Recombinant CD2-binding proteins and their use

Abstract

The present invention relates to recombinant binding proteins comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to CD2. Furthermore, the present 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 mammals, including humans.

Description

Technical Field

[0001] Field of Disclosure The present invention relates to a recombinant binding protein comprising a designed ankyrin repeat domain having binding specificity for CD2. Furthermore, the present invention relates to a nucleic acid encoding such a recombinant binding protein, a pharmaceutical composition comprising such a recombinant binding protein or nucleic acid, and the use of such a recombinant binding protein, nucleic acid or pharmaceutical composition in a method for treating diseases such as cancer in mammals including humans.

Background Art

[0002] Background Very effective cancer therapeutics that utilize redirected T cells have been developed. These therapeutics include bispecific T cell engagers and CAR-T therapies. For example, a bispecific T cell engager (blinatumomab) that targets the CD19 antigen expressed in B cell malignancies (such as acute lymphoblastic 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).

[0003] Despite the initial effectiveness of such immunotherapies, T cell exhaustion has contributed to treatment failure after bispecific T cell engagers and CAR-T therapies. One molecule thought to be useful in avoiding T cell exhaustion is CD2. CD2 is a surface antigen found on all peripheral blood T cells and functions as a costimulatory receptor. The CD2 protein interacts with CD58 on antigen-presenting cells to optimize immune recognition. There are two isoforms of human CD2 (precursor of isoform 1: NCBI reference sequence NP_001315538.1; precursor of isoform 2: NCBI reference sequence NP_001758.2).

[0004] CD2 signaling is associated with the phenotype of non-exhausted T cells. The interaction between CD2 and its ligand, CD58, has been shown to be crucial for CAR-T cell-mediated tumor cell death, while loss of CD58 expression on cancer cells (such as lymphoma cells) is correlated with resistance and relapse in patients treated with CAR-T therapy. Therefore, CD2 costimulation may overcome T cell exhaustion, and therapies utilizing CD2 costimulation may achieve deeper and more sustained responses compared to similar therapies that do not utilize CD2 costimulation.

[0005] Therefore, there is a need for novel CD2-specific binding proteins with beneficial properties that can be used to co-stimulate T cells. Such binding proteins may be useful in therapeutic approaches for treating diseases, including cancer. [Overview of the Initiative]

[0006] overview The present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to CD2. Furthermore, the present 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 mammals, including humans.

[0007] The recombinant binding proteins of the present invention specifically bind to or target CD2 expressed by immune cells such as T cells. Such recombinant binding proteins of the present invention can function as tools or building blocks for creating new therapeutic agents such as T cell engagers. Also disclosed herein are recombinant binding proteins in which a CD2-specific ankyrin repeat domain is bound to one or more other functional moieties within a single molecule. Such other functional moieties include CD3-specific binding moieties and / or binding moieties having binding specificity to disease-related antigens (DAAs), such as tumor-associated antigens (TAAs). When bound to CD3-specific binding moieties and TAA-specific binding moieties, the CD2-specific ankyrin repeat domain of the present invention can form an enhanced multispecific T cell engager that can simultaneously bind to TAAs on tumor cells, as well as CD3 (a T cell receptor signaling component) and CD2 on T cells. The CD2-specific ankyrin repeat domain may also be further bound to other moieties, such as half-life extension moieties. The functional moiety bound to the CD2-specific ankyrin repeat domain is also preferably an ankyrin repeat domain.

[0008] Thus, the recombinant binding protein of the present invention, which has binding specificity to CD2, is useful for generating novel therapeutic molecules that can provide an improved response (e.g., a deeper and more sustained response) compared to current therapeutic modes.

[0009] In one embodiment, the present invention provides a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD2, wherein the 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: 7-14, 16, and 17, and (2) a sequence in which up to nine amino acids in any one of SEQ ID NOs: 7-14, 16, and 17 are substituted with other amino acids.

[0010] In one embodiment, the present invention provides a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD2, wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 1-3, 15, wherein the second-to-last A in SEQ ID NOs: 1-3, 15 is optionally substituted with L, and / or the last A in SEQ ID NOs: 1-3, 15 is optionally substituted with N.

[0011] In one embodiment, the present invention provides a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD2, wherein the binding protein further comprises a binding moiety having binding specificity to CD3. In one embodiment, the binding moiety having binding specificity to CD3 is an ankyrin repeat domain. In a further embodiment, the binding moiety having binding specificity to CD3 is an ankyrin repeat domain, wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NOs: 4, 26-28, wherein the second-to-last A in SEQ ID NOs: 4, 26-28 is optionally substituted with L, and / or the last A in SEQ ID NOs: 4, 26-28 is optionally substituted with N.

[0012] In one embodiment, the present invention provides a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD2, wherein the recombinant binding protein further comprises one, two, or three binding sites, each of which has binding specificity to a disease-related antigen (such as TAA). In one embodiment, any of the binding sites having binding specificity to a disease-related antigen is an ankyrin repeat domain.

[0013] In one embodiment, the present invention provides a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD2 as described herein, wherein the recombinant binding protein further comprises a half-life extension portion. In one embodiment, the half-life extension portion is an ankyrin repeat domain having binding specificity to human serum albumin.

[0014] Within the recombinant binding protein of the present invention, an ankyrin repeat domain having binding specificity to CD2 and any other functional moiety can be linked to one another via a peptide linker. An example of such a peptide linker is provided by SEQ ID NO: 5 or SEQ ID NO: 55.

[0015] In one embodiment, the present invention provides a nucleic acid encoding the recombinant binding protein of the present invention, and a pharmaceutical composition comprising the recombinant binding protein of the present invention or the nucleic acid of the present invention and an optionally pharmaceutically acceptable carrier and / or diluent.

[0016] In one embodiment, the present invention provides a method for activating T cells, the method comprising administering an effective amount of the recombinant binding protein of the present invention, the nucleic acid of the present invention, or the pharmaceutical composition of the present invention to a subject requiring such action.

[0017] In one embodiment, the present invention provides recombinant binding proteins, nucleic acids, or pharmaceutical compositions of the present invention for use in methods of treating a medical condition. In one embodiment, the medical condition is cancer.

[0018] In one embodiment, the present invention provides a method for treating a medical condition in a human subject, the method comprising administering a therapeutically effective amount of the recombinant binding protein of the present invention, the nucleic acid of the present invention, or the pharmaceutical composition of the present invention to the subject. In one embodiment, the medical condition is cancer.

[0019] Based on the disclosures provided herein, those skilled in the art will recognize many equivalents to the specific embodiments of the invention described herein, or can confirm them by routine experimentation alone. Such equivalents are intended to be encompassed in the following embodiment (E).

[0020] Specifically, this disclosure provides the following aspects, advantageous features, and specific embodiments, each individually or in combination:

[0021] E1. Recombinant binding protein containing an ankyrin repeat domain with binding specificity to CD2.

[0022] E2. The recombinant binding protein according to E1, wherein the ankyrin repeat domain comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) one of sequence numbers 7-14, 16, and 17, and (2) an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in one of sequence numbers 7-14, 16, and 17 is substituted with other amino acids.

[0023] E3. The recombinant binding protein according to E1 or E2, wherein the ankyrin repeat domain comprises a first ankyrin repeat module and a second ankyrin repeat module, and optionally, the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module within the ankyrin repeat domain.

[0024] E4. The recombinant binding protein according to E3, wherein the first ankyrin repeat module and the second ankyrin repeat module each independently have an amino acid sequence selected from the group consisting of (1) one of sequence numbers 7-14, 16, and 17, and (2) an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in one of sequence numbers 7-14, 16, and 17 are substituted with other amino acids.

[0025] E5. i. The first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 7 and (2) an amino acid sequence in which 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 of SEQ ID NO: 7 is substituted by other amino acids; and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 8 and (2) an amino acid sequence in which 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 of SEQ ID NO: 8 is substituted by other amino acids; ii. The first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 16 and (2) an amino acid sequence in which 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 of SEQ ID NO: 16 is substituted by other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 17 and (2) an amino acid sequence in which 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 of SEQ ID NO: 17 is substituted by other amino acids, Recombinant binding protein as described in E3 or E4.

[0026] E6. The recombinant binding protein according to E1 or E2, wherein the ankyrin repeat domain comprises a first ankyrin repeat module, a second ankyrin repeat module, and a third ankyrin repeat module, wherein optionally, the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module, and the second ankyrin repeat module is located at the N-terminus of the third ankyrin repeat module within the ankyrin repeat domain.

[0027] E7. The recombinant binding protein according to E6, wherein each of the first, second, and third ankyrin repeat modules independently has an amino acid sequence selected from the group consisting of (1) one of sequence numbers 7-14, 16, and 17, and (2) an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in one of sequence numbers 7-14, 16, and 17 is substituted by other amino acids.

[0028] E8. i. The first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 9 and (2) an amino acid sequence in which 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 of SEQ ID NO: 9 is substituted by other amino acids; the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 10 and (2) an amino acid sequence in which 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 of SEQ ID NO: 10 is substituted by other amino acids; the third ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 11 and (2) an amino acid sequence in which 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 of SEQ ID NO: 11 is substituted by other amino acids; ii. The first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 12 and (2) an amino acid sequence in which 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 of SEQ ID NO: 12 is substituted by other amino acids; the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 13 and (2) an amino acid sequence in which 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 of SEQ ID NO: 13 is substituted by other amino acids; and the third ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 14 and (2) an amino acid sequence in which 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 of SEQ ID NO: 14 is substituted by other amino acids. Recombinant binding protein as described in E6 or E7.

[0029] E10. The recombinant binding protein according to any one of E1 to E9, wherein the ankyrin repeat domain contains an amino acid sequence that is 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: 1-3, 15.

[0030] E11. Recombinant binding protein according to any one of E1 to E10, wherein the CD2 is human CD2.

[0031] E12. The ankyrin repeat domain is approximately 10 -6 M or less, or about 10 -7 M or less, or about 10 -8A recombinant binding protein described in any one of items E1 to E11, which has a dissociation constant (KD) of M or less and binds to human CD2 in PBS.

[0032] E13. A recombinant binding protein according to any one of E1 to E12, further comprising at least one binding site having binding specificity to a protein expressed on the surface of an immune cell, preferably the immune cell being a T lymphocyte (T cell), and preferably the protein expressed on the surface of the immune cell being CD3.

[0033] E14. The recombinant binding protein according to E13, wherein the binding portion comprises an ankyrin repeat domain having binding specificity to CD3.

[0034] E15. The recombinant binding protein according to E14, wherein the ankyrin repeat domain having binding specificity to CD3 contains an amino acid sequence that is 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, 26-28.

[0035] E16. A recombinant binding protein according to any one of items E1 to E15, further comprising at least one half-life extension moiety.

[0036] E17. The recombinant binding protein according to E16, wherein the half-life extension portion comprises an ankyrin repeat domain having binding specificity to human serum albumin.

[0037] E18. The recombinant binding protein according to E17, wherein the ankyrin repeat domain having binding specificity to human serum albumin contains an amino acid sequence that is 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. 29-31.

[0038] E19. Recombinant binding protein according to any one of E1 to E18, further comprising a binder having binding specificity to disease-related antigens, preferably tumor-related antigens.

[0039] E20. A nucleic acid encoding a recombinant binding protein as described in any one of items E1 to E19.

[0040] E21. A vector containing the nucleic acid described in E20.

[0041] E22. The vector described in E20, wherein the vector is a DNA vector, RNA vector, plasmid, cosmid, or viral vector.

[0042] E23. Cells containing nucleic acids as described in E20 or vectors as described in E21 or E22.

[0043] E24. A pharmaceutical composition comprising a recombinant binding protein as described in any one of items E1 to E19, a nucleic acid as described in E20, a vector as described in E21 or E22, or a cell as described in E23, and a pharmaceutically acceptable carrier and / or diluent.

[0044] E25. A method for producing recombinant binding proteins, comprising culturing the cells described in E23 and collecting recombinant binding proteins from the cells and / or culture medium.

[0045] E26. Recombinant binding proteins as described in any one of E1 to E19, nucleic acids as described in E20, vectors as described in E21 or E22, cells as described in E23, or pharmaceutical compositions as described in E24, for use as pharmaceuticals.

[0046] E27. Use in the manufacture of pharmaceuticals a recombinant binding protein as described in any one of items E1 to E19, nucleic acid as described in E20, vector as described in E21 or E22, cell as described in E23, or pharmaceutical composition as described in E24.

[0047] E28. The use described in E26, which is a recombinant binding protein, nucleic acid, vector, cell, or pharmaceutical composition described in E26, or which is a pharmaceutical for the treatment of cancer, or which is described in E27.

[0048] E29. A method for treating a subject in need of treatment, comprising administering a therapeutically effective amount of a recombinant binding protein described in any one of E1 to E19, a nucleic acid described in E20, a vector described in E21 or E22, a cell described in E23, or a pharmaceutical composition described in E24 to the subject.

[0049] E30. The method according to E29, wherein the treatment is for cancer.

[0050] E31. The method according to E30, wherein the cancer is a humoral tumor.

[0051] E32. A method for activating T cells, comprising administering a therapeutically effective amount of a recombinant binding protein described in any one of E1 to E19, a nucleic acid described in E20, a vector described in E21 or E22, a cell described in E23, or a pharmaceutical composition described in E24 to a subject in need thereof. [Brief explanation of the drawing]

[0052] [Figure 1]T cell proliferation assay. See Example 2 for detailed instructions. [Figure 2A-B] In vitro binding of CD2-specific DARPin (SEQ ID NOs: 1, 3, and 15) to CD2 targets expressed by Jurkat cell lines. Figure 2A shows the binding of tested DARPin to CD2-expressing Jurkat wild-type cells; Figure 2B shows that when the same experiment was performed using CD2-nonexpressing knockout Jurkat cells (KO), the same tested DARPin showed no binding activity (FLU: fluorescence intensity). [Figure 3] Efficacy evaluation of selected DARPin proteins (DARPin protein #42, DARPin protein #43, and DARPin protein #44) containing CD19, CD2, and CD3-specific ankyrin repeat domains in the presence of T cells and OCI-Ly19-CD58 knockout cells. The absence of CD58, the innate ligand for CD2, leads to disruption of the CD2-CD58 interaction, resulting in a loss of T cell activation. All tested proteins induced T cell proliferation and were able to rescue the loss of T cell response caused by CD58 deficiency in CD58 knockout cells. All tested proteins showed increased T cell proliferation compared to DARPin #45, which does not contain a CD2-binding domain. [Figure 4] Efficacy evaluation of selected DARPin proteins (DARPin protein #42, DARPin protein #43, and DARPin protein #44) containing CD19, CD2, and CD3-specific ankyrin repeat domains in the presence of T cells and OCI-Ly19-CD58 knockout cells. The absence of CD58, the natural ligand for CD2, leads to disruption of the CD2-CD58 interaction, resulting in a loss of T cell activation. All tested proteins induced tumor cell death and could rescue the loss of T cell response caused by CD58 absence in CD58 knockout cells. All tested proteins showed increased tumor cell death compared to DARPin #45, which does not contain a CD2-binding domain. [Figure 5]Efficacy evaluation of selected DARPin proteins (DARPin protein #42, DARPin protein #43, and DARPin protein #44) containing CD19, CD2, and CD3-specific ankyrin repeat domains in the presence of T cells and OCI-Ly19-CD58 expressing cells. All tested proteins showed increased T cell proliferation compared to DARPin #45, which does not contain a CD2-binding domain. [Figure 6] Efficacy evaluation of selected DARPin proteins (DARPin protein #46, DARPin protein #47, DARPin protein #48, DARPin protein #49, DARPin protein #50, and DARPin protein #51) containing CD22, CD19, CD2, and CD3-specific ankyrin repeat domains in a T cell proliferation assay in the presence of T cells and OCI-Ly19-CD58 knockout cells. [Figure 7] Efficacy evaluation of selected DARPin proteins (DARPin protein #46, DARPin protein #47, DARPin protein #48, DARPin protein #49, DARPin protein #50, and DARPin protein #51) containing CD22, CD19, CD2, and CD3-specific ankyrin repeat domains in a T cell proliferation assay in the presence of T cells and OCI-Ly19-CD58 expressing cells. [Figure 8A-B]Efficacy evaluation of selected DARPin proteins containing 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 (Figure 8A), or in the presence of T cells and OCI-Ly19-CD58 wild-type cells (Figure 8B). DARPin protein #59 and DARPin protein #60 represent proteins that have binding specificity to CD20 and CD2 but not to CD3, while DARPin protein #58 is a protein that has binding specificity to CD20 and CD3 but not to CD2; these were used as control groups. All proteins tested were able to induce T cell proliferation and rescue the loss of T cell response caused by CD58 deficiency in CD58 knockout cells. [Figure 9A-B] Efficacy evaluation of selected DARPin proteins containing 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 (Figure 9A), or in the presence of T cells and OCI-Ly19-CD58 wild-type cells (Figure 9B). DARPin protein #59 and DARPin protein #60 represent proteins with binding specificity to CD20 and CD2 but not to CD3, while DARPin protein #58 is a protein with binding specificity to CD20 and CD3 but not to CD2; these were used as control groups. All tested proteins were able to induce tumor cell death and rescue the loss of T cell response caused by CD58 deficiency in CD58 knockout cells. [Figure 10]Efficacy evaluation of selected proteins containing CD70, CD2, and CD3-specific ankyrin repeat domains (DARPin protein #61, DARPin protein #62, and DARPin protein #63) in the presence of purified PBMC pan-T cells and human CD70 target proteins. After 5 rounds of stimulation (S1-S5), T cells were collected and flow cytometry analysis was performed to calculate the number of T cells. [Figure 11] Efficacy evaluation of selected proteins containing CD70, CD2, and CD3-specific ankyrin repeat domains (DARPin protein #61, DARPin protein #62, and DARPin protein #63) in the presence of purified PBMC pan-T cells and human CD70 target proteins. After 5 rounds of stimulation (S1-S5), T cells were collected and flow cytometry analysis was performed to measure T cell activation (median fluorescence intensity (MFI)). [Modes for carrying out the invention]

