Dual variable domain immunoglobulins and uses thereof

1. A binding protein comprising first and second polypeptide chains, each independently comprising VD1-(X1)n-VD2-C-(X2)n, wherein

VD1 is a first variable domain;

VD2 is a second variable domain;

C is a constant domain;

X1 is a linker with the proviso that it is not CH1;

X2 is an Fc region;

n is 0 or 1;

wherein the VD1 domains on the first and second polypeptide chains form a first functional target binding site and the VD2 domains on the first and second polypeptide chains form a second functional target binding site, and wherein the binding protein is capable of binding:

(a) methotrexate (MTX) and CD3, wherein

(i) the variable domains that form a functional target binding site for MTX comprise:

three CDRs from SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105; and

(ii) the variable domains that form a functional target binding site for CD3 comprise:

three CDRs from SEQ ID NO: 32 and a three CDRs from SEQ ID NO: 33,

three CDRs from SEQ ID NO: 68 and three CDRs from SEQ ID NO: 69, or

three CDRs from SEQ ID NO: 100 and three CDRs from SEQ ID NO: 101; and

(iii) the IC 50 of the binding pair is at most 4.0 nM as measured in a methotrexate neutralization assay;

(b) MTX and EGFR, wherein

(i) the variable domains that form a functional target binding site for MTX comprise:

three CDRs from SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105; and

ii) the variable domains that form a functional target binding site for EGFR comprise:

three CDRs from SEQ ID NO: 34 and three CDRs from SEQ ID NO: 35,

three CDRs from SEQ ID NO: 62 and three CDRs from SEQ ID NO: 63, or

three CDRs from SEQ ID NO: 76 and three CDRs from SEQ ID NO: 77; and

(iii) the IC 50 of the binding pair is at most 3.9 nM as measured in a methotrexate neutralization assay;

or

(c) MTX and HER-2, wherein

(i) the variable domains that form a functional target binding site for MTX comprise:

three CDRs from SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105, and

(ii) the variable domains that form a functional target binding site for HER-2 comprise:

three CDRs from SEQ ID NO: 36 and three CDRs from SEQ ID NO: 37; or

three CDRs from SEQ ID NO: 98 and three CDRs from SEQ ID NO: 99; and

(iii) the IC 50 of the binding pair is at most 3.9 nM as measured in a methotrexate neutralization assay.

2. The binding protein according to claim 1 , wherein the X1 comprises a sequence from SEQ ID NOs 1-29 or a G/S based sequence.

3. The binding protein according to claim 1 , wherein the binding protein comprises two first polypeptide chains and two second polypeptide chains.

4. The binding protein according to claim 1 , wherein the Fc region is a variant sequence Fc region.

5. The binding protein according to claim 4 , wherein the Fc region is from an IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

6. The binding protein according to claim 1 , wherein said VD1 variable domains bind to a first target with an affinity and/or potency different from the affinity and/or potency with which said VD2 variable domains bind to a second target.

7. A binding protein comprising four polypeptide chains, wherein each polypeptide chain independently comprises VD1-(X1)n-VD2-C-(X2)n, wherein

VD1 is a first variable domain;

VD2 is a second variable domain;

C is a constant domain;

X1 is a linker with the proviso that it is not CH1;

X2 is an Fc region;

n is 0 or 1; and

wherein

the VD1 domains on the first and second polypeptide chains form a first functional target binding site and the VD2 domains on the first and second polypeptide chains form a second functional target binding site, and the VD1 domains on the third and fourth polypeptide chains form a third functional target binding site and the VD2 domains on the third and fourth polypeptide chains form a fourth functional target binding site, and wherein the binding protein is capable of binding:

(a) methotrexate (MTX) and CD3, wherein

(i) the variable domains that form a functional target binding site for MTX comprise:

three CDRs from SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105; and

(ii) the variable domains that form a functional target binding site for CD3 comprise:

three CDRs from SEQ ID NO: 32 and a three CDRs from SEQ ID NO: 33,

three CDRs from SEQ ID NO: 68 and three CDRs from SEQ ID NO: 69, or

three CDRs from SEQ ID NO: 100 and three CDRs from SEQ ID NO: 101; and

(iii) the IC 50 of the binding pair is at most 4.0 nM as measured in a methotrexate neutralization assay;

(b) MTX and EGFR, wherein

(i) the variable domains that form a functional target binding site for MTX comprise:

three CDRs from SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105; and

(ii) the variable domains that form a functional target binding site for EGFR comprise:

three CDRs from SEQ ID NO: 34 and three CDRs from SEQ ID NO: 35,

three CDRs from SEQ ID NO: 62 and three CDRs from SEQ ID NO: 63, or

three CDRs from SEQ ID NO: 76 and three CDRs from SEQ ID NO: 77; and

(ii) the IC 50 of the binding pair is at most 3.9 nM as measured in a methotrexate neutralization assay;

or

(c) MTX and HER-2, wherein

(i) the variable domains that form a functional target binding site for MTX comprise:

three CDRs from SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105, and

(ii) the variable domains that form a functional target binding site for HER-2 comprise:

three CDRs from SEQ ID NO: 36 and three CDRs from SEQ ID NO: 37; or

three CDRs from SEQ ID NO: 98 and three CDRs from SEQ ID NO: 99; and

(iii) the IC 50 of the binding pair is at most 3.9 nM as measured in a methotrexate neutralization assay.

