Multiselective protein

Abstract

The present invention relates to multispecific proteins comprising engineered ankyrin repeat domains with binding specificities for different targets, such as CD40 and FAR. In addition, the invention relates to nucleic acids encoding such multispecific proteins, pharmaceutical compositions, pharmaceutical compositions comprising such multispecific proteins or nucleic acids, and the use of such binding proteins, nucleic acids, or pharmaceutical compositions in mammals, including humans, in methods for treating or diagnosing diseases, such as cancer.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit and priority of European Patent Application No. 20174847, filed with the European Patent Office on 14 May 2020, and European Patent Application No. 20181498, filed with the European Patent Office on 22 June 2020. The contents of European Patent Application Publications No. 20174847 and No. 20181498, including all tables, figures, and claims, are incorporated herein by reference in their entirety.

[0002] Field of Invention The present invention relates to a multiselective protein comprising an ankyrin repeat domain designed to have binding specificity to different targets, such as CD40 and FAP. In addition, the present invention relates to such multiselective proteins, nucleic acids encoding pharmaceutical compositions, pharmaceutical compositions comprising such multiselective proteins or nucleic acids, and the use of such binding proteins, nucleic acids, or pharmaceutical compositions in mammals, including humans, in methods for treating or diagnosing diseases such as cancer. [Background technology]

[0003] The tumor necrosis factor receptor (TNFR) superfamily member CD40 is a crucial costimulatory receptor that, when engaged by its ligand (CD40L) or agonist antibody, is involved in regulating a wide range of molecular and cellular processes, including the initiation and progression of cellular and humoral adaptive immunity. For example, CD40 engagement on the surface of dendritic cells has been demonstrated to promote their cytokine production, induce the expression of costimulatory molecules on their surface, and facilitate antigen presentation. Overall, the effects of CD40 signaling "allow" dendritic cells to mature and achieve all the characteristics necessary to effectively induce T cell activation and differentiation. CD40 signaling in B cells promotes germinal center formation, immunoglobulin (Ig) isotype switching, somatic high-frequency mutations in Ig to enhance affinity for antigens, and finally, the formation of long-lived plasma cells and memory B cells. Furthermore, the CD40 pathway has been shown to be important for the survival of many cell types, including germinal center B cells, dendritic cells, and endothelial cells, under normal and inflammatory conditions. Deregulation of CD40 signaling has been observed in various autoimmune diseases. Furthermore, this broad function highlights the importance of the CD40 receptor for the production of adaptive immune responses.

[0004] CD40 was first characterized on B cells, but is also expressed on dendritic cells, monocytes, platelets, and macrophages, as well as on non-hematopoietic cells such as myofibroblasts, fibroblasts, epithelial cells, and endothelial cells. The ligand for CD40, known as CD154 or CD40L, is primarily expressed on activated T cells, as well as activated B cells and platelets, and is also induced in monocytes, natural killer cells, mast cells, and basophils under inflammatory conditions.

[0005] CD40 is recognized as a desirable target for tumor immunotherapy because it can activate both the innate and adaptive immune systems. Several reports have confirmed that CD40 stimulation can enhance the antitumor immune response through dendritic cell maturation. Activation of dendritic cells by CD40 agonists leads to increased survival and secretion of IL-1, IL-6, IL-8, IL-12, TNF-α, and macrophage inflammatory protein-1α. In addition, CD40 activation induces upregulation of co-stimulatory molecules such as MHC class II, LFA-3, CD80, and CD86, which promote antigen presentation, priming, and cross-priming of T helper cells (Th) and cytotoxic T lymphocytes (CTLs), respectively. Agonist antibodies against CD40 have proven effective in preclinical mouse tumor models. However, while their clinical use has shown some antitumor efficacy, the clinical development of agonist anti-CD40 antibodies is likely hindered by dose-limiting toxicity and the resulting low efficacy.