[0053] Detailed description of the invention As disclosed and illustrated herein, this disclosure provides ankyrin repeat proteins that specifically bind to target CD2. The designed ankyrin repeat protein library (International Publication No. 2002 / 020565; Binz et al., Nat. Biotechnol. 22, 575-582, 2004; Stumpp et al., Drug Discov. Today 13, 695-701, 2008) can be used to select target-specific ankyrin repeat domains that bind to targets with high affinity. Such target-specific ankyrin repeat domains can be used as valuable components of recombinant binding proteins for the treatment of diseases. Designed ankyrin repeat proteins represent a class of binding molecules that have the potential to overcome the limitations of monoclonal antibodies, thus enabling novel therapeutic approaches. Such ankyrin repeat proteins may contain a single designed ankyrin repeat domain or a combination of two or more designed ankyrin repeat domains having the same or different target specificities (Stumpp et al., Drug Discov. Today 13, 695-701, 2008; U.S. Patent No. 9,458,211). Ankyrin repeat proteins containing only a single designed domain are small proteins (14 kDa) that can be selected to bind to a given target protein with high affinity and specificity. These properties, and the possibility of combining two, three, four or more designed ankyrin repeat domains in a single binding protein to result in binding proteins with two, three, four or more different specificities, make designed ankyrin repeat proteins ideal candidates for agonists, antagonists and / or inhibitors, enabling novel drug designs with improved safety profiles. These include, for example, multispecific drugs comprising a CD2-specific conjugate and at least one tumor localizer, such as a tumor-associated antigen (TAA) conjugate and / or a conjugate having binding specificity to proteins expressed on the surface of immune cells, preferably T cells.Furthermore, these designed ankyrin repeat proteins can be engineered to carry various effector functions, such as cytotoxic agents or half-life extenders, enabling entirely new drug formats. Taken together, these engineered ankyrin repeat proteins represent an example of next-generation protein-based therapeutics with the potential to surpass existing antibody drugs.

[0054] i. Ankyrin repeat domains with binding specificity to CD2 In one embodiment, a recombinant binding protein is provided that includes an ankyrin repeat domain having binding specificity to CD2. Specifically, the ankyrin repeat domain includes an ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) one of sequence numbers 7-14, 16, and 17, and (2) an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in one of sequence numbers 7-14, 16, and 17 are substituted with other amino acids. Therefore, in one embodiment, the ankyrin repeat sequence has an amino acid sequence selected from the group consisting of (1) one of sequence numbers 7-14, 16, and 17, and (2) an amino acid sequence in which up to 9 amino acids in one of sequence numbers 7-14, 16, and 17 are substituted with other amino acids. In one embodiment, the ankyrin repeat module has an amino acid sequence selected from the group consisting of (1) one of sequence numbers 7-14, 16, and 17, and (2) an amino acid sequence in which up to eight amino acids in one of sequence numbers 7-14, 16, and 17 are substituted with other amino acids. In one embodiment, the ankyrin repeat module has an amino acid sequence selected from the group consisting of (1) one of sequence numbers 7-14, 16, and 17, and (2) an amino acid sequence in which up to seven amino acids in one of sequence numbers 7-14, 16, and 17 are substituted with other amino acids. In one embodiment, the ankyrin repeat module has an amino acid sequence selected from the group consisting of (1) one of sequence numbers 7-14, 16, and 17, and (2) an amino acid sequence in which up to six amino acids in one of sequence numbers 7-14, 16, and 17 are substituted with other amino acids. In one embodiment, the ankyrin repeat module has an amino acid sequence selected from the group consisting of (1) one of sequence numbers 7-14, 16, and 17, and (2) an amino acid sequence in which up to five amino acids in one of sequence numbers 7-14, 16, and 17 are substituted with other amino acids.In one embodiment, the ankyrin repeat module has an amino acid sequence selected from the group consisting of (1) one of sequence numbers 7-14, 16, and 17, and (2) an amino acid sequence in which up to four amino acids in one of sequence numbers 7-14, 16, and 17 are substituted with other amino acids. In one embodiment, the ankyrin repeat module has an amino acid sequence selected from the group consisting of (1) one of sequence numbers 7-14, 16, and 17, and (2) an amino acid sequence in which up to three amino acids in one of sequence numbers 7-14, 16, and 17 are substituted with other amino acids. In one embodiment, the ankyrin repeat module has an amino acid sequence selected from the group consisting of (1) one of sequence numbers 7-14, 16, and 17, and (2) an amino acid sequence in which up to two amino acids in one of sequence numbers 7-14, 16, and 17 are substituted with other amino acids. In one embodiment, the ankyrin repeat module has an amino acid sequence selected from the group consisting of (1) one of sequence numbers 7-14, 16, and 17, and an amino acid sequence in which at most one amino acid in one of sequence numbers 7-14, 16, and 17 is substituted with another amino acid. In one embodiment, all of the nine, eight, seven, six, five, four, three, two, or one amino acid substitutions occur at framework positions of the ankyrin repeat module (may be multiple). In one embodiment, the ankyrin repeat module includes or consists of one of sequence numbers 7-14, 16, and 17.

[0055] In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 7, or a sequence in which one or two amino acids in SEQ ID NO: 7 are substituted with other amino acids. In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 8, or a sequence in which one or two amino acids in SEQ ID NO: 8 are substituted with other amino acids. In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 9, or a sequence in which one or two amino acids in SEQ ID NO: 9 are substituted with other amino acids. In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 10, or a sequence in which one or two amino acids in SEQ ID NO: 10 are substituted with other amino acids. In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 11, or a sequence in which one or two amino acids in SEQ ID NO: 11 are substituted with other amino acids. In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 12, or a sequence in which one or two amino acids in SEQ ID NO: 12 are substituted with other amino acids. In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 13, or a sequence in which one or two amino acids in SEQ ID NO: 13 are substituted with other amino acids. In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 14, or a sequence in which one or two amino acids in SEQ ID NO: 14 are substituted with other amino acids. In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 16, or a sequence in which one or two amino acids in SEQ ID NO: 16 are substituted with other amino acids. In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 17, or a sequence in which one or two amino acids in SEQ ID NO: 17 are substituted with other amino acids.

[0056] In one embodiment, the ankyrin repeat module includes or consists of the amino acid sequence of SEQ ID NO: 7. In one embodiment, the ankyrin repeat module includes or consists of the amino acid sequence of SEQ ID NO: 8. In one embodiment, the ankyrin repeat module includes or consists of the amino acid sequence of SEQ ID NO: 9. In one embodiment, the ankyrin repeat module includes or consists of the amino acid sequence of SEQ ID NO: 10. In one embodiment, the ankyrin repeat module includes or consists of the amino acid sequence of SEQ ID NO: 11. In one embodiment, the ankyrin repeat module includes or consists of the amino acid sequence of SEQ ID NO: 12. In one embodiment, the ankyrin repeat module includes or consists of the amino acid sequence of SEQ ID NO: 13. In one embodiment, the ankyrin repeat module includes or consists of the amino acid sequence of SEQ ID NO: 14. In one embodiment, the ankyrin repeat module includes or consists of the amino acid sequence of SEQ ID NO: 16. In one embodiment, the ankyrin repeat module includes or consists of the amino acid sequence of SEQ ID NO: 17.

[0057] In one embodiment, the ankyrin repeat domain that specifically binds to CD2 includes a first ankyrin repeat module and a second ankyrin repeat module. In one embodiment, the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module within the ankyrin repeat domain. In one embodiment, the first ankyrin repeat module and the second ankyrin repeat module each independently have an amino acid sequence selected from the group consisting of (1) one of sequence numbers 7-14, 16, and 17, and (2) an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in one of sequence numbers 7-14, 16, and 17 is substituted with other amino acids. Accordingly, in one embodiment, the ankyrin repeat domain that specifically binds to CD2 includes a first ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 7-14, 16, or 17 and (2) an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in any one of SEQ ID NOs: 7-14, 16, or 17 is substituted with another amino acid, and further includes a second ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 7-14, 16, or 17 and (2) an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in any one of SEQ ID NOs: 7-14, 16, or 17 is substituted with another amino acid.

[0058] In one particular embodiment, the ankyrin repeat domain that specifically binds to CD2 includes 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 in which 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 of SEQ ID NO: 7 is replaced by another amino acid, 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 in which 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 of SEQ ID NO: 8 is replaced by another amino acid. In one embodiment, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 7 and (2) an amino acid sequence in which up to 3, up to 2, or up to 1 amino acid of SEQ ID NO: 7 is replaced by other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 8 and (2) an amino acid sequence in which up to 3, up to 2, or up to 1 amino acid of SEQ ID NO: 8 is replaced by other amino acids, wherein the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module. In one embodiment, the first ankyrin repeat module includes or consists of the amino acid sequence of SEQ ID NO: 7, and the second ankyrin repeat module includes or consists of the amino acid sequence of SEQ ID NO: 8, wherein the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module.

[0059] In one particular embodiment, the ankyrin repeat domain that specifically binds to CD2 includes 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 in which 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 of SEQ ID NO: 16 is replaced by 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 in which 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 of SEQ ID NO: 17 is replaced by other amino acids. In one embodiment, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 16 and (2) an amino acid sequence in which up to three, up to two, or up to one amino acid of SEQ ID NO: 16 is replaced by other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 17 and (2) an amino acid sequence in which up to three, up to two, or up to one amino acid of SEQ ID NO: 17 is replaced by other amino acids, wherein the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module. In one embodiment, the first ankyrin repeat module includes or consists of the amino acid sequence of SEQ ID NO: 16, and the second ankyrin repeat module includes or consists of the amino acid sequence of SEQ ID NO: 17, wherein the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module.

[0060] In one embodiment, the ankyrin repeat domain that specifically binds to CD2 includes a first ankyrin repeat module, a second ankyrin repeat module, and a third ankyrin repeat module. In a preferred embodiment, the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module within the ankyrin repeat domain, and the second ankyrin repeat module is located at the N-terminus of the third ankyrin repeat module within the ankyrin repeat domain.

[0061] In one embodiment, the first, second, and third ankyrin repeat modules each independently have an amino acid sequence selected from the group consisting of (1) one of sequence numbers 7-14, 16, and 17, and (2) an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in one of sequence numbers 7-14, 16, and 17 is substituted with other amino acids. Therefore, in one embodiment, the ankyrin repeat domain that specifically binds to CD2 is a first ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) one of sequence numbers 7-14, 16, and 17, and (2) an amino acid sequence in which 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 in one of sequence numbers 7-14, 16, and 17 is substituted with another amino acid. The present invention includes a second ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) an amino acid sequence in which up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid is substituted with another amino acid, and a third ankyrin repeat module having an amino acid sequence selected from the group consisting of (2) any one of SEQ ID NOs: 7-14, 16, 17, and (3) an amino acid sequence in which 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 in any one of SEQ ID NOs: 7-14, 16, 17 is substituted with another amino acid.

[0062] In one particular embodiment, the ankyrin repeat domain that specifically binds to CD2 includes 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 in which 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 of SEQ ID NO: 9 is replaced by another amino acid; 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 in which 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 of SEQ ID NO: 10 is replaced by another amino acid; 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 in which 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 of SEQ ID NO: 11 is replaced by another amino acid. In one embodiment, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 9 and (2) an amino acid sequence in which up to 3, up to 2, or up to 1 amino acid of SEQ ID NO: 9 is replaced by other amino acids; the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 10 and (2) an amino acid sequence in which up to 3, up to 2, or up to 1 amino acid of SEQ ID NO: 10 is replaced by other amino acids; and the third ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 11 and (2) an amino acid sequence in which up to 3, up to 2, or up to 1 amino acid of SEQ ID NO: 11 is replaced by other amino acids. Here, the first, second, and third ankyrin repeat modules are arranged within the ankyrin repeat domain from the N-terminus to the C-terminus as the first module - second module - third module.In one embodiment, the ankyrin repeat domain includes (i) a first ankyrin repeat module containing or comprising the amino acid sequence of SEQ ID NO: 9, (ii) a second ankyrin repeat module containing or comprising the amino acid sequence of SEQ ID NO: 10, and (iii) a third ankyrin repeat module containing or comprising the amino acid sequence of SEQ ID NO: 11, wherein the first, second, and third ankyrin repeat modules are arranged within the ankyrin repeat domain in the direction from the N-terminus to the C-terminus as the first module - second module - third module.

[0063] In one particular embodiment, the ankyrin repeat domain that specifically binds to CD2 is 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 in which 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 of SEQ ID NO: 12 is replaced by other amino acids, and (1) SEQ ID NO: 13 and (2) up to 9, up to 8, up to 7, up to 6, up to 5, up to 4 of SEQ ID NO: 13 The present invention includes a second 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 in which 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 of SEQ ID NO: 14 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 in which 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 of SEQ ID NO: 14 are substituted with other amino acids. In one embodiment, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 12 and (2) an amino acid sequence in which up to three, up to two, or up to one amino acid of SEQ ID NO: 12 is replaced by other amino acids; the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 13 and (2) an amino acid sequence in which up to three, up to two, or up to one amino acid of SEQ ID NO: 13 is replaced by other amino acids; and the third ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 14 and (2) an amino acid sequence in which up to three, up to two, or up to one amino acid of SEQ ID NO: 14 is replaced by other amino acids, wherein the first, second, and third ankyrin repeat modules are arranged within the ankyrin repeat domain in the direction from the N-terminus to the C-terminus as the first module - second module - third module.In one embodiment, the ankyrin repeat domain includes (i) a first ankyrin repeat module containing or comprising the amino acid sequence of SEQ ID NO: 12, (ii) a second ankyrin repeat module containing or comprising the amino acid sequence of SEQ ID NO: 13, and (iii) a third ankyrin repeat module containing or comprising the amino acid sequence of SEQ ID NO: 14, wherein the first, second, and third ankyrin repeat modules are arranged within the ankyrin repeat domain in the direction from the N-terminus to the C-terminus as the first module - second module - third module.

[0064] In one embodiment, all of the amino acid substitutions of the ankyrin repeat module(s) described and referenced herein occur at framing locations of the ankyrin repeat module(s), and typically the overall structure of the module(s) is not affected by the substitutions.

