WO2024251742A1

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Publication number: WO2024251742A1
Application number: PCT/EP2024/065350
Authority: WO
Inventors: Stefanie RIESENBERG; Kenneth Crook; Nina RESCHKE; Mischa Roland MÜLLER
Original assignee: Molecular Partners AG
Current assignee: Molecular Partners AG
Priority date: 2023-06-06
Filing date: 2024-06-04
Publication date: 2024-12-12
Anticipated expiration: 2025-12-06

Abstract

The present invention relates to recombinant binding proteins comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for CD2. In addition, the invention relates to nucleic acids encoding such recombinant binding proteins, pharmaceutical compositions comprising such proteins or nucleic acids, and the use of such binding proteins, nucleic acids or pharmaceutical compositions in methods for treating diseases, such as cancer, in a mammal, including a human.

Description

RECOMBINANT CD2 BINDING PROTEINS AND THEIR USE

FIELD OF THE DISCLOSURE

The present invention relates to recombinant binding proteins comprising a designed ankyrin repeat domain with binding specificity for CD2. In addition, the invention relates to nucleic acids encoding such recombinant binding proteins, pharmaceutical compositions comprising such recombinant binding proteins or nucleic acids, and the use of such recombinant binding proteins, nucleic acids or pharmaceutical compositions in methods of treating diseases, such as cancer, in a mammal, including a human.

BACKGROUND

Highly effective cancer therapeutics have been developed, which leverage redirected T cells. These therapeutics include bispecific T-cell engagers and CAR-T therapies. For example, a bispecific T-cell engager (blinatumomab) targeting the CD19 antigen expressed in B-cell malignancies (such as Acute Lymphocytic Leukemia (ALL) and Non-Hodgkin Lymphoma (NHL)) has been developed. Similarly, CD19- directed CAR-T therapies have also been developed (such as axicabtagene ciloleucel, tisagenlecleucel, and lisocabtagene maraleucel).

Despite initial effectiveness of such immunotherapies, T cell exhaustion contributes to treatment failure following bispecific T-cell engager and CAR-T therapies. One molecule that has been implicated in helping to avoid T cell exhaustion is CD2. CD2 is a surface antigen found on all peripheral blood T-cells, where it functions as a co-stimulatory receptor. The CD2 protein interacts with CD58 on antigen presenting cells to optimize immune recognition. Human CD2 exists in two isoforms (precursor of isoform 1 : NCBI Reference Sequence NP_001315538.1 ; precursor of isoform 2: NCBI Reference Sequence NP_001758.2).

CD2 signaling has been associated with a non-exhausted T cell phenotype. The interaction between CD2 and its ligand, CD58, has been found to be important for tumour cell killing by CAR-T cells, while the loss of CD58 expression on cancer cells (such as lymphoma cells) correlates with resistance and relapse in patients treated with CAR-T therapy. Therefore, CD2 co-stimulation may overcome T cell exhaustion and therapies utilizing CD2 co-stimulation may have the potential to achieve deeper and more durable responses compared to similar therapies not utilizing CD2 co-stimulation.

Thus, there remains a need for new CD2-specific binding proteins with beneficial properties, which may be used to co-stimulate T cells. Such binding proteins may be useful for therapeutic approaches for the treatment of diseases, including cancer.

SUMMARY

The present invention relates to recombinant binding proteins comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for CD2. In addition, the invention relates to nucleic acids encoding such recombinant binding proteins, pharmaceutical compositions comprising such recombinant binding proteins or nucleic acids, and the use of such recombinant binding proteins, nucleic acids, or pharmaceutical compositions in methods for treating diseases, such as cancer, in a mammal, including a human.

Recombinant binding proteins of the invention specifically bind to or target CD2 expressed by immune cells such as T cells. Such recombinant binding proteins of the invention can serve as a tool or as a building block for the generation of new therapeutic agents, such as T cell engagers. Also disclosed herein are recombinant binding proteins, in which the CD2-specific ankyrin repeat domains are combined with one or more other functional moieties in one molecule. Such other functional moieties include a CD3-specific binding moiety and/or a binding moiety with binding specificity for a Disease-Associated Antigen (DAA), such as a Tumor Associated Antigen (TAA). When combined with a CD3-specific binding moiety and a TAA-specific binding moiety, the CD2-specific ankyrin repeat domain of the invention can form an enhanced, multi-specific T cell engager that can simultaneously engage the TAA on tumor cells as well as CD3 (a T cell receptor signalling component) and CD2 on T cells. The CD2-specific ankyrin repeat domains may be further combined other moieties, such as, e.g., a half-life extending moiety. The functional moieties combined with the CD2-specific ankyrin repeat domains are preferably also ankyrin repeat domains.

As such, recombinant binding proteins of the invention with binding specificity for CD2 are useful for the generation of novel therapeutic molecules, which may provide improved responses (e.g. deeper and more durable responses) as compared to current therapeutic modalities.

In one aspect, the invention provides a recombinant binding protein comprising an ankyrin repeat domain having binding specificity for CD2, wherein said ankyrin repeat domain comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 7 to 14, 16, 17 and (2) sequences in which up to 9 amino acids in any one of SEQ ID NOs: 7 to 14, 16, 17 are substituted by other amino acids.

In one aspect, the invention provides a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for CD2, wherein said ankyrin repeat domain comprises an amino acid sequence with at least 80% amino acid sequence identity with any one of SEQ ID NOs: 1 to 3, 15 and wherein A at the second last position of SEQ ID NOs: 1 to 3, 15 is optionally substituted by L, and/or A at the last position of SEQ ID NOs: 1 to 3, 15 is optionally substituted by N.

In one aspect, the invention provides a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for CD2, wherein said binding protein further comprises a binding moiety with binding specificity for CD3. In one embodiment, said binding moiety with binding specificity for CD3 is an ankyrin repeat domain. In a further embodiment, said binding moiety with binding specificity for CD3 is an ankyrin repeat domain, wherein said ankyrin repeat domain comprises an amino acid sequence with at least 80% amino acid sequence identity with SEQ ID NOs: 4, 26 to 28, and wherein A at the second last position of SEQ ID NOs: 4, 26 to 28 is optionally substituted by L, and/or A at the last position of SEQ ID NO: 4, 26 to 28 is optionally substituted by N.

In one aspect, the invention provides a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for CD2, wherein said recombinant binding protein further comprises one, two or three binding moieties, wherein each of said binding moieties has binding specificity for a disease- associated antigen (such as a TAA). In one embodiment, any of said binding moieties with binding specificity for a disease-associated antigen is an ankyrin repeat domain.

In one aspect, the invention provides a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for CD2 as described herein, wherein said recombinant binding protein further comprises a half-life extending moiety. In one embodiment, said half-life extending moiety is an ankyrin repeat domain with binding specificity for human serum albumin.

Within the recombinant binding proteins of the invention, the ankyrin repeat domain with binding specificity for CD2 and any other functional moieties may be linked to each other via a peptide linker. One example of such a peptide linker is provided by SEQ ID NO: 5 or by SEQ ID NO: 55.

In one aspect, the invention provides nucleic acids encoding the recombinant binding proteins of the invention and pharmaceutical compositions comprising the recombinant binding protein of the invention or the nucleic acid of the invention and optionally a pharmaceutically acceptable carrier and/or diluent.

In one aspect, the invention provides a method of activating T cells, the method comprising the step of administering to a subject in need thereof an effective amount of the recombinant binding protein of the invention, the nucleic acid of the invention or the pharmaceutical composition of the invention.

In one aspect, the invention provides the recombinant binding protein of the invention, the nucleic acid of the invention or the pharmaceutical composition of the invention for use in a method of treating a medical condition. In one embodiment, said medical condition is a cancer.

In one aspect, the invention provides a method of treating a medical condition in a human subject, the method comprising administering to said subject a therapeutically effective amount of the recombinant binding protein of the invention, the nucleic acid of the invention or the pharmaceutical composition of the invention. In one embodiment, said medical condition is a cancer.

Based on the disclosure provided herein, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following embodiments (E).

Specifically, the present disclosure provides the following aspects, advantageous features and specific embodiments, respectively alone or in combination:

E1 . A recombinant binding protein comprising an ankyrin repeat domain with binding specificity for CD2.

E2. The recombinant binding protein of E1 , wherein said ankyrin repeat domain comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 7 to 14, 16, 17, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acid(s) in any one of SEQ ID NOs: 7 to 14, 16, 17 are substituted with other amino acids.

E3. The recombinant binding protein of any one of E1 to E2, wherein said ankyrin repeat domain comprises a first ankyrin repeat module and a second ankyrin repeat module, optionally wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module within said ankyrin repeat domain.

E4. The recombinant binding protein of E3, wherein said first and said second ankyrin repeat module each independently has an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 7 to 14, 16, 17, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 7 to 14, 16, 17 are substituted with other amino acids.

E5. The recombinant binding protein of any one of E3 to E4, wherein i. said first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 7, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 7 are substituted by other amino acids, and wherein said second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 8, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 8 are substituted by other amino acids; ii. said first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 16, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to

6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 16 are substituted by other amino acids, and wherein said second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 17, and (2) an amino acid sequence wherein up to 9, up to 8, up to

7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 17 are substituted by other amino acids;

E6. The recombinant binding protein of any one of E1 to E2, wherein said ankyrin repeat domain comprises a first ankyrin repeat module, a second ankyrin repeat module, and a third ankyrin repeat module, optionally wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module, and wherein said second ankyrin repeat module is located N-terminally of said third ankyrin repeat module within said ankyrin repeat domain.

E7. The recombinant binding protein of E6, wherein said first, second and third ankyrin repeat module each independently has an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 7 to 14, 16, 17 and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 7 to 14, 16, 17 are substituted by other amino acids.

E10. The recombinant binding protein of any one of E1 to E9, wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 1 to 3, 15.

E11. The recombinant binding protein of any one of E1 to E10, wherein said CD2 is human CD2.

E12. The recombinant binding protein of any one of E1 to E11 , wherein said ankyrin repeat domain binds human CD2 in PBS with a dissociation constant (KD) of or below about 10^-6M or of or below about 10^-7M, or of or below about 10^-8M.

E13. The recombinant binding protein of any one of E1 to E12, further comprising at least one binding moiety with binding specificity for a protein expressed on the surface of an immune cell, suitably wherein the immune cell is a T lymphocyte (T cell), suitably wherein said protein expressed on the surface of an immune cell is CD3.

E14. The recombinant binding protein of E13, wherein said binding moiety comprises an ankyrin repeat domain with binding specificity for CD3.

E15. The recombinant binding protein of E14, wherein said ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 4, 26 to 28. E16. The recombinant binding protein of any one of E1 to E15, further comprising at least one half-life extending moiety.

E17. The recombinant binding protein of E16, wherein said half-life extending moiety comprises an ankyrin repeat domain with binding specificity for human serum albumin.

E18. The recombinant binding protein of E17, wherein said ankyrin repeat domain with binding specificity for human serum albumin comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 29 to 31 .

E19. The recombinant binding protein of any one of E1 to E18, further comprising a binding agent with binding specificity for disease-associated antigen, suitably a tumor-associated antigen.

E20. A nucleic acid encoding the recombinant binding protein of any one of E1 to E19.

E21 . A vector comprising the nucleic acid of E20.

E22. The vector of E20, wherein the vector is a DNA vector, an RNA vector, a plasmid, a cosmid, or a viral vector.

E23. A cell comprising the nucleic acid of E20 or the vector of any one of E21 to E22.

E24. A pharmaceutical composition comprising the recombinant binding protein of any one of E1 to E19, the nucleic acid of E20, the vector of any one of E21 to E22, or the cell of E23, and a pharmaceutically acceptable carrier and/or diluent.

E25. A method of producing a recombinant binding protein, the method comprising culturing the cell of E23 and collecting the recombinant binding protein from the cell and/or the culture medium.

E26. The recombinant binding protein of any one of E1 to E19, the nucleic acid of E20, the vector of any one of E21 to E22, the cell of E23, or the pharmaceutical composition of E24, for use as a medicament.

E27. Use of the recombinant binding protein of any one of E1 to E19, the nucleic acid of E20, the vector of any one of E21 to E22, the cell of E23, or the pharmaceutical composition of E24, in the manufacture of a medicament.

E28. The recombinant binding protein of E26, the nucleic acid of E26, the vector of E26, the cell of E26, or the pharmaceutical composition of E26, or the use of E27, wherein said medicament is a medicament for the treatment of cancer.

E29. A method of treatment of a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the recombinant binding protein of any one of E1 to E19, the nucleic acid of E20, the vector of any one of E21 to E22, the cell of E23, or the pharmaceutical composition of E24.

E30. The method of E29, wherein said treatment is the treatment of cancer.

E31 . The method according to E30, wherein said cancer is a liquid tumor. E32. A method of activating T cells, the method comprising administering to the subject in need thereof a therapeutically effective amount of the recombinant binding protein of any one of E1 to E19, the nucleic acid of E20, the vector of any one of E21 to E22, the cell of E23, or the pharmaceutical composition of E24.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : T cell proliferation assay. See Example 2 for detailed description.

Figure 2 (A-B): In vitro binding of CD2 specific DARPins (SEQ ID NO: 1 , 3 and 15) to CD2 target expressed by a Jurkat cell line. Fig, 2A shows binding of the tested DARPins to Jurkat wild type cells, expressing CD2; Fig.2B shows that the same tested DARPins do not exhibit any binding activity when the same experiment is performed with knock-out Jurkat cells (KO), which do not express CD2 (FLU: Fluorescence intensity).