[0006] Therefore, novel CD40-specific binding proteins, as well as therapeutic and diagnostic approaches for the treatment and characterization of diseases, including cancer, remain necessary and beneficial for CD40-specific binding and activation. In particular, there is a need for a new generation of agonists that can effectively engage CD40 while avoiding undesirable side effects. [Overview of the Initiative]

[0007] Based on the disclosures provided herein, those skilled in the art will recognize many equivalents to the particular embodiments of the invention described herein, or can confirm them by routine experimentation alone. Such equivalents are intended to be encompassed by the following embodiment (E). E1. A recombinant protein comprising a first ankyrin repeat domain that specifically binds to fibroblast activation protein (FAP) and a second ankyrin repeat domain that specifically binds to CD40. The recombinant protein described in E1 further comprises a third ankyrin repeat domain that specifically binds to E2.CD40. E3. The recombinant protein described in E2, wherein the ankyrin repeat domain is arranged from the N-terminus to the C-terminus according to the following formula: (FAP binding domain)-(CD40 binding domain)-(CD40 binding domain). E4 Recombinant protein as described in any one of E1 to E3, further comprising a half-life extension portion. E5. The recombinant protein according to E4, wherein the half-life extension portion includes a fourth ankyrin repeat domain that specifically binds to serum albumin. E6. The recombinant protein described in E5, wherein the ankyrin repeat domain is arranged from the N-terminus to the C-terminus according to the following formula: (serum albumin binding domain)-(FAP binding domain)-(CD40 binding domain)-(CD40 binding domain). E7. A recombinant protein according to any one of E1 to E6, further comprising a linker between any of the FAP-binding domains, the CD40-binding domain(s), and the half-life extension portion. E8. A recombinant protein described in any one of E1 to E7, comprising the following formula from the N-terminus to the C-terminus: (FAP-binding domain)-(linker)-(CD40-binding domain)-(linker)-(CD40-binding domain). E9. A recombinant protein described in any one of E1 to E8, comprising the following formula from the N-terminus to the C-terminus: (serum albumin-binding domain)-(linker)-(FAP-binding domain)-(linker)-(CD40-binding domain)-(linker)-(CD40-binding domain). E10. The recombinant protein described in E4, wherein the half-life extension portion includes an immunoglobulin heavy chain constant domain. E11. The recombinant protein described in E10, wherein the immunoglobulin domain is the Fc domain of IgA1, IgA2, IgD, IgE, IgM, IgG1, IgG2, IgG3, or IgG4 immunoglobulin. E12. The recombinant protein described in E11, wherein the Fc domain is the Fc domain of human IgG1 immunoglobulin. E13. The recombinant protein described in E12, wherein the Fc domain is modified to reduce effector function. E14. A recombinant protein described in any one of E1 to E13, wherein the FAP is human FAP. E15. A recombinant protein described in any one of E1 to E14, wherein the CD40 is human CD40. E16. A recombinant protein as described in any one of E5 to E19, wherein the serum albumin in question is human serum albumin (HSA). E17. A recombinant protein described in any one of E10 to E13, wherein the immunoglobulin heavy chain constant domain is a human immunoglobulin heavy chain constant domain. E18. A recombinant protein according to any one of E1 to E17, wherein the binding of the recombinant protein to FAP does not reduce the protease activity of FAP by more than 25%, more than 20%, more than 15%, more than 10%, or more than 5%. E19. A recombinant protein according to any one of E1 to E18, wherein the FAP-binding domain comprises an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 2, and optionally, the A at the second to last position of SEQ ID NO: 2 is replaced with L, and / or the A at the last position of SEQ ID NO: 2 is replaced with N. E20. The recombinant protein described in E19, wherein the FAP-binding domain contains the amino acid sequence of Sequence ID No. 2. E21. The recombinant protein according to any one of E1 to E18, wherein the FAP-binding domain comprises an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 8, and optionally, the A at the second to last position of SEQ ID NO: 2 is replaced with L, and / or the A at the last position of SEQ ID NO: 8 is replaced with N. E22. The recombinant protein described in E21, wherein the FAP-binding domain contains the amino acid sequence of Sequence ID No. 8. E23. A recombinant protein according to any one of E1 to E18, wherein the FAP-binding domain contains an amino acid sequence that is at least 80%, at least 81%, 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 to any one of Sequence ID No. 9 and 28 to 38. E24. The recombinant protein described in E23, wherein the FAP-binding domain contains one of the amino acid sequences of SEQ ID NO: 9 and 28-38. E25. The recombinant protein according to E23, wherein the FAP-binding domain comprises an amino acid sequence that is at least 80%, at least 81%, 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 to any one of Sequence IDs 9, 28-31, and 38, and optionally, the second-to-last A is replaced with L, and / or the last A is replaced with N. E26. The recombinant protein according to E25, wherein the FAP-binding domain comprises an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 28, and optionally, the second-to-last A is replaced with L, and / or the last A is replaced with N. E27. The recombinant protein according to E23, wherein the FAP-binding domain comprises an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs. 32-37, and optionally, the second-to-last L is replaced with A, and / or the last N is replaced with A. E28. The