Bispecific single chain antibodies with specificity for high molecular weight target antigens

1. A method for identifying a bispecific single chain antibody that mediates T cell lysis of a target cell expressing human prostate-specific membrane antigen (PSMA) by binding to a membrane proximal epitope of human PSMA but does not mediate lysis of a target cell expressing rat PSMA, wherein said membrane proximal epitope is formed only by amino acid residues of the extracellular domain of human PSMA, the alpha C-atom of which has a distance of less than 60 Angstroms (Å) from the alpha C-atom of the 13th amino acid residue as counted from the junction of transmembrane and extracellular regions, the bispecific single chain antibody comprising a first binding domain capable of binding to an epitope of human and/or non-chimpanzee primate cluster of differentiation 3 (CD3) on a T cell surface and a second binding domain capable of binding to the extracellular domain of human PSMA, the method comprising the steps of:

(a) providing:

(i) at least a first type of host cell expressing on its surface an extracellular domain of wild-type (wt) human PSMA comprising the amino acid sequence set forth in SEQ ID NO: 202, wherein said extracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 447;

(ii) at least a second type of host cell expressing on its surface an extracellular domain of a mutated form of said wt human PSMA, said mutated form comprising amino acids 1-750 of the amino acid sequence set forth in SEQ ID NO: 208;

(iii) at least a third type of host cell expressing on its surface an extracellular domain of wt rat PSMA comprising the amino acid sequence set forth in SEQ ID NO: 866;

(b) contacting said at least first, second and third types of host cells with a bispecific single chain antibody comprising a first binding domain capable of binding to an epitope of human and/or non-chimpanzee primate CD3 on the surface of a T cell and a second binding domain capable of binding to the extracellular domain of human PSMA and an effector T cell; and

(c) identifying a bispecific single chain antibody comprising a first binding domain capable of binding to an epitope of human and/or non-chimpanzee primate CD3 on the surface of a T cell and a second binding domain capable of binding to the extracellular domain of human PSMA that mediates lysis of said at least first and second types of host cells, but does not mediate lysis of said at least third type of host cell, thereby identifying said bispecific single chain antibody that mediates lysis of a target cell expressing human PSMA by binding to a membrane proximal epitope of human PSMA but does not mediate lysis of a target cell expressing rat PSMA.

2. The method of claim 1 , wherein the first binding domain binds to CD3 epsilon (CD3ε) of human or non-chimpanzee primate CD3.

3. The method of claim 2 , wherein the first binding domain comprises a VL region comprising CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of:

(a) CDR-L1 set forth in SEQ ID NO. 27, CDR-L2 set forth in SEQ ID NO. 28 and CDR-L3 set forth in SEQ ID NO. 29;

(b) CDR-L1 set forth in SEQ ID NO. 117, CDR-L2 set forth in SEQ ID NO. 118 and CDR-L3 set forth in SEQ ID NO. 119; and

(c) CDR-L1 set forth in SEQ ID NO. 153, CDR-L2 set forth in SEQ ID NO. 154 and CDR-L3 set forth in SEQ ID NO. 155.

4. The method of claim 2 , wherein the first binding domain comprises a VH region comprising CDR-H1, CDR-H2 and CDR-H3 selected from the group consisting of:

(a) CDR-H1 set forth in SEQ ID NO. 12, CDR-H2 set forth in SEQ ID NO. 13 and CDR-H3 set forth in SEQ ID NO. 14;

(b) CDR-H1 set forth in SEQ ID NO. 30, CDR-H2 set forth in SEQ ID NO. 31 and CDR-H3 set forth in SEQ ID NO. 32;

(c) CDR-H1 set forth in SEQ ID NO. 48, CDR-H2 set forth in SEQ ID NO. 49 and CDR-H3 set forth in SEQ ID NO. 50;

(d) CDR-H1 set forth in SEQ ID NO. 66, CDR-H2 set forth in SEQ ID NO. 67 and CDR-H3 set forth in SEQ ID NO. 68;

(e) CDR-H1 set forth in SEQ ID NO. 84, CDR-H2 set forth in SEQ ID NO. 85 and CDR-H3 set forth in SEQ ID NO. 86;

(f) CDR-H1 set forth in SEQ ID NO. 102, CDR-H2 set forth in SEQ ID NO. 103 and CDR-H3 set forth in SEQ ID NO. 104;

(g) CDR-H1 set forth in SEQ ID NO. 120, CDR-H2 set forth in SEQ ID NO. 121 and CDR-H3 set forth in SEQ ID NO. 122;

(h) CDR-H1 set forth in SEQ ID NO. 138, CDR-H2 set forth in SEQ ID NO. 139 and CDR-H3 set forth in SEQ ID NO. 140;

(i) CDR-H1 set forth in SEQ ID NO. 156, CDR-H2 set forth in SEQ ID NO. 157 and CDR-H3 set forth in SEQ ID NO. 158; and

(j) CDR-H1 set forth in SEQ ID NO. 174, CDR-H2 set forth in SEQ ID NO. 175 and CDR-H3 set forth in SEQ ID NO. 176.