[0065] The ankyrin repeat domains disclosed herein that specifically bind to CD2 preferably include N-terminal and / or C-terminal capping modules (hereinafter also referred to as "capping repeats"). The capping modules are located at the N-terminus and / or C-terminus of the ankyrin repeat domain and typically form close tertiary interactions (i.e., tertiary structural interactions) with the ankyrin repeat module(s) between them, thereby providing a cap that shields the hydrophobic core on the sides of the ankyrin repeat domain from exposure to the solvent. Examples of capping sequences are described in International Patent Publication Nos. WO2002 / 020565 and WO2012 / 069655, U.S. Patent Publication No. US20130296221, and J Mol Biol. 2008 Jan 18;375(3):837-54 by Interlandi et al. Examples of amino acid sequences for the N-terminal capping module (i.e., N-terminal capping repeats) are provided in SEQ ID NOs: 18-20, and examples of amino acid sequences for the C-terminal capping module (i.e., C-terminal capping repeats) are provided in SEQ ID NOs: 22-24.

[0066] Accordingly, in some embodiments, recombinant binding proteins are provided that include an ankyrin repeat domain having binding specificity to CD2 as described herein, wherein the ankyrin repeat domain includes any one amino acid sequence of SEQ ID NOs. 18-20, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in any one of SEQ ID NOs. 18-20 is substituted with other amino acids, or includes an N-terminal capping module consisting of such amino acid sequence. Alternatively or additionally, the ankyrin repeat domain includes any one amino acid sequence of SEQ ID NOs. 22-24, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in any one of SEQ ID NOs. 22-24 is substituted with other amino acids, or includes a C-terminal capping module consisting of such amino acid sequence.

[0067] In one preferred embodiment, the ankyrin repeat domain that specifically binds to CD2 comprises: an N-terminal capping module; at least one, at least two, at least three or more ankyrin repeat modules as specifically described herein; and a C-terminal capping module.

[0068] In one embodiment, the ankyrin repeat domain includes or comprises an N-terminal capping module consisting of any one amino acid sequence of SEQ ID NOs: 18-20, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in any one of SEQ ID NOs: 18-20 is substituted with other amino acids; or any one amino acid sequence of SEQ ID NOs: 7-14, 16, 17, or an amino acid sequence in which up to 9, 8, 7, 6, 5, or 4 amino acids in any one of SEQ ID NOs: 7-14, 16, 17 is substituted with other amino acids; At least one, at least two, or at least three ankyrin repeat modules comprising, independently, an amino acid sequence in which three major, up to two, or up to one amino acid is substituted by another amino acid; comprising, any one of the amino acid sequences of SEQ ID NOs. 22-24, or an amino acid sequence in any one of SEQ ID NOs. 22-24 in which up to nine, up to eight, up to seven, up to six, up to five, up to four, up to three, up to two, or up to one amino acid is substituted by another amino acid, or comprising a C-terminal capping module comprising such amino acid sequence.

[0069] In another embodiment, a recombinant binding protein is provided comprising an ankyrin repeat domain having binding specificity to CD2, wherein the ankyrin repeat domain comprises an amino acid sequence that is 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: 1-3, 15. Optionally, the K at the third-to-last position in SEQ ID NOs: 1-3, 15 is replaced by Q, and / or optionally, the A at the second-to-last position is replaced by L, and / or optionally, the A at the last position in SEQ ID NOs: 1-3, 15 is replaced by N.

[0070] Therefore, in one embodiment, the ankyrin repeat domain having binding specificity to CD2 contains an amino acid sequence that is at least about 80% identical to any one of SEQ ID NOs: 1-3 and 15. In one embodiment, the ankyrin repeat domain having binding specificity to CD2 contains an amino acid sequence that is at least about 90% identical to any one of SEQ ID NOs: 1-3 and 15. In another embodiment, the ankyrin repeat domain having binding specificity to CD2 contains an amino acid sequence that is at least about 93% identical to any one of SEQ ID NOs: 1-3 and 15. In a further embodiment, the ankyrin repeat domain having binding specificity to CD2 contains an amino acid sequence that is at least about 95% identical to any one of SEQ ID NOs: 1-3 and 15. In one embodiment, the ankyrin repeat domain having binding specificity to CD2 contains an amino acid sequence that is at least about 98% identical to any one of SEQ ID NOs: 1-3 and 15. In one embodiment, the ankyrin repeat domain having binding specificity to CD2 contains an amino acid sequence that is at least about 99% identical to any one of SEQ ID NOs: 1-3 and 15. In one embodiment, the ankyrin repeat domain having binding specificity to CD2 includes or consists of one of the amino acid sequences of SEQ ID NOs: 1-3 and 15.

[0071] In one embodiment, the ankyrin repeat domain having binding specificity to CD2 contains 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% the same amino acid sequence as SEQ ID NO: 1. In one embodiment, the ankyrin repeat domain contains or consists of the amino acid sequence of SEQ ID NO: 1.

[0072] In one embodiment, the ankyrin repeat domain having binding specificity to CD2 contains an amino acid sequence that is 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, the ankyrin repeat domain contains or consists of the amino acid sequence of SEQ ID NO: 2.

[0073] In one embodiment, the ankyrin repeat domain having binding specificity to CD2 contains 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% the same amino acid sequence as SEQ ID NO: 3. In one embodiment, the ankyrin repeat domain contains or consists of the amino acid sequence of SEQ ID NO: 3.

[0074] In one embodiment, the ankyrin repeat domain having binding specificity for CD2 comprises an amino acid sequence that is 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, the ankyrin repeat domain comprises, or consists of, the amino acid sequence of SEQ ID NO: 15.

[0075] Also provided is a recombinant binding protein having binding specificity for CD2 that competes with a reference binding protein comprising an ankyrin repeat domain that comprises, or consists of, any one of the amino acid sequences of SEQ ID NOs: 1-3, 15. In one embodiment thereof, the competing recombinant binding protein having binding specificity for CD2 comprises an ankyrin repeat domain. Also provided is a recombinant binding protein having binding specificity for CD2 that binds to the same epitope as a reference binding protein comprising an ankyrin repeat domain that comprises any one of the amino acid sequences of SEQ ID NOs: 1-3, 15. In one embodiment thereof, the competing recombinant binding protein having binding specificity for CD2 comprises an ankyrin repeat domain.

[0076] In one embodiment, a recombinant binding protein comprising an ankyrin repeat domain having binding specificity for CD2 has a dissociation constant (K D ) of about 10 -6 M or less, or about 10 -7 M or less, or about 10 -8 M or less, or about 10 -9 M or less and binds to human CD2. Thus, in one embodiment, the recombinant binding protein binds to CD2 with a K D of about 10 -6 M or less. In another embodiment, the recombinant binding protein has a K -7K below M D It binds to CD2. In another embodiment, the recombinant binding protein is about 10 -8 K below M D It binds to CD2. In another embodiment, the recombinant binding protein is about 10 -9 K below M D It binds to CD2. In one embodiment, the recombinant binding protein, which includes an ankyrin repeat domain having binding specificity to human CD2, is approximately 10 -7 M or less, or about 10 -8 M or less, or about 10 9 M or less, or about 10 10 M or less, or about 10 11 Dissociation constants (K) less than or equal to M D It binds to soluble cynomolgus monkey CD2. Preferably, the dissociation constant is determined in PBS using surface plasmon resonance (SPR), for example, as described in Example 5.

[0077] Therefore, in one embodiment, the recombinant binding protein comprises an ankyrin repeat domain, where the binding protein comprises about 10 -6 M or less, or about 10 -7 M or less, or about 10 -8 K below M D The ankyrin repeat domain then binds to human CD2 in PBS and contains an amino acid sequence that is 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 sequence numbers 1-3, 15.

[0078] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain, where the recombinant binding protein comprises about 10 -8 K below M DThe ankyrin repeat domain then binds to soluble human CD2 in PBS, and contains 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% the same amino acid sequence as SEQ ID NO: 1. -8 K below M D It then binds to soluble human CD2 in PBS, where the ankyrin repeat domain contains the amino acid sequence of SEQ ID NO: 1.

[0079] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain, where the recombinant binding protein comprises about 10 -6 K below M D The ankyrin repeat domain then binds to soluble human CD2 in PBS, and contains 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% the same amino acid sequence as SEQ ID NO: 2. -6 K below M D It then binds to soluble human CD2 in PBS, where the ankyrin repeat domain contains the amino acid sequence of SEQ ID NO: 2.

[0080] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain, where the recombinant binding protein comprises about 10-7 K below M D The ankyrin repeat domain then binds to soluble human CD2 in PBS, and contains 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% the same amino acid sequence as SEQ ID NO: 3. -7 K below M D It then binds to soluble human CD2 in PBS, where the ankyrin repeat domain contains the amino acid sequence of SEQ ID NO: 3.

[0081] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain, where the recombinant binding protein comprises about 10 -7 K below M D The ankyrin repeat domain then binds to soluble human CD2 in PBS, and contains 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% the same amino acid sequence as SEQ ID NO: 15. -7 K below M D It then binds to soluble human CD2 in PBS, where the ankyrin repeat domain contains the amino acid sequence of SEQ ID NO: 15.

[0082] ii. Molecules that bind to immune cells CD2-specific binding proteins may be useful as components of multispecific binding molecules that are targeted to disease-associated cells. CD2-specific binding proteins can be used in multispecific binding proteins that further include at least one binding site specific to immune cells, such as CD3. Such multispecific binding proteins can recruit immune cells, such as T cells, to cancer cells.

[0083] In further embodiments, recombinant binding proteins having CD2 binding specificity as described herein are provided, further comprising at least one binding moiety having binding specificity to a protein expressed on the surface of an immune cell, preferably a T lymphocyte (T cell). Preferably, the protein expressed on the surface of an immune cell, such as a T cell, is an immune cell activation receptor. An example of such an immune cell activation receptor is CD3. Accordingly, in certain embodiments, recombinant binding proteins are provided, comprising an ankyrin repeat domain having CD2 binding specificity as described herein, and further comprising a binding moiety having CD3 binding specificity. Preferably, the recombinant binding protein comprises a CD3 binding moiety and an ankyrin repeat domain having CD2 binding specificity, wherein the CD3 binding moiety is located at the C-terminus of the ankyrin repeat domain having CD2 binding specificity. In one embodiment, the recombinant binding protein comprises a CD3 binding moiety and an ankyrin repeat domain having CD2 binding specificity, wherein the CD3 binding moiety is located at the N-terminus of the ankyrin repeat domain having CD2 binding specificity.

[0084] In one embodiment, the binder having binding specificity to CD3 is an ankyrin repeat domain. In one embodiment, the ankyrin repeat domain having binding specificity to CD3 contains an amino acid sequence that is 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, 26-28. In one embodiment, the ankyrin repeat domain having binding specificity to CD3 contains an amino acid sequence that is 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: 4. In one embodiment, the ankyrin repeat domain having binding specificity to CD3 contains an amino acid sequence that is 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: 26.In one embodiment, the ankyrin repeat domain having binding specificity to CD3 contains an amino acid sequence that is 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: 27. In one embodiment, the ankyrin repeat domain having binding specificity to CD3 contains an amino acid sequence that is 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: 28.

[0085] iii. Tumor-associated antigen binding site In another embodiment, a recombinant binding protein is provided comprising an ankyrin repeat domain having binding specificity to CD2, wherein the binding protein further comprises a binder having binding specificity to a disease-related antigen (DAA). In one embodiment, the DAA is a tumor-related antigen (TAA). In one embodiment, such a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD2 comprises a binder having binding specificity to a disease-related antigen and a binder having binding specificity to a protein expressed on the surface of immune cells, preferably T cells. In one embodiment, such a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD2 comprises a binder having binding specificity to a tumor-related antigen and a binder having binding specificity to a protein expressed on the surface of immune cells, preferably T cells. In a preferred embodiment, such a binder is a designed ankyrin repeat domain. Examples of disease-related antigens are CD19, CD20, and CD22. In one particular embodiment of the present invention, a binder having binding specificity to disease-related antigens, such as tumor-related antigens, and / or binding specificity to proteins expressed on the surface of immune cells, preferably T cells, is covalently bonded or fused to a designed ankyrin repeat domain having binding specificity to CD2. In one particular embodiment, a binder having binding specificity to disease-related antigens, such as tumor-related antigens, and / or binding specificity to proteins expressed on the surface of immune cells, preferably T cells, is covalently bonded to the ankyrin repeat domain having binding specificity to CD2 by a peptide linker, preferably a proline-threonine-rich peptide linker or a glycine-serine-rich peptide linker. In one embodiment, the amino acid sequence of the peptide linker has a length of 1 to 50 amino acids, preferably 6 to 38 amino acids.

[0086] Therefore, in one embodiment, a recombinant binding protein is provided comprising a first ankyrin repeat domain having binding specificity to CD19, a second ankyrin repeat domain having binding specificity to CD2, and a third ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domains are optionally arranged in the order of the first-second-third ankyrin repeat domains from the N-terminus to the C-terminus.

[0087] In another embodiment, a recombinant binding protein is provided comprising a first ankyrin repeat domain having binding specificity to CD19, a second ankyrin repeat domain having binding specificity to CD19, a third ankyrin repeat domain having binding specificity to CD2, and a fourth ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domains are optionally arranged in the order of the first-second-third-fourth ankyrin repeat domains from the N-terminus to the C-terminus.

[0088] In another embodiment, a recombinant binding protein is provided comprising a first ankyrin repeat domain having binding specificity to CD19, a second ankyrin repeat domain having binding specificity to CD22, a third ankyrin repeat domain having binding specificity to CD2, and a fourth ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domains are optionally arranged in the order of the first-second-third-fourth ankyrin repeat domains from the N-terminus to the C-terminus.

[0089] Another embodiment provides a recombinant binding protein comprising a first ankyrin repeat domain having binding specificity to CD20, a second ankyrin repeat domain having binding specificity to CD2, and a third ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domains are optionally arranged in the order of the first-second-third ankyrin repeat domains from the N-terminus to the C-terminus.

[0090] Therefore, in one embodiment, an ankyrin repeat domain having binding specificity to CD2, wherein it binds to 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%, and at least about 9 An ankyrin repeat domain having binding specificity to CD2, containing 6%, at least about 97%, at least about 98%, at least about 99%, or 100% identical amino acid sequences, and an ankyrin repeat domain having binding specificity to CD3, wherein 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%, and less than one of sequence numbers 4, 26-28. An ankyrin repeat domain having binding specificity to CD3, comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences, and an ankyrin repeat domain having binding specificity to CD19, wherein at least 80%, at least 81%, at least 82%, and at least one of sequence numbers 36-38 are identical. A recombinant binding protein is provided which contains an ankyrin repeat domain having binding specificity to CD19 and which contains approximately 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences.

[0091] In another embodiment, the recombinant binding protein is an ankyrin repeat domain having binding specificity to CD2, and is bound to 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 An ankyrin repeat domain having binding specificity to CD2, comprising approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identical amino acid sequences; an ankyrin repeat domain having binding specificity to CD3, wherein it comprises at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, and at least approximately An ankyrin repeat domain having binding specificity to CD3, comprising 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 amino acid sequences; an ankyrin repeat domain having binding specificity to CD19, comprising at least about 80%, at least about 81% of any one of SEQ ID NOs. 36-38. Ankyrin repeat domains having binding specificity to CD19, containing at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identical amino acid sequences;Furthermore, the present invention includes an ankyrin repeat domain having binding specificity to CD22, comprising an amino acid sequence that is at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identical to that of SEQ ID NO: 39.

[0092] In yet another embodiment, the recombinant binding protein is an ankyrin repeat domain having binding specificity to CD2, and is bound to 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%, and at least An ankyrin repeat domain having binding specificity to CD2, containing approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identical amino acid sequences; an ankyrin repeat domain having binding specificity to CD3, wherein it contains at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, or at least approximately 88% identical amino acid sequences to any one of sequence numbers 4, 26-28. An ankyrin repeat domain having binding specificity to CD3, comprising at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identical amino acid sequences; and an ankyrin repeat domain having binding specificity to CD20, comprising at least approximately 80%, at least approximately 81% of any one of SEQ ID NOs. 40-41. It comprises an ankyrin repeat domain having binding specificity to CD20, containing at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identical amino acid sequences.