recombinant protein according to E27, wherein the FAP-binding domain comprises an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 34, and optionally, the second-to-last L is replaced with A and / or the last N is replaced with A. E29. The recombinant protein according to any one of E1 to E28, wherein the FAP-binding domain comprises an amino acid sequence that is at least 80%, at least 81%, 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 to any one of Sequence IDs 2, 8, 9, and 28-37, and (ii) further comprises G, S, or GS at its N-terminus. E30. The recombinant protein according to any one of E1 to E29, wherein the FAP-binding domain comprises an amino acid sequence that is at least 90% identical to any one of Sequence IDs 2, 8, 9, and 28-37, and (ii) further comprises G, S, or GS at its N-terminus. E31. The recombinant protein according to any one of E1 to E30, wherein the CD40 binding domain or each of the CD40 binding domains independently comprises an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 3, and optionally, the A at the second to last position of SEQ ID NO: 3 is replaced with L, and / or the A at the last position of SEQ ID NO: 3 is replaced with N. E32. A recombinant protein according to any one of E1 to E31, wherein the CD40 binding domain or each of the CD40 binding domains contains the amino acid sequence of SEQ ID NO: 3. E33. A recombinant protein according to any one of E1 to E30, wherein the CD40 binding domain or each of the CD40 binding domains independently contains an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs. E34. A recombinant protein according to any one of E1 to E30 and E33, wherein the CD40-binding domain or each of the CD40-binding domains contains one of the amino acid sequences of SEQ ID NOs. 10 and 43-50. E35. The recombinant protein according to any one of E1-E30 and E33, wherein the CD40 binding domain or each of the CD40 binding domains independently comprises an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs: 10, 43, 44, and 48-50, and optionally, the second-to-last A is replaced with L and / or the last A is replaced with N. E36. The recombinant protein according to E35, wherein the CD40-binding domain or each of the CD40-binding domains independently comprises an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 43, and optionally, the second-to-last A is substituted with L, and / or the last A is substituted with N. E37. The recombinant protein according to any one of E1 to E30 and E33, wherein the CD40 binding domain or each of the CD40 binding domains independently comprises an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs. E38. The recombinant protein according to E37, wherein the CD40-binding domain or each of the CD40-binding domains independently comprises an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 47, and optionally, the second-to-last L is replaced with A and / or the last N is replaced with A. E39. The recombinant protein according to any one of E1 to E38, wherein the CD40 binding domain or each of the CD40 binding domains independently comprises an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs: 3, 10, and 43-49, and further comprises G, S, or GS at its N-terminus. E40. The recombinant protein according to any one of E1 to E39, wherein the CD40-binding domain or each of the CD40-binding domains independently comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 3, 10, and 43-49, and (ii) further comprises G, S, or GS at its N-terminus. E41. The recombinant protein described in any one of E1 to E40, wherein the CD40 binding domain or each of the CD40 binding domains independently contains Q at position 8, L at position 15, R at position 143, and / or Q at position 147, where the position numbers correspond to the positions of SEQ ID NO: 3. E42. The recombinant protein described in any one of E1 to E41, wherein the CD40 binding domain or each of the CD40 binding domains independently contains Q at position 8, L at position 15, R at position 143, and Q at position 147, where the position numbers correspond to the positions of SEQ ID NO: 3. E43. (a) The FAP-binding domain comprises an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs: 2, 8, 9, and 28-37, wherein its N-terminus optionally further comprises G, S, or GS, the second to last position may be L or A, and the last position may be N or A. (b) The recombinant protein according to any one of E1 to E42, wherein the CD40 binding domain or each of the CD40 binding domains independently comprises an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs: 3, 10, and 43-49, wherein the N-terminus optionally further comprises G, S, or GS, the second to last position may be L or A, and the last position may be N or A. E44. (a) The FAP-binding domain comprises an amino acid sequence that is at least 90% identical to any one of sequence numbers 2, 8, 9, and 28-37, wherein its N-terminus optionally further comprises G, S, or GS, the second to last position may be L or A, and the last position may be N or A. (b) The recombinant protein according to E43, wherein the CD40 binding domain or each of the CD40 binding domains independently comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NOs: 3, 10, and 43-49, and the N-terminus optionally further comprises G, S, or GS, the second to last position may be L or A, and the last position may be N or A. E45. (a) The FAP-binding domain comprises an amino acid sequence that is at least 95% identical to one of sequence numbers 2, 8, 9, and 28-37, wherein its N-terminus optionally further comprises G, S, or GS, the second to last position may be L or A, and the last position may be N or A. (b) The recombinant protein according to E43, wherein the CD40 binding domain or each of the CD40 binding domains independently comprises an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 3, 10, and 43-49, and