Figure 3: Potency assessment of selected DARPin proteins comprising CD19, CD2 and CD3 specific ankyrin repeat domains (DARPin protein #42, DARPin protein #43 and DARPin protein #44) in the presence of T cells and OCI-Ly19-CD58 knockout cells. Absence of CD58, which is a natural ligand of CD2 leads to disruption of the CD2-CD58 interaction and hence to loss of T cell activation. All tested proteins are able to induce T cell proliferation and rescue the loss of T cell response, which is caused by the absence of CD58 in CD58 knock-out cells. All tested proteins show increased T cell proliferation compared to DARPin #45, which does not comprise a CD2 binding domain.

Figure 4: Potency assessment of selected DARPin proteins comprising CD19, CD2 and CD3 specific ankyrin repeat domains (DARPin protein #42, DARPin protein #43 and DARPin protein #44) in the presence of T cells and OCI-Ly19-CD58 knockout cells. Absence of CD58, which is a natural ligand of CD2 leads to disruption of the CD2-CD58 interaction and hence to loss of T cell activation. All tested proteins are able to induce tumour cell killing and rescue the loss of T cell response, which is caused by the absence of CD58 in CD58 knock-out cells. All tested proteins show increased tumour cell killing compared to DARPin #45, which does not comprise a CD2 binding domain.

Figure 5: Potency assessment of selected DARPin proteins comprising CD19, CD2 and CD3 specific ankyrin repeat domains (DARPin protein #42, DARPin protein #43 and DARPin protein #44) in the presence of T cells and OCI-Ly19-CD58 expressing cells. All tested proteins show increased T cell proliferation compared to DARPin #45, which does not comprise a CD2 binding domain.

Figure 6: Potency assessment of selected DARPin proteins comprising CD22, CD19, CD2 and CD3 specific ankyrin repeat domains (DARPin protein #46, DARPin protein #47, DARPin protein #48, DARPin protein #49, DARPin protein #50 and DARPin protein #51) in the presence of T cells and OCI-Ly19-CD58 knock out cells, in a T cell proliferation assay.

Figure 7: Potency assessment of selected DARPin proteins comprising CD22, CD19, CD2 and CD3 specific ankyrin repeat domains (DARPin protein #46, DARPin protein #47, DARPin protein #48, DARPin protein #49, DARPin protein #50 and DARPin protein #51) in the presence of T cells and OCI-Ly19-CD58 expressing cells, in a T cell proliferation assay.

Figure 8 (A-B): Potency assessment of selected DARPin proteins comprising CD20, CD2 and CD3 specific ankyrin repeat domains (DARPin protein #56 and DARPin protein #57) in the presence of T cells and OCI- Ly19-CD58 knockout cells (Fig. 8A), or in the presence of T cells and OCI-Ly19-CD58 wild type cells (Fig. 8B). DARPin protein #59 and DARPin protein #60 represent proteins with binding specificity to CD20 and CD2 but not to CD3, while DAPRin protein #58 is a protein with binding specificity for CD20 and CD3 but not to CD2, and these were used as comparative controls. All tested proteins are able to induce T cell proliferation and rescue the loss of T cell response, which is caused by the absence of CD58 in CD58 knock-out cells.

Figure 9 (A-B): Potency assessment of selected DARPin proteins comprising CD20, CD2 and CD3 specific ankyrin repeat domains (DARPin protein #56 and DARPin protein #57) in the presence of T cells and OCI- Ly19-CD58 knockout cells (Fig. 9A), or the presence of T cells and OCI-Ly19-CD58 wild type cells (Fig. 9B). DARPin protein #59 and DARPin protein #60 represent proteins with binding specificity to CD20 and CD2 but not to CD3, while DAPRin protein #58 is a protein with binding specificity for CD20 and CD3 but not to CD2, and these were used as comparative controls. All tested proteins are able to induce tumor cell killing and rescue the loss of T cell response, which is caused by the absence of CD58 in CD58 knock-out cells.

Figure 10: Potency assessment of selected proteins comprising CD70, CD2 and CD3 specific ankyrin repeat domains (DARPin protein #61 , DARPin protein #62 and DARPin protein #63) in the presence of PBMC-purified pan T cells and human CD70 target protein. After five rounds of stimulation (S1 to S5), T cells were collected and flow cytometry analysis performed for calculation of T cell counts.

Figure 11 : Potency assessment of selected proteins comprising CD70, CD2 and CD3 specific ankyrin repeat domains (DARPin protein #61 , DARPin protein #62 and DARPin protein #63) in the presence of PBMC-purified pan T cells and human CD70 target protein. After five rounds of stimulation (S1 to S5), T cells were collected and flow cytometry analysis performed for measurement of T cell activation (median fluorescent intensity (MFI)).

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, said ankyrin repeat domain specifically binding CD2 comprises a first ankyrin repeat module and a second ankyrin repeat module. In one embodiment, said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module within said ankyrin repeat domain. In one embodiment, said first and said second ankyrin repeat module each independently has an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 7 to 14, 16, 17, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 7 to 14, 16, 17 are substituted with other amino acids. Thus, in one embodiment, said ankyrin repeat domain specifically binding CD2 comprises a first ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 7 to 14, 16, 17, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to

1 amino acids in any one of SEQ ID NOs:7 to 14, 16, 17 are substituted with other amino acids and further comprises a second ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 7 to 14, 16, 17, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 7 to 14, 16, 17 are substituted with other amino acids.

In one particular embodiment, the ankyrin repeat domain specifically binding CD2 comprises a first ankyrin repeat module comprising an amino acid sequence selected from the group consisting of (1) SEQ ID NO:

7, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to

2 or up to 1 amino acids of SEQ ID NO: 7 are substituted with other amino acids, and a second ankyrin repeat module comprising an amino acid sequence selected from the group consisting of (1) SEQ ID NO:

8, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 8 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 7, and (2) an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 7 are substituted with other amino acids and said second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 8, and (2) an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 8 are substituted with other amino acids, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module. In one embodiment, said first ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 7 and said second ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 8, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module.

16, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 16 are substituted with other amino acids, and a second ankyrin repeat module comprising an amino acid sequence selected from the group consisting of (1) SEQ ID NO:

17, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 17 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 16, and (2) an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 16 are substituted with other amino acids and said second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 17, and (2) an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 17 are substituted with other amino acids, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module. In one embodiment, said first ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 16 and said second ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 17, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module.

In one embodiment, said ankyrin repeat domain specifically binding CD2 comprises a first ankyrin repeat module, a second ankyrin repeat module and a third ankyrin repeat module. In one preferred embodiment, said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module within said ankyrin repeat domain, and said second ankyrin repeat module is located N-terminally of said third ankyrin repeat module within said ankyrin repeat domain.

In one embodiment, said first, second and third ankyrin repeat module each independently has an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 7 to 14, 16, 17, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to

1 amino acids in any one of SEQ ID NOs: 7 to 14, 16, 17 are substituted with other amino acids. Thus, in one embodiment, said ankyrin repeat domain specifically binding CD2 comprises a first ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 7 to 14, 16, 17, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 7 to 14, 16, 17 are substituted with other amino acids, wherein said second ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 7 to 14, 16, 17, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 7 to 14, 16, 17 are substituted with other amino acids and wherein said third ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 7 to 14, 16, 17, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 7 to 14, 16, 17 are substituted with other amino acids.

In one particular embodiment, the ankyrin repeat domain specifically binding CD2 comprises a first ankyrin repeat module comprising an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 9, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to

12, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO:12 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence selected from the group consisting of (1) SEQ ID NO:

13, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 13 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence selected from the group consisting of (1) SEQ ID NO:

14, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 14 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 12, and (2) an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 12 are substituted with other amino acids, said second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 13, and (2) an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 13 are substituted with other amino acids, and said third ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 14, and (2) an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 14 are substituted with other amino acids, wherein said first, second and third ankyrin repeat modules are arranged as first module-second module-third module in N-terminal to C-terminal direction within said ankyrin repeat domain. In one embodiment, said ankyrin repeat domain comprises (i) a first ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 12, (ii) a second ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 13 and (iii) a third ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 14, wherein said first, second and third ankyrin repeat modules are arranged as first module-second module-third module in N-terminal to C-terminal direction within said ankyrin repeat domain.

In one embodiment, all of said amino acid substitutions of said ankyrin repeat module(s) as described and referred to herein occur in framework positions of said ankyrin repeat module(s), wherein typically the overall structure of the module(s) is not affected by the substitutions.

The ankyrin repeat domains specifically binding CD2 as disclosed herein preferably comprise a N-terminal and/or a C-terminal capping module (thereafter also referred to as “capping repeats”). Capping modules are located at the N-and/or C-terminal end of an ankyrin repeat domain, typically forming tight tertiary interactions (i.e. tertiary structure interactions) with the ankyrin repeat module(s) in between, thereby providing a cap that shields the hydrophobic core of the ankyrin repeat domain at the side from exposure to the solvent. Examples of capping sequences are described in International Patent Publication Nos. WO 2002/020565 and WO 2012/069655, in U.S. Patent Publication No. US20130296221 , and by Interlandi et al., J Mol Biol. 2008 Jan 18;375(3):837-54. Examples of amino acid sequences of N-terminal capping modules (i.e. N-terminal capping repeats) are provided in SEQ ID NOs:18 to 20 and examples of amino acid sequences of C-terminal capping modules (i.e. C-terminal capping repeats) are provided in SEQ ID NOs: 22 to 24.

Accordingly, in some embodiments, provided is a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for CD2 as described herein, wherein said ankyrin repeat domain comprises an N-terminal capping module comprising or consisting of an amino acid sequence of any one of SEQ ID NOs:18 to 20, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 18 to 20 are substituted by other amino acids. Alternatively or in addition, said ankyrin repeat domain comprises a C-terminal capping module comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 22 to 24, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 22 to 24 are substituted by other amino acids.

In one preferred embodiment, said ankyrin repeat domain specifically binding CD2 comprises from N- terminus to C-terminus: an N-terminal capping module; at least one, at least two, at least three or more ankyrin repeat module(s) as described more specifically herein; and a C-terminal capping module.

In one embodiment, said ankyrin repeat domain comprises an N-terminal capping module comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 18 to 20, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 18 to 20 are substituted by other amino acids; at least one, at least two, or at least three ankyrin repeat module independently comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 7-14, 16, 17, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 7-14, 16, 17 are substituted by other amino acids; a C-terminal capping module comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 22 to 24, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 22 to 24 are substituted by other amino acids.

In another aspect, provided is a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for CD2, wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 1 to 3, 15. Optionally, K at the third last position of SEQ ID NOs: 1 to 3, 15 is substituted by Q, and/or optionally A at the second last position is substituted with L, and/or optionally A at the last position of SEQ ID NOs: 1 to 3, 15 is substituted with N.

Thus, in one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80% identical with any one of SEQ ID NOs: 1 to 3, 15. In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 90% identical with any one of SEQ ID NOs: 1 to 3, 15. In another embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 93% identical with any one of SEQ ID NOs: 1 to 3, 15. In a further embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 95% identical with any one of SEQ ID NOs: 1 to 3, 15. In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 98% identical with any one of SEQ ID NOs: 1 to 3, 15. In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 99% identical with any one of SEQ ID NOs: 1 to 3, 15. In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 3, 15.

In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with SEQ ID NO: 1. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 1 .

In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with SEQ ID NO: 2. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 2.

In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with SEQ ID NO: 3. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 3.

In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with SEQ ID NO: 15. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 15.

Also provided is a recombinant binding protein with binding specificity for CD2 that competes with a reference binding protein comprising an ankyrin repeat domain comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 3, 15. In one embodiment thereof, said competing recombinant binding protein with binding specificity for CD2 comprises an ankyrin repeat domain. Also provided is a recombinant binding protein with binding specificity for CD2 that binds to the same epitope as a reference binding protein comprising an ankyrin repeat domain comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 3, 15. In one embodiment thereof, said competing recombinant binding protein with binding specificity for CD2 comprises an ankyrin repeat domain.

In one embodiment, the recombinant binding protein comprising an ankyrin repeat domain with binding specificity for CD2 binds to human CD2 with a dissociation constant (KD) of or below about 10^-6M, or of or below about 10^-7M, or of or below about 10^-8M, or of or below about 10^-9M. Thus, in one embodiment, said recombinant binding protein binds to CD2 with a KD of or below about 10^-6M. In another embodiment, said recombinant binding protein binds to CD2 with a KD of or below about 10^-7M. In another embodiment, said recombinant binding protein binds to CD2 with a KD of or below about 10^-8M. In another embodiment, said recombinant binding protein binds to CD2 with a KD of or below about 10^-9M. In one embodiment, said recombinant binding protein comprising an ankyrin repeat domain with binding specificity for human CD2 binds to soluble cynomolgus CD2 with a dissociation constant (KD) of or below about 10^-7M, or of or below about 10^-8M, or of or below about 10⁹M, or of or below about 10¹°M, or of or below about 10¹¹M. Suitably, said dissociation constant is determined in PBS, suitably using surface plasmon resonance (SPR), e.g. as described in Example 5.

Accordingly, in one embodiment, said recombinant binding protein comprises an ankyrin repeat domain, wherein said binding protein binds human CD2 in PBS with a KD of or below about 10^-6M, or of or below about 10^-7M, or of or below about 10^-8M, and wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 1 to 3, 15.

In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain, wherein said recombinant binding protein binds soluble human CD2 in PBS with a KD of or below about 10^-8M, and wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 1. In one embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds soluble human CD2 in PBS with a KD of or below about 10^-8M, wherein said ankyrin repeat domain comprises the amino acid sequence with SEQ ID NO: 1.