[0093] In yet another embodiment, the recombinant binding protein is an ankyrin repeat domain having binding specificity to CD2, and is bound to one of SEQ ID NOs: 1-3, 15 in an amount of 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%, and less An ankyrin repeat domain having binding specificity to CD2, containing at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences; an ankyrin repeat domain having binding specificity to CD3, wherein at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, and at least one of sequence numbers 4, 26-28. An ankyrin repeat domain having binding specificity to CD3, containing approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% the same amino acid sequence as SEQ ID NO. 65; and an ankyrin repeat domain having binding specificity to CD70, containing at least approximately 80%, at least approximately 81%, or less the same amino acid sequence as SEQ ID NO. 65. It contains an ankyrin repeat domain having binding specificity to CD70, comprising at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences.

[0094] In certain embodiments, each ankyrin repeat domain can bind to its respective target while each of the other ankyrin repeat domains binds to its respective target.

[0095] iv. Half-life extension portion The "half-life extension portion" extends the in vivo serum half-life of the recombinant binding protein described herein compared to the same protein without the half-life extension portion. Examples of half-life extension portions include, but are not limited to, polyhistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, immunoglobulin domain, maltose-binding protein (MBP), human serum albumin (HSA)-binding domain, or polyethylene glycol (PEG).

[0096] In one embodiment, the recombinant binding protein provided herein further comprises one or more half-life extension moieties. Thus, in a particular embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to CD2 as described herein, and further comprises one or more half-life extension moieties. Preferably, the half-life extension moieties bind to human serum albumin.

[0097] In one embodiment, the half-life extension portion includes an ankyrin repeat domain that binds to human serum albumin, wherein the ankyrin repeat domain contains an amino acid sequence that is 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. In one embodiment, the half-life extension portion includes an ankyrin repeat domain containing an amino acid sequence that is 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 extension portion includes an ankyrin repeat domain containing an amino acid sequence that is 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 extension portion includes an ankyrin repeat domain containing an amino acid sequence that is 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.

[0098] Preferably, the ankyrin repeat domain-bound human serum albumin is located at the C-terminus of the ankyrin repeat domain having binding specificity to CD2. Preferably, the ankyrin repeat domain-bound human serum albumin is located at the N-terminus of the ankyrin repeat domain having binding specificity to CD2. In some embodiments, the recombinant binding protein provided herein comprises two or more serum albumin-bound ankyrin repeat domains. In some embodiments, two serum albumin-bound ankyrin repeat domains are located at the N-terminus of the recombinant binding protein provided herein. In some embodiments, two serum albumin-bound ankyrin repeat domains are located at the C-terminus of the recombinant binding protein provided herein. In some embodiments, a first serum albumin-bound ankyrin repeat domain is located at the N-terminus of the recombinant binding protein provided herein, and a second serum albumin-bound ankyrin repeat domain is located at the C-terminus of the recombinant binding protein provided herein.

[0099] In some embodiments, the half-life extension portion includes an immunoglobulin domain. In some embodiments, the immunoglobulin domain includes an Fc domain. In some embodiments, the Fc domain is derived from one of the known heavy chain isotypes: IgG(γ), IgM(μ), IgD(δ), IgE(ε), or IgA(α). In some embodiments, the Fc domain is derived from one of the known heavy chain isotypes or subtypes: IgG1(γ1), IgG2(γ2), IgG3(γ3), IgG4(γ4), IgA1(α1), or IgA2(α2). In some embodiments, the Fc domain is the Fc domain of human IgG1.

[0100] In some embodiments, the Fc domain includes the uninterrupted native sequence of the Fc domain (i.e., the wild-type sequence). In some embodiments, the immunoglobulin Fc domain includes a variant Fc domain that results in altered biological activity. For example, at least one point mutation or deletion may be introduced into the Fc domain to reduce or eliminate effector activity (e.g., International Patent Publication No. WO2005 / 063815) and / or to increase homogeneity during the production of recombinant binding proteins. In some embodiments, the Fc domain is the Fc domain of human IgG1 and includes one or more of the following effector null substitutions: L234A, L235A, and G237A (Eu numbered). In some embodiments, the Fc domain does not include lysine located at the C-terminal position of human IgG1 (i.e., K447 by Eu numbering). The absence of lysine may increase homogeneity during the production of recombinant binding proteins. In some embodiments, the Fc domain includes a lysine molecule (K447, Eu-numbered) located at the C-terminal position.

[0101] I. Nucleic acids and vectors Furthermore, this specification also provides nucleic acids encoding recombinant binding proteins or ankyrin repeat domains described herein. Such nucleic acids may encode polypeptides containing segments or domains of the recombinant binding proteins or ankyrin repeat domains described herein. Due to coding degeneracy, various nucleic acid sequences will encode each of the ankyrin repeat domain sequences described herein. The nucleic acids may contain the nucleotide sequence described in any one of SEQ ID NOs. 32-35, or a substantially identical sequence (for example, a sequence having at least about 85%, 90%, 95%, or 99% sequence identity, or a sequence that differs from any one of SEQ ID NOs. 32-35 by only 3, 6, 15, 30, or 45 nucleotides or less).

[0102] In certain embodiments, the nucleic acid comprises a nucleotide sequence encoding one of the amino acid sequences of SEQ ID NOs: 1-3, 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 one of the ankyrin repeat-binding domains or one of the ankyrin repeat-binding modules disclosed herein.

[0103] Furthermore, nucleic acid molecules derived from any one of SEQ ID NOs. 32-35, optimized for protein expression in suitable prokaryotic host cells such as Escherichia coli, are also provided. The nucleic acids may be present in vectors such as phage display vectors or recombinant plasmid vectors. Accordingly, cloning vectors or expression vectors containing one or more nucleic acid sequences including any one of SEQ ID NOs. 32-35 are also provided, which are suitable for recombinant production of the recombinant-binding proteins described herein.

[0104] For the expression of the recombinant binding proteins described herein, standard techniques can be applied to preferably transfect host cells with an expression vector containing one of the nucleic acid sequences of SEQ ID NOs. 32-35 encoding the recombinant proteins 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 prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, and DEAE-dextran transfection.

[0105] In one embodiment, the cloning or expression vector described herein comprises one nucleic acid sequence from SEQ ID NOs. 32 to 35, operably ligated to a suitable promoter sequence.

[0106] Also provided are vectors comprising nucleic acids encoding any of the recombinant binding proteins described herein, wherein the vector is a DNA vector, an RNA vector, a plasmid, a cosmid, or a viral vector.

[0107] When a recombinant expression vector encoding a recombinant binding protein disclosed herein is introduced into a suitable host cell, such as a prokaryotic host cell, the recombinant binding protein is produced by culturing the host cell for a period sufficient to allow expression of the recombinant binding protein in the host cell or secretion of the recombinant binding protein into the culture medium in which the host cell is grown. The recombinant binding protein can be recovered from the host cell and / or culture medium using standard protein purification methods. In one embodiment, the method includes culturing a suitable host cell, such as a prokaryotic host cell described herein, and collecting the recombinant binding protein from the host cell.

[0108] II. Composition, Use, and Treatment Method In one embodiment, a pharmaceutical composition is also provided comprising a recombinant binding protein described herein and / or a nucleic acid encoding a recombinant binding protein described herein, as well as a pharmaceutically acceptable carrier, stabilizer and / or diluent. Examples of pharmaceutically acceptable carriers, additives and stabilizers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980.

[0109] Accordingly, in one embodiment, a recombinant binding protein is provided, comprising an ankyrin repeat domain having binding specificity to CD2, wherein the ankyrin repeat domain has an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 1-3, 15, and a pharmaceutically acceptable carrier, stabilizer and / or diluent. The pharmaceutically acceptable carrier and / or diluent is known to those skilled in the art and will be described in detail below.

[0110] Suitable carriers, diluents, additives, or stabilizers include, for example, physiological saline, Ringer's solution, dextrose solution, Hanks' solution, fixative oil, ethyl oleate, 5% glucose in physiological saline, substances that enhance isotonicity and chemical stability, buffers, and preservatives. Pharmaceutically acceptable additives generally do not cause serious adverse effects to patients receiving compositions such as proteins, polysaccharides, polylactic acid, polyglycolic acid, high molecular weight amino acids, and amino acid copolymers. Pharmaceutical compositions may also be complex formulations containing additional activators, such as anticancer agents or anti-angiogenic agents, or additional bioactive compounds. Compositions used for in vivo administration must be sterile or sterile. This can be easily achieved by filtration through a sterile filtration membrane.

[0111] In one embodiment, the pharmaceutical composition comprises at least one recombinant binding protein described herein, a surfactant, such as a nonionic surfactant such as Tween-20, a buffer such as phosphate buffer, and a sugar such as sucrose. In one embodiment, such a pharmaceutical composition comprises the recombinant binding protein described herein and PBS.

[0112] The recombinant binding proteins having CD2 binding specificity described herein have numerous in vitro and in vivo therapeutic uses in the treatment of disorders. For example, these molecules can be administered to cells in culture in vitro or ex vivo, or to human subjects, for example, in vivo, to treat, prevent, and / or diagnose various disorders. Accordingly, in one embodiment, the herein provides a method for treating a medical condition, for example a disease, such as cancer, in a subject requiring treatment, comprising administering a therapeutically effective amount of the recombinant binding protein described herein, a nucleic acid encoding such a recombinant binding protein, a vector containing such a nucleic acid encoding such a recombinant binding protein, cells containing such a nucleic acid or vector, or a pharmaceutical composition containing such a recombinant binding protein, nucleic acid, vector or cells.

[0113] In another embodiment, methods are provided for treating a subject, for example, a hyperproliferative state or disorder (e.g., cancer), such as solid tumors, hematological malignancies, soft tissue tumors, or metastatic lesions. Thus, one embodiment provides a method for inhibiting the proliferation of tumor cells, comprising administering a therapeutically effective amount of the recombinant binding protein having CD2 binding specificity described herein, alone or in combination with other agents, such as therapeutic agents or therapeutic modes, to a subject in need. The recombinant binding protein having CD2 binding specificity described herein and one or more additional agents may be administered simultaneously, in the same composition, in separate compositions, or sequentially. In the case of sequential administration, the recombinant binding protein having CD2 binding specificity described herein may be administered first, followed by the additional agents, or the order of administration may be reversed. In one embodiment, the other agents, such as therapeutic agents or therapeutic modes, are administered separately or sequentially with the recombinant binding protein having CD2 binding specificity described herein. In one embodiment, the therapeutic agent comprises a checkpoint inhibitor molecule. In one embodiment, the therapeutic agent comprises a binder specific to DAA, for example TAA, and further comprises a binder specific to a protein expressed on the surface of immune cells, preferably T lymphocytes (T cells). A suitable protein expressed on the surface of immune cells is CD3. In one embodiment, the therapeutic agent is a chimeric antigen receptor T cell (CAR-T). In one experiment, the therapeutic agent is a binder.

[0114] Also provided are recombinant binding proteins described herein, nucleic acids encoding such recombinant binding proteins, vectors containing nucleic acids encoding such recombinant binding proteins, cells containing such nucleic acids or vectors, or pharmaceutical compositions containing such recombinant binding proteins, nucleic acids, vectors or cells, for use in the treatment of medical conditions, such as diseases, such as cancer.

[0115] In one embodiment, the use of recombinant binding proteins described herein, nucleic acids encoding such recombinant binding proteins, vectors containing nucleic acids encoding such recombinant binding proteins, cells containing such nucleic acids or vectors, or pharmaceutical compositions containing such recombinant binding proteins, nucleic acids, vectors or cells is provided for the manufacture of pharmaceuticals, for example, for the treatment of cancer.

[0116] In one embodiment, the recombinant binding protein having binding specificity to CD2 as described herein further comprises an immune cell activating moiety, preferably a CD3-binding ankyrin repeat domain as described herein, a nucleic acid encoding the recombinant binding protein, or a pharmaceutical composition comprising the recombinant binding protein. In one embodiment, the activation is tumor-localized. In one embodiment, the immune cells are adaptive immune cells. In one embodiment, the adaptive immune cells are T cells.

[0117] Administration may include topical, oral, or parenteral administration. The typical route of administration is parenteral administration. In parenteral administration, the pharmaceutical composition is formulated in a unit-dosage injectable form, such as a solution, suspension, or emulsion, in combination with appropriate pharmaceutically acceptable additives, such as those mentioned above. The dosage and mode of administration are determined by the individual being treated and the disease.

[0118] Recombinant binding proteins having CD2 binding specificity as described herein, nucleic acids encoding such recombinant binding proteins, vectors or cells containing nucleic acids encoding such recombinant binding proteins, or pharmaceutical compositions containing such recombinant binding proteins, nucleic acids, vectors or cells can be administered parenterally to a subject, for example, intravenously, intratumorally, or subcutaneously. In some embodiments, such recombinant binding proteins, nucleic acids, vectors, cells, or pharmaceutical compositions are administered intravenously.

[0119] The drug regimen is adjusted to obtain the optimal desired response (e.g., therapeutic response). For example, a single bolus dose may be administered, divided doses may be administered over time, or the dose may be proportionally reduced or increased depending on the urgency of the treatment situation. Parenteral compositions are particularly advantageous to be formulated in unit dose forms for ease of administration and uniformity of dosage. As used herein, a drug unit form refers to a physically distinct unit suitable as a unit dose for the subject being treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in combination with the necessary pharmaceutical carrier. The specifications of the drug unit forms in this disclosure are defined and directly depend on the inherent properties of the active compound and the specific therapeutic effect to be achieved, as well as the limitations inherent in the techniques for formulating such active compounds for the treatment of hypersensitivity in an individual.

[0120] A “medical condition” may be characterized by inappropriate cell proliferation. A medical cellular condition may be a hyperproliferative state. In one embodiment, the medical condition is a neoplastic disease. As used herein, the term “neoplastic disease” refers to an abnormal condition or pathology of cells or tissues characterized by rapidly growing cell proliferation or neoplasms. In one embodiment, the medical condition is a malignant neoplastic disease. Preferably, the medical condition or disease is cancer. In one embodiment, the cancer is ovarian cancer, fallopian tube cancer, peritoneal cancer, mesothelioma, or pancreatic ductal adenocarcinoma (PDAC). In one embodiment, the cancer is ovarian cancer. In one embodiment, the cancer is mesothelioma. In one embodiment, the cancer is pancreatic ductal adenocarcinoma (PDAC). Preferably, the medical condition is a B-cell malignancy. Exemplary B-cell malignancies include non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and certain types of multiple myeloma (MM) and Hodgkin lymphoma (HL). In one embodiment, the B-cell malignancy is a B-cell lymphoma. In one embodiment, the B-cell malignancy is a Hodgkin lymphoma. In one embodiment, the B-cell malignancy is a non-Hodgkin lymphoma.

[0121] The recombinant binding proteins described herein, nucleic acids encoding such recombinant binding proteins, vectors containing nucleic acids encoding such recombinant binding proteins, cells containing such nucleic acids or vectors, or pharmaceutical compositions containing such recombinant binding proteins, nucleic acids, vectors or cells may also be used in combination with one or more other therapies known in the Art. In this specification, the term “combined use” refers to co-administration carried out under a given regimen. This includes simultaneous administration of different compounds, as well as time-shifted administration of different compounds (for example, compound A is administered once and compound B is administered several times thereafter, or vice versa, or both compounds are administered simultaneously and one of them is administered at a later stage).