the N-terminus optionally further comprises G, S, or GS, the second to last position may be L or A, and the last position may be N or A. E46. (a) The FAP-binding domain comprises one of the amino acid sequences of SEQ ID NOs: 2, 8, 9, and 28-37, wherein its N-terminus optionally further comprises G, S, or GS, the second to last position may be L or A, and the last position may be N or A. (b) The recombinant protein according to E43, wherein the CD40 binding domain or each of the CD40 binding domains independently comprises one amino acid sequence of SEQ ID NOs: 3, 10, and 43-49, and its N-terminus optionally further comprises G, S, or GS, the second to last position may be L or A, and the last position may be N or A. E47. A recombinant protein according to any one of E5-E9, E14-E16, and E18-E46, wherein the serum albumin-binding domain comprises an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 1, and optionally, the second-to-last A is replaced with L, and / or the last A is replaced with N. E48. A recombinant protein according to any one of E5-E9, E14-E16, and E18-E47, wherein the serum albumin-binding domain contains the amino acid sequence of SEQ ID NO: 1. E49. The serum albumin-binding domain is at least 80%, at least 81%, 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%, and Recombinant proteins according to any one of E5-E9, E14-E16, and E18-E46, wherein each contains an amino acid sequence that is 100% identical, and optionally, the second-to-last A in any one of SEQ ID NOs. 39-40 and 42 is replaced with L, and / or the last A in any one of SEQ ID NOs. 39-40 and 42 is replaced with N, and optionally, the second-to-last L in SEQ ID NO. 41 is replaced with A, and / or the last N in SEQ ID NO. 41 is replaced with A. E50. A recombinant protein according to any one of E5-E9, E14-E16, E18-E45, and E49, wherein the serum albumin-binding domain contains one amino acid sequence from any one of SEQ ID NOs. 39-42. E51. The serum albumin-binding domain contains an amino acid sequence that is (i) at least 80%, at least 81%, 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 to any one of SEQ ID NOs. 1 and 39-41, or (ii) at least identical to SEQ ID NOs. 42. Recombinant proteins according to any one of E5-E9, E14-E16, and E18-E50, comprising amino acid sequences that are identical by 80%, at least 81%, 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%, and further comprising G, S, or GS at its N-terminus. E52. A recombinant protein according to any one of E5-E9, E14-E16, and E18-E51, wherein the serum albumin-binding domain comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 1 and 39-41, or (ii) an amino acid sequence that is at least 90% identical to SEQ ID NOs: 42, and further comprises G, S, or GS at its N-terminus. E53. A recombinant protein according to any one of E5-E9, E14-E16, and E18-E52, wherein the serum albumin-binding domain comprises (i) one amino acid sequence of SEQ ID NO: 1 and 39-41, or (ii) the amino acid sequence of SEQ ID NO: 42, further comprising G, S, or GS at its N-terminus. E54. The linker contains the amino acid sequence of SEQ ID NO: 4, and is a recombinant protein as described in any one of E8 to E53. E55. A recombinant protein described in any one of E1 to E54, wherein the protein contains exactly four ankyrin repeat domains. E56. Recombinant protein comprising an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and wherein the protein specifically binds to FAP and CD40. E57. The recombinant protein according to E56, wherein the recombinant protein contains an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 5. E58. A recombinant protein according to any one of E56 to E57, wherein the protein contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 5. E59. A recombinant protein according to any one of E56 to E58, wherein the protein contains the amino acid sequence of SEQ ID NO: 5. E60. The recombinant protein according to E56, wherein the protein contains an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 6. E61. The recombinant protein according to E56 or E60, wherein the protein contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 6. E62. A recombinant protein according to any one of E56 and E60-E61, wherein the protein contains the amino acid sequence of Sequence ID No. 6. E63. A recombinant protein as described in any one of E56 to E62, wherein the FAP is a human FAP. E64. A recombinant protein according to any one of E56 to E63, wherein the CD40 is human CD40. E65. A recombinant protein according to any one of E1 to E64, wherein the FAP-binding domain binds to human FAP with a KD value of 100 nM, 90 nM, 80 nM, 75 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 250 pM, 200 pM, 150 pM, 140 pM, 130 pM, or 120 pM or less. E66. A recombinant protein described in any one of E1 to E65, wherein the FAP-binding domain binds to human FAP with a KD value of 100 nM or less. E67. A recombinant protein according to any one of E1 to E66, wherein the FAP-binding domain binds to human FAP with a KD value of 1 nM or less. E68. A recombinant protein described in any one of E1 to E67, wherein the FAP-binding domain binds to human FAP with a KD value of 120 pM or less. E69. A recombinant protein according to any one of E1 to E68, wherein the CD40-binding domain or each of the CD40-binding domains independently binds to human CD40 with a KD value of 100 nM, 90 nM, 80 nM, or 75 nM or less. E70. A recombinant protein according to any one of E1 to E69, wherein the CD40-binding domain or each of the CD40-binding domains independently binds to human CD40 with a KD value of 100 nM or less. E71. A recombinant protein according to any one of E1 to E70, wherein the CD40-binding domain or each of the CD40-binding domains independently binds to human CD40 with a KD value of 75 nM or less. E72. A recombinant protein according to any one of E1 to E71, wherein the