8. A binding protein comprising first and second polypeptide chains, wherein said first polypeptide chain comprises a first VD1 -(X1)n-VD2 -C-(X2)n, wherein

VD1 is a first heavy chain variable domain;

VD2 is a second heavy chain variable domain;

C is a heavy chain constant domain;

X1 is a linker with the proviso that it is not CH1; and

X2 is an Fc region; and

n is 0 or 1;

wherein said second polypeptide chain comprises a second VD1-(X1)n-VD2-C-(X2)n, wherein

VD1 is a first light chain variable domain;

VD2 is a second light chain variable domain;

C is a light chain constant domain;

X1 is a linker with the proviso that it is not CH1;

X2 does not comprise an Fc region; and

n is 0 or 1,

wherein the variable domains of the binding protein form two functional target binding site, and wherein the binding protein is capable of binding:

(a) methotrexate (MTX) and CD3, wherein

(i) the heavy and light chain variable domains that form a functional target binding site for MTX comprise:

three CDRs from SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105; and

(ii) the heavy and light chain variable domains that form a functional target binding site for CD3 comprise:

three CDRs from SEQ ID NO: 32 and a three CDRs from SEQ ID NO: 33,

three CDRs from SEQ ID NO: 68 and three CDRs from SEQ ID NO: 69, or

three CDRs from SEQ ID NO: 100 and three CDRs from SEQ ID NO: 101; and

(iii) the IC 50 of the binding pair is at most 4.0 nM as measured in a methotrexate neutralization assay;

(b) MTX and EGFR, wherein

(i) the heavy and light chain variable domains that form a functional target binding site for MTX comprise:

three CDRs from SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105, and

(ii) the heavy and light variable domains that form a functional target binding site for EGFR comprise:

three CDRs from SEQ ID NO: 34 and three CDRs from SEQ ID NO: 35,

three CDRs from SEQ ID NO: 62 and three CDRs from SEQ ID NO: 63, or

three CDRs from SEQ ID NO: 76 and three CDRs from SEQ ID NO: 77; and

(iii) the IC 50 of the binding pair is at most 3.9 nM as measured in a methotrexate neutralization assay;

or

(c) MTX and HER-2, wherein

(i) the heavy and light chain variable domains that form a functional target binding site for MTX comprise:

three CDRs from SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105; and

(ii) the heavy and light chain variable domains that form a functional target binding site for HER-2 comprise:

three CDRs from SEQ ID NO: 36 and three CDRs from SEQ ID NO: 37, or

three CDRs from SEQ ID NO: 98 and three CDRs from SEQ ID NO: 99; and

(iii) the IC 50 of the binding pair is at most 3.9 nM as measured in a methotrexate neutralization assay.

9. The binding protein according to claim 8 , wherein the binding protein comprises two first polypeptide chains and two second polypeptide chains.

10. The binding protein according to claim 1 , 7 , 8 , or 9 , wherein the binding protein comprises: DVD1011 (comprising SEQ ID NOs: 110 and 111); DVD1012 (comprising SEQ ID NOs: 112 and 113); DVD1013 (comprising SEQ ID NOs: 114 and 115); DVD1014 (comprising SEQ ID NOs: 116 and 117); DVD1015 (comprising SEQ ID NOs: 118 and 119); DVD1016 (comprising SEQ ID NOs: 120 and 121); DVD1017 (comprising SEQ ID NOs: 122 and 123); DVD1018 (comprising SEQ ID NOs: 124 and 125); DVD1027 (comprising SEQ ID NOs: 142 and 143); DVD1028 (comprising SEQ ID NOs: 144 and 145); DVD1029 (comprising SEQ ID NOs: 146 and 147); DVD1030 (comprising SEQ ID NOs: 148 and 149); DVD1031 (comprising SEQ ID NOs: 150 and 151); DVD1032 (comprising SEQ ID NOs: 152 and 153); DVD1033 (comprising SEQ ID NOs: 154 and 155); DVD1034 (comprising SEQ ID NOs: 156 and 157); DVD1210 (comprising SEQ ID NOs: 178 and 179); DVD1211 (comprising SEQ ID NOs: 180 and 181); DVD1212 (comprising SEQ ID NOs: 182 and 183); DVD1213 (comprising SEQ ID NOs: 184 and 185); DVD1216 (comprising SEQ ID NOs: 186 and 187); or DVD1217 (comprising SEQ ID NOs: 188 and 189).

11. The binding protein according to claim 1 or claim 7 , wherein the binding protein is DVD1211, DVD1213, or DVD1217.

12. A binding protein conjugate comprising a binding protein according to claim 1 , claim 7 , or claim 8 , said binding protein conjugate further comprising an immunoadhesion molecule, an imaging agent, a therapeutic agent, or a cytotoxic agent.

13. The binding protein conjugate according to claim 12 , wherein said imaging agent is a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, or biotin.

14. The binding protein conjugate according to claim 13 , wherein said radiolabel is 3 H, 14 C, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153 Sm.

15. The binding protein conjugate according to claim 13 , wherein said therapeutic or cytotoxic agent is an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, a toxin, or an apoptotic agent.

16. The binding protein according to claim 1 , claim 7 , or claim 8 , wherein said binding protein is a crystallized binding protein.

17. A pharmaceutical composition comprising the binding protein according to claim 1 , claim 7 , or claim 8 , and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition according to claim 17 further comprising at least one additional therapeutic agent.

19. The pharmaceutical composition according to claim 18 , wherein said additional therapeutic agent is an imaging agent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, a co-stimulation molecule blocker, an adhesion molecule blocker, an anti-cytokine antibody or functional fragment thereof, methotrexate, cyclosporin, rapamycin, FK506, a detectable label or reporter, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressive, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an epinephrine or analog thereof, a cytokine, or a cytokine antagonist.

20. The binding protein according to claim 1 , claim 7 , or claim 8 , wherein the binding protein binds to a prophylactic or therapeutic drug and to a specific location, tissue, or cell type in the body.

21. The binding protein according to claim 20 , wherein the binding protein binds methotrexate.