In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain, wherein said recombinant binding protein binds soluble human CD2 in PBS with a KD of or below about 10^-6M, and wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 2. In one embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds soluble human CD2 in PBS with a KD of or below about 10^-6M, wherein said ankyrin repeat domain comprises the amino acid sequence with SEQ ID NO: 2.

In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain, wherein said recombinant binding protein binds soluble human CD2 in PBS with a KD of or below about 10^-7M, and wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 3. In one embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds soluble human CD2 in PBS with a KD of or below about 10^-7M, wherein said ankyrin repeat domain comprises the amino acid sequence with SEQ ID NO: 3.

In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain, wherein said recombinant binding protein binds soluble human CD2 in PBS with a KD of or below about 10^-7M, and wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 15. In one embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds soluble human CD2 in PBS with a KD of or below about 10^-7M, wherein said ankyrin repeat domain comprises the amino acid sequence with SEQ ID NO: 15. ii. Immune Cell Engaging Moieties

Binding proteins specific for CD2 may be useful as a component of a multi-specific binding molecule that is directed against disease-associated cells. Binding proteins specific for CD2 may be used in a multispecific binding protein further comprising at least one binding moiety specific for immune cells, e.g. CD3. Such a multi-specific binding protein may recruit immune cells, such as T cells, to the cancer cells.

Hi. Tumor associated antigen binding moieties

Thus, in one embodiment, provided is a recombinant binding protein comprising a first ankyrin repeat domain with binding specificity for CD19, a second ankyrin repeat domain with binding specificity for CD2, and a third ankyrin repeat domain with binding specificity for CD3, optionally wherein said ankyrin repeat domains are arranged from N terminus to C terminus in the order of first-second-third ankyrin repeat domain.

In another embodiment, provided is a recombinant binding protein comprising a first ankyrin repeat domain with binding specificity for CD19, a second ankyrin repeat domain with binding specificity for CD19, a third ankyrin repeat domain with binding specificity for CD2, and a fourth ankyrin repeat domain with binding specificity for CD3, optionally wherein said ankyrin repeat domains are arranged from N terminus to C terminus in the order of first-second-third-fourth ankyrin repeat domain.

In another embodiment, provided is a recombinant binding protein comprising a first ankyrin repeat domain with binding specificity for CD19, a second ankyrin repeat domain with binding specificity for CD22, a third ankyrin repeat domain with binding specificity for CD2, and a fourth ankyrin repeat domain with binding specificity for CD3, optionally wherein said ankyrin repeat domains are arranged from N terminus to C terminus in the order of first-second-third-fourth ankyrin repeat domain.

In another embodiment, provided is a recombinant binding protein comprising a first ankyrin repeat domain with binding specificity for CD20, a second ankyrin repeat domain with binding specificity for CD2, and a third ankyrin repeat domain with binding specificity for CD3, optionally wherein said ankyrin repeat domains are arranged from N terminus to C terminus in the order of first-second-third ankyrin repeat domain.

Thus, in one embodiment provided is a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for CD2 wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 1 to 3, 15, an ankyrin repeat domain with binding specificity for CD3 wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 4, 26 to 28, and an ankyrin repeat domain with binding specificity for CD19 wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 36 to 38.

In an even further embodiment said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for CD2 wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 1 to 3, 15; an ankyrin repeat domain with binding specificity for CD3 wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 4, 26 to 28; and ankyrin repeat domain with binding specificity for CD20 wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 40 to 41 .

In an even further embodiment said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for CD2 wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 1 to 3, 15; an ankyrin repeat domain with binding specificity for CD3 wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 4, 26 to 28; and ankyrin repeat domain with binding specificity for CD70 wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 65.

In certain embodiments, each ankyrin repeat domain is capable of binding its respective target at the same time as each of the other ankyrin repeat domain is bound to its respective target. iv. Half-Life Extending Moieties

A “half-life extending moiety” extends the serum half-life in vivo of the recombinant binding proteins described herein, compared to the same protein without the half-life extending moiety. Examples of halflife extending moieties include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin domain, maltose binding protein (MBP), a human serum albumin (HSA) binding domain, or polyethylene glycol (PEG).

In one embodiment, the recombinant binding proteins provided herein further comprise one or more halflife extending moieties. Thus, in a particular embodiment, the recombinant binding protein comprises an ankyrin repeat domain with binding specificity for CD2 as described herein and further comprises one or more half-life extending moieties. Preferably, said half-life extending moiety binds to human serum albumin. In one embodiment, the half-life extending moiety comprises an ankyrin repeat domain binding human serum albumin, said ankyrin repeat domain comprising an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 29 to 31 . In one embodiment, the half-life extending moiety comprises an ankyrin repeat domain comprising an amino acid sequence at least about 90% identical to any one of SEQ ID NOs: 29 to 31. In one embodiment, the half-life extending moiety comprises an ankyrin repeat domain comprising an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 29. In one embodiment, the half-life extending moiety comprises an ankyrin repeat domain comprising an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 30. In one embodiment, the half-life extending moiety comprises an ankyrin repeat domain comprising an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 31 .

In some embodiments, the half-life extending moiety comprises an immunoglobulin domain. In some embodiments, the immunoglobulin domain comprises an Fc domain. In some embodiments, the Fc domain is derived from any one of the known heavy chain isotypes: IgG (y), IgM (p), IgD (6), IgE (s), or IgA (a). In some embodiments, the Fc domain is derived from any one of the known heavy chain isotypes or subtypes: IgGi (y1), lgG2 (y2), lgG3 (y3), lgG4 (y4), IgAi (a1), lgA2 (a2). In some embodiments, the Fc domain is the Fc domain of human IgGi.

In some embodiments, the Fc domain comprises an uninterrupted native sequence (i.e., wild type sequence) of an Fc domain. In some embodiments, the immunoglobulin Fc domain comprises a variant Fc domain resulting in altered biological activity. For example, at least one point mutation or deletion may be introduced into the Fc domain so as to reduce or eliminate the effector activity (e.g., International Patent Publication No. WO 2005/063815), and/or to increase the homogeneity during the production of the recombinant binding protein. In some embodiments, the Fc domain is the Fc domain of human IgGi and comprises one or more of the following effector-null substitutions: L234A, L235A, and G237A (Eu numbering). In some embodiments, the Fc domain does not comprise the lysine located at the C-terminal position of human lgG1 (i.e., K447 by Eu numbering). The absence of the lysine may increase homogeneity during the production of the recombinant binding protein. In some embodiments, the Fc domain comprises the lysine located at the C-terminal position (K447, Eu numbering).

I. Nucleic acids and vectors

Also provided herein are nucleic acids encoding a recombinant binding protein or ankyrin repeat domain described herein. Such nucleic acids can encode polypeptides comprising segments or domains of the recombinant binding protein or ankyrin repeat domain described above. Because of the degeneracy of the code, a variety of nucleic acid sequences will encode each of the ankyrin repeat domain sequences described herein. The nucleic acid can comprise a nucleotide sequence as set forth in any one of SEQ ID NOs: 32 to 35, or a sequence substantially identical thereto (e.g., a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, orwhich differs by no more than 3, 6, 15, 30, or 45 nucleotides from the sequences in any one of SEQ ID NOs: 32 to 35).

In certain embodiments, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOs: 1 to 3, 15. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 15. In another embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of any one of the ankyrin repeat binding domains or any one of the ankyrin repeat binding modules disclosed herein.

Also provided are nucleic acid molecules that derive from any one of SEQ ID NOs: 32 to 35, having been optimized for protein expression in a suitable host cell, such as a prokaryotic host cell, e.g., E.coli. The nucleic acid may be present in a vector such as a phage display vector, or in a recombinant plasmid vector. Accordingly, also provided is a cloning or expression vector comprising one or more nucleic acid sequences comprising any one of SEQ ID NOs:32 to 35, wherein the vector is suitable for the recombinant production of the recombinant binding protein as described herein.

For expression of the recombinant binding protein described herein, standard techniques can be applied to transfect a host cell with the expression vector, suitably comprising the nucleic acid sequence of any one of SEQ ID NOs: 32 to 35 encoding a recombinant protein as described herein. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calciumphosphate precipitation, DEAE-dextran transfection and the like.

In one embodiment, a cloning or expression vector as described herein comprises the nucleic acid sequence of any one of SEQ ID NOs: 32 to 35, operatively linked to suitable promoter sequences. Also provided is a vector comprising the nucleic acid encoding any of the recombinant binding proteins described herein, wherein said vector is a DNA vector, an RNA vector, a plasmid, a cosmid, or a viral vector.

When recombinant expression vectors encoding the recombinant binding proteins disclosed herein are introduced into suitable host cells, e.g., prokaryotic host cells, the recombinant binding proteins are produced by culturing the host cells for a period of time sufficient to allow for expression of the recombinant binding protein in the host cells or secretion of the recombinant binding proteins into the culture medium in which the host cells are grown. Recombinant binding proteins can be recovered from the host cells and/or the culture medium using standard protein purification methods. In one embodiment, the method comprises culturing a suitable host cell, e.g., a prokaryotic host cell as described herein and collecting the recombinant binding protein from the host cells.

II. Compositions, Uses and Methods of Treatment

In one aspect, also provided is a pharmaceutical composition comprising a recombinant binding protein as described herein, and/or a nucleic acid encoding a recombinant binding protein as described herein, and a pharmaceutically acceptable carrier, stabilizer and/or diluent. Pharmaceutically acceptable carriers, excipients, and stabilizers, are, for example, described in Remington's Pharmaceutical Sciences 16^th edition, Osol, A. Ed., 1980.

Accordingly, in one aspect, provided is a pharmaceutical composition comprising a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for CD2, wherein said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with any one of SEQ ID NOs: 1 to 3, 15, and a pharmaceutically acceptable carrier, stabilizer and/or diluent. Pharmaceutically acceptable carriers and/or diluents are known to the person skilled in the art and are explained in more detail below.

Suitable carriers, diluents, excipients or stabilizers include, for example, saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers, and preservatives. A pharmaceutically acceptable excipient typically has no significant adverse effect on the patient receiving the composition such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, and amino acid copolymers. A pharmaceutical composition may also be a combination formulation, comprising an additional active agent, such as an anticancer agent or an anti-angiogenic agent, or an additional bioactive compound. The compositions to be used for in vivo administration must be aseptic or sterile. This is readily accomplished by filtration through sterile filtration membranes.

In one embodiment, the pharmaceutical composition comprises at least one recombinant binding protein as described herein and a detergent, such as non-ionic detergent, e.g. Tween-20, a buffer, such as phosphate buffer, and a sugar, such as sucrose. In one aspect, such a pharmaceutical composition comprises a recombinant binding protein as described herein and PBS.

The recombinant binding proteins with binding specificity for CD2 described herein have numerous in vitro and in vivo therapeutic utilities involving the treatment of disorders. For example, these molecules can be administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g., in vivo, to treat, prevent and/or to diagnose a variety of disorders with. Accordingly, in one aspect provided herein are methods of treating a medical condition, e.g., a disease, e.g., cancer, in a subject in need thereof, comprising administering a therapeutically effective amount of the recombinant binding protein described herein, a nucleic acid encoding such a recombinant binding protein, a vector comprising a nucleic acid encoding such a recombinant binding protein, a cell comprising such a nucleic acid or vector, or a pharmaceutical composition comprising such a recombinant binding protein, nucleic acid, vector or cell.

In another aspect, a method of treating a subject, e.g., reducing or ameliorating, a hyperproliferative condition or disorder (e.g., a cancer), e.g., solid tumor, a hematological cancer, soft tissue tumor, or a metastatic lesion, is provided. Accordingly, in one embodiment, provided is a method of inhibiting the growth of tumor cells, comprising administering to a subject in need thereof a therapeutically effective amount of the recombinant binding proteins with binding specificity for CD2 as described herein alone or in combination with other agents, e.g., therapeutic agents, or therapeutic modalities. The recombinant binding proteins with binding specificity for CD2 as described herein and one or more additional agents can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the recombinant binding proteins with binding specificity for CD2 as described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed. In one embodiment, said other agents, e.g., therapeutic agents, or therapeutic modalities, are administered separately or sequentially with the recombinant binding proteins with binding specificity for CD2 described herein. In one embodiment, said therapeutic agent comprises a checkpoint inhibitor molecule. In one embodiment, said therapeutic agent comprises a binding agent specific for a DAA, e.g., a TAA, and further comprises a binding agent specific for a protein expressed on the surface of an immune cell, suitably a T lymphocyte (T cell). A suitable protein expressed on the surface of an immune cell is CD3. In one embodiment, said therapeutic agent is a chimeric antigen receptor T cell (CAR-T). In one experiment, said therapeutic agent is a binding agent

Also provided is a recombinant binding protein described herein, a nucleic acid encoding such a recombinant binding protein, a vector comprising a nucleic acid encoding such a recombinant binding protein, a cell comprising such a nucleic acid or vector, or a pharmaceutical composition comprising such a recombinant binding protein, nucleic acid, vector or cell, for use in the treatment of a medical condition, e.g., a disease, e.g., cancer.

In one aspect, provided is the use of recombinant binding protein described herein, a nucleic acid encoding such a recombinant binding protein, a vector comprising a nucleic acid encoding such a recombinant binding protein, a cell comprising such a nucleic acid or vector, or a pharmaceutical composition comprising such a recombinant binding protein, nucleic acid, vector or cell, in the manufacture of a medicament, e.g., for treatment of cancer.

In one embodiment, the recombinant binding protein with binding specificity for CD2 as described herein further comprises an immune-cell activating moiety, suitably a CD3-binding ankyrin repeat domain as described herein, a nucleic acid encoding said recombinant binding protein or a pharmaceutical composition comprising said recombinant binding protein. In one embodiment, said activation is tumor- localized. In one embodiment, said immune cells are adaptive immune cells. In one embodiment, said adaptive immune cells are T cells.