serum albumin-binding domain binds to human serum albumin having a KD value of 100 nM, 90 nM, 80 nM, 75 nM, 70 nM, 60 nM, 50 nM, 40 nM, or 35 nM or less. E73. A recombinant protein according to any one of E1 to E72, wherein the serum albumin-binding domain binds to human serum albumin having a KD value of 100 nM or less. E74. A recombinant protein according to any one of E1 to E73, wherein the serum albumin-binding domain binds to human serum albumin having a KD value of 50 nM or less. E75. A recombinant protein according to any one of E1 to E74, wherein the serum albumin-binding domain binds to human serum albumin having a KD value of 35 nM or less. E76. A recombinant protein according to any one of E1 to E75, wherein the recombinant protein binds to human FAP with a KD value of 100nM, 90nM, 80nM, 75nM, 70nM, 60nM, 50nM, 40nM, 30nM, 20nM, 10nM, 5nM, 2nM, 1nM, 900pM, 800pM, 700pM, 600pM, 500pM, 400pM, or 300pM or less. E77. A recombinant protein described in any one of E1 to E76, wherein the recombinant protein binds to human FAP with a KD value of 100 nM or less. E78. A recombinant protein described in any one of E1 to E77, wherein the recombinant protein binds to human FAP with a KD value of 1 nM or less. E79. A recombinant protein described in any one of E1 to E78, wherein the recombinant protein binds to human FAP with a KD value of 500 pM or less. E80. A recombinant protein described in any one of E1 to E79, wherein the recombinant protein binds to human FAP with a KD value of 300 pM or less. E81. A recombinant protein according to any one of E1 to E80, wherein the recombinant protein binds to human CD40 with a KD value of 100 nM, 90 nM, 80 nM, 75 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 250 pM, 200 pM, 150 pM, 140 pM, 130 pM, 120 pM, 115 pM, 110 pM, 105 pM, or 100 pM or less. E82. The recombinant protein described in any one of E1 to E81, wherein the recombinant protein binds to human CD40 with a KD value of 100 nM or less. E83. The recombinant protein described in any one of E1 to E82, wherein the recombinant protein binds to human CD40 with a KD value of 1 nM or less. E84. A recombinant protein described in any one of E1 to E83, wherein the recombinant protein binds to human CD40 with a KD value of 500 pM or less. E85. The recombinant protein described in any one of E1 to E84, wherein the recombinant protein binds to human CD40 with a KD value of 100 pM or less. E86. The recombinant protein described in any one of E1 to E85, wherein the recombinant protein binds to human serum albumin with a KD value of 100 nM, 90 nM, 80 nM, 75 nM, 70 nM, 60 nM, or 50 nM or less. E87. A recombinant protein described in any one of E1 to E86, wherein the recombinant protein binds to human serum albumin with a KD value of 100 nM or less. E88. A recombinant protein described in any one of E1 to E87, wherein the recombinant protein binds to human serum albumin with a KD value of 75 nM or less. E89. A recombinant protein described in any one of E1 to E88, wherein the recombinant protein binds to human serum albumin with a KD value of 50 nM or less. E90. The KD is a recombinant protein as described in any one of E65-E89, measured in PBS by surface plasmon resonance (SPR). E91. The recombinant protein described in E90, whose KD is measured using a Biacore T200 instrument. E92. The KD is a recombinant protein as described in any one of E65 to E89, measured by bio-layer interferometry (BLI). E93. The recombinant protein described in E92, whose KD is measured using a ForteBio Octet instrument. E94. The recombinant protein is evaluated by an in vitro B cell activation assay, with a molecular weight of approximately 100 nM or less, approximately 75 nM or less, approximately 65 nM or less, approximately 55 nM or less, approximately 45 nM or less, approximately 35 nM or less, approximately 25 nM or less, approximately 15 nM or less, approximately 10 nM or less, approximately 5 nM or less, approximately 4 nM or less, approximately 3 nM or less, approximately 2 nM or less, approximately 1 nM, or approximately 0.1 nM or less, approximately 0.01 nM to approximately 50 nM, approximately 0.01 nM to approximately 25 nM, approximately 0.01 nM to approximately 10 nM, approximately 0.01 nM to approximately 5 nM, approximately 0.01 nM to approximately 1 nM, approximately 0.01 nM to approximately 0.1nM, about 0.01nM to about 0.07nM, about 0.04nM to about 50nM, about 0.04nM to about 25nM, about 0.04nM to about 10nM, about 0.04nM to about 5nM, about 0.04nM to about 1nM, about 0.04nM to about 0.1nM, about 0.04nM to about 0.07n M, about 0.1nM to about 50nM, about 0.1nM to about 25nM, about 0.1nM to about 10nM, about 0.1nM to about 5nM, about 0.1nM to about 1nM, about 0.1nM to about 0.9nM, about 0.1nM to about 0.85nM, about 0.18nM to about 0.85nM half-effective concentration (EC 50 A recombinant protein described in any one of E1 to E93, having the following characteristics: E95. A recombinant protein according to any one of E1 to E94, wherein the recombinant protein has an EC50 of approximately 10 nM or less, as can be evaluated by an in vitro B cell activation assay. E96. A recombinant protein according to any one of E1 to E95, wherein the recombinant protein has an EC50 of approximately 1 nM or less, as can be evaluated by an in vitro B cell activation assay. E97. The recombinant protein according to any one of E1 to E96, wherein the recombinant protein has an EC50 of about 0.1 nM to about 1 nM, preferably about 0.18 nM to about 0.85 nM, as evaluated by an in vitro B cell activation assay. E98. The recombinant protein according to any one of E1 to E97, wherein the recombinant protein has an EC50 of about 0.01 nM to about 0.1 nM, preferably about 0.04 nM to about 0.07 nM, as evaluated by an in vitro B cell activation assay. E99. A recombinant protein according to any one of E94 to E98, wherein the B cell activation assay is a human