[0122] In one embodiment, a kit is also provided comprising a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD2 as described herein. In one embodiment, a kit is provided comprising a nucleic acid encoding a recombinant binding protein disclosed herein. In one embodiment, a kit is provided comprising a pharmaceutical composition as described herein. In one embodiment, a kit is provided comprising a recombinant protein and / or a nucleic acid and / or a pharmaceutical composition as described herein. In one embodiment, a kit is provided comprising a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD2, for example, a recombinant binding protein comprising an ankyrin repeat domain containing one amino acid from any of SEQ ID NOs: 1-3, 15, and / or a nucleic acid encoding a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD2, for example, an ankyrin repeat domain containing one amino acid from any of SEQ ID NOs: 1-3, 15, and / or a pharmaceutical composition comprising a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD2, for example, a recombinant binding protein comprising an ankyrin repeat domain containing one amino acid from any of SEQ ID NOs: 1-3, 15. In one embodiment, a kit is provided comprising a recombinant binding protein comprising an ankyrin repeat domain containing any one of the amino acid sequences described herein, such as SEQ ID NOs: 1-3, 15, and / or a nucleic acid encoding the recombinant protein, and / or a pharmaceutical composition comprising the recombinant protein.

[0123] definition The selected terms are defined below and throughout this specification. Unless otherwise defined, all technical and scientific terms used herein are understood to have the same meaning as those generally understood by those skilled in the art in the field of the present invention.

[0124] All publications, patents, and accession numbers referenced herein are incorporated herein by reference in whole, as if each individual publication or patent had been specifically and individually indicated to be invoked by reference.

[0125] All examples or illustrative statements provided herein (e.g., "etc.") are intended solely to better illustrate this disclosure and, unless otherwise claimed, do not limit its scope.

[0126] As used herein, the articles "a" and "an" may mean "one," but their use also coincides with the meanings of "one or more," "at least one," and "one or more."

[0127] In this specification, the term “or” means the term “and / or” unless the context clearly indicates otherwise, and is used interchangeably with “and / or” and is interpreted as a comprehensive “or” meaning any one or any combination.

[0128] Throughout this specification and the subsequent claims, unless otherwise required by context, the terms “comprising,” “having,” and “including” can be used interchangeably. “Comprising” and “including” are to be interpreted as specifying the presence of the mentioned feature or component, but not as excluding the presence or addition of one or more features, components, or groups thereof. Furthermore, “comprising” and “including” are intended to include examples that are encompassed by the term “consisting of.” Therefore, to provide more specific embodiments of the present invention, the term “consisting of” can be used instead of “comprising” and “including.”

[0129] "Approximately" and "about" generally refer to an acceptable degree of error in a measured quantity, taking into account the nature or precision of the measurement. An exemplary degree of error is within 10% of a given value or range of values. For example, when a dosage is stated as "approximately" a specific value, it is intended to include a range of plus or minus 10% around the specified value. While not always explicitly stated, please understand that all numerical notations are preceded by the term "approximately."

[0130] Furthermore, although not necessarily explicitly stated, it should be understood that the reagents described herein are merely examples, and equivalents of such reagents are known in the art.

[0131] In this specification, 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 includes peptides modified by, for example, glycosylation, and proteins comprising two or more polypeptide chains crosslinked by, for example, disulfide bonds.

[0132] As used herein, the term “protein” refers to a molecule containing polypeptides, at least a portion of which may have, or acquire, a defined three-dimensional configuration by forming secondary, tertiary, and / or quaternary structures within a single polypeptide chain and / or between multiple polypeptide chains. If a protein contains two or more polypeptide chains, the individual polypeptide chains may be linked by non-covalent or covalent bonds, for example, by disulfide bonds between the two polypeptides. A portion of a protein that may have, or acquire, a defined three-dimensional configuration by forming secondary and / or tertiary structures is called a “protein domain.”

[0133] The term "recombinant" as used in "recombinant protein," "recombinant polypeptide," etc., means that the protein or polypeptide is produced by the use of recombinant DNA technology known to those skilled in the art. For example, a "recombinant DNA molecule" encoding a polypeptide (e.g., produced by gene synthesis) can be cloned into a bacterial expression plasmid (e.g., pQE30, QIAgen), a yeast expression plasmid, a mammalian expression plasmid, or a plant expression plasmid, or into DNA that enables in vitro expression. For example, when such a recombinant bacterial expression plasmid is inserted into a suitable bacterium (e.g., E. coli), these bacteria can produce the polypeptide(s) encoded by this recombinant DNA. The polypeptide or protein produced in this manner is called a "recombinant polypeptide" or "recombinant protein."

[0134] 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, the binding protein is a recombinant binding protein. The recombinant binding proteins of this disclosure comprise an ankyrin repeat domain having binding specificity to human CD2. Furthermore, any such binding protein may comprise additional polypeptides known to those skilled in the art (e.g., polypeptide tags, peptide linkers, fusion with other proteinaceous domains having binding specificity, cytokines, hormones, or antagonists), or chemical modifications (e.g., coupling with polyethylene glycol, toxins (e.g., DM1), small molecules, antibiotics, etc.).

[0135] The term "binding domain" refers to a protein domain that has binding specificity to a target. Preferably, the binding domain is a recombinant binding domain.

[0136] As used herein, the term “target” refers to individual molecules (including any part of such individual molecules), such as nucleic acid molecules, polypeptides or proteins, carbohydrates, or other naturally occurring molecules, or complexes of two or more such molecules, or whole cells or tissue samples, or any non-natural compounds. Preferably, targets are natural or non-natural polypeptides or proteins, or polypeptides or proteins that include chemical modifications, such as natural or non-natural phosphorylation, acetylation, or methylation. In the context of this disclosure, the CD2 protein expressed on T cells is a target of the CD2-specific binding protein. Preferably, the target is human CD2 (UniProt ID: P06729). CD2 functions as a cell adhesion and costimulatory molecule. It is known to bind to lymphocyte function-associated antigen-3 (LFA-3 / CD58), a surface molecule expressed by antigen-presenting cells and epithelial cells. The CD2 / CD58 interaction facilitates the initial stages of cell contact and facilitates the triggering of the T cell receptor (TCR). CD2 is used as a specific marker for T cells and NK cells, and is used to distinguish B cell neoplasms from T cell lymphoma and leukemia.

[0137] The term "CD3" or "Cluster of Differentiation 3" refers to a multimeric protein complex consisting of four distinct chains. In mammals, this complex includes the CD3γ (gamma) chain, the CD3δ (delta) chain, and two CD3ε (epsilon) chains. These chains bind to the T cell receptor (TCR) and the CD3ζ (zeta) chain, generating activation signals within T cells. CD3 is crucial for T cell activation and the initiation of T cell effector functions such as cytokine production, cytotoxicity, and the release of other immune mediators. This enables T cells to recognize and respond to specific antigens, thereby modulating adaptive immune responses. The amino acid sequences of the 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. An ankyrin repeat domain that specifically binds to CD3 is disclosed in International Publication No. 2022129428 (as referenced).

[0138] The term "CD19" or "Cluster of Differentiation 19" (UniProt ID Nr:Q71UW0) refers to a 95kd type I transmembrane glycoprotein of the immunoglobulin superfamily (IgSF) that has two extracellular C2 set Ig-like domains and a relatively large 240-amino acid cytoplasmic tail that is highly conserved across mammalian species and widely expressed at all stages of B cell development up to terminal differentiation into plasma cells.

[0139] The term "CD22" or "Cluster of Differentiation 22" (UniProt ID Nr:P20273) refers to a 140 kDa membrane glycoprotein expressed on the surface of B cells. CD22 may mediate interactions between B cells and be involved in the localization of B cells in lymphoid tissues.

[0140] 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 regulating the influx of cellular calcium necessary for the development, differentiation, and activation of B lymphocytes.

[0141] The term "CD70" or "Cluster of Differentiation 70" (UniProt ID Nr: P32970) refers to a cytokine that is a ligand for TNFRSF27 / CD27. It is a surface antigen of activated T lymphocytes and B lymphocytes, but not of quiescent T lymphocytes and B lymphocytes. It induces the proliferation of costimulated T cells, promotes the generation of cytolytic T cells, and contributes to T cell activation. CD70 is overexpressed in several types of cancer, including Hodgkin lymphoma and non-Hodgkin lymphoma. CD70 has also been found to be overexpressed in several types of solid tumors.

[0142] International Patent Application Publication No. 2002 / 020565 and Forrer et al. (FEBS Letters 539, 2-6, 2003) provide a general description of the characteristics, techniques, and applications of repeat proteins and repeat domains. The term “repeat protein” refers to a protein containing one or more repeat domains. Preferably, a repeat protein contains one, two, three, four, five, or six repeat domains. Furthermore, the repeat protein may contain additional non-repeat protein domains, polypeptide tags, and / or peptide linkers. Repeat domains can be binding domains.

[0143] The term “repeatable domain” refers to a protein domain comprising two or more consecutive repeating modules as structural units, wherein the repeating modules have structural and sequence homology. Preferably, the repeatable domain further comprises N-terminal and / or C-terminal capping modules. For clarity, the capping modules may also be repeating modules. Such repeatable domains, repeating modules, and capping modules, sequence motifs, and their structural and sequence homology are known to those skilled in the art from examples such as the ankyrin repeatable domain (International Publication No. 2002 / 020565), the leucine-rich repeatable domain (International Publication No. 2002 / 020565), the tetratricopeptide repeatable domain (Main, ER, et al., Structure 11(5), 497-508, 2003), and the armadillo repeatable domain (International Publication No. 2009 / 040338). Furthermore, it is known to those skilled in the art that, unlike proteins containing repeated amino acid sequences, all repeated amino acid sequences in such repeating domains can form individual domains (e.g., the FN3 domain of fibronectin).

[0144] The term “ankyrin repeat domain” refers to a repeat domain that contains two or more consecutive ankyrin repeat modules as structural units. Ankyrin repeat domains can be modularly assembled using standard recombinant DNA techniques to optionally have larger ankyrin repeat proteins with half-life extension domains (see, e.g., Forrer, P., et al., FEBS letters 539, 2-6, 2003; International Publication No. 2002 / 020565, International Publication No. 2016 / 156596; International Publication No. 2018 / 054971). As used herein, the term “construct” refers to a recombinant binding protein that optionally includes one or more designed ankyrin repeat domains and a peptide linker and / or tag sequence. Examples of peptide linkers are shown in SEQ ID NO: 5 or SEQ ID NO: 55, and examples of tag sequences are shown in SEQ ID NO: 6. For clarity, the term “ankyrin repeat domain” includes the N-terminal and C-terminal capping modules.

[0145] The term "designed," as used in phrases like "designed repeat protein" and "designed repeat domain," refers to the fact that such repeat proteins and domains are artificially created and do not exist in nature.

[0146] The term "target interaction residue" refers to amino acid residues in a repeating module that contribute to direct interaction with the target.

[0147] The term "framework residue" refers to an amino acid residue of a repeating module that contributes to the folding topology, i.e., contributes to the folding of the repeating module or to interactions with adjacent modules. Such contributions may include interactions with other residues of the repeating module, influences on polypeptide backbone conformation as seen in α-helices or β-sheets, or involvement in amino acid elongation to form linear polypeptides or loops. Such framework and target interaction residues can be identified by analyzing structural data obtained by physicochemical methods such as X-ray crystallography, NMR and / or CD spectroscopy, or by comparing them with known related structural information.

[0148] The term “repeatable module” refers to the repeated amino acid sequences and structural units of a designed repeatable domain, which are derived from repeatable units of naturally occurring repeatable proteins. Each repeatable module contained within a repeatable domain is derived from one or more repeatable units of a family or subfamily of naturally occurring repeatable proteins, preferably the family of ankyrin repeatable proteins. Furthermore, each repeatable module contained within a repeatable domain may contain “repeatable sequence motifs” inferred from homologous repeatable modules with the same target specificity obtained from a repeatable domain selected on the target. As used herein, “repeatable module” includes internal repeatable modules as well as capping modules such as N-terminal and C-terminal capping modules. An “internal repeatable module” refers to a repeatable module adjacent to two other repeatable modules. In other words, an internal repeatable module is adjacent at its N-terminus by one repeatable module and at its C-terminus by another repeatable module.

[0149] Therefore, the term "ankyrin repeat module" originally refers to a repeat module derived from the repeat unit of an existing ankyrin repeat protein. Ankyrin repeat proteins are known to those skilled in the art.

[0150] The term “repetitive sequence motif” refers to an amino acid sequence inferred from one or more repetitive modules. Preferably, the repetitive modules are from repetitive domains having binding specificity to the same target. Such a repetitive sequence motif includes framework residue positions and target interaction residue positions. The framework residue positions correspond to the framework residue positions of the repetitive module. Similarly, the target interaction residue positions correspond to the target interaction residue positions of the repetitive module. The repetitive sequence motif includes non-randomized and randomized positions.

[0151] The term “repeating unit” refers to an amino acid sequence containing one or more naturally occurring protein sequence motifs, where the “repeating unit” is found in multiple copies and exhibits a defined folding topology common to all such motifs that determine the protein's folding. Examples of such repeating units include leucine-rich repeating units, ankyrin repeating units, armadillo repeating units, tetratricopeptide repeating units, HEAT repeating units, and leucine-rich variant repeating units.

[0152] The repeating module may include positions with amino acid residues that are not randomized within the library ("unrandomized positions") for the purpose of selecting target-specific repeating domains, and positions with amino acid residues that are randomized within the library ("randomized positions") for the purpose of selecting target-specific repeating domains. Unrandomized positions include framework residues. Randomized positions include target interaction residues. "Randomized" means that two or more amino acids are permitted at the amino acid position of the repeating module, for example, any of the 20 common natural amino acids are permitted, or most of the 20 naturally occurring amino acids are permitted, such as amino acids other than cysteine, or amino acids other than glycine, cysteine, and proline. For the purposes of this disclosure, amino acid residues 3, 4, 6, 11, 14, and 15 of SEQ ID NOs: 7-14, 16, and 17 are randomized positions in the internal repeat module; amino acid residues 4, 8, 11, and 12 of SEQ ID NOs: 18-20 are randomized positions in the N-capping repeat module; amino acid residues 3, 4, 6, 14, and 15 of SEQ ID NOs: 22-24 are randomized positions in the C-capping repeat module. Amino acid residues 1-3, 5-7, 9, 10, 13-30 of SEQ ID NOs: 18-20 are framework residues included in the N-capping repeat module; amino acid residues 1, 2, 5, 7-10, 12, 13, and 16-33 of SEQ ID NOs: 7-14, 16, and 17 are framework residues included in the internal repeat module; and amino acid residues 1, 2, 5, 7-13, and 16-28 of SEQ ID NOs: 22-24 are framework residues included in the C-capping repeat module. For the purposes of this disclosure, substitutions at framework locations shall apply to all embodiments, regardless of whether such substitutions are explicitly described or not.

[0153] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are 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), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-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.

[0154] TIFF2026524079000001.tif111170

[0155] In addition, (a) the second-to-last position of any ankyrin repeat domain or any C-capping module described herein may be "A" or "L" and / or the last position may be "A" or "N", or (b) the third-to-last position may be "Q". Furthermore, each ankyrin repeat domain described herein may optionally contain a "G", "S", or "GS" sequence at its N-terminus.

[0156] To determine the percentage of identity between two sequences (e.g., polynucleotides or polypeptides), the sequences are aligned for optimal comparison. A position in the first sequence is considered identical if it has the same nucleotide or amino acid as the corresponding position in the second sequence. The percentage of 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, alignment is usually performed over the length of the reference sequence. For example, to determine whether a test sequence is at least 80% identical to SEQ ID NO: 1 (an example of a reference sequence), alignment is performed 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 deletions in short sequences are considered non-identical positions. Various computer programs are available to determine sequence homology. The Needleman and Wunsch algorithm can calculate the identity percentage between amino acids or nucleic acid sequences using either a Blosum62 matrix or a PAM250 matrix, and specific parameters such as gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. Examples of suitable parameters include the Blosum62 scoring matrix, a gap penalty of 12, a gap expansion penalty of 4, and a frameshift gap penalty of 5.

[0157] Individual binding domains may also be covalently bonded by a peptide linker. A suitable linker may contain 1 to 50 amino acids, preferably 6 to 38 amino acids. The peptide linker may be a proline-threonine (PT) rich peptide linker. An example of a suitable PT rich linker in the context of this disclosure includes the amino acid sequence of SEQ ID NO: 5. The peptide linker may also be a glycine-serine (GS) rich peptide linker. An example of a suitable GS rich linker in the context of this disclosure includes the amino acid sequence of SEQ ID NO: 55.

