T cell recruiting polypeptides based on CD3 reactivity

1. A polypeptide comprising a first and a second immunoglobulin single variable domain (ISV), wherein

said first ISV has high affinity for/binds to cluster of differentiation 3 (CD3) present on a T cell;

said second ISV has high affinity for/binds to a first antigen on a target cell;

wherein said first antigen is different from said CD3; and

wherein said target cell is different from said T cell;

wherein said first ISV 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: 81; and

(b) amino acid sequences that have 1, 2, 3 or 4 amino acid difference(s) with SEQ ID NO: 81, wherein

at position 1 the G has been changed into R;

at position 3 the T has been changed into A;

at position 4 the Y has been changed into F;

at position 8 the S has been changed into G; and/or

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

(ii) CDR2 is chosen from the group consisting of:

(a) SEQ ID NO: 101; and

(b) amino acid sequences that have 1, 2, 3 or 4 amino acid difference(s) with SEQ ID NO: 101, wherein

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

at position 5 the S has been changed into T;

at position 6 the G has been changed into D or E; and/or

at position 9 the T has been changed into S, A or P; and

(iii) CDR3 is chosen from the group consisting of:

(a) SEQ ID NO: 123; and

(b) amino acid sequences that have 1, 2 or 3 amino acid difference(s) with SEQ ID NO: 123, wherein

at position 2 the I has been changed into T;

at position 9 the I has been changed into V; and/or

at position 10 the A has been changed into P.

2. The polypeptide according to claim 1 , wherein said first antigen on a target cell is a tumor antigen.

3. The polypeptide according to claim 2 , wherein the tumor antigen is a tumor associated antigen (TAA).

4. The polypeptide according to claim 1 , further comprising a third ISV, which has high affinity for/binds to a second antigen on a target cell, wherein said second antigen is different from said first antigen.

5. The polypeptide according to claim 4 , wherein said second antigen on a target cell is a tumor antigen.

6. The polypeptide according to claim 4 , wherein said first antigen and said second antigen are chosen from the group consisting of:

EGFR as a first antigen and CEA as a second antigen;

CD19 as a first antigen and CD20 as a second antigen;

CD19 as a first antigen and CD22 as a second antigen;

CD123 as a first antigen and Tim-3 as a second antigen; and

CD132 as a first antigen and CD69 as a second antigen.

7. The polypeptide according to claim 5 , wherein the tumor antigen is a tumor associated antigen (TAA).

8. The polypeptide according to claim 1 , further comprising a serum protein binding moiety.

9. The polypeptide according to claim 8 , wherein said serum protein binding moiety is an ISV binding serum albumin.

10. A polypeptide that specifically binds CD3 and that comprises or 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: 81; or

(b) amino acid sequences that have 1 or 2 amino acid(s) difference with SEQ ID NO: 81, wherein

at position 1 the G has been changed into R;

at position 3 the T has been changed into A;

at position 4 the Y has been changed into F;

at position 8 the S has been changed into G; and/or

at position 10 the G has been changed into A,

 provided that the polypeptide comprising the CDR1 with 1 or 2 amino acid(s) difference binds CD3 with about the same or a higher affinity compared to the binding by the polypeptide comprising the CDR1 without the 1 or 2 amino acid(s) difference, said affinity as measured by surface plasmon resonance; and

(ii) CDR2 is chosen from the group consisting of:

(c) SEQ ID NO: 101; or

(d) amino acid sequences that have 1, 2 or 3 amino acid(s) difference(s) with SEQ ID NO: 101, wherein

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

at position 5 the S has been changed into T;

at position 6 the G has been changed into D or E; and/or

at position 9 the T has been changed into S, A or P,

 provided that the polypeptide comprising the CDR2 with 1, 2 or 3 amino acid(s) difference binds CD3 with about the same or a higher affinity compared to the binding by the polypeptide comprising the CDR2 without the 1, 2 or 3 amino acid(s) difference, said affinity as measured by surface plasmon resonance; and

(iii) CDR3 is chosen from the group consisting of:

(e) SEQ ID NO: 123; or

(f) amino acid sequences that have 4, 3, 2, or 1 or 2 amino acid(s) difference with SEQ ID NO: 123, wherein

at position 2 the I has been changed into T;

at position 9 the I has been changed into V; and/or

at position 10 the A has been changed into P,

 provided that the polypeptide comprising the CDR3 with or 1 or 2 amino acid(s) difference binds CD3 with about the same or a higher affinity compared to the binding by the polypeptide comprising the CDR3 without the 1 or 2 amino acid(s) difference, said affinity as measured by surface plasmon resonance.

11. The polypeptide according to claim 10 , further comprising a serum protein binding moiety.

12. The polypeptide according to claim 11 , wherein said serum protein binding moiety is an ISV that binds serum albumin.

13. The polypeptide according to claim 1 , wherein CDR1 is the amino acid sequence of SEQ ID NO: 81, CDR2 is the amino acid sequence of SEQ ID NO: 101, and CDR3 is the amino acid sequence of SEQ ID NO: 123.