B cell activation assay. E100. The relevant EC 50 However, it is a recombinant protein listed in one of the following categories, E94-E99, as measured using GraphPad Prism (version 8.1.2). E101. Recombinant protein, It comprises a first ankyrin repeat domain that specifically binds to serum albumin, a second ankyrin repeat domain that specifically binds to fibroblast-activating protein (FAP), a third ankyrin repeat domain that specifically binds to CD40, and a fourth ankyrin repeat domain that specifically binds to CD40. Here, the ankyrin repeat domain is a recombinant protein in which the following is arranged from the N-terminus to the C-terminus: (serum albumin binding domain)-(FAP binding domain)-(CD40 binding domain)-(CD40 binding domain). E102. The FAP-binding domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, and has a K content of 100 nM or less, preferably 1 nM or less, more preferably 120 pM or less. D A recombinant protein described in E101 that binds to human FAP at a specific value. E103. The recombinant protein described in E101 or E102, wherein the FAP-binding domain contains the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 8. E104. Each of the CD40 binding domains independently contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, and has a K content of 100 nM or less, preferably 75 nM or less. D A recombinant protein listed in any of E101-E103 that binds to human CD40 by a specific value. E105. A recombinant protein according to any one of E101 to E104, wherein each of the CD40-binding domains independently contains Q at position 8, L at position 15, R at position 143, and / or Q at position 147, where the position numbers correspond to the positions of SEQ ID NO: 3, preferably each of the CD40-binding domains independently contains Q at position 8, L at position 15, R at position 143, and Q at position 147, where the position numbers correspond to the positions of SEQ ID NO: 3. E106. A recombinant protein according to any one of E101 to E105, wherein each of the CD40-binding domains contains the amino acid sequence of SEQ ID NO: 3. E107. The serum albumin-binding domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, and has a K content of 100 nM or less, preferably 50 nM or less, more preferably 35 nM or less. D A recombinant protein listed in any of E101-E106 that binds to human serum albumin at a specific value. E108. A recombinant protein described in any one of E101 to E107, wherein the serum albumin domain contains the amino acid sequence of SEQ ID NO: 1. E109. A recombinant protein described in any one of E101 to E108, comprising the following formula from the N-terminus to the C-terminus: (serum albumin-binding domain)-(linker)-(FAP-binding domain)-(linker)-(CD40-binding domain), wherein the linker contains the amino acid sequence of SEQ ID NO: 4. E110. A recombinant protein described in any one of E101 to E110, wherein the protein contains exactly four ankyrin repeat domains. E111. Recombinant protein containing the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. E112. Recombinant protein containing the amino acid sequence of SEQ ID NO: 5. E113. Recombinant protein containing the amino acid sequence of SEQ ID NO: 6. E114. Recombinant protein containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, with a K content of 100 nM or less. D Recombinant proteins that bind to human FAP, human CD40, and human serum albumin. E115. A recombinant protein according to any one of E5 to E114, wherein the protein can simultaneously bind to CD40, FAP, and serum albumin. E116. A recombinant protein according to any one of E1 to E115, wherein the protein has a half-effect concentration (EC50) of approximately 0.1 nM to approximately 5 nM, as evaluated by an in vitro human B cell activation assay. E117.A recombinant protein described in any one of E1 to E116, wherein the binding of the protein to FAP does not inhibit the prolyl endopeptidase activity of FAP by more than 25%. E117a. The recombinant protein described in any one of E1 to E117, wherein the recombinant protein specifically binds to the N-terminal cysteine-rich domain 1 (CRD1) of the CD40 receptor (amino acids 23-59 of SEQ ID NO: 51). E118. A recombinant protein described in any one of E1 to E117, or a nucleic acid encoding an ankyrin repeat domain as defined in any one of E1 to E117. E119. The nucleic acid described in E118, containing the nucleotide sequence of sequence number 58. E120. A recombinant protein containing the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 58. E121. Recombinant protein comprising an amino acid sequence encoded by a nucleotide sequence that is at least 85%, 90%, 95%, or 99% identical to the sequence of Sequence ID No. 58. E122. A recombinant protein containing an amino acid sequence encoded by a nucleic acid sequence that can hybridize to the nucleotide sequence of SEQ ID NO: 58 under very stringent conditions. A vector containing nucleic acids as described in E123.E118 or E119. A host cell containing the nucleic acid described in E124.E118 or E119. Host cells containing the E125.E123 vector. E126. The host cell described in E124 or E125, wherein the cell is a bacterial cell. E127. A host cell described in any one of E124 to E126, wherein the host cell is Escherichia coli. E128. The host cell described in E124 or E125, wherein the cell is a eukaryotic cell. E129. A method for producing a recombinant protein according to any one of E1 to E117 and E120 to E122, comprising culturing a host cell according to any one of E124 to E128 under conditions in which the recombinant protein is expressed. E130. The method according to E129, further comprising isolating the recombinant protein. A pharmaceutical composition comprising a recombinant protein described in any one of E1 to E117 and E120 to E122, or a nucleic acid described in E118 or E119, and optionally a pharmaceutically