[0158] Terms such as "binding specificity," "having binding specificity to the target," "binding specifically to the target," "binding to the target with high specificity," "specific to the target," "target specificity," or "binding specifically" mean that the binding protein or binding domain binds to the target with a lower dissociation constant (i.e., binds with high affinity) than it would to an unrelated protein such as E. coli maltose-binding protein (MBP). Preferably, the dissociation constant to the target is ("K D ) is at least 10 of the corresponding dissociation constant for MBP. 2 times; more preferably, at least 10 3 times; more preferably, at least 10 4 times; more preferably, at least 10 5 It is twice as low. Methods for determining the dissociation constant of protein-protein interactions, such as surface plasmon resonance (SPR) based techniques (e.g., SPR equilibrium analysis) or isothermal titration calorimetry (ITC), are known to those skilled in the art. The K of a particular protein-protein interaction D The measured value may vary when measured under different conditions (e.g., salt concentration, pH). Therefore, K D The measurement of the values ​​is preferably carried out using a protein standardization solution and a standardization buffer such as PBS. The term "PBS" refers to a phosphate-buffered aqueous solution containing 137 mM NaCl, 10 mM phosphate, and 2.7 mM KCl, with a pH of 7.4.

[0159] The bonding of any molecule to another molecule is due to two forces, namely the association rate (k on ) and dissociation rate (k off ) is governed by the equilibrium dissociation constant K. Then, the affinity of any binder [B] to the target [T] is determined by the equilibrium dissociation constant K. D This can be expressed by k off / k on It is the quotient. TIFF2026524079000002.tif15170k on is, in units of M -1 s -1 The second-order rate constant of a bonding reaction having k, and the second-order rate constant of a dissociation reaction k offis, in units of s -1 This is a first-order rate constant. From this, it becomes clear that association reactions depend on the concentration of the reactants, while dissociation does not depend on the concentration and follows a simple exponential decay function.

[0160] Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of this disclosure. For example, as illustrated herein, the binding affinity of a particular binding site to a drug molecular target is K D It can be expressed as a value, which refers to the dissociation constant between the binding site and the drug molecular target. K D is, "off rate (k off The dissociation rate, also called the "on rate (k)", and the association rate, or "on rate (k)", are also referred to as the dissociation rate and the association rate. on This is the ratio of ) to . Therefore, K D is k off / k on It is equal to and expressed as molar concentration (M), K D The smaller the value, the stronger the affinity of the bond.

[0161] K D The value can be determined using any appropriate method. K D One exemplary method for measuring this is surface plasmon resonance (SPR) (see, for example, Nguyen et al. Sensors (Basel). 2015 May 5;15(5):10481-510). D The values ​​can be measured by SPR using a biosensor system such as the BIACORE® system. BIAcore dynamics analysis includes, for example, analyzing the binding and dissociation of antigens from a chip having immobilized molecules (e.g., molecules containing epitope-binding domains) on its surface. D Another method for determining this is to use bio-layer interferometry (see, for example, Shah et al. J Vis Exp. 2014;(84):51383). DThe values ​​can be measured using OCTET® technology (Octet QKe system, ForteBio). Alternatively or additionally, the KinExA® (binding equilibrium exclusion method) assay, available from Sapidyne Instruments (Boise, Id.), can also be used. Any method suitable for evaluating the binding affinity between two binding partners is incorporated herein.

[0162] Dissociation constant (K) of recombinant binding proteins having binding specificity to CD2 as disclosed herein D A typical and preferred determination of ) is by surface plasmon resonance (SPR) analysis, as described in Example 5.

[0163] The terms “binding agent” or “binding site” refer to any molecule capable of binding to 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 mimes, repeat proteins, such as designed ankyrin repeat proteins, receptor proteins, and other naturally occurring interaction partners of the target molecule. They may include native proteins and modified or genetically engineered proteins, for example, those containing non-native residues and / or lacking native residues.

[0164] The term "therapeutic portion" refers to a chemical part that, when administered to or otherwise provided to a patient or subject, can function as a therapeutic agent (or perform a therapeutic function) for purposes such as treating a disease or disorder.

[0165] The terms "linked" or "linked" refer to any covalent or noncovalent bond between a chemical portion and a protein, such as an ankyrin repeat domain or a designed repeat protein.

[0166] Preferably, clearance, and / or exposure, and / or terminal phase half-life are evaluated in mammals, preferably mice and / or cynomolgus monkeys. Preferably, when measuring clearance, and / or exposure, and / or terminal phase half-life in mice, the evaluation is made considering data up to 48 hours after injection. More preferably, the evaluation of terminal phase half-life in mice is calculated over 24 to 48 hours. Preferably, when measuring clearance, and / or exposure, and / or terminal phase half-life in cynomolgus monkeys, the evaluation is made considering data up to 7 days after injection. Those skilled in the art can further identify effects such as target-mediated clearance and take them into consideration when calculating terminal phase half-life. The term “terminal phase 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 (e.g., calculated over 24 to 48 hours in mice and over 1 to 5 days in cynomolgus monkeys). Terminal phase half-life is not defined as the time required to eliminate half of the dose of a drug administered to a mammal. Preferably, pharmacokinetic comparisons are performed at any dose, preferably equivalent dose (i.e., the same mg / kg dose) or equimolar dose (i.e., the same mol / kg dose), preferably equimolar dose (i.e., the same mol / kg dose). It will be understood by those skilled in the art that equivalent and / or equimolar doses in animals are subject to experimental dose variability 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, the dose used for pharmacokinetic measurements 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, or about 0.5 to about 10 mg / kg.

[0167] "Tumor-localized activation" of immune cells means that immune cells are preferentially activated in tumor tissue compared to non-tumor tissue.

[0168] The term "antibody" refers not only to intact antibody molecules but also to fragments and variants of antibody molecules that retain immunogenicity. Such fragments and variants are known in the art and are commonly used both in vitro and in vivo. Therefore, the term "antibody" encompasses intact immunoglobulin molecules, antibody fragments such as Fab, Fab', F(ab')2, and single-chain V-region fragments (scFv), bispecific antibodies, chimeric antibodies, antibody-fusion polypeptides, and unconventional antibodies.

[0169] The terms “subject,” “patient,” “subject in need,” and “patient in need” are used interchangeably herein and refer to a person suffering from one or more of the diseases described herein (e.g., cancer). A subject “needs” treatment if the subject would benefit from such treatment in terms of biological, medical, and / or quality of life.

[0170] The term "dosage" refers to the prescribed amount of a therapeutic agent (drug), such as the CD2-specific binding protein described herein, administered to a subject requiring it on a particular day of treatment. The dosage is indicated, for example, on the product packaging or in the product information leaflet.

[0171] As used herein, “administer” or “dosage” refers to the act of physically delivering a substance to a target that is outside the body. Administration includes all forms suitable for delivering the therapeutic agents described herein.

[0172] In this specification, the terms “cancer” and “malignant” are used to refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell proliferation. Cancer includes solid tumors and humoral tumors, as well as primary tumors and metastases. A “tumor” includes one or more cancer cells. A solid tumor usually also includes the tumor stroma. [Examples]

[0173] Examples The 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.

[0174] material Chemicals were purchased from Sigma-Aldrich (USA). Oligonucleotides were obtained from Microsynth (Switzerland). Unless otherwise specified, DNA polymerase, restriction enzymes, and buffers were obtained from New England Biolabs (USA) or Fermentas / Thermo Fisher Scientific (USA). Inducible E. coli expression strains, such as E. coli XL1-blue (Stratagene, USA) or BL21 (Novagen, USA), were used for cloning and protein production.

[0175] molecular biology Unless otherwise specified, the method will follow known protocols (see, for example, Sambrook J., Fritsch EF and Maniatis T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory 1989, New York).

[0176] Designed an ankyrin repeat protein library Methods for generating designed ankyrin repeat protein libraries are described, for example, 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 may be assembled as appropriate 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 so that none of the amino acids C, G, M, N (before the G residue) and P are present at the randomized positions of the repeat modules or capping modules.

[0177] Furthermore, such randomization modules within such libraries may include additional polypeptide loop inserts having randomized amino acid positions. Examples of such polypeptide loop inserts include complement-determining region (CDR) loop libraries of antibodies or de novo peptide libraries. For example, such loop inserts can 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, KT, EMBO J.23(30), 3929-3938, 2004) as a guideline. Similar to this ankyrin repeat domain, in which 10 amino acids are inserted during a β-turn located near the boundary of two ankyrin repeats, an ankyrin repeat protein library may include variable-length (e.g., 1 to 20 amino acids) randomization loops (with fixed and randomized positions) inserted during one or more β-turns of the ankyrin repeat domain. Any such N-terminal capping module of an ankyrin repeat protein library preferably has a RILLAA, RILLKA, or RELLKA motif (e.g., located at positions 19 to 24 in SEQ ID NO: 1), and any such C-terminal capping module of an ankyrin repeat protein library preferably has a KLN, KLA, or KAA motif (e.g., located at the last three amino acids in SEQ ID NO: 1).

[0178] The design of such ankyrin repeat protein libraries can be guided by known structures of ankyrin repeat domains that interact with targets. Examples of such structures are identified by Protein Databank (PDB) specific accession codes or identification codes (PDB-IDs), including 1WDY, 3V31, 3V30, 3V2X, 3V2O, 3UXG, 3TWQ-3TWX, 1N11, 1S70, and 2ZGD.

[0179] Examples of designed ankyrin repeat protein libraries, such as N2C and N3C designed ankyrin repeat protein libraries, are described (U.S. Patent No. 7,417,130; Binz et al. 2003, op. cit.; Binz et al. 2004, op. cit.). The numbers N2C and N3C describe the number of randomized repeat modules present between the N-terminal and C-terminal capping modules.

[0180] The nomenclature used to define the positions within repeating units and modules is based on Binz et al. 2004 (cited above), but with a modification in which the boundary between ankyrin repeating modules and ankyrin repeating units is shifted by one amino acid position. For example, position 1 of the ankyrin repeating module in Binz et al. 2004 (cited above) corresponds to position 2 of the ankyrin repeating module in this disclosure, and consequently, position 33 of the ankyrin repeating module in Binz et al. 2004 (cited above) corresponds to position 1 of the next ankyrin repeating module in this disclosure.

[0181] All DNA sequences were confirmed by sequencing, and the calculated molecular weights of the selected proteins were confirmed by mass spectrometry.

[0182] Example 1: Selection and characterization of binding proteins containing ankyrin repeat domains with binding specificity to CD2. Using a ribosome display (Hanes, J. and Plueckthun, A., PNAS 94, 4937-42, 1997), several ankyrin repeat proteins with binding specificity to human CD2 or the CD2:CD58 complex were selected from a DARPin library similar to that described by Binz et al. 2004 (cited above). Binding of the selected clones to recombinant human CD2 target proteins was evaluated by homogeneous time-resolved fluorescence (HTRF) of the crude extract, demonstrating successful selection of human CD2-specific binding proteins. For example, the ankyrin repeat domains of SEQ ID NOs. 1-3, 15 constitute the amino acid sequence of the selected binding protein containing an ankyrin repeat domain with binding specificity to CD2. Individual ankyrin repeat modules from such ankyrin repeat domains with binding specificity to CD2 are provided, for example, in SEQ ID NOs. 7-14, 16, 17.

[0183] Human recombinant CD2 target preparation Biotinylated human CD2 / SRBC protein, His, Avitag TM The Fc-tagged human CD58 / LFA-3 protein was purchased from ACRO Biosystems. The recombinant biotin-labeled human CD2-Fc fusion protein with a C-terminal Avi tag was purchased from BPS Bioscience.

[0184] Selection of CD2-specific ankyrin repeat proteins by ribosome display Selection of CD2-specific ankyrin repeat proteins was performed using ribosome display (Hanes and Plueckthun, op. cit.) with an established protocol (see Zahnd, C., Amstutz, P. and Plueckthun, A., Nat. Methods 4, 69-79, 2007) as the target protein, with or without complexation with CD58. The number of reverse transcription (RT)-PCR cycles after the first selection round was 45, and thereafter it was always 30. The four rounds of selection were performed using a standard ribosome display selection method with decreasing target concentrations (400 nM CD2 or 300 nM CD2 for the CD2:CD58 complex, 100 nM, 25 nM, and 5 nM, respectively).

[0185] The selected clones show binding to the CD2 target (indicated by homogeneous time-resolved fluorescence-HTRF). In the first screening step, a pool from the ribosome display was subcloned into derivatives 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. Over 2000 DARPin proteins from ribosome display round 4 were expressed in E. coli cells with His tags (as CD2 mono-DARPin or in TAA-CD2 multidomain form). The crude extract was prepared to test the binding of His-tagged DARPin proteins to biotinylated human CD2-Fc recombinant protein using an HTRF assay. The crude extract was diluted in PBS-TB (PBS supplemented with 0.1% (w / v) BSA and 0.1% Tween 20, pH 7.4) and used in the assay at a 1:500 dilution (final). Binding was performed against 6 nM (final concentration) human biotinylated CD2-Fc. FRET donor (streptavidin-Tb) and acceptor (MAb anti-His-d2) conjugate (Cisbio) were used in 1:400 dilutions (final) in the wells of a 384-well plate and incubated overnight at 4°C (refrigerated room). HTRF was read using a Tecan M1000 with an excitation wavelength of 340 nm and an emission filter of 665 ± 10 nm. Four selected binders were subcloned into derivatives of the pQE30 (Qiagen) expression vector containing an N-terminal His tag (SEQ ID NO: 6) and further characterized using AKTAxpress for detailed characterization. TM It was refined by the system.

[0186] For example, an expression vector encoding the following ankyrin repeat protein was constructed: DARPin protein #1 (Sequence ID 1, which has a His tag (Sequence ID 6) fused to its N-terminus) DARPin protein #2 (with a His tag (SEQ ID NO: 6) fused to its N-terminus) DARPin protein #3 (with a His tag (SEQ ID NO: 6) fused to its N-terminus) DARPin protein #15 (sequence number 15, which has a His tag (sequence number 6) fused to its N-terminus)

[0187] Example 2: Functional testing of CD2-specific binding proteins Experiment A The CD2-specific ankyrin repeat domain (SEQ ID NO: 1) according to the present invention was formatted as a triple-specific T cell engager molecule (TCE#1) further comprising an ankyrin repeat domain having binding specificity to CD3 (SEQ ID NO: 4) and an ankyrin repeat domain having binding specificity to TAA. The three ankyrin repeat domains are linked to each other via a peptide linker (SEQ ID NO: 5). At the N-terminus, the triple-specific T cell engager molecule (TCE#1) contained a His tag (SEQ ID NO: 6) to facilitate purification.

[0188] Another T cell engager molecule (TCE#2) was generated, which was identical to TCE#1 except that the CD2-specific ankyrin repeat domain was absent in TCE#2. Therefore, TCE#2 contained only two ankyrin repeat domains: an ankyrin repeat domain with binding specificity to CD3 (SEQ ID NO: 4; the selection of the DARPin protein with binding specificity to CD3 is disclosed in International Publication No. 2022129428(A1) incorporated herein by reference) and an ankyrin repeat domain with binding specificity to TAA.

[0189] Evaluation of the efficacy of CD2-binding DARPin protein against OCI-Ly19 CD58-KO target cells in co-culture with human pan-T cells. In an in vitro short-term assay measuring T cell proliferation by evaluating CellTrace Violet (CTV) using flow cytometry, the efficacy of CD2-engaged T cell-binding DARPin [TCE#1] was evaluated compared to CD2-inengaged T cell-binding DARPin protein [TCE#2].

[0190] 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 (wild-type) cells) [effector:target ratio 5:1] per well of a 96-well plate were co-incubated in double serial dilutions of selected DARPin protein at 37°C for 72 hours. The cells were washed and stained with 1:3600 Zombie NIR Viability dye (BioLegend) and 1:200 anti-TAA antibody (BD Biosciences) at 4°C for 30 minutes. After washing and fixation, the cells were analyzed using an Attune NxT cytometer. T cell proliferation was assessed by measuring the dilution of CTV in the CTV+ / TAA-negative population compared to the untreated population. Flow cytometry data was analyzed using FlowJo software and plotted using GraphPad Prism 8.