5. The method of claim 2 , wherein the first binding domain comprises a VL region comprising the amino acid sequence set forth in SEQ ID NO. 35, 39, 125, 129, 161 or 165.

6. The method of claim 2 , wherein the first binding domain comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO. 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105, 109, 123, 127, 141, 145, 159, 163, 177 or 181.

7. The method of claim 2 , wherein the first binding domain comprises a VL region and a VH region selected from the group consisting of:

(a) a VL region comprising the amino acid sequence set forth in SEQ ID NO. 17 or 21 and a VH region comprising the amino acid sequence set forth in SEQ ID NO. 15 or 19;

(b) a VL region comprising the amino acid sequence set forth in SEQ ID NO. 35 or 39 and a VH region comprising the amino acid sequence set forth in SEQ ID NO. 33 or 37;

(c) a VL region comprising the amino acid sequence set forth in SEQ ID NO. 53 or 57 and a VH region comprising the amino acid sequence set forth in SEQ ID NO. 51 or 55;

(d) a VL region comprising the amino acid sequence set forth in SEQ ID NO. 71 or 75 and a VH region comprising the amino acid sequence set forth in SEQ ID NO. 69 or 73;

(e) a VL region comprising the amino acid sequence set forth in SEQ ID NO. 89 or 93 and a VH region comprising the amino acid sequence set forth in SEQ ID NO. 87 or 91;

(f) a VL region comprising the amino acid sequence set forth in SEQ ID NO. 107 or 111 and a VH region comprising the amino acid sequence set forth in SEQ ID NO. 105 or 109;

(g) a VL region comprising the amino acid sequence set forth in SEQ ID NO. 125 or 129 and a VH region comprising the amino acid sequence set forth in SEQ ID NO. 123 or 127;

(h) a VL region comprising the amino acid sequence set forth in SEQ ID NO. 143 or 147 and a VH region comprising the amino acid sequence set forth in SEQ ID NO. 141 or 145;

(i) a VL region comprising the amino acid sequence set forth in SEQ ID NO. 161 or 165 and a VH region comprising the amino acid sequence set forth in SEQ ID NO. 159 or 163; and

(j) a VL region comprising the amino acid sequence set forth in SEQ ID NO. 179 or 183 and a VH region comprising the amino acid sequence set forth in SEQ ID NO. 177 or 181.

8. The method of claim 7 , wherein the first binding domain comprises the amino acid sequence set forth in SEQ ID NO: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185 or 187.

9. The method of claim 1 , wherein the first binding domain binds to an epitope of CD3 epsilon which is part of a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, or 8.

10. The method of claim 1 , wherein the target cell expressing human PSMA is a tumor cell.

11. The method of claim 10 , wherein the tumor cell is a cancer cell.

12. The method of claim 11 , wherein the cancer cell is a prostate cancer cell.

13. A method for identifying a bispecific single chain antibody that binds human and/or primate cluster of differentiation 3 (CD3) and a membrane proximal epitope of human prostate-specific membrane antigen (PSMA), wherein the antibody comprises a first binding domain that binds to an epitope of human and/or primate CD3 on a T cell surface and a second binding domain that binds to the extracellular domain of human PSMA, and wherein the antibody mediates T cell lysis of a target cell expressing human PSMA but does not mediate lysis of a target cell expressing rat PSMA, the method comprising the steps of:

(a) providing:

(i) at least a first type of host cell expressing on its surface an extracellular domain of wild-type (wt) human PSMA comprising the amino acid sequence set forth in SEQ ID NO: 202, wherein said extracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 447;

(ii) at least a second type of host cell expressing on its surface an extracellular domain of a mutated form of said wt human PSMA, said mutated form comprising amino acids 1-750 of the amino acid sequence set forth in SEQ ID NO: 208; and

(iii) at least a third type of host cell expressing on its surface an extracellular domain of wt rat PSMA comprising the amino acid sequence set forth in SEQ ID NO: 866;

(b) contacting said at least first, second and third types of host cells with a test bispecific single chain antibody comprising a first binding domain capable of binding to an epitope of human and/or primate CD3 on the surface of a T cell and a second binding domain capable of binding to the extracellular domain of human PSMA and an effector T cell; and

(c) identifying said test antibody that mediates lysis of said at least first and second types of host cells, but does not mediate lysis of said at least third type of host cell, as a bispecific single chain antibody that binds CD3 and a membrane proximal epitope of human PSMA.

14. The method of claim 13 , wherein the first binding domain binds to CD3 epsilon (CD3ε) of human or non-chimpanzee primate CD3.

15. The method of claim 13 , wherein the first binding domain binds to an epitope of CD3 epsilon which is part of a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, or 8.

16. The method of claim 13 , wherein the target cell expressing human PSMA is a tumor cell.

17. The method of claim 16 , wherein the tumor cell is a cancer cell.

18. The method of claim 17 , wherein the cancer cell is a prostate cancer cell.