acceptable carrier or excipient. E132. A method for locally activating CD40 in CD40-expressing cells in a mammal, including a human, comprising the steps of administering a recombinant protein described in any one of E1 to E117 and E120 to E122 to the mammal, a nucleic acid described in E118 or E119, or a pharmaceutical composition described in E131. E133. The method according to E132, wherein the CD40-expressing cells are located in a tumor, preferably a solid tumor. E134. The method according to E133, wherein the tumor contains cells that express FAP. E135. A method for treating a medical condition, comprising administering to a subject in need of a method for treating the medical condition a therapeutically effective amount of a recombinant protein according to any one of E1-E117 and E120-E122, a nucleic acid according to E118 or E119, or a pharmaceutical composition according to E131. E136. The method described in E134, wherein the subject is a human. E137. The method described in E134 or E135, wherein the medical condition is cancer. E138. The method according to E136, wherein the cancer is a solid tumor. E139. The method according to E136 or E137, wherein the cancer includes cells that express FAP. E140. The method according to any one of E137 to E139, wherein the cancer is brain cancer, bladder cancer, breast cancer, clear cell renal cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, gastric cancer, head and neck cancer, head and neck squamous cell carcinoma, lip cancer, oral cancer, liver cancer, cervical cancer, lung squamous cell carcinoma, melanoma, mesothelioma, non-small cell lung cancer (NSCLC), non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, urothelial carcinoma, sarcoma, small cell lung cancer (SCLC), head and neck squamous cell carcinoma (SCCHN), triple-negative breast cancer, or thyroid cancer. E141. The method according to any one of E137 to E140, wherein the cancer is an adrenocortical tumor, hydatidiform soft part sarcoma, carcinoma, chondrosarcoma, colorectal cancer, tendonoid, fibroplastic round cell tumor, endocrine tumor, yolk sac tumor, epithelioid hemangioendothelioma, Ewing's sarcoma, germ cell tumor, hepatoblastoma, hepatocellular carcinoma, melanoma, renal tumor, neuroblastoma, non-rhabdomyosarcoma soft part sarcoma (NRSTS), osteosarcoma, paravertebral sarcoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, synovial sarcoma, or Wilms' tumor. E142. The method according to E137, wherein the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or chronic myeloid leukemia (CML). E143. The method according to E137, wherein the cancer is diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), or small lymphocytic lymphoma (SLL). E144. The method according to any one of E132 to E143, wherein the recombinant protein, nucleic acid, or pharmaceutical composition is administered intravenously. E145. The method according to any one of E132 to E144, wherein the recombinant protein, nucleic acid, or pharmaceutical composition is administered subcutaneously. E146. The method according to any one of E132 to E145, wherein the recombinant protein, nucleic acid, or pharmaceutical composition is administered approximately twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, twice a month, once a month, once every two months, once every three months, or once every four months. E147. Recombinant proteins according to any one of E1 to E117 and E120 to E122, nucleic acids according to E118 or E119, or pharmaceutical compositions according to E131, for use as pharmaceuticals. E148. Recombinant proteins according to any one of E1-E117 and E120-E122, nucleic acids according to E118 or E119, or pharmaceutical compositions according to E131, for use in treating a medical condition in a subject. E149. Recombinant protein for use as described in E148, where the medical condition is cancer. E150. Use of recombinant proteins described in any one of E1-E117 and E120-E122, nucleic acids described in E118 or E119, or pharmaceutical compositions described in E131 in the manufacture of drugs for the treatment of cancer in a subject. E151. Use of recombinant proteins described in any one of E1-E117 and E120-E122, nucleic acids described in E118 or E119, or pharmaceutical compositions described in E131 for treating a medical condition in a subject. E152. The method described in E151, wherein the medical condition is cancer. E153. A kit comprising a container, a composition in the container comprising a recombinant protein described in any one of E1 to E117 and E120 to E122, or a nucleic acid described in E118 or E119, or a pharmaceutical composition described in E131, and an accompanying document containing instructions for administering a therapeutically effective amount of recombinant protein, nucleic acid, or pharmaceutical composition to treat a patient in need of treatment. E154. A method for inducing antitumor immunological memory in a mammal, including a human, comprising the steps of administering to the mammal a recombinant protein described in any one of E1 to E117 and E120 to E122, a nucleic acid described in E118 or E119, or a pharmaceutical composition described in E131. E155. The method according to E154, wherein the immunological memory is not limited to FAP-related antigens. E156. A recombinant protein according to any one of E1-E117 and E120-E122, wherein the recombinant protein is capable of preferential localization and / or accumulation in tumors in mammals, including humans. E157. The tumor contains cells expressing FAP, wherein the recombinant protein is as described in E156. E158. A recombinant protein according to any one of E1-E117, E120-E122, and E156-E157, which is capable of inducing antitumor immunological memory in mammals, including humans. E159. The recombinant protein described in E158, wherein the immunological memory is not limited to FAP-related antigens.