[0191] As shown in Figure 1, TCE#2 induces less T cell proliferation in OCI-Ly19 WT cells when co-cultured with OCI-Ly19 CD58-KO cells. CD2-bound TCE#1 induces T cell proliferation in OCI-Ly19 CD58-KO cells to a similar level as TCE#2 in OCI-Ly19 WT cells.

[0192] These results indicate that the presence of a CD2 binding moiety within the TCE rescues the loss of T cell response caused by the absence of CD58 in CD58 knockout cells.

[0193] Experiment B In a similar experimental setup, three selected DARPin proteins, each containing an ankyrin repeat domain with binding specificity to CD2, an ankyrin repeat domain with binding specificity to CD3, and an ankyrin repeat domain with binding specificity to CD19, were evaluated for their efficacy in T cell proliferation assays and tumor cell death assays against OCI-Ly19 CD58-KO or OCI-Ly19 CD58-targeted cells in co-culture with human pan-T cells. As a control molecule, a DARPin protein containing ankyrin repeat domains with binding specificity to CD3 and CD19, but without an ankyrin repeat domain that binds to human CD2, was used. The three ankyrin repeat domains were linked to each other via a peptide linker (SEQ ID NO: 5), but included a His tag (SEQ ID NO: 6) at the N-terminus to facilitate purification.

[0194] In short, pan-T cells and OCI-LY19 wt cells or OCI-LY19 CD58 KO cells were incubated in an E:T ratio of 5:1 and co-cultured for 72 hours in the presence of serial dilutions of the tested protein. T cell proliferation was then evaluated by FACS. To evaluate T cell proliferation and tumor cell death, the co-culture was stained with an anti-CD2 antibody (Brilliant Violet 711). TM Cell tracing was performed at 4°C for 30 minutes using anti-human CD2 antibody (Biolegend 300232, 1:200) and Cell Trace Violet (CTV, ThermoFisher, 1:1000). After washing and fixation, cells were analyzed using an AttuneNxt flow cytometer (Thermo Fisher). Flow cytometry data were analyzed using FlowJo software, and the data were plotted using GraphPad Prism. T cell proliferation was gated as live CD2+ / CTV+ cells. Tumor cell death was gated as live CD22+ cells normalized to co-culture.

[0195] TIFF2026524079000003.tif42170

[0196] As shown in Figure 3 (showing T cell proliferation induced by the tested protein) and Figure 4 (showing tumor cell death induced by the tested protein), the presence of CD2-specific ankyrin repeat domains in proteins DARPin#42, DARPin#43, and DARPin#44 can rescue the loss of efficacy against OCI-Ly19 CD58-KO target cells caused by the absence of CD58. For reference, OCI-Ly19 wild-type cells were used to demonstrate the loss of efficacy of DARPin#45, which lacks a CD2-binding domain. That is, the T cell engager protein DARPin#45 induces T cell proliferation and tumor cell death, but a decrease in efficacy is observed when OCI-Ly19-CD58 knockout cells are used as target cells. Thus, this indicates that the presence of a CD2-binding domain rescues such loss.

[0197] When the same protein was evaluated in the presence of OCI-Ly19 wild-type cells, it could still induce increased potency, expressed as increased T cell proliferation, compared to a control DARPin protein (DARPin protein #45) that lacked the CD2 binding domain, as shown in Figure 5.

[0198] Experiment C Similar to Experiment B, six selected DARPin proteins, formatted as quadruplespecificity proteins containing an ankyrin repeat domain with binding specificity to CD2, an ankyrin repeat domain with binding specificity to CD3, an ankyrin repeat domain with binding specificity to CD19, and an ankyrin repeat domain with binding specificity to CD22, were evaluated for efficacy in a T cell proliferation assay against OCI-Ly19 CD58-KO or OCI-Ly19 CD58-expressing target cells in co-culture with human pan-T cells. The selected proteins contained the same ankyrin repeat domain, but differed in their arrangement from the N-terminus to the C-terminus, as shown in Table 3 below. As control molecules, two DARPin proteins containing ankyrin repeat domains with binding specificity to CD3, CD19, and CD22, but without an ankyrin repeat domain with binding specificity to human CD2, as well as unbound DARPin (SEQ ID NO: 54), were used. The three ankyrin repeat domains were linked to each other via a peptide linker (SEQ ID NO: 55), but at the N-terminus, they contained a His tag (SEQ ID NO: 6) to facilitate purification.

[0199] In short, pan-T cells and OCI-LY19 wt cells or OCI-LY19 CD58 KO cells were incubated in an E:T ratio of 5:1 and co-cultured for 72 hours in the presence of serial dilutions of the tested protein. T cell proliferation was then evaluated by FACS. To assess T cell proliferation, the co-culture was stained with an anti-CD2 antibody (Brilliant Violet 711). TM Cell tracing was performed at 4°C for 30 minutes using anti-human CD2 antibody (Biolegend 300232, 1:200) and Cell Trace Violet (CTV, ThermoFisher, 1:1000). After washing and fixation, cells were analyzed using an AttuneNxt flow cytometer (Thermo Fisher). Flow cytometry data were analyzed using FlowJo software, and the data were plotted using GraphPad Prism. T cell proliferation was gated as live CD2+ / CTV+ cells.

[0200] TIFF2026524079000004.tif82170

[0201] 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), the inclusion of a CD2-binding domain along with a CD3-binding domain (DARPin#50, DARPin#51) results in increased potency, as demonstrated by increased T cell proliferation (e.g., compared to DARPin#52 which contains a CD3-binding domain but does not contain a CD2-binding domain).

[0202] Experiment D In experimental settings similar to those described in Experiments A-C, two selected DARPin proteins, each containing an ankyrin repeat domain with binding specificity to CD2, an ankyrin repeat domain with binding specificity to CD3, and two ankyrin repeat domains with binding specificity to CD20, were evaluated for their efficacy in T cell proliferation and tumor cell death assays against OCI-Ly19 CD58-KO or OCI-Ly19 CD58-expressing cells in co-culture with human pan-T cells. As control molecules, a DARPin protein containing an ankyrin repeat domain with binding specificity to CD3 and CD20 but without an ankyrin repeat domain that binds to human CD2 (DARPin#58), or DARPin proteins containing an ankyrin repeat domain with binding specificity to CD2 and CD20 but without an ankyrin repeat domain that binds 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), but at the N-terminus, they contained a His tag (SEQ ID NO: 6) to facilitate purification.

[0203] To evaluate T cell proliferation and tumor cell death, the co-culture was stained with an anti-CD2 antibody (Brilliant Violet 711). TMCell tracing was performed at 4°C for 30 minutes using anti-human CD2 antibody (Biolegend 300232, 1:200) and Cell Trace Violet (CTV, ThermoFisher, 1:1000). After washing and fixation, cells were analyzed using an AttuneNxt flow cytometer (Thermo Fisher). Flow cytometry data were analyzed using FlowJo software, and the data were plotted using GraphPad Prism. T cell proliferation was gated as live CD2+ / CTV+ cells. Tumor cell death was gated as the number of live CD22+ cells normalized to untreated co-culture controls.

[0204] TIFF2026524079000005.tif48170

[0205] As shown in Figures 8(AB) and 9(AB), the presence of CD2-binding ankyrin repeat domains increases T cell proliferation and target cell death, respectively, in co-culture assays of healthy donor T cells with OCI-Ly19 or OCI-Ly29 CD58-KO cells.

[0206] Experiment E Evaluation of the efficacy of CD2-binding DARPin on human pan-T cells in a T cell activation assay. To evaluate the ability of CD2-binding DARPin to induce sustained T cell activation, three selected binding proteins (see Table 5 below) with binding specificity to CD70 (tumor-associated antigen; the selection of DARPin with binding specificity to CD70 is disclosed in International Publication No. 2022215032(A1) incorporated herein by reference), CD2, and CD3 were cultured with isolated PBMC T cells and compared with DARPin T cell engagers (without the 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 neutraavidin (Thermo Fisher Scientific) in phosphate-buffered saline (PBS, Gibco) at final concentrations of 70 μg / ml and 8 μg / ml, respectively. After incubation overnight at 4°C and continuous stirring at 450 rpm, the plates were washed with PBS to remove unbound neutraavidin or anti-CD3 antibody, and then 20 nM biotinylated CD70 (UniProt ID Nr: P32970; ACROBiosystems) in PBS was added to the neutraavidin-coated wells. The plates were incubated at room temperature (RT) at 450 rpm for 30 minutes, and then washed to remove excess unbound biotinylated CD70. Finally, 500 nM of each tested DARPin in PBS was added to the neutraavidin / CD70-coated wells, and the plates were incubated again at RT at 450 rpm for 30 minutes, and washed again with PBS to remove unbound DARPin. Then, 200,000 purified PBMC pan-T cells (Miltenyi, negative selected) were added to each well and incubated at 37°C at 5% CO2. After 2-3 days of incubation, T cells were collected from the wells and replated in fresh medium on newly coated 96-well plates for a total of five stimulation rounds (referred to as S1-S5). After each stimulation round, the T cells were harvested for flow cytometry analysis.First, T cells were washed with PBS and stained with a viability dye (Live / Dead Zombie NIR, BioLegend) for 30 minutes at 4°C to allow for the exclusion of dead cells. 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 μg / ml in FACS buffer) was added to the wells and incubated for another 30 minutes at 4°C. Subsequently, the 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 fluorescence intensity (MFI) and cell count of CD8+CD25+ T cells were plotted using GraphPad Prism (Version 10).

[0207] TIFF2026524079000006.tif42170

[0208] As shown in Figures 10 and 11, the presence of CD2-binding ankyrin repeat domains in DARPin#61, DARPin#62, and DARPin#63 induced sustained T cell proliferation and T cell activation over time, respectively, compared to the benchmark antibody or a DARPin T cell engager (SEQ ID NO: 64) that did not contain CD2-binding domains.

[0209] Example 3: Selection of binding proteins containing ankyrin repeat domains with binding specificity for CD19, CD22, and CD20, respectively. Using ribosome display (Hanes, J. and Plueckthun, A., PNAS 94, 4937-42, 1997), many ankyrin repeat proteins with binding specificity to human CD117 were selected from a DARPin library similar to that described by Binz et al. 2004 (cited above). Binding of the selected clones to recombinant human CD19, CD22, and CD20 targets was independently evaluated by homogeneous time-resolved fluorescence (HTRF) of crude extracts, demonstrating successful selection of human CD19-specific, CD22-specific, and CD20-specific binding proteins. For example, the ankyrin repeat domains of SEQ ID NOs. 36-38 constitute the amino acid sequence of a recombinant binding protein containing an ankyrin repeat domain with binding specificity to CD19, the ankyrin repeat domain of SEQ ID NOs. 39 constitutes the amino acid sequence of a recombinant binding protein containing an ankyrin repeat domain with binding specificity to CD22, and the ankyrin repeat domains of SEQ ID NOs. 40-41 constitute the amino acid sequence of a recombinant binding protein containing an ankyrin repeat domain with binding specificity to CD20.

[0210] Selection of binding proteins containing ankyrin repeat domains with binding specificity for A.CD19 Human recombinant CD19 target preparation Two target formats were used in the selection procedure. Both were based on a target single-chain target polypeptide consisting of the extracellular domain of the human CD19 protein (UniProt accession number: Q71UW0, residues 20-279). In the first format, the extracellular domain was ligated to the human IgG1 Fc domain (purchased from Acro Biosystems), and in the second format, it was ligated to a polyhistidine tag, followed by a C-terminal Avi tag (SEQ ID NO: 67; purchased from R&D Systems).

[0211] Selection of CD19-specific ankyrin repeat proteins by ribosome display Selection of CD19-specific ankyrin repeat proteins was performed using the extracellular domain of CD19 as the target protein, a library of ankyrin repeat proteins as described above, and ribosome display (Hanes and Plueckthun, op. cit.) with an established protocol (see Zahnd, C., Amstutz, P. and Plueckthun, A., Nat. Methods 4, 69-79, 2007). The number of reverse transcription (RT)-PCR cycles after each selection round was always 30. The first four rounds of selection were performed using a standard ribosome display selection method with decreasing target concentrations (400 nM, 100 nM, 25 nM, and 5 nM, respectively), followed by a fifth off-rate round using a target concentration of 1 nM and a 600-fold excess of non-biotinylated target as competitor to select high-affinity binding domains, and a sixth recovery round at a target concentration of 5 nM.

[0212] In selection rounds 2-4, additional selection branches were performed, and a monoclonal mouse anti-human CD19 antibody (clone FMC63; purchased from Novus Biologicals) was used for competitive elution.

[0213] The selected clones exhibit binding to the CD19 target expressed on the cell. Selected CD19-specific DARPin was expressed and purified by 96-well IMAC. Briefly, E. coli XL1 blue cells were transformed with selected ankyrin repeat proteins, plated on LB-agar (containing 1% glucose and 50 μg / mL ampicillin), and incubated overnight at 37°C. For each protein, a single colony was collected in an individual well of a 96-deep-well plate containing 1.2 mL of TB medium (containing 1% glucose and 50 μg / mL ampicillin) and incubated overnight at 37°C with shaking at 850 rpm at 80% humidity using a Multitron Pro microplate shaker. The culture (1:10) was inoculated overnight into fresh TB medium (containing 50 μg / mL ampicillin; 0.99 mL per well of a 96-deep-well plate) and incubated at 37°C at 850 rpm. After 2 hours, the culture was induced by the addition of IPTG (final concentration 0.5 mM) and incubated for a further 5-6 hours at 37°C at 850 rpm. Harvest was performed by centrifugation (6 minutes 3200xg). 50 μl of B-PER was collected. TM Cells were disrupted according to the manufacturer's protocol using Bacterial Protein Extraction Reagent (catalog number 78260; Thermo Fisher Scientific, Waltham, Massachusetts, USA; supplemented with DNAseI (200 units / ml) and lysozyme (0.4 mg / ml)), and 60 μl of IMAC preparation buffer (Na2HP04x2H2O 50 mM, NaCl 300 mM, pH 7.4) was added. Up to 8 clones were then pooled and plated in a 96-well column IMAC plate (HisPur TMThe DARPin was purified using Cobalt Spin Plates (catalog number: 90095; Thermo Fisher Scientific, Waltham, Massachusetts, USA) and rebuffered to PBS pH 7.4 using Zeba Spin desalted 96-well plates (catalog number: 89807; Thermo Fisher Scientific, Waltham, Massachusetts, USA). All procedures followed the manufacturer's protocol. The pooled DARPin was tested for binding to DAUDI cells at final dilutions of 1:1,000 and / or 1:10,000. Binding was detected using an anti-DARPin antibody. Pools showing a positive binding signal were deconvoluted; i.e., wells containing the glycerol stock of DARPin originally pooled for the purification step described above were used for the reexpression and purification of individual DARPin. These reexpressed and purified individual DARPin were retested at 10 nM for binding to cell expression targets to isolate the DARPin(s) responsible for the positive pool signal.

[0214] For example, an expression vector encoding the following ankyrin repeat protein was constructed: DARPin protein #36 (Sequence ID #36, which has a His tag (Sequence ID #6) fused to its N-terminus) DARPin protein #37 (Sequence ID #37, which has a His tag (Sequence ID #6) fused to its N-terminus) DARPin protein #38 (sequence number 38, which has a His tag (sequence number 6) fused to its N-terminus)

[0215] Selection of binding proteins containing ankyrin repeat domains with binding specificity for B CD22 Human recombinant CD22 target preparation Three target formats were used in the selection procedure: Target a: The extracellular domain of CD22 (residues 20-687 of human CD2, UniProt ID Nr: P20273) was ligated to the human IgG1 Fc domain, followed by a C-terminal Avi tag (purchased from Acro Biosystems); Target b: CD22 lacking domains 5 and 6 (residues 20-504 of Uniprot P20273) followed by a C-terminal Avi tag (SEQ ID NO: 67; purchased from Evitria); and Target c: CD22 domains 4, 5, and 6 (residues 416-687 of Uniprot P20273) were fused to the Fc-kih domain (knob-in-hole format), followed by a C-terminal Avi tag (SEQ ID NO: 67; purchased from Evitria), and subsequently enzymatically biotinylated in vitro via BirA-GST ligase (purchased from BPS Bioscience).