Administration may include topical administration, oral administration, or parenteral administration. Atypical route of administration is parenteral administration. In parental administration, the pharmaceutical composition is formulated in a unit dosage injectable form such as a solution, suspension, or emulsion, in association with suitable pharmaceutically acceptable excipients, e.g., as described above. The dosage and mode of administration depends on the individual to be treated and the disease.

The recombinant binding protein with binding specificity for CD2 described herein, the nucleic acid encoding such a recombinant binding protein, or the vector or cell comprising a nucleic acid encoding such a recombinant binding protein, or the pharmaceutical composition comprising such a recombinant binding protein, nucleic acid, vector, or cell, can be administered to the subject parenterally, e.g., through intravenous, intratumoral or subcutaneous route. In some embodiments, such a recombinant binding protein, nucleic acid, vector, cell, or pharmaceutical composition, is administered intravenously.

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

A “medical condition” may be one that is characterized by inappropriate cell proliferation. A medical cell condition may be a hyperproliferative condition. In one embodiment said medical condition is a neoplastic disease. The term "neoplastic disease", as used herein, refers to an abnormal state or condition of cells or tissue characterized by rapidly proliferating cell growth or neoplasm. In one embodiment said medical condition is a malignant neoplastic disease. Suitably, said medical condition or disease is a cancer. In one embodiment, said cancer is ovarian cancer, fallopian tube cancer, peritoneal cancer, mesothelioma or pancreatic ductal adenocarcinoma (PDAC). In one embodiment, said cancer is ovarian cancer. In one embodiment, said cancer is mesothelioma. In one embodiment, said cancer is pancreatic ductal adenocarcinoma (PDAC). Suitably said medical condition is a B cell malignancy. Exemplary B cell malignancies include Non-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle-cell lymphoma (MCL), marginal zone lymphoma (MZL), as well as some types of Multiple myeloma (MM) and Hodgkin lymphoma (HL). In one embodiment said B cell malignancy is a B cell lymphoma. In one embodiment said B cell malignancy is Hodgkin’s lymphoma. In one embodiment said B cell malignancy is a non-Hodgkin lymphoma.

The recombinant binding protein described herein, nucleic acid encoding such a recombinant binding protein, vector comprising a nucleic acid encoding such a recombinant binding protein, cell comprising such a nucleic acid or vector, or pharmaceutical composition comprising such a recombinant binding protein, nucleic acid, vector or cell may also be used in combination with one or more other therapies known in the art. The term “use in combination with”, as used herein, refers to a co-administration, which is carried out under a given regimen. This includes synchronous administration of the different compounds as well as time-shifted administration of the different compounds (e.g., compound A is given once and compound B is given several times thereafter, or vice versa, or both compounds are given synchronously and one of the two is also given at later stages).

DEFINITIONS

Selected terms are defined below and throughout the specification. It is understood that unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one skilled in the art in the technical field of the invention.

All publications, patents, and accession numbers mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

The use of any and all examples, or exemplary language (e g., "such as") provided herein, is intended merely to better illustrate the disclosure and does not pose a limitation on the scope unless otherwise claimed.

As used herein, the articles "a" and "an” can mean “one”, but their use is also consistent with the meaning of “one or more”, “at least one” and “one or more than one”.

The term "or" is used herein to mean, and is used interchangeably with, the term "and/or", unless context clearly indicates otherwise and is to be interpreted as an inclusive “or” meaning any one or any combination. Throughout this specification and the claims which follow, and unless the context requires otherwise, the terms “comprising”, “having” and “including” can be used interchangeably. The terms “comprising” and “including” are to be interpreted as specifying the presence of the stated features or components as referred to, but does not preclude the presence or addition of one or more features, or components, or groups thereof. Additionally, the terms “comprising” and “including” are intended to include examples encompassed by the term “consisting of’. Consequently, the term “consisting of’ can be used in place of the terms “comprising” and “including” to provide for more specific embodiments of the invention.

"About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 10% of a given value or range of values. For example, where a dosage is mentioned as “about” a particular value, it is intended to include a range around the specified value of plus or minus 10%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”.

It is also to be understood, although not always explicitly stated, that the reagents described herein are merely examples and that equivalents of such are known in the art.

As used herein, the term “polypeptide” refers to a molecule comprising a chain of multiple amino acids linked via peptide bonds. Preferably, a polypeptide consists of more than eight amino acids linked via peptide bonds. Furthermore, the term also encompasses peptides modified by, e.g., glycosylation, and proteins comprising two or more polypeptide chains, cross-linked by, e.g., disulfide bonds.

As used herein, the term "protein" refers to a molecule comprising a polypeptide, wherein at least part of the polypeptide has, or is able to acquire, a defined three-dimensional arrangement by forming secondary, tertiary, and/or quaternary structures within a single polypeptide chain and/or between multiple polypeptide chains. If a protein comprises two or more polypeptide chains, the individual polypeptide chains may be linked non-covalently or covalently, e.g., by a disulfide bond between two polypeptides. A part of a protein, which individually has, or is able to acquire, a defined three-dimensional arrangement by forming secondary and/or tertiary structure, is termed "protein domain”.

The term “recombinant” as used in “recombinant protein”, “recombinant polypeptide” and the like, means that said protein or polypeptide is produced by the use of recombinant DNA technologies known to the practitioner skilled in the art. For example, a “recombinant DNA molecule” (e.g., produced by gene synthesis) encoding a polypeptide can be cloned into a bacterial expression plasmid (e.g., pQE30, QIAgen), yeast expression plasmid, mammalian expression plasmid, or plant expression plasmid, or a DNA enabling in vitro expression. If, for example, such a recombinant bacterial expression plasmid is inserted into appropriate bacteria (e.g., Escherichia coli), these bacteria can produce the polypeptide(s) encoded by this recombinant DNA. The correspondingly produced polypeptide or protein is called a “recombinant polypeptide” or “recombinant protein”.

As used herein, the term "binding protein" refers to a protein comprising at least one binding domain. A binding protein may also comprise two, three, four, five or more binding domains. Preferably, said binding protein is a recombinant binding protein. Recombinant binding proteins of the present disclosure comprise an ankyrin repeat domain with binding specificity for human CD2. Furthermore, any such binding protein may comprise additional polypeptides (such as e.g., polypeptide tags, peptide linkers, fusion to other proteinaceous domains with binding specificity, cytokines, hormones, or antagonists), or chemical modifications (such as coupling to polyethylene-glycol, toxins (e.g., DM1), small molecules, antibiotics and alike) known to the person skilled in the art.

The term “binding domain” refers to a protein domain with binding specificity for a target. Preferably, said binding domain is a recombinant binding domain.

As used herein, the term "target" refers to an individual molecule such as a nucleic acid molecule, a polypeptide or protein, a carbohydrate, or any other naturally occurring molecule, including any part of such individual molecule, orto complexes of two or more of such molecules, orto a whole cell or a tissue sample, or to any non-natural compound. Preferably, a target is a naturally occurring or non-natural polypeptide or protein, or a polypeptide or protein containing chemical modifications, for example, naturally occurring or non-natural phosphorylation, acetylation, or methylation. In the context of the present disclosure, CD2 protein expressed on T cells is a target of CD2-specific binding proteins. Preferably, the target is human CD2 (UniProt ID: P06729). CD2 functions as a cell adhesion and co-stimulatory molecule. It is known to bind lymphocyte function-associated antigen-3 (LFA-3/CD58), a surface molecule expressed by antigen presenting cells and epithelial cells. The CD2/CD58 interaction promotes the initial stages of cell contact and facilitates T-cell receptor (TCR) triggering. CD2 is used as a specific marker for T cells and NK cells, and to distinguish B cell neoplasms from T cell lymphomas and leukemias.

The term “CD3" or "Cluster of Differentiation 3" refers to a multimeric protein complex composed of four distinct chains. In mammals, the complex contains a CD3y (gamma) chain, a CD36 (delta) chain, and two CD3s (epsilon) chains. These chains associate with the T cell receptor (TCR) and the CD3 (zeta) chain to generate an activation signal in T cells. CD3 is critical for T cell activation and the initiation of T cell effector functions, such as cytokine production, cytotoxicity, and the release of other immune mediators. It enables T cells to recognize and respond to specific antigens, thereby orchestrating adaptive immune responses. The amino acid sequences of human CD3 gamma, delta, epsilon and zeta chains are shown in NCBI Ref. Seq. NP_ 000064.1 , NP_000723.1 , NP_000724.1 and NP_932170.1 , respectively. Ankyrin repeat domains specifically binding CD3 are disclosed in WO2022129428 (incorporated by reference).

The term “CD19” or "Cluster of Differentiation 19" (UniProt ID Nr: Q71 UW0) refers to a 95 kd Type I transmembrane glycoprotein in the immunoglobulin superfamily (IgSF) with two extracellular C2-set Ig-like domains and a relatively large, 240 amino acid, cytoplasmic tail that is highly conserved among mammalian species and is widely expressed during all phases of B cell development until terminal differentiation into plasma cells.

The term “CD22” or "Cluster of Differentiation 22" (UniProt ID Nr: P20273) refers to a membrane glycoprotein of 140 kDa expressed on the surface of B cells. CD22 mediates B-cell B-cell interactions and may be involved in the localization of B-cells in lymphoid tissues.

The term “CD20” or "Cluster of Differentiation 20" (UniProt ID Nr: P11836) refers to a B-lymphocyte-specific membrane protein that plays a role in the regulation of cellular calcium influx necessary for the development, differentiation, and activation of B-lymphocytes.

The term “CD70” or "Cluster of Differentiation 70" (UniProt ID Nr: P32970) refers to a cytokine, ligand for TNFRSF27/CD27. It is a surface antigen on activated, but not on resting, T and B lymphocytes. It induces proliferation of costimulated T cells, enhances the generation of cytolytic T cells, and contributes to T cell activation. CD70 is overexpressed in several types of cancer, including Hodgkin's lymphoma and nonHodgkin's lymphoma. CD70 is also found to be overexpressed in some types of solid tumors.

Patent application W02002/020565 and Forrer et al., (FEBS Leters 539, 2-6, 2003), contain a general description of repeat protein features and repeat domain features, techniques and applications. The term "repeat protein" refers to a protein comprising one or more repeat domains. Preferably, a repeat protein comprises one, two, three, four, five or six repeat domains. Furthermore, said repeat protein may comprise additional non-repeat protein domains, polypeptide tags and/or peptide linkers. The repeat domains can be binding domains. The term "repeat domain" refers to a protein domain comprising two or more consecutive repeat modules as structural units, wherein said repeat modules have structural and sequence homology. Preferably, a repeat domain further comprises an N-terminal and/or a C-terminal capping module. For clarity, a capping module can be a repeat module. Such repeat domains, repeat modules, and capping modules, sequence motives, as well as structural homology and sequence homology are known to the practitioner in the art from examples of ankyrin repeat domains (W02002/020565), leucine-rich repeat domains (W02002/020565), tetratricopeptide repeat domains (Main, E.R., et al., Structure 11 (5), 497-508, 2003), and armadillo repeat domains (W02009/040338). It is further known to the practitioner in the art that such repeat domains are different from proteins comprising repeated amino acid sequences, where every repeated amino acid sequence is able to form an individual domain (for example FN3 domains of Fibronectin).

The term "ankyrin repeat domain" refers to a repeat domain comprising two or more consecutive ankyrin repeat modules as structural units. Ankyrin repeat domains may be modularly assembled into larger ankyrin repeat proteins, optionally with half-life extension domains, using standard recombinant DNA technologies (see, e.g., Forrer, P„ et al., FEBS letters 539, 2-6, 2003); W02002/020565, WO2016/156596; WO2018/054971). The term “construct” as used herein refers to a recombinant binding protein comprising one or more designed ankyrin repeat domain and optionally a peptide linker and/or tag sequence. An example of a peptide linker is provided in SEQ ID NO: 5 or in SEQ ID NO:55 and an example of a tag sequence is provided in SEQ ID NO: 6. For clarity, the term “ankyrin repeat domain” includes N-terminal and C-terminal capping modules.

The term "designed" as used in “designed repeat protein”, “designed repeat domain” and the like refers to the property that such repeat proteins and repeat domains, respectively, are man-made and do not occur in nature.

The term "target interaction residue(s)" refers to amino acid residue(s) of a repeat module, which contribute to the direct interaction with a target.

The term "framework residue(s)" refers to amino acid residue(s) of a repeat module, which contribute to the folding topology, i.e. which contribute to the fold of said repeat module or which contribute to the interaction with a neighboring module. Such contribution may be the interaction with other residues in the repeat module, or the influence on the polypeptide backbone conformation as found in a-helices or p-sheets, or the participation in amino acid stretches forming linear polypeptides or loops. Such framework and target interaction residues may be identified by analysis of the structural data obtained by physicochemical methods, such as X-ray crystallography, NMR and/or CD spectroscopy, or by comparison with known and related structural information.

The term "repeat modules" refers to the repeated amino acid sequence and structural units of the designed repeat domains, which are originally derived from the repeat units of naturally occurring repeat proteins. Each repeat module comprised in a repeat domain is derived from one or more repeat units of a family or subfamily of naturally occurring repeat proteins, preferably the family of ankyrin repeat proteins. Furthermore, each repeat module comprised in a repeat domain may comprise a “repeat sequence motif’ deduced from homologous repeat modules obtained from repeat domains selected on a target and having the same target specificity. A “repeat module” as used herein encompasses internal repeat modules and capping modules such as N-terminal and C-terminal capping modules. An “internal repeat module” refers to a repeat module that is flanked by two repeat modules. In other words, an internal repeat module is N- terminally flanked by one repeat module and C-terminally flanked by another repeat module.

