T cell recruiting polypeptides capable of binding CD123 and TCR α/β

1 . A nucleic acid encoding a polypeptide comprising a first immunoglobulin single variable domain (ISV) and a second ISV, wherein the first ISV specifically binds TCR and essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which:

i) CDR1 is chosen from the group consisting of:

a) SEQ ID NO: 181; and

b) amino acid sequences that have 3, 2 or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 181, wherein

at position 2 the D has been changed into A, S, E, or G;

at position 4 the H has been changed into Y;

at position 5 the K has been changed into L;

at position 6 the I has been changed into L;

at position 8 the F has been changed into I or V; and/or

at position 10 the G has been changed into S; and

ii) CDR2 is chosen from the group consisting of:

c) SEQ ID NO: 192; and

d) amino acid sequences that have 3, 2 or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 192, wherein

at position 1 the D has been changed into T or R;

at position 3 the S has been changed into T or A;

at position 5 the G has been changed into S or A;

at position 7 the Q has been changed into D, E, T, A or V;

at position 8 the T has been changed into A or V; and/or

at position 9 the D has been changed into A, Q, N, V or S;

and

iii) CDR3 is chosen from the group consisting of:

e) SEQ ID NO: 218; and

f) amino acid sequences that have 3, 2 or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 218, wherein

at position 1 the F has been changed into Y, L or G;

at position 4 the I has been changed into L;

at position 5 the Y has been changed into W; and/or

at position 8 the D has been changed into N or S;

and wherein the second ISV specifically binds CD123 and essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which:

i) CDR1 is chosen from the group consisting of:

a) SEQ ID NO: 11; and

b) amino acid sequences that have 3, 2 or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 11, wherein

at position 3 the T has been changed into S or P;

at position 6 the I has been changed into S;

at position 7 the N has been changed into D; and/or

at position 8 the D has been changed into V or A;

and

ii) CDR2 is SEQ ID NO: 17;

and

iii) CDR3 is chosen from the group consisting of:

c) SEQ ID NO: 21; and

d) amino acid sequences that have 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 21, wherein at position 3 the P has been changed into A.

2 . The nucleic acid according to claim 1 , which is in the form of a genetic construct.

3 . An expression vector comprising the nucleic acid according to claim 1 .

4 . A host or host cell comprising the nucleic acid according to claim 1 .

5 . A method for the production of a polypeptide, said method at least comprising the steps of:

a) expressing, in a suitable host cell or in another suitable expression system, the nucleic acid according to claim 1 ; optionally followed by

b) isolating and/or purifying the polypeptide.

6 . A composition comprising the nucleic acid according to claim 1 .

7 . The composition according to claim 6 , which is a pharmaceutical composition.

8 . The composition according to claim 7 , which further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant.

9 . A kit comprising the nucleic acid according to claim 1 .

10 . The nucleic acid of claim 1 , wherein the first ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which CDR1 is SEQ ID NO: 181, CDR2 is SEQ ID NO: 192, and CDR3 is SEQ ID NO: 218.

11 . The nucleic acid of claim 1 , wherein the first ISV is chosen from the group consisting of SEQ ID NOs: 42 and 78-180 and an amino acid sequence having a sequence identity of more than 90% with one of SEQ ID NOs: 42 and 78-180.

12 . The nucleic acid of claim 1 , comprising a third ISV, wherein the third ISV specifically binds CD123 and essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which:

i) CDR1 is SEQ ID NO: 16;

and

ii) CDR2 is chosen from the group consisting of:

a) SEQ ID NO: 18; and

b) amino acid sequences that have 3, 2 or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 18, wherein

at position 3 the Y has been changed into W;

at position 6 the N has been changed into S; and/or

at position 10 the Q has been changed into E;

and

iii) CDR3 is chosen from the group consisting of:

c) SEQ ID NO: 23; and

d) amino acid sequences that have 2 or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 23, wherein

at position 4 the E has been changed into R; and/or

at position 5 the T has been changed into D or Y.

13 . The nucleic acid of claim 1 , wherein the second ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which CDR1 is SEQ ID NO: 11, CDR2 is SEQ ID NO: 17, and CDR3 is SEQ ID NO: 21.

14 . The nucleic acid of claim 1 , wherein the second ISV is chosen from the group consisting of SEQ ID NOs: 1-6 and an amino acid sequence having a sequence identity of more than 90% with one of SEQ ID NOs: 1-6.

15 . The nucleic acid of claim 1 , further comprising a third ISV, wherein the third ISV specifically binds CD123, wherein the second ISV binds to an epitope on CD123 that is different from the epitope on CD123 bound by the third ISV.

16 . The nucleic acid of claim 15 , wherein the second ISV is chosen from the group consisting of SEQ ID NOs: 1-6 and an amino acid sequence having more than 90% identity with one of SEQ ID NOs: 1-6, and wherein the third ISV is chosen from the group consisting of SEQ ID NOs: 7-10 and an amino acid sequence having more than 90% identity with one of SEQ ID NOs: 7-10.

17 . The nucleic acid of claim 1 , wherein each of the first ISV and the second ISV essentially consist of a single domain antibody, a dAb, a Nanobody, a VHH, a humanized VHH, a camelized VH or a VHH which has been obtained by affinity maturation.

18 . The nucleic acid of claim 1 , wherein the polypeptide is chosen from the group consisting of SEQ ID NOs: 47, 49, 52, 53, 55, 56 and 58-61 and an amino acid sequence having a sequence identity of more than 90% with one of SEQ ID NOs: 47, 49, 52, 53, 55, 56 and 58-61.

19 . A nucleic acid encoding a polypeptide comprising a first immunoglobulin single variable domain (ISV) and a second ISV, wherein the first ISV specifically binds TCR and essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which:

i) CDR1 is chosen from the group consisting of:

a) SEQ ID NO: 181; and

b) amino acid sequences that have 1 amino acid difference with the amino acid sequence of SEQ ID NO: 181;

and

ii) CDR2 is chosen from the group consisting of:

c) SEQ ID NO: 192; and

d) amino acid sequences that have 1 amino acid difference with the amino acid sequence of SEQ ID NO: 192;

and

iii) CDR3 is chosen from the group consisting of:

e) SEQ ID NO: 218; and

f) amino acid sequences that have 1 amino acid difference with the amino acid sequence of SEQ ID NO: 218;

and wherein the second ISV specifically binds CD123 and essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which:

i) CDR1 is chosen from the group consisting of:

a) SEQ ID NO: 11; and

b) amino acid sequences that have 1 amino acid difference with the amino acid sequence of SEQ ID NO: 11;

and

ii) CDR2 is chosen from the group consisting of:

c) SEQ ID NO: 17; and

d) amino acid sequences that have 1 amino acid difference with the amino acid sequence of SEQ ID NO: 17;

and

iii) CDR3 is chosen from the group consisting of:

e) SEQ ID NO: 21; and

f) amino acid sequences that have 1 amino acid difference with the amino acid sequence of SEQ ID NO: 21.