[0216] Selection of CD22-specific ankyrin repeat proteins by ribosome display Selection of CD22-specific ankyrin repeat proteins was performed by ribosome display (Hanes and Plueckthun, op. cit.) using the extracellular domain of CD22 as the target protein, a library of ankyrin repeat proteins as described above, and an established protocol (see, e.g., Zahnd, C., Amstutz, P., and Plueckthun, A., Nat. Methods 4, 69-79, 2007). Two different selection branches were applied. In one branch, target b was used for deselection against the distal membrane domain, prioritizing binder selection against the proximal membrane Ig-fold domains C5 and C6. In the other branch, selection was applied to target c, which includes the proximal CD22 membrane domains C4-6. The number of reverse transcription (RT)-PCR cycles for selection rounds 1 through 6 were 45, 35, 30, 28, 30, and 35, respectively. The first four rounds of selection were performed using a standard ribosome display selection method with decreasing target concentrations (400 nM, 100 nM, 25 nM, and 5 nM, respectively). A fifth off-rate round was then performed to select high-affinity binders, using a target concentration of 1 nM and non-biotinylated targets in 289-fold or 900-fold excess, respectively, as competitors. A sixth recovery round was performed at a target concentration of 5 nM.

[0217] The selected clones show binding to the CD22 target (indicated by HTRF). Ribosome display products were subcloned into derivatives 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 crude extracts of ankyrin repeat proteins expressed in E. coli. Selected hits were rearranged on four plates, purified using their His tags in a 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 used for detailed characterization in AKTAxpress. TMIt was refined by the system.

[0218] For example, an expression vector encoding the following ankyrin repeat protein was constructed: DARPin protein #39 (sequence number 39, which has a His tag (sequence number 6) fused to its N-terminus)

[0219] Selection of binding proteins containing ankyrin repeat domains with binding specificity for C. CD20 Human recombinant CD20 target preparation To select ankyrin repeat domains with binding specificity to CD20, full-length human CD20 protein (UniProt ID Nr: P11836) was used as a target, ligated to a polyhistidine tag, and subsequently to a C-terminal Avi tag (purchased from Acro Biosystems).

[0220] Selection of CD20-specific ankyrin repeat proteins by ribosome display Selection of CD20-specific ankyrin repeat proteins was performed by ribosome display (Hanes and Plueckthun, op. cit.) using full-length CD20 protein, a library of ankyrin repeat proteins as described above, and an established protocol (see, e.g., Zahnd, C., Amstutz, P., and Plueckthun, A., Nat. Methods 4, 69-79, 2007). The number of reverse transcription (RT)-PCR cycles after each selection round was always 30. Four rounds of selection were performed using a standard ribosome display selection method with decreasing target concentrations (400 nM, 100 nM, 25 nM, and 5 nM, respectively). Additional selection steps were performed in selection rounds 2–4, using monoclonal human anti-CD20 antibody for competitive elution.

[0221] The selected clones show binding to the CD20 target (indicated by HTRF). In the first approach, a pool from ribosome displays was subcloned into derivatives of a pQE30(Qiagen) expression vector containing an N-terminal His tag followed by a Flag tag, and expressed in E. coli cells in a 96-well plate. The crude extract was prepared to test the binding of the His-tagged DARPin protein to biotinylated human CD20-Fc recombinant protein using an HTRF assay. Extracts from each lysed clone were applied to the wells of a 384-well plate at a 1:500 dilution in PBSTB (PBS supplemented with 0.1% Tween20® and 0.2% (w / v) BSA, pH 7.4) along with 4 nM (final concentration) biotinylated full-length target hCD20 His, 1:400 (final concentration) anti-strep-Tb HTRF antibody-FRET donor conjugate (Cisbio), and 1:400 (final concentration) anti-Flag-d2 antibody-FRET acceptor conjugate (Cisbio), and incubated at RT for 1 hour. HTRF was read using a Tecan M1000pro with an excitation wavelength of 340 nm and an emission filter of 665 ± 10 nm. In the second screening campaign, up to seven pooled bivalent DARPin (MRGS-His6-GS-DAPRin-PT1n-leucine-zipper-MYC tag) were first screened for binding on cells using sequential deconvolution instead of HTRF. Briefly, E. coli XL1 blue cells were transformed with ankyrin repeat protein, plated on LB-agar (containing 1% glucose and 50 μg / mL ampicillin), and incubated overnight at 37°C. For each construct, a single colony was collected in an individual well of a 96-deep-well plate containing 1.2 mL of TB medium (containing 1% glucose and 50 μg / mL ampicillin) and incubated overnight at 37°C with shaking at 850 rpm at 80% humidity using a Multitron Pro microplate shaker. The cultures (1:10) were inoculated overnight into fresh TB medium (containing 50 μg / ml ampicillin; 0.99 mL per well in a 96-deep-well plate) and incubated at 37°C and 850 rpm.After 2 hours, the culture was induced by the addition of IPTG (final concentration 0.5 mM) and incubated for a further 5-6 hours at 37°C at 850 rpm. Harvest was performed by centrifugation (6 minutes 3200xg). 50 μl of B-PER was collected. TM Cells were disrupted according to the manufacturer's protocol using Bacterial Protein Extraction Reagent (catalog number 78260; Thermo Fisher Scientific, Waltham, Massachusetts, USA; supplemented with DNAseI (200 units / ml) and lysozyme (0.4 mg / ml)), and 60 μl of IMAC preparation buffer (Na2HP04x2H2O 50 mM, NaCl 300 mM, pH 7.4) was added. Up to 8 clones were then pooled and plated in a 96-well column IMAC plate (HisPur TMThe DARPin was purified using Cobalt Spin Plates (catalog number: 90095; Thermo Fisher Scientific, Waltham, Massachusetts, USA) and rebuffered to PBS pH 7.4 using Zeba Spin desalted 96-well plates (catalog number: 89807; Thermo Fisher Scientific, Waltham, Massachusetts, USA). All procedures followed the manufacturer's protocol. The pooled DARPin was tested for binding to DAUDI cells at final dilutions of 1:1'00 and / or 1:10'000. Binding was detected by flow cytometry. For this purpose, cells were incubated with each dilution of pooled DARPin at 4°C for 45 minutes to 1 hour, followed by two washes, and then incubated with a fluorescently labeled detection antibody (anti-DARPin-1.1.1-AF488) at 4°C for 30 minutes. During this process, Live / Dead Aqua (Thermo Fisher, L34957, 1:1000) was included to allow for the exclusion of dead cells during analysis. After two washing steps, cells were fixed at 4°C for 20 minutes using BD Cytofix CellFix Fixation Buffer (catalog no. 554655). Fluorescence signals were acquired using an Attune Nxt flow cytometer (Thermo Fisher), and data were analyzed using FlowJo and GraphPad software. Pools showing positive binding signals were deconvolved, and wells containing the original pooled DARPin glycerol stocks were used for the expression and purification of individual DARPin. These purified individual DARPin were tested at 10 nM for binding to DAUDI cells using the same cell binding protocol as above to isolate the DARPin(s) responsible for the positive signals observed in the pool.

[0222] For example, an expression vector encoding the following ankyrin repeat protein was constructed: DARPin protein #40 (Sequence ID #40, which has a His tag (Sequence ID #6) fused to its N-terminus) DARPin protein #41 (SEQ ID NO: 41, which has a His tag (SEQ ID NO: 6) fused to its N-terminus)

[0223] Example 4: Cell-binding specificity profile of selected CD2-specific DARPin The binding specificity of the selected CD2 ankyrin repeat protein generated in Example 1 was evaluated in reporter cell binding experiments using CD2-expressing Jurkat E6-1 cells and Jurkat CD2 knockout cells. Briefly: 0.5 x 10 5 Each cell / well was resuspended in 50 μl of each tested DARPin dilution (1000 nM, 1:5 serial dilution with FACS buffer (PBS + 2% FBS)) and incubated at 4°C for 1 hour. After washing twice with FACS buffer, the cells were resuspended in 50 μl of anti-DARPin-AF488 (AF488-labeled anti-DARPin rabbit monoclonal AB 1.1.1) and incubated at 4°C for 30 minutes. After washing twice with FACS buffer, the cell pellet was diluted 1 / 10 with BD Cytofix TM Cells were fixed in (BD) at RT for 20 minutes. 5000 fixed cells were counterstained red with 5uM DRAQ5 (Abcam) and acquired using a Mirrorball (SPTlabtech). Using Cellista software, data for DRAQ5 counterstaining and Alexa Fluor 488 signaling were collected in the red (FL-4) and green (FL-2) channels of the Mirrorball, respectively. The amount of anti-DARPin 1.1.1 (AF488) antibody bound to each cell was measured in green fluorescence intensity [median (mean intensity of FL-2)] and plotted using GraphPad Prism software. As seen in Figure 2(AB), all tested proteins with binding specificity to CD2 (DARPin protein #1, DARPin protein #3, DAPRin protein #15) bound to Jurkat E6-1 (CD2 positive; Figure 2A) but not to Jurkat CD2 KO (knockout cells not expressing the CD2 target protein; Figure 2B).

[0224] TIFF2026524079000007.tif42170

[0225] Example 5: Dissociation constant (K) of ankyrin repeat protein with binding specificity to human CD2 determined by surface plasmon resonance (SPR) analysis. D ) decision The binding affinity of four purified ankyrin repeat proteins to biotinylated recombinant human CD2 targets was analyzed by multi-trace SPR using a Bruker Sierra SPR-32 instrument (Bruker) with PBS-T (PBS containing 0.005% Tween-20) as the running buffer. The Bruker Biotin-Tag Capture Sensor chip was prepared according to the manufacturer's instructions. Biotinylated human CD2 targets were captured in channels 1 and 2 at 437 and 469 RU, respectively (10 μg / mL, 30 sec). The running buffer was PBS pH 7.4 containing 0.005% Tween-20 (PBST). DARPin proteins with binding specificity to CD2 were injected into channel 1 at 25 μl / min for 240 seconds each, from the lowest to the highest concentration (270 nM - 0.041 nM, 3-fold dilution), and association was observed. Dissociation was recorded for 500 seconds (25 μL / min). Ligands were regenerated with a 60-second pulse (25 μL / min) of 10 mM HCl. Data were dual-referenced (injection control (Ch1B) and buffer injection (Ch2)) and fitted to a 1:1 Langmuir model. Table 7 below shows the binding parameters of selected DARPin proteins #1 to #4 as measured by multi-trace PCR.

[0226] TIFF2026524079000008.tif41170

[0227] This specification is best understood in light of the teachings of the references cited herein. The embodiments herein are illustrative of embodiments of the invention and should not be construed as limiting the scope of the invention. Those skilled in the art will readily recognize that many other embodiments are encompassed within the invention. All publications, patents and GenBank sequences cited herein are incorporated by reference in their entirety. To the extent that any material incorporated by reference is inconsistent with or contradicts this specification, this specification shall prevail. Any reference herein is not an admission that such reference is prior art of the invention.

[0228] Those skilled in the art will recognize, or can confirm by routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed in the following claims.

[0229] TIFF2026524079000009.tif255170TIFF2026524079000010.tif255170TIFF2026524079000011.tif255170TIFF20265240790 00012.tif255170TIFF2026524079000013.tif255170TIFF2026524079000014.tif255170TIFF2026524079000015.tif255170

Claims

1. A recombinant binding protein containing an ankyrin repeat domain that has binding specificity to CD2.

2. The recombinant binding protein according to claim 1, wherein the 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: 7-14, 16, and 17, and (2) a sequence in which up to nine amino acids in any one of SEQ ID NOs: 7-14, 16, and 17 are replaced by other amino acids.

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

4. A recombinant binding protein according to any one of claims 1 to 3, i. The 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 in which 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 of SEQ ID NO: 7 is replaced by another amino acid; 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 in which 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 of SEQ ID NO: 8 is replaced by another amino acid; ii. The ankyrin repeat domain comprises a first ankyrin repeat module including an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 16 and (2) an amino acid sequence in which 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 of SEQ ID NO: 16 is replaced by another amino acid, and a second ankyrin repeat module including an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 17 and (2) an amino acid sequence in which 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 of SEQ ID NO: 17 is replaced by another amino acid, Recombinant binding protein.

5. The recombinant binding protein according to any one of claims 1 to 4, wherein the ankyrin repeat domain comprises a first ankyrin repeat module, a second ankyrin repeat module, and a third ankyrin repeat module, and optionally, the first, second, and third ankyrin repeat modules each independently include an amino acid sequence selected from the group consisting of (1) one of sequence numbers 7-14, 16, and 17, and (2) an amino acid sequence in which 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 in one of sequence numbers 7-14, 16, and 17 is substituted with other amino acids.

6. A recombinant binding protein according to any one of claims 1, 2, and 5, i. The ankyrin repeat domain comprises a first ankyrin repeat module including an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 9 and (2) an amino acid sequence in which 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 of SEQ ID NO: 9 is replaced by another amino acid; a second ankyrin repeat module including an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 10 and (2) an amino acid sequence in which 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 of SEQ ID NO: 10 is replaced by another amino acid; and a third ankyrin repeat module including an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 11 and (2) an amino acid sequence in which 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 of SEQ ID NO: 11 is replaced by another amino acid; ii. The ankyrin repeat domain comprises a first ankyrin repeat module including an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 12 and (2) an amino acid sequence in which 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 of SEQ ID NO: 12 is replaced by another amino acid; a second ankyrin repeat module including an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 13 and (2) an amino acid sequence in which 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 of SEQ ID NO: 13 is replaced by another amino acid; and a third ankyrin repeat module including an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 14 and (2) an amino acid sequence in which 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 of SEQ ID NO: 14 is replaced by another amino acid. Recombinant binding protein.

7. A recombinant binding protein according to any one of claims 2 to 6, further comprising an N-terminal capping module containing an amino acid sequence in which 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 in any one of sequence numbers 18 to 20 is substituted by other amino acids, and / or a C-terminal capping module containing an amino acid sequence in which 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 in any one of sequence numbers 22 to 24 is substituted by other amino acids.

8. The recombinant binding protein according to any one of claims 1 to 7, wherein the ankyrin repeat domain contains an amino acid sequence that is 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 sequence numbers 1 to 3, 15.

9. The recombinant binding protein according to any one of claims 1 to 8, wherein the CD2 is human CD2.

10. The recombinant binding protein is approximately 10 －７ M or less, or about 10 －６ M or less, or about 10 －７ M or less, or about 10 －８ A recombinant binding protein according to any one of claims 1 to 9, which binds to human soluble CD2 in PBS with a dissociation constant (KD) of M or less.

11. The recombinant binding protein according to any one of claims 1 to 10, further comprising at least one binding site having binding specificity to a protein expressed on the surface of immune cells, preferably T lymphocytes (T cells).

12. The recombinant binding protein according to claim 11, wherein the binding portion comprises an ankyrin repeat domain having binding specificity to CD3, and optionally, the ankyrin repeat domain comprises an amino acid sequence that is 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, 26-28.

13. The recombinant binding protein according to any one of claims 1 to 12, further comprising at least one half-life extension portion.

14. The recombinant binding protein according to claim 13, wherein the half-life extension portion comprises an ankyrin repeat domain that binds to human serum albumin, and optionally, the ankyrin repeat domain comprises an amino acid sequence that is 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.

15. The recombinant binding protein according to any one of claims 1 to 14, further comprising a tumor-associated binding protein, preferably an ankyrin repeat protein having binding specificity to a tumor-associated antigen.

16. A nucleic acid encoding a recombinant binding protein according to any one of claims 1 to 15.

17. A nucleic acid-containing vector according to claim 28, wherein the vector is optionally a DNA vector, an RNA vector, a plasmid, a cosmid, or a viral vector.

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

19. A method for producing recombinant binding protein, comprising culturing the cells described in claim 18 and collecting recombinant binding protein from the cells and / or culture medium.

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

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

22. The method according to claim 21, wherein the cancer is a humoral tumor.

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