Accordingly, the term "ankyrin repeat module" refers to a repeat module, which is originally derived from the repeat units of occurring ankyrin repeat proteins. Ankyrin repeat proteins are known to the person skilled in the art.

The term "repeat sequence motif refers to an amino acid sequence, which is deduced from one or more repeat modules. Preferably, said repeat modules are from repeat domains with binding specificity for the same target. Such repeat sequence motifs comprise framework residue positions and target interaction residue positions. Said framework residue positions correspond to the positions of framework residues of the repeat modules. Likewise, said target interaction residue positions correspond to the positions of target interaction residues of the repeat modules. Repeat sequence motifs comprise non-randomized positions and randomized positions.

The term “repeat unit” refers to amino acid sequences comprising sequence motifs of one or more naturally occurring proteins, wherein said "repeat units" are found in multiple copies, and exhibit a defined folding topology common to all said motifs determining the fold of the protein. Examples of such repeat units include leucine-rich repeat units, ankyrin repeat units, armadillo repeat units, tetratricopeptide repeat units, HEAT repeat units, and leucine-rich variant repeat units.

Repeat modules may comprise positions with amino acid residues which have not been randomized in a library for the purpose of selecting target-specific repeat domains ("non-randomized positions") and positions with amino acid residues which have been randomized in the library for the purpose of selecting target-specific repeat domains ("randomized positions"). The non-randomized positions comprise framework residues. The randomized positions comprise target interaction residues. “Have been randomized” means that two or more amino acids were allowed at an amino acid position of a repeat module, for example, wherein any of the usual twenty naturally occurring amino acids were allowed, or wherein most of the twenty naturally occurring amino acids were allowed, such as amino acids other than cysteine, or amino acids otherthan glycine, cysteine and proline. Forthe purpose of the present disclosure, amino acid residues 3, 4, 6, 11 , 14 and 15 of SEQ ID NOs: 7 to 14, 16, 17 are randomized positions of the internal repeat modules; amino acid residues 4, 8, 11 and 12 of SEQ ID NOs: 18 to 20, are randomized positions of the N-capping repeat modules; and amino acid residues 3, 4, 6, 14 and 15 of SEQ ID NOs: 22 to 24 are randomized positions of the C-capping repeat modules. Amino acid residues 1 to 3, 5 to 7, 9, 10, 13 to 30 of SEQ ID NOs: 18 to 20 are framework residues comprised in the N-capping repeat modules, amino acid residues 1 , 2, 5, 7 to 10, 12, 13, 16 to 33 of SEQ ID NOs: 7 to 14, 16, 17 are framework residues comprised in the internal repeat modules and amino acid residues of 1 , 2, 5, 7 to 13, 16 to 28 of SEQ ID NOs: 22 to 24 are framework residues comprised in the C-capping repeat modules. For the purposes of the present disclosure, a substitution in framework positions shall apply to all embodiments irrespective of whether such substitution is explicitly described.

A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Exemplary and conservative amino acid substitutions are shown in Table 1 below.

Table 1 : Exemplary and conservative Amino Acid Substitutions

In addition, (a) the second last position of any ankyrin repeat domain or any C-capping module described herein can be “A” or “L”, and/or the last position can be “A” or “N” or (b) the third last position can be a “Q”. Furthermore, each ankyrin repeat domain described herein may optionally comprise a “G,” an “S,” or a “GS” sequence at its N-terminus. For determining the percent identity between two sequences (e.g., polynucleotide or polypeptide) the sequences are aligned for optimal comparison. A position in the first sequence is considered identical to the corresponding position in the second sequence when they have the same nucleotide or amino acid. The percent identity is calculated by dividing the number of identical positions by the total number of positions in the reference sequence and multiplying by 100. To assess similarity, the alignment is typically performed over the length of the reference sequence. For example, to determine if a test sequence is at least 80% identical to SEQ ID NO: 1 (an example of a reference sequence), the alignment is carried out against SEQ ID NO: 1 , and the number of identical positions is compared. If at least 80% of the positions are identical, the test sequence is considered at least 80% identical to SEQ ID NO: 1. Gaps or missing positions in a shorter sequence are considered non-identical positions. Various computer programs are available to determine sequence homology. The Needleman and Wunsch algorithm can be used with specific parameters such as using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6 to calculate percent identity between amino acid or nucleic acid sequences. One example of suitable parameters is a Blosum 62 scoring matrix, a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Individual binding domains may be linked covalently with a peptide linker. A suitable linker may comprise from 1 to 50 amino acids, suitably from 6 to 38 amino acids. A peptide linker may be a proline-threonine (PT) rich peptide linker. An example of a suitable PT-rich linker in the context of the present disclosure comprises the amino acid sequence of SEQ ID NO: 5. A peptide linker may also be a glycine-serine (GS) rich peptide linker. An example of a suitable GS-rich linker in the context of the present disclosure comprises the amino acid sequence of SEQ ID NO: 55.

The terms “binding specificity”, “has binding specificity for a target”, “specifically binding to a target”, “binding to a target with high specificity”, “specific for a target”, “target specificity”, or “specifically binds” and the like means that a binding protein or binding domain binds to a target with a lower dissociation constant (i.e. it binds with higher affinity) than it binds to an unrelated protein such as the E. coli maltose binding protein (MBP). Preferably, the dissociation constant (“KD”) for the target is at least 10²; more preferably, at least 10³; more preferably, at least 10⁴; or more preferably, at least 10⁵ times lower than the corresponding dissociation constant for MBP. Methods to determine dissociation constants of protein-protein interactions, such as surface plasmon resonance (SPR) based technologies (e.g., SPR equilibrium analysis) or isothermal titration calorimetry (ITC) are known to the person skilled in the art. The measured KD values of a particular protein-protein interaction can vary if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of KD values are preferably made with standardized solutions of protein and a standardized buffer, such as PBS. The term “PBS” means a phosphate buffered water solution comprising 137 mM NaCI, 10 mM phosphate and 2.7 mM KCI and having a pH of 7.4.

Binding of any molecule to another is governed by two forces, namely the association rate (k_on) and the dissociation rate (koff). The affinity of any binder [B] to a target [T] can then be expressed by the equilibrium dissociation constant KD, which is the quotient of koir/kon.

kon is a second-order rate constant of the binding reaction, with the unit

whereas the dissociation reaction koff is a first-order rate constant with the unit s~¹. From this it becomes clear that the association reaction depends on the concentration of the reactants, whereas the dissociation is independent of the concentration, following a simple exponential decay function.

A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. For example, as exemplified herein, the binding affinity of a particular binding moiety to a drug molecule target can be expressed as KD value, which refers to the dissociation constant of the binding moiety and the drug molecule target. KD is the ratio of the rate of dissociation, also called the “off-rate (koff)”, to the association rate, or “on-rate (kon)”. Thus, KD equals k_Off/k_On and is expressed as a molar concentration (M), and the smaller the KD, the stronger the affinity of binding. KD values can be determined using any suitable method. One exemplary method for measuring KD is surface plasmon resonance (SPR) (see, e.g., Nguyen et al. Sensors (Basel). 2015 May 5; 15(5):10481- 510). KD value may be measured by SPR using a biosensor system such as a BIACORE® system. BIAcore kinetic analysis comprises, e.g., analysing the binding and dissociation of an antigen from chips with immobilized molecules (e.g., molecules comprising epitope binding domains), on their surface. Another method for determining the KD of a protein is by using Bio-Layer Interferometry (see, e.g., Shah et al. J Vis Exp. 2014; (84): 51383). A KD value may be measured using OCTET® technology (Octet QKe system, ForteBio). Alternatively, or in addition, a KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Id.) can also be used. Any method suitable for assessing the binding affinity between two binding partners is encompassed herein.

A typical and preferred determination of dissociation constants (KD) of the recombinant binding proteins with binding specificity for CD2 as disclosed herein is by Surface Plasmon Resonance (SPR) analysis as described in Example 5.

The term “binding agent” or “binding moiety” refers to any molecule capable of binding a target molecule. Binding agents include, for example, antibodies, antibody fragments, aptamers, peptides (e.g., Williams et al., J Biol Chem 266:5182-5190 (1991)), alternative scaffolds, antibody mimics, repeat proteins, e.g., designed ankyrin repeat proteins, receptor proteins and any other naturally occurring interaction partners of the target molecule, and can comprise natural proteins and proteins modified or genetically engineered, e.g., to include non-natural residues and/or to lack natural residues.

The term “therapeutic moiety” refers to a chemical moiety that can function as a therapeutic agent (or perform a therapeutic function), such as for a treatment of a disease or disorder when administered to or otherwise provided to a patient or subject.

The term “linked” or “linkage” refers to any covalent or non-covalent linkage between a chemical moiety and a protein such as a ankyrin repeat domain or a designed repeat protein.

Preferably, clearance, and/or exposure, and/or terminal half-life are assessed in a mammal, suitably mouse and/or cynomolgus monkey. Suitably, when measuring the clearance, and/or exposure, and/or terminal half-life in mouse, the evaluation is done considering the data up to 48 h post-injection. More preferably, the evaluation of terminal half-life in mouse is calculated from 24 h to 48 h. Suitably, when measuring the clearance, and/or exposure, and/or terminal half-life in cynomolgus monkey, the evaluation is done considering the data up to day 7 post-injection. The person skilled in the art further is able to identify effects such as target-mediated clearance and consider them when calculating the terminal half-life. The term “terminal half-life” of a drug refers to the time required to reach half the plasma concentration of the drug applied to a mammal after reaching pseudo-equilibrium (for example calculated from 24 hours to 48 hours in mouse or calculated from day 1 to day 5 in cynomolgus monkey). Terminal half-life is not defined as the time required to eliminate half the dose of the drug administered to the mammal. Suitably, pharmacokinetic comparison is done at any dose, suitably at equivalent dose (i.e., same mg/kg dose) or equimolar dose (i.e., same mol/kg dose), suitably at equimolar dose (i.e., same mol/kg dose). It is understood by the person skilled in the art that equivalent and/or equimolar dosing in animals is subject to experimental dose variations of at least about 20%, more preferably about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. Preferably, a dose used for pharmacokinetic measurement is selected from about 0.001 to about 1000 mg/kg, about 0.01 to about 100 mg/kg, about 0.1 to about 50 mg/kg, about 0.5 to about 10 mg/kg.

The term “tumor-localized activation of immune cells” means that immune cells are activated preferentially in tumor tissue as compared to a non-tumor tissue.

The term "antibody" means not only intact antibody molecules, but also any fragments and variants of antibody molecules that retain immunogen-binding ability. Such fragments and variants are known in the art and are regularly employed both in vitro and in vivo. Accordingly, the term "antibody" encompasses intact immunoglobulin molecules, antibody fragments such as, e.g., Fab, Fab', F(ab')2, and single chain V region fragments (scFv), bispecific antibodies, chimeric antibodies, antibody fusion polypeptides, and unconventional antibodies.

The terms “subject”, “patient”, “subject in need thereof”, and “patient in need thereof” are used interchangeably herein and refer to a human suffering from one or more of the diseases described herein (e.g., cancer). A subject is “in need of’ a treatment if such subject would benefit biologically, medically and/or in quality of life from such treatment.

The term “dose” refers to a specified amount of a therapeutic agent (drug), e.g. the CD2-specific binding protein described herein, administered to the subject in need thereof on the particular treatment day. The dose would, for example, be declared on a product package or in a product information leaflet.

As used herein, “administer” or “administration” refers to the act of physically delivering a substance as it exists outside the body into a subject. Administration includes all forms suitable for delivering the therapeutic agent(s) described herein.

The terms "cancer" and "cancerous" are used herein to refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer encompasses solid tumors and liquid tumors, as well as primary tumors and metastases. A "tumor" comprises one or more cancerous cells. Solid tumors typically also comprise tumor stroma.

EXAMPLES

Starting materials and reagents disclosed below are known to those skilled in the art, are commercially available and/or can be prepared using well-known techniques.

Materials

Chemicals were purchased from Sigma-Aldrich (USA). Oligonucleotides were from Microsynth (Switzerland). Unless stated otherwise, DNA polymerases, restriction enzymes and buffers were from New England Biolabs (USA) or Fermentas/Thermo Fisher Scientific (USA). Inducible E. coli expression strains were used for cloning and protein production, e.g. E. coli XL1-blue (Stratagene, USA) or BL21 (Novagen, USA).

Molecular Biology

Unless stated otherwise, methods are performed according to known protocols (see, e.g., Sambrook J., Fritsch E.F. and Maniatis T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory 1989, New York). Designed ankyrin repeat protein libraries

Methods to generate designed ankyrin repeat protein libraries have been described, e.g., in U.S. Patent No. 7,417,130; Binz et al., J. Mol. Biol. 332, 489-503, 2003; Binz et al., Nat. Biotechnol. 22, 575-582, 2004. By such methods designed ankyrin repeat protein libraries having randomized ankyrin repeat modules and/or randomized capping modules can be constructed. For example, such libraries could accordingly be assembled based on a fixed N-terminal capping module and a fixed C-terminal capping module or a randomized C-terminal capping module. Preferably, such libraries are assembled to not have any of the amino acids C, G, M, N (in front of a G residue) and P at randomized positions of repeat or capping modules. Furthermore, such randomized modules in such libraries may comprise additional polypeptide loop insertions with randomized amino acid positions. Examples of such polypeptide loop insertions are complement determining region (CDR) loop libraries of antibodies or de novo generated peptide libraries. For example, such a loop insertion could be designed using the structure of the N-terminal ankyrin repeat domain of human ribonuclease L (Tanaka, N., Nakanishi, M, Kusakabe, Y, Goto, Y., Kitade, Y, Nakamura, K.T., EMBO J. 23(30), 3929-3938, 2004) as guidance. In analogy to this ankyrin repeat domain where ten amino acids are inserted in the beta-turn present close to the boarder of two ankyrin repeats, ankyrin repeat proteins libraries may contain randomized loops (with fixed and randomized positions) of variable length (e.g., 1 to 20 amino acids) inserted in one or more beta-turns of an ankyrin repeat domain. Any such N- terminal capping module of an ankyrin repeat protein library preferably possesses the RILLAA, RILLKA or RELLKA motif (e.g., present from position 19 to 24 in SEQ ID NO: 1) and any such C-terminal capping module of an ankyrin repeat protein library preferably possesses the KLN, KLA or KAA motif (e.g., present at the last three amino acids in SEQ ID NO: 1).