14. The polypeptide according to claim 1 , wherein said first antigen is selected from the group consisting of Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), Fibroblast Activation Protein (FAP), MART-1, carcinoembryonic antigen (CEA), gp100, MAGE-1, HER-2, and LewisY antigens, CD123, CD44, CLL-1, CD96, CD47, CD32, CXCR4, Tim-3, CD25, TAG-72, Ep-CAM, PSMA, PSA, GD2, GD3, CD4, CD5, CD19, CD20, CD22, CD33, CD36, CD45, CD52, and CD147, growth factor receptors, including ErbB3 and ErbB4, and Cytokine receptors including Interleukin-2 receptor gamma chain (CD132 antigen), Interleukin-10 receptor alpha chain (IL-10R-A), Interleukin-10 receptor beta chain (IL-10R-B), Interleukin-12 receptor beta-1 chain (IL-12R-beta1), Interleukin-12 receptor beta-2 chain (IL-12 receptor beta-2), Interleukin-13 receptor alpha-1 chain (IL-13R-alpha-1) (CD213a1 antigen), Interleukin-13 receptor alpha-2 chain (Interleukin-13 binding protein), Interleukin-17 receptor (IL-17 receptor), Interleukin-17B receptor (IL-17B receptor), Interleukin 21 receptor precursor (IL-21R), Interleukin-1 receptor type I (IL-1R-1) (CD121a), Interleukin-1 receptor type II (IL-1R-beta) (CDw121b), Interleukin-1 receptor antagonist protein (IL-1ra), Interleukin-2 receptor alpha chain (CD25 antigen), Interleukin-2 receptor beta chain (CD122 antigen), Interleukin-3 receptor alpha chain (IL-3R-alpha) (CD123 antigen), CD30, IL23R, IGF-1R, IL5R, IgE, CD248 (endosialin), CD44v6, gpA33, Ron, Trop2, PSCA, claudin 6, claudin 18.2, CLEC12A, CD38, ephA2, c-Met, CD56, MUC16, EGFRvIII, AGS-16, CD27L, Nectin-4, SLITRK6, mesothelin, folate receptor, tissue factor, axl, glypican-3, CA9, Cripto, CD138, CD37, MUC1, CD70, gastrin releasing peptide receptor, PAP, CEACAM5, CEACAM6, CXCR7, N-cadherin, FXYD2 gamma a, CD21, CD133, Na/K-ATPase, mIgM (membrane-bound IgM), mIgA (membrane-bound IgA), Mer, Tyro2, CD120, CD95, CA 195, DR5, DR6, DcR3 and CAIX.

15. The polypeptide according to claim 5 , wherein said second antigen is selected from the group consisting of Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), Fibroblast Activation Protein (FAP), MART-1, carcinoembryonic antigen (CEA), gp100, MAGE-1, HER-2, and LewisY antigens, CD123, CD44, CLL-1, CD96, CD47, CD32, CXCR4, Tim-3, CD25, TAG-72, Ep-CAM, PSMA, PSA, GD2, GD3, CD4, CD5, CD19, CD20, CD22, CD33, CD36, CD45, CD52, and CD147, growth factor receptors, including ErbB3 and ErbB4, and Cytokine receptors including Interleukin-2 receptor gamma chain (CD132 antigen), Interleukin-10 receptor alpha chain (IL-10R-A), Interleukin-10 receptor beta chain (IL-10R-B), Interleukin-12 receptor beta-1 chain (IL-12R-beta1), Interleukin-12 receptor beta-2 chain (IL-12 receptor beta-2), Interleukin-13 receptor alpha-1 chain (IL-13R-alpha-1) (CD213a1 antigen), Interleukin-13 receptor alpha-2 chain (Interleukin-13 binding protein), Interleukin-17 receptor (IL-17 receptor), Interleukin-17B receptor (IL-17B receptor), Interleukin 21 receptor precursor (IL-21R), Interleukin-1 receptor type I (IL-1R-1) (CD121a), Interleukin-1 receptor type II (IL-1R-beta) (CDw121b), Interleukin-1 receptor antagonist protein (IL-1ra), Interleukin-2 receptor alpha chain (CD25 antigen), Interleukin-2 receptor beta chain (CD122 antigen), Interleukin-3 receptor alpha chain (IL-3R-alpha) (CD123 antigen), CD30, IL23R, IGF-1R, IL5R, IgE, CD248 (endosialin), CD44v6, gpA33, Ron, Trop2, PSCA, claudin 6, claudin 18.2, CLEC12A, CD38, ephA2, c-Met, CD56, MUC16, EGFRvIII, AGS-16, CD27L, Nectin-4, SLITRK6, mesothelin, folate receptor, tissue factor, axl, glypican-3, CA9, Cripto, CD138, CD37, MUC1, CD70, gastrin releasing peptide receptor, PAP, CEACAM5, CEACAM6, CXCR7, N-cadherin, FXYD2 gamma a, CD21, CD133, Na/K-ATPase, mIgM (membrane-bound IgM), mIgA (membrane-bound IgA), Mer, Tyro2, CD120, CD95, CA 195, DR5, DR6, DcR3 and CAIX.

16. The polypeptide according to claim 10 , wherein CDR1 is the amino acid sequence of SEQ ID NO: 81, CDR2 is the amino acid sequence of SEQ ID NO: 101, and CDR3 is the amino acid sequence of SEQ ID NO: 123.

17. A pharmaceutical composition comprising a polypeptide according to claim 1 .