprises a humanized variable heavy (VH) region and a humanized variable light (VL) region and wherein the humanized VH region and the humanized VL region comprise framework regions;
wherein the VH region comprises SEQ ID NO: 28, and the VL region comprises SEQ ID NO: 34 or an amino acid sequence from about 90% to less than 100% identical to SEQ ID NO: 34;
wherein the VL region comprises a CDR1 having a sequence of SEQ ID NO: 52, a CDR2 region having a sequence of SEQ ID NO: 53, and a CDR3 having a sequence of SEQ ID NO: 54; and
wherein the CD3 binding polypeptide further comprises a second binding domain.
2. The CD3 binding polypeptide of claim 1 , wherein two or more amino acids in the framework regions of VL are modified compared to SEQ ID NO: 34 by substituting positively charged amino acids with neutral amino acids and/or substituting neutral amino acids with negatively charged amino acids.
3. The CD3 binding polypeptide of claim 1 , wherein at least one of the framework regions comprises an amino acid sequence from a human germline IgG sequence.
4. The CD3 binding polypeptide of claim 3 , wherein the human germline IgG sequence comprises SEQ ID NO:43.
5. The CD3 binding polypeptide of claim 3 , wherein the human germline IgG sequence comprises SEQ ID NO:44.
6. The CD3 binding polypeptide of claim 1 further comprising a prehinge region.
7. The CD3 binding polypeptide of claim 6 , wherein the prehinge region has the sequence SSS, and the CD3 binding polypeptide has a reduced isoelectric point as compared to a CD3 binding polypeptide that is identical except for having a prehinge region having the sequence RRT.
8. A CD3 binding polypeptide comprising a CD3 binding domain with framework regions, a prehinge region and a hinge region,
wherein the CD3 binding domain has a reduced isoelectric point as compared to the isoelectric point of the binding domain with the amino acid sequence of SEQ ID NO:41;
wherein the CD3 binding domain comprises a humanized VH region and a humanized VL region;
wherein the VH region comprises SEQ ID NO: 28, and the VL region comprises SEQ ID NO: 34 or an amino acid sequence from about 90% to less than 100% identical to SEQ ID NO: 34;
wherein the VL region comprises a CDR1 having a sequence of SEQ ID NO: 52, a CDR2 region having a sequence of SEQ ID NO: 53, and a CDR3 having a sequence of SEQ ID NO: 54; and
wherein the CD3 binding polypeptide further comprises a second binding domain.
9. The CD3 binding polypeptide of claim 8 , wherein at least two or more amino acids in the framework regions of VL are modified as compared to SEQ ID NO: 34 by substituting positively charged amino acids with neutral amino acids and/or substituting neutral amino acids with negatively charged amino acids.
10. The CD3 binding polypeptide of claim 8 , wherein at least four or more amino acids in the framework regions of VL are modified as compared to SEQ ID NO: 34 by substituting positively charged amino acids with neutral amino acids and/or substituting neutral amino acids with negatively charged amino acids.
11. The CD3 binding polypeptide of claim 8 , wherein three to five amino acids in the framework regions of VL are modified as compared to SEQ ID NO: 34 by substituting positively charged amino acids with neutral amino acids and/or substituting neutral amino acids with negatively charged amino acids.
12. The CD3 binding polypeptide of claim 8 , wherein three to ten amino acids in the framework regions of VL are modified as compared to SEQ ID NO: 34 by substituting positively charged amino acids with neutral amino acids and/or substituting neutral amino acids with negatively charged amino acids.
13. The CD3 binding polypeptide of claim 8 , wherein the CD3 binding polypeptide prehinge region comprises the amino acid sequence SSS or SST.
14. The CD3 binding polypeptide of claim 8 , wherein the CD3 binding polypeptide has an empirical isoelectric point of at least 1 pI unit less than the empirical isoelectric point of a polypeptide of SEQ ID NO:4.
15. The CD3 binding polypeptide of claim 8 wherein the second binding domain binds or interacts with a target molecule and the CD3 binding polypeptide induces T-cell cytotoxicity.
16. The CD3 binding polypeptide of claim 15 , wherein the second binding domain binds or interacts with a tumor associated antigen.
17. The CD3 binding polypeptide of claim 16 , wherein the CD3 binding polypeptide induces T-cells to lyse tumor cells.
18. The CD3 binding polypeptide of claim 16 , wherein the CD3 binding polypeptide induces polyclonal T-cell activation and expansion in the vicinity of a tumor.
19. The CD3 binding polypeptide of claim 8 , wherein the CD3 binding domain comprises a VL region selected from the group consisting of SEQ ID NO:34, SEQ ID NO:36 and SEQ ID NO:38.
20. The CD3 binding polypeptide of claim 19 , wherein the VL region comprises SEQ ID NO:34.
21. The CD3 binding polypeptide of claim 19 , wherein the VH region comprises SEQ ID NO:28 and the VL region comprises SEQ ID NO:38.
22. The CD3 binding polypeptide of claim 8 , wherein the CD3 binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:20.
23. A nucleic acid encoding a polypeptide of claim 8 .
24. An expression vector comprising the nucleic acid of claim 23 .
25. A recombinant host cell comprising the expression vector of claim 24 .
26. A composition comprising the CD3 binding polypeptide of claim 8 and a pharmaceutically acceptable carrier, diluent or excipient.
27. A method for treating prostate cancer comprising administering a therapeutically effective amount of the composition of claim 26 to a patient in need thereof;
wherein the second binding domain binds or interacts with a tumor associated antigen.
28. The CD3 binding polypeptide of claim 8 , wherein the CD3 binding polypeptide further comprises an immunoglobulin dimerization domain.
29. The CD3 binding polypeptide of claim 28 , wherein the CD3 binding polypeptide forms a homodimer.
30. The CD3 binding polypeptide of claim 28 , wherein the CD3 binding polypeptide forms a heterodimer.
31. The CD3 binding polypeptide of claim 8 , wherein the CD3 binding domain is about 90% to about 99% identical to SEQ ID NO:41.
32. A CD3 binding polypeptide comprising a CD3 binding domain,
wherein the CD3 binding domain comprises a humanized VH region and a humanized VL region;
wherein the VH region comprises an amino acid sequence of SEQ ID NO: 28, and the VL region comprises an amino acid sequence of SEQ ID NO: 34.
33. The CD3 binding polypeptide of claim 6 , wherein the prehinge region is a short amino acid sequence that connects the binding domain to the hinge region.