The design of such an ankyrin repeat protein library may be guided by known structures of an ankyrin repeat domain interacting with a target. Examples of such structures, identified by their Protein Data Bank (PDB) unique accession or identification codes (PDB-IDs), are 1WDY, 3V31 , 3V30, 3V2X, 3V2O, 3UXG, 3TWQ-3TWX, 1 N11 , 1 S70 and 2ZGD.

Examples of designed ankyrin repeat protein libraries, such as N2C and N3C designed ankyrin repeat protein libraries, have been described (U.S. Patent No. 7,417,130; Binz et al. 2003, loc. cit.; Binz et al. 2004, loc. cit.). The digit in N2C and N3C describes the number of randomized repeat modules present between the N-terminal and C-terminal capping modules.

The nomenclature used to define the positions inside the repeat units and modules is based on Binz et al. 2004, loc. cit. with the modification that borders of the ankyrin repeat modules and ankyrin repeat units are shifted by one amino acid position. For example, position 1 of an ankyrin repeat module of Binz et al. 2004 (loc. cit.) corresponds to position 2 of an ankyrin repeat module of the current disclosure and consequently position 33 of an ankyrin repeat module of Binz et al. 2004, loc. cit. corresponds to position 1 of a following ankyrin repeat module of the current disclosure.

All the DNA sequences were confirmed by sequencing, and the calculated molecular weight of selected proteins was confirmed by mass spectrometry.

Example 1 : Selection and characterization of binding proteins comprising an ankyrin repeat domain with binding specificity for CD2 Using ribosome display (Hanes, J. and Pluckthun, A., PNAS 94, 4937-42, 1997), several ankyrin repeat proteins with binding specificity for human CD2 or CD2:CD58 complex were selected from DARPin libraries similar as described by Binz et al. 2004 (loc. cit.). The binding of the selected clones towards recombinant human CD2 target protein was assessed by crude extract Homogeneous Time Resolved Fluorescence (HTRF), indicating that human CD2-specific binding proteins were successfully selected. For example, the ankyrin repeat domains of SEQ ID NOs: 1 to 3, 15 constitute amino acid sequences of selected binding proteins comprising an ankyrin repeat domain with binding specificity for CD2. Individual ankyrin repeat modules from such ankyrin repeat domains with binding specificity for CD2 are provided, e.g., in SEQ ID NOs: 7 to 14, 16, 17.

Human recombinant CD2 target preparation

Biotinylated Human CD2/SRBC Protein, His, Avitag™ as well as Human CD58/LFA-3 Protein with an Fc tag were purchased from ACRO Biosystems. Recombinant, biotin-labeled human CD2-Fc fusion protein with C-terminal Avi-tag was purchased from BPS Bioscience.

Selection of CD2-specific ankyrin repeat proteins by ribosome display

The selection of CD2-specific ankyrin repeat proteins was performed by ribosome display (Hanes and Pluckthun, loc. cit.) using the extracellular domain of CD2 (UniProt ID: P06729, residues 25-209) as target protein either with or without being in complex with CD58, libraries of ankyrin repeat proteins as described above, and established protocols (see, e.g., Zahnd, C., Amstutz, P. and Pluckthun, A., Nat. Methods 4, 69- 79, 2007). The number of reverse transcription (RT)-PCR cycles after the 1^st selection round was 45 and then constantly thirty. The four rounds of selection employed standard ribosome display selection, using decreasing target concentration (400 nM CD2 or 300 nM CD2 in the CD2:CD58 complex, 100 nM, 25 nM and 5 nM, respectively).

Selected clones show binding to CD2 target (shown by Homogeneous Time Resolved Fluorescence -HTRF)

In a first screening step, the pools from the ribosome display were subcloned into a derivative of the pQE30 (Qiagen) expression vector containing an N-terminal His-tag (SEQ ID NO: 6) followed by a Flag-tag (SEQ ID NO: 66) and expressed in E. coli cells in 96 well plates. More than 2000 DARPin proteins from ribosome display round 4 were expressed in E. coli cells with a His-tag (either as CD2 mono-DARPins or in a TAA- CD2 multi-domain format). Crude extracts thereof were prepared to test binding of the His-tagged DARPin proteins to biotinylated human CD2-Fc recombinant protein in an HTRF assay. The crude extracts were diluted in PBS-TB (PBS supplemented with 0.1 % (w/v) BSA and 0.1 % Tween20, pH 7.4) and used at 1 :500 dilution (final) in the assay. Binding was performed against 6 nM (final concentration) of human biotinylated CD2-Fc. FRET donor (Streptavidin-Tb) and acceptor (MAb anti-His-d2) conjugate (Cisbio) were used at a 1 :400 dilution (final) in a well of a 384-well plate and incubated over night at 4°C (cold room). The HTRF was read-out on a Tecan M1000 using a 340 nm excitation wavelength and a 665 ±10 nm emission filter. Four selected binders were subcloned into a derivative of the pQE30 (Qiagen) expression vector containing an N-terminal His-tag (SEQ ID NO: 6) and purified on an AKTAxpress™ system for in-depth characterization.

For example, expression vectors encoding the following ankyrin repeat proteins were constructed:

DARPin protein #1 (SEQ ID NO: 1 with a His-tag (SEQ ID NO: 6) fused to its N terminus)

DARPin protein #2 (SEQ ID NO: 2 with a His-tag (SEQ ID NO: 6) fused to its N terminus)

DARPin protein #3 (SEQ ID NO: 3 with a His-tag (SEQ ID NO: 6) fused to its N terminus)

DARPin protein #15 (SEQ ID NO: 15 with a His-tag (SEQ ID NO: 6) fused to its N terminus)

Example 2: Functional testing of CD2-specific binding proteins

Experiment A

A CD2-specific ankyrin repeat domain according to the invention (SEQ ID NO: 1) was formatted as a trispecific T cell engager molecule (TCE #1), further comprising an ankyrin repeat domain with binding specificity for CD3 (SEQ ID NO: 4) and an ankyrin repeat domain with binding specificity for a TAA. The three ankyrin repeat domains were linked to each other via a peptide linker (SEQ ID NO: 5). At the N- terminus, the tri-specific T cell engager molecule (TCE #1) comprised a His-tag (SEQ ID NO: 6) for ease of purification.

Another T cell engager molecule (TCE #2) was generated, which was identical to TCE #1 except that the CD2-specific ankyrin repeat domain was not present in TCE #2. Thus, TCE #2 comprised only two of the ankyrin repeat domains, namely the ankyrin repeat domain with binding specificity for CD3 (SEQ ID NO: 4; selection of DARPin proteins with binding specificity for CD3 are disclosed in WO2022129428 A1 which is incorporated herein by reference) and the ankyrin repeat domain with binding specificity for a TAA.

Assessment of the potency of a CD2-engaging DARPin protein on OCI-Ly19 CD58-KO target cells in coculture with human Pan-T cells

The potency of a CD2-engaging T cell engaging DARPin [TCE #1] was assessed in comparison to a T cell engaging DARPin protein not engaging CD2 [TCE #2] in an in vitro short-term assay measuring T cell proliferation by assessing CellTrace Violet (CTV) by Flow Cytometry.

For this purpose, frozen purified pan-T effector cells were thawed, washed, and stained with 1 :1 ’000 Cell Trace Violet (Thermo Fisher). 50,000 cells and 10,000 OCI-Ly19 CD58-KO [knockout] cells (or 10,000 OCI- Ly19 WT [wildtype] cells) [Effector:Target ratio 5:1] per well of a 96-well plate were co-incubated with serial dilutions of selected DARPin proteins in duplicates for 72h at 37°C. Cells were washed and stained with 1 :3600 Zombie NIR Viability dye (BioLegend) and 1 :200 anti-TAA antibody (BD Biosciences) for 30 minutes at 4 °C. After washing and fixation, cells were analysed on a Attune NxT Cytometer. T cell proliferation was assessed by measuring the dilution of CTV on CTV+/TAA-negative population in comparison to the untreated population. Flow Cytometry data was analysed using FlowJo Software and plotted using GraphPad Prism 8.

As shown in Figure 1 , TCE #2 induces less T cell proliferation when co-cultured with OCI-Ly19 CD58-KO cells compared to OCI-Ly19 WT cells. The CD2-engaging TCE #1 induces T cell proliferation on OCI-Ly19 CD58-KO cells up to a similar level as TCE #2 on OCI-Ly19 WT cells. These results indicate that the presence of the CD2-engaging moiety in the TCE rescues the loss of T cell response, which is caused by the absence of CD58 in CD58 knock-out cells.

Experiment B

In a similar experimental set up, three selected DARPin proteins, comprising an ankyrin repeat domain with binding specificity for CD2, an ankyrin repeat domain with binding specificity for CD3 and an ankyrin repeat domain with binding specificity for CD19, were assessed fortheir efficacy in a T cell proliferation assay and tumor cell killing assay on OCI-Ly19 CD58-KO or OCI-Ly19 CD58 target expressing cells in co-culture with human Pan-T cells. As a control molecule, a DARPin protein comprising ankyrin repeat domains with binding specificity for CD3 and CD19, but not an ankyrin repeat domain binding to human CD2 was used. The three ankyrin repeat domains were linked to each other via a peptide linker (SEQ ID NO: 5), while at the N-terminus, each tested protein molecule comprised a His-tag (SEQ ID NO: 6) for ease of purification.

Briefly, Pan-T and OCI-LY19 wt or OCI-LY19 CD58 KO cells were incubated at an E:T ratio of 5:1 and T- cell proliferation was assessed by FACS after 72 hours of co-culture, in the presence of serial dilutions of the tested proteins. To assess T cell proliferation and tumor cell killing, staining of the co-cultures was performed using anti-CD2 antibody (Brilliant Violet 711 ™ anti-human CD2 Antibody, Biolegend 300232, 1 :200) and Cell Trace Violet (CTV, ThermoFisher, ThermoFisher, 1 :1000) for 30 minutes at 4°C. After washing and fixation, cells were analyzed on a AttuneNxt Flow Cytometer (Thermo Fisher). Flow Cytometry data was analyzed using FlowJo software and data was plotted using GraphPad Prism. T cell proliferation was gated as living CD2+/CTV+ cells. Tumor cell killing was gated as living counts CD22+ normalized to co-culture.

Table 2: Tested DARPin proteins

As shown in Figure 3 (showing T cell proliferation induced by tested proteins) and Figure 4 (showing tumour cell killing induced by tested proteins), the presence of a CD2 specific ankyrin repeat domain in proteins DARPin #42, DARPin #43 and DARPin #44 can rescue the loss of potency on OCI-Ly19 CD58-KO target cells, which is caused by the absence of CD58. As a reference, OCI-Ly19 wildtype cells were used in order to show the loss of potency of DARPin #45, which does not comprise a CD2-binding domain. Namely, T cell engager protein DARPin #45 induces T cell proliferation and tumor cell killing, with a potency drop observed when OCI-Ly19-CD58 knockout cells are used as target cells. Therefore, this indicates that the presence of a CD2 binding domain rescues such a loss.

The same proteins, when assessed in the presence of OCI-Ly19 wild type cells, as shown in Figure 5, can still induce increased potency, expressed as increased T cell proliferation, when compared to a control DARPin protein (DARPin protein #45) which does not comprise a CD2 binding domain.

Experiment C

Similarly to experiment B, six selected DARPins, formatted as tetra-specific proteins, comprising an ankyrin repeat domain with binding specificity for CD2, an ankyrin repeat domain with binding specificity for CD3, an ankyrin repeat domain with binding specificity for CD19 and additionally comprising an ankyrin repeat domain with binding specificity for CD22, were assessed for their efficacy in a T cell proliferation assay on OCI-Ly19 CD58-KO or OCI-Ly19 CD58 expressing target cells in co-culture with human Pan-T cells. The selected proteins comprised the same ankyrin repeat domains, but in a different arrangement from N- terminus to C-terminus, as shown in Table 3 below. As control molecules, two DARPin proteins were used comprising ankyrin repeat domains with binding specificity for CD3, CD19, and CD22, but not comprising an ankyrin repeat domain with binding specificity for human CD2, and a non-binding DARPin (SEQ ID NO: 54). The three ankyrin repeat domains were linked to each other via a peptide linker (SEQ ID NO: 55), while at the N-terminus, each tested protein molecule comprised a His-tag (SEQ ID NO: 6) for ease of purification.

Briefly, Pan-T and OCI-LY19 wt or OCI-LY19 CD58 KO cells were incubated at an E:T ratio of 5:1 and T- cell proliferation was assessed by FACS after 72 hours of co-culture, in the presence of serial dilutions of the tested proteins. To assess T cell proliferation, staining of the co-cultures was performed using anti-CD2 antibody (Brilliant Violet 711 ™ anti-human CD2 Antibody, Biolegend 300232, 1 :200) and Cell Trace Violet (CTV, ThermoFisher, ThermoFisher, 1 :1000) for 30 minutes at 4°C. After washing and fixation, cells were analyzed on a AttuneNxt Flow Cytometer (Thermo Fisher). Flow Cytometry data were analyzed using FlowJo software and data were plotted using GraphPad Prism. T cell proliferation was gated as living CD2+/CTV+ cells.

Table 3: Tested DARPin proteins

As shown in Figure 6 (OCI-Ly19 CD58-wild type target expressing cells in co-culture with human Pan-T cells) and Figure 7 (OCI-Ly19 CD58-KO cells in co-culture with human Pan-T cells), including a CD2 binding domain along with a CD3 binding domain (DARPin #50, DARPin #51) leads to increased potency, as demonstrated by increased T cell proliferation (when compared e.g. to DARPin #52 which comprises a CD3 binding domain but not a CD2 binding domain).

Experiment D

In a similar experimental set up to the one described in Experiments A to C, two selected DARPin proteins, comprising an ankyrin repeat domain with binding specificity for CD2, an ankyrin repeat domain with binding specificity for CD3 and two ankyrin repeat domains with binding specificity for CD20, were assessed for their efficacy in a T cell proliferation and tumor cell killing assay, on OCI-Ly19 CD58-KO or OCI-Ly19 CD58- expressing cells in co-culture with human Pan-T cells. As control molecules, a DARPin protein comprising ankyrin repeat domains with binding specificity for CD3 and CD20, but not comprising an ankyrin repeat domain binding to human CD2 (DARPin #58), or a DARPin protein comprising ankyrin repeat domains with binding specificity for CD2 and CD20, but not comprising an ankyrin repeat domain binding to human CD3 (DARPin #59 and #60), were used (see Table 4 below). The three ankyrin repeat domains were linked to each other via a peptide linker (SEQ ID NO: 5), while at the N-terminus each tested protein molecule comprised a His-tag (SEQ ID NO: 6) for ease of purification.

To assess T cell proliferation and tumor cell killing, staining of the co-cultures was performed using anti- CD2 antibody (Brilliant Violet 711 ™ anti-human CD2 Antibody, Biolegend 300232, 1 :200) and Cell Trace Violet (CTV, ThermoFisher, ThermoFisher, 1 :1000) for 30 minutes at 4°C. After washing and fixation, cells were analyzed on a AttuneNxt Flow Cytometer (Thermo Fisher). Flow Cytometry data was analyzed using FlowJo software and data was plotted using GraphPad Prism. T cell proliferation was gated as living CD2+/CTV+ cells. Tumor cell killing was gated as living counts CD22+ normalized to the untreated coculture control.

Table 4: Tested DARPin proteins

As shown in Figure 8(A-B) and Figure 9(A-B), the presence of a CD2-engaging ankyrin repeat domain increases T cell proliferation and target cell killing respectively, in co-culture assays of healthy donor T cells and OCI-Ly19 or OCI-Ly29 CD58-KO cells.

Experiment E

Assessment of the potency CD2-engaging DARPins on human Pan-T cells in a T cell activation assay

In order to assess the ability of CD2-engaging DARPins to induce sustained T cell activation, three selected binding proteins with binding specificity for CD70 (tumor associated antigen; selection of DAPRins with binding specificity to CD70 are disclosed in WO2022215032 A1 , which is incorporated herein by reference), CD2 and CD3 (see Table 5 below) were cultured with isolated PBMC T cells and compared with a DARPin T cell engager (not comprising a CD2-binding domain) and a benchmark anti-CD3 monoclonal antibody (OKT3). Briefly, 96-well flat-bottom high-binding plates (Thermo Scientific MaxiSorp) were coated with anti- CD3 antibody (OKT3, BioLegend) or neutravidin (Thermo Fisher Scientific) in phosphate-buffered saline (PBS, Gibco) at a final concentration of 70 pg/ml and 8 pg/ml, respectively. After overnight incubation at 4°C and continuous agitation at 450 rpm, plates were washed with PBS to remove any unbound neutravidin or anti-CD3 antibody and then 20 nM biotinylated CD70 (UniProt ID Nr: P32970; ACROBiosystems) in PBS was added to the neutravidin-coated wells. Plates were incubated for 30 min at room temperature (RT) and 450 rpm and then washed to remove the excess of unbound biotinylated CD70. Finally, 500 nM of each tested DARPin in PBS were added to the neutravidin/CD70 coated wells, further incubated for 30 min at RT and 450 rpm, and then washed again with PBS to remove unbound DARPins. 200,000 PBMC-purified pan T cells (Miltenyi, negative selection) were then added to each well and incubated at 37°C, 5% CO2. After 2-3 days of incubation, T cells were collected from the wells and re-plated in fresh medium on newly coated 96-well plates for a total of 5 stimulation rounds (referred to as S1 to S5). After each stimulation round, T cells were harvested to perform flow cytometry analysis. Firstly, T cells were washed with PBS and stained with a viability dye (Live/Dead Zombie NIR, BioLegend) for 30 min at 4°C to enable dead cell exclusion. After a second wash with PBS + 1 % Fetal Bovine Serum (FACS buffer), an antibody cocktail containing anti-human CD8-PE (SK1) and anti-human CD25-PerCP/Cy5.5 (BC96) (both from BioLegend, used at 1 pg/ml in FACS buffer) was added to the wells and incubated at 4°C for additional 30 min. Subsequently, cells were washed with cold FACS buffer and finally fixed with 4% paraformaldehyde (PFA, HiMedia). Stained cells were analyzed using Attune NxT (Thermo Fisher Scientific) and the median fluorescent intensity (MFI) values and cell counts of CD8+CD25+ T cells were plotted using GraphPad Prism (Version 10).

Table 5: Tested DARPin proteins

As shown in Figures 10 and 11 , the presence of a CD2-engaging ankyrin repeat domain comprised in DARPin #61 , DARPin #62 and DARPin #63 induced a sustained T cell expansion and T cell activation respectively over the course of time, compared to the benchmark antibody or the DARPin T cell engager without CD2 binding domain (SEQ ID NO: 64).

Example 3: Selection of binding proteins comprising an ankyrin repeat domain with binding specificity for CD19, CD22 and CD20 respectively

Using ribosome display (Hanes, J. and Pluckthun, A., PNAS 94, 4937-42, 1997), many ankyrin repeat proteins with binding specificity for human CD117 were selected from DARPin libraries similar as described by Binz et al. 2004 (loc. cit.). The binding of the selected clones towards recombinant human CD19, CD22 and CD20 targets was assessed independently by crude extract Homogeneous Time Resolved Fluorescence (HTRF), indicating that human CD19-specific, CD22-specific and CD20-specific binding proteins were successfully selected. For example, the ankyrin repeat domains of SEQ ID NOs: 36 to 38 constitutes an amino acid sequence of a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for CD19, the ankyrin repeat domain of SEQ ID NOs: 39 constitutes an amino acid sequence of a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for CD22, and the ankyrin repeat domains of SEQ ID NOs: 40 to 41 constitute an amino acid sequence of a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for CD20.

A. Selection of binding proteins comprising an ankyrin repeat domain with binding specificity for

CD19 Human recombinant CD19 target preparation

Two target formats were used for the selection procedure. Both are based on target single chain target polypeptide, consisting of the extracellular domain of human CD19 protein (UniProt Accession Nr: Q71 UW0, residues 20 to 279). The extracellular domain was linked in the first format to a human lgG1 Fc domain (purchased from Aero Biosystems) and in the second format to a polyhistidine tag followed by a C- terminal Avi-tag (SEQ ID NO: 67; purchased from R&D Systems)

Selection of CD19-specific ankyrin repeat proteins by ribosome display

The selection of CD19-specific ankyrin repeat proteins was performed by ribosome display (Hanes and Pluckthun, loc. cit.) using the extracellular domain of CD19 as target protein, libraries of ankyrin repeat proteins as described above, and established protocols (see, e.g., Zahnd, C., Amstutz, P. and Pluckthun, A., Nat. Methods 4, 69-79, 2007). The number of reverse transcription (RT)-PCR cycles after each selection round was constantly thirty. The first four rounds of selection employed standard ribosome display selection, using decreasing target concentration (400 nM, 100 nM, 25 nM and 5 nM, respectively), followed by a 5^th off rate round using a target concentration of 1 nM and non-biotinylated target as competitor at 600 fold excess in order to select high affinity binding domains, and a 6th recovery round at a target concentration of 5 nM.

An additional selection branch was performed on selection rounds 2-4, where a monoclonal mouse anti human CD19 antibody (clone FMC63; purchased from Novus Biologicals) was used for competitive elution.

Selected clones show binding to CD19 target expressed on cells

Selected CD19 specific DARPins were expressed and purified by 96-well IMAC. Briefly, E. coli XL1 blue cells were transformed with the selected ankyrin repeat proteins, plated on LB-agar (containing 1 % glucose and 50 pg/ml ampicillin) and then incubated overnight at 37°C. For each protein, a single colony was picked into an individual well of a 96-deep-well plate with 1 .2 mL TB medium (containing 1 % glucose and 50 pg/ml ampicillin) and incubated overnight at 37°C, shaking at 850 rpm, 80% humidity in Multitron Pro microplate shaker. Fresh TB medium (containing 50 pg/ml ampicillin; 0.99 mL per well of a 96-deep-well plate) was inoculated with overnight culture (1 :10) and incubated at 37°C at 850 rpm. After2 h the culture was induced by addition of IPTG (0.5 mM final concentration) and incubated for further 5-6 h at 37°C 850 rpm. Harvest was performed by centrifugation (6 min 3200 x g). After cell disruption with 50 pl B-PER™ II Bacterial Protein Extraction Reagent (Cat. No. 78260; Thermo Fisher Scientific, Waltham, Massachusetts, United States); supplemented with DNAse I (200 Units/ml) and lysozyme (0.4 mg/ml)) according to the manufacturer’s protocol and addition of 60 pl IMAC adjusting buffer (Na2HP04 x 2H2O 50 mM, NaCI 300 mM, pH7.4), up to 8 clones were pooled and purified with via 96-well column IMAC plate (HisPur™ Cobalt Spin Plates, Cat. No: 90095; Thermo Fisher Scientific, Waltham, Massachusetts, United States) and rebuffered into PBS pH7.4 with a Zeba Spin de-salting 96-well plate (Cat. No: 89807; Thermo Fisher Scientific, Waltham, Massachusetts, United States), all according to the manufacturer’s protocol. Pooled DARPins were tested for binding to DAUDI cells at a final dilution of 1 :1 ’000 and/or 1 :10’000. Binding was detected using an anti-DARPin antibody. Pools delivering a positive binding signal were de-convoluted, namely the wells comprising the glycerol-stocks of originally pooled DARPins for the above mentioned purification step were used for re-expression and purification of individual DARPins. These re-expressed, purified individual DARPins were re-tested at 10 nM for binding to cellular expressed target in order to isolate the DARPin(s) responsible for the positive pool signal.

DARPin protein #36 (SEQ ID NO: 36 with a His-tag (SEQ ID NO: 6) fused to its N terminus)

DARPin protein #37 (SEQ ID NO: 37 with a His-tag (SEQ ID NO: 6) fused to its N terminus)

DARPin protein #38 (SEQ ID NO: 38 with a His-tag (SEQ ID NO: 6) fused to its N terminus)

B. Selection of binding proteins comprising an ankyrin repeat domain with binding specificity for

CD22

Human recombinant CD22 target preparation

For the selection procedure three target formats were used: Target a: the extracellular domain of CD22 (residues 20 to 687 of human CD2, UniProt ID Nr: P20273) linked to a human IgG 1 Fc domain followed by a C-terminal an Avi-tag (purchased from Aero Biosystems), Target b: CD22 lacking domains 5 and 6 (Uniprot P20273 residues 20-504) followed by a C-terminal Avi-tag (SEQ ID NO: 67; purchased from Evitria) and target c: CD22 domains 4, 5 and 6 (Uniprot P20273 residues 416-687) fused to an Fc-kih domain (knob-in-hole format) followed by a C-terminal Avi-tag (SEQ ID NO: 67 purchased from Evitria), later enzymatically biotinylated in vitro, catalysed by the BirA-GST ligase (purchased from BPS Bioscience).

Selection of CD22-specific ankyrin repeat proteins by ribosome display

The selection of CD22-specific ankyrin repeat proteins was performed by ribosome display (Hanes and Pluckthun, loc. cit.) using the above described extracellular domains of CD22 as target protein, libraries of ankyrin repeat proteins as described above, and established protocols (see, e.g., Zahnd, C., Amstutz, P. and Pluckthun, A., Nat. Methods 4, 69-79, 2007). Two different selection branches were applied. In one branch, target b was used to deselect against the membrane distal domains, favouring selection of binders against membrane-proximal Ig-fold domains C5 and C6. In the other branch, selection was applied on target c comprising CD22 membrane proximal domains C4-6. The number of reverse transcription (RT)- PCR cycles were 45, 35, 30, 28, 30 and 35 in the selection rounds 1 to 6, respectively. The first four rounds of selection employed standard ribosome display selection, using decreasing target concentration (400 nM, 100 nM, 25 nM and 5 nM, respectively), followed by a 5^th off rate round using a target concentration of 1 nM and non-biotinylated target as competitor at 289 or 900fold excess, respectively in order to select high affinity binders, and a 6th recovery round at a target concentration of 5 nM.

Selected clones show binding to CD22 target (shown by HTRF)

The ribosome display output was subcloned into a derivative of the pQE30 (Qiagen) expression vector containing an N-terminal His-tag followed by a Flag-tag. Ribosome display rounds 4 and 6 were screened by HTRF of ankyrin repeat proteins crude extracts expressed in E. coli. Selected hits were re-arrayed on 4 plates, purified using their His-tag in 96 well format and binding was confirmed by HTRF. One selected binder was subcloned into a derivative of the pQE30 (Qiagen) expression vector containing an N-terminal His-tag (SEQ ID NO: 6) and purified on an AKTAxpress™ system for in-depth characterization.

For example, an expression vector encoding the following ankyrin repeat protein were constructed:

DARPin protein #39 (SEQ ID NO: 39 with a His-tag (SEQ ID NO: 6) fused to its N terminus)

C. Selection of binding proteins comprising an ankyrin repeat domain with binding specificity for CD20

Human recombinant CD20 target preparation

For the selection of ankyrin repat domains with binding specificity for CD20, the full length, human CD20 protein (UniProt ID Nr:P11836) was used as a target, linked to a polyhistidine tag followed by a C-terminal Avi-tag (purchased from Aero Biosystems).

Selection of CD20-specific ankyrin repeat proteins by ribosome display

The selection of CD20-specific ankyrin repeat proteins was performed by ribosome display (Hanes and Pluckthun, loc. cit.) using the full length CD20 protein as target protein, libraries of ankyrin repeat proteins as described above, and established protocols (see, e.g., Zahnd, C., Amstutz, P. and Pluckthun, A., Nat. Methods 4, 69-79, 2007). The number of reverse transcription (RT)-PCR cycles after each selection round was constantly thirty. The four rounds of selection employed standard ribosome display selection, using decreasing target concentration (400 nM, 100 nM, 25 nM and 5 nM, respectively). An additional selection step was performed on selection rounds 2-4, where the monoclonal human anti-CD20 antibody Rituximab was used for competitive elution.

Selected clones show binding to CD20 target (shown by HTRF)

In a first approach, the pools from the ribosome display were subcloned into a derivative of the pQE30 (Qiagen) expression vector containing an N-terminal His-tag followed by a Flag-tag and expressed in E. coli cells in 96 well plates. Crude extracts thereof were prepared to test binding of the His-tagged DARPin proteins to biotinylated human CD20-Fc recombinant protein in an HTRF assay. The extract of each lysed clone was applied as a 1 :500 dilution in PBSTB (PBS supplemented with 0.1 % Tween 20® and 0.2% (w/v) BSA, pH 7.4) together with 4 nM (final concentration) biotinylated full length target hCD20 His, 1 :400 (final concentration) of anti-strep-Tb HTRF antibody - FRET donor conjugate (Cisbio) and 1 :400 (final concentration) of anti-Flag-d2 antibody FRET acceptor conjugate (Cisbio) to a well of 384 well plate and incubated for 1 h at RT. The HTRF was read-out on a Tecan M1000pro using a 340 nm excitation wavelength and a 665 ±10 nm emission filter. In a 2nd screening campaign, up to 7 pooled DARPins in a bi-valent format (MRGS-His6-GS-DAPRin-PT1 n-Leucin-Zipper-MYC-Tag) were screened for binding on cells first, with consecutive deconvolution instead of HTRF. Briefly, E. coli XL1 blue cells were transformed with the ankyrin repeat proteins, plated on LB-agar (containing 1 % glucose and 50 pg/ml ampicillin) and then incubated overnight at 37°C. For each construct, a single colony was picked into an individual well of a 96-deep-well plate with 1 .2 mL TB medium (containing 1 % glucose and 50 pg/ml ampicillin) and incubated overnight at 37°C, shaking at 850 rpm, 80% humidity in Multitron Pro microplate shaker. Fresh TB medium (containing 50 pg/ml ampicillin; 0.99 mL per well of a 96-deep-well plate) was inoculated with overnight culture (1 :10) and incubated at 37°C at 850 rpm. After 2 h the culture was induced by addition of IPTG (0.5 mM final concentration) and incubated for further 5-6 h at 37°C 850 rpm. Harvest was done by centrifugation (6 min 3200 x g). After cell disruption with 50 pl B-PER™ II Bacterial Protein Extraction Reagent (Cat. No. 78260; Thermo Fisher Scientific, Waltham, Massachusetts, United States); supplemented with DNAse I (200 Units/ml) and lysozyme (0.4 mg/ml)) according to the manufacturer’s protocol and addition of 60 pl IMAC adjusting buffer (Na2HP04 x 2H2O 50 mM, NaCI 300 mM, pH7.4), up to 8 clones were pooled and purified with via 96-well column IMAC plate (HisPur™ Cobalt Spin Plates, Cat. No: 90095; Thermo Fisher Scientific, Waltham, Massachusetts, United States) and re-buffered into PBS pH7.4 with a Zeba Spin desalting 96-well plate (Cat. No: 89807; Thermo Fisher Scientific, Waltham, Massachusetts, United States), all according to the manufacturer’s protocol. Pooled DARPins were tested for binding to DAUDI cells at a final dilution of 1 :100 and/or 1 :10’000. Binding was detected by Flow Cytometry. For this purpose, cells were incubated with the respective dilutions of pooled DARPins for 45min to 1 h at 4°C, followed by two wash steps and incubation with the fluorescently labelled detection antibody (anti-DARPin-1 .1 .1-AF488) for 30 minutes at 4 °C. During this step, Live/Dead Aqua (Thermo Fisher, L34957, 1 :1000) was included in order to enable exclusion of dead cells during analysis. Following two wash steps, cells were fixed with BD Cytofix CellFix Fixation buffer (Cat nr 554655) for 20 min at 4 °C. The fluorescent signal was acquired using the Attune Nxt Flow Cytometer (ThermoFisher) and data analysed using FlowJo and GraphPad software. Pools delivering a positive binding signal were de-convoluted, meaning the wells comprising the glycerolstocks of originally pooled DARPins were used for expression and purification of individual DARPins. These purified individual DARPins were tested at 10 nM for binding to DAUDI cells in order to isolate the DARPin(s) responsible for the positive signal observed in the pools using the same cell binding protocol as described above.

DARPin protein #40 (SEQ ID NO: 40 with a His-tag (SEQ ID NO: 6) fused to its N terminus)

DARPin protein #41 (SEQ ID NO: 41 with a His-tag (SEQ ID NO: 6) fused to its N terminus)

Example 4: Cell binding specificity profile of selected CD2-specific DARPins

Binding specificity of selected CD2 ankyrin repeat proteins, as generated in Example 1 , was assessed in a reporter cell binding experiment, using Jurkat E6-1 cells, expressing CD2 and Jurkat CD2 knock-out cells, In brief: 0.5x10⁵ cells/well were resuspended in 50 pl of each tested DARPin dilution (1000 nM, 1 :5 serial diluted in FACS buffer (PBS + 2% FBS)) and incubated for 1 h at 4°C. Cells were washed twice in FACS buffer before resuspending in 50 pL anti-DARPin-AF488 (Anti-DARPin rabbit monoclonal AB 1 .1.1 labelled with AF488) and incubated for 30 min at 4°C. After washing twice with FACS buffer, cell pellets were fixed with 1/10 diluted BD Cytofix™ (BD) for 20 min at RT. 5000 fixed cells were counterstained in red with 5 uM DRAQ5 (Abeam) and acquired at Mirrorball (SPTIabtech). Using Cellista software, data was collected in the red (FL-4) and green (FL-2) channels of Mirrorball for DRAQ5 counterstain and Alexa Fluor 488 signal, respectively. The amount of anti-DARPin 1.1.1 (AF488) antibody binding to each cell is the measure of the green fluorescence intensity [median (mean intensity FL-2)] and was plotted using GraphPad Prism software. As it can be seen in Figure 2(A-B), all tested proteins with binding specificity for CD2 (DARPin protein #1 , DARPin protein #3 and DAPRin protein #15 show binding to Jurkat E6-1 (CD2 positive; Fig. 2A but not to Jurkat CD2 KO (knock- out cells, not expressing CD2 target protein; Fig. 2B).

Table 6: Cell binding of selected CD2-specific DARPins

Example 5: Determination of dissociation constants (KD) of ankyrin repeat proteins with binding specificity for human CD2 by Surface Plasmon Resonance (SPR) analysis

The binding affinities of four purified ankyrin repeat proteins to biotinylated recombinant human CD2 target were analysed by multi-trace SPR using a Bruker Sierra SPR-32 instrument (Bruker) with PBS-T (PBS containing 0.005% Tween-20) as running buffer. A Bruker Biotin-Tag Capture Sensor chip was conditioned according to manufacturer’s manual. Biotinylated human CD2 target material was captured to 437 and 469 RUs on channel 1 and 2, respectively (10 pg/mL, 30 s). The running buffer was PBS pH 7.4 containing 0.005% Tween-20(PBST). DARPin proteins with binding specificity for CD2 were injected from lowest to highest concentration on Channel 1 for 240 s each (at 270 nM-0.041 nM, 3-fold dilutions) for 240 s each at 25 pl/min for association and dissociation was recorded for 500 s (at 25 pL/min). The ligand was regenerated with a 60 s pulse of 10 mM HCI (25 pL/min). The data were double referenced (injection control (Ch1 B) and buffer injection (Ch2)) and fitted to a 1 :1 Langmuir model. Table 7 below shows the binding parameters of the selected DARPin proteins #1 to #4 as measured by multi-trace PCR.

Table 7: Binding parameters for ankyrin repeat proteins with binding specificity for human CD2 by Surface Plasmon Resonance (SPR)

The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The aspects within the specification provide an illustration of aspects of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other aspects are encompassed by the invention. All publications, patents, and GenBank sequences cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. The citation of any references herein is not an admission that such references are prior art to the present invention.

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific aspects of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

SEQUENCES

Claims

1 . A recombinant binding protein comprising an ankyrin repeat domain with binding specificity for CD2.

2. The recombinant binding protein of claim 1 , wherein said ankyrin repeat domain comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 7 to 14, 16, 17 and (2) sequences in which up to 9 amino acids in any one of SEQ ID NOs: 7 to 14, 16, 17 are substituted by other amino acids.

3. The recombinant binding protein of claim 1 to 2, wherein said ankyrin repeat domain comprises a first ankyrin repeat module and a second ankyrin repeat module, optionally wherein said first and said second ankyrin repeat module each independently comprises an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 7 to 14, 16, 17 and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 7 to 14, 16, 17 are substituted with other amino acids.

4. The recombinant binding protein of any one of claims 1 to 3, wherein i. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 7, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 7 are substituted with other amino acids and a second ankyrin repeat module comprising an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 8, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 8 are substituted with other amino acids; ii. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 16, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 16 are substituted with other amino acids and a second ankyrin repeat module comprising an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 17, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 17 are substituted with other amino acids;

5. The recombinant binding protein of any one of claims 1 to 4, wherein said ankyrin repeat domain comprises a first ankyrin repeat module, a second ankyrin repeat module, and a third ankyrin repeat module, optionally wherein said first, second and third ankyrin repeat module each independently comprises an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 7 to 14, 16, 17, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 7 to 14, 16, 17 are substituted with other amino acids.

6. The recombinant binding protein of any one of claims 1 to 2 and claim 5, wherein i. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 9, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 9 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 10, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 10 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 11 , and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 11 are substituted with other amino acids; ii. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 12, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 12 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 13, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 13 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 14, and (2) an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 14 are substituted with other amino acids; or

7. The recombinant binding protein of any one of claims 2 to 6, further comprising an N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 18 to 20, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 18 to 20 are substituted by other amino acids and/or a C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 22 to 24, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 22 to 24 are substituted by other amino acids.

8. The recombinant binding protein of any one of the preceding claims, wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 1 to 3, 15.

9. The recombinant binding protein of any one of the preceding claims, wherein said CD2 is human CD2.

10. The recombinant binding protein of any one of the preceding claims, wherein said recombinant binding protein binds human soluble CD2 in PBS with a dissociation constant (KD) of or below about 10^_⁷M or of or below about 10^-6M, or of or below about 10^-7M, or of or below about 10^-8M.

11. The recombinant binding protein of any one of the preceding claims, further comprising at least one binding moiety with binding specificity for a protein expressed on the surface of an immune cell, suitably a T lymphocyte (T cell).

12. The recombinant binding protein of claim 11 , wherein said binding moiety comprises a ankyrin repeat domain with binding specificity for CD3, optionally comprising an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 4, 26 to 28.

13. The recombinant binding protein of any one of the preceding claims, further comprising at least one half-life extending moiety.

14. The recombinant binding protein of claim 13, wherein said half-life extending moiety comprises an ankyrin repeat domain binding human serum albumin, optionally comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 29 to 31 .

15. The recombinant binding protein of any one of the preceding claims, further comprising a tumor- associated binding protein, suitably an ankyrin repeat protein with binding specificity for a tumor associated antigen.

16. A nucleic acid encoding the recombinant binding protein of any one of the preceding claims.

17. A vector comprising the nucleic acid of claim 28, optionally wherein the vector is a DNA vector, an RNA vector, a plasmid, a cosmid, or a viral vector.

18. A cell comprising the nucleic acid of claim 16 or the vector of claim 17.

19. A method of producing a recombinant binding protein, the method comprising culturing the cell of claim 18 and collecting the recombinant binding protein from the cell and/or the culture medium.

20. A pharmaceutical composition comprising the recombinant binding protein of any one of claims 1 to 15, the nucleic acid of claim 16, the vector of claim 17, or the cell of claim 18, and a pharmaceutically acceptable carrier and/or diluent.

21 . A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the recombinant binding protein of any one of claims 1 to 15, the nucleic acid of claim 16, the vector of claim 17, the cell of claim 18, or the pharmaceutical composition of claim 20.

22. The method according to claim 21 , wherein said cancer is a liquid tumor.