[0008] The use of section headings in this specification is solely for the convenience of reading and is not intended to limit the scope. The entire document is intended to be considered a unified disclosure, and it should be understood that all combinations of the features described herein are contemplated. [Brief explanation of the drawing]

[0009] [Figure 1]An illustration illustrating an in vitro B cell activation assay. The assay was performed using purified primary human B cells and FAP-expressing (+FAP) or non-FAP-expressing (-FAP) CHO cells. [Figure 2-1] An overview of the gating strategy used to measure the proportion of MFI and CD86-positive cells. The following settings were used: FSC: 200; SSC: 400; Acquisition: 200 uL / min, 100,000 events. Abbreviations: FMO = Fluorescence minus 1, SSC = Side scattering, FSC = Forward scattering, FSC-A = Forward scatter area, FSC-H = Forward scattering height. [Figure 2-2] An overview of the gating strategy used to measure the proportion of MFI and CD86-positive cells. The following settings were used: FSC: 200; SSC: 400; Acquisition: 200 uL / min, 100,000 events. Abbreviations: FMO = Fluorescence minus 1, SSC = Side scattering, FSC = Forward scattering, FSC-A = Forward scatter area, FSC-H = Forward scattering height. [Figure 3-1] HSA-binding domains(s) impair the potency and efficacy of bispecific FAPxCD40 ankyrin repeat-binding protein. Human B cells were co-cultured in the presence of FAP-expressing CHO cells (black symbols) and treated with escalating concentrations of SMA014 (upward triangle), SMA087 (downward triangle), SMA095 (diamond), and the agonist anti-CD40 mAb (square). As a control, B cells were co-cultured in the presence of FAP-negative CHO cells and treated with only the highest concentration of each construct, shown as white symbols. Human B cell activation was evaluated in terms of the upregulation of CD86 (measured as mean fluorescence intensity (MFI) and cell percentage (%)) in the absence (A) and presence (B) of 600 μM HSA. Each value represents the average of overlapping measurements. The data shown are representative of two independent experiments. Error bars indicate ±SEM. The EC50 values ​​and efficacy values ​​(nM) for all constructs in the presence of FAP-expressing CHO cells are shown in the table within the graph. [Figure 3-2]HSA-binding domains(s) impair the potency and efficacy of bispecific FAPxCD40 ankyrin repeat-binding protein. Human B cells were co-cultured in the presence of FAP-expressing CHO cells (black symbols) and treated with escalating concentrations of SMA014 (upward triangle), SMA087 (downward triangle), SMA095 (diamond), and the agonist anti-CD40 mAb (square). As a control, B cells were co-cultured in the presence of FAP-negative CHO cells and treated with only the highest concentration of each construct, shown as white symbols. Human B cell activation was evaluated in terms of the upregulation of CD86 (measured as mean fluorescence intensity (MFI) and cell percentage (%)) in the absence (A) and presence (B) of 600 μM HSA. Each value represents the average of overlapping measurements. The data shown are representative of two independent experiments. Error bars indicate ±SEM. The EC50 values ​​and efficacy values ​​(nM) for all constructs in the presence of FAP-expressing CHO cells are shown in the table within the graph. [Figure 4] CD40 bivalentity strongly increases the potency and efficacy of the bispecific FAPxCD40 ankyrin repeat-binding protein. Human B cells were cultured in the presence of FAP-expressing CHO cells and treated with escalating concentrations of SMA014 (upward triangle), SMA104 (downward triangle), SMA105 (diamond), and the agonist anti-CD40 mAb (square). As a control, B cells were co-cultured in the presence of FAP-negative CHO cells and treated with only the highest concentration of each construct, indicated by white-filled symbols. Human B cell activation was evaluated in terms of the upregulation of CD86 in the absence of HSA (measured as mean fluorescence intensity (MFI) and cell percentage (%)). Each value represents the average of overlapping measurements. The data shown are representative of two independent experiments. Error bars indicate ±SEM. EC50 values ​​and efficacy values ​​(nM) for all constructs in the presence of FAP-expressing CHO cells are shown in the table in the graph. [Figure 5-1]The bivalent nature of CD40 rescues the inhibitory effect induced by the HSA-binding domain. Human B cells were cultured in the presence of FAP-expressing CHO cells and treated with escalating concentrations of SMA014 (upward triangle), SMA104 (downward triangle), SMA091 (circle), SMA099 (diamond), AS579 (hexagon), and the agonist anti-CD40 mAb (square). As a control, B cells were co-cultured in the presence of FAP-negative CHO cells and treated with only the highest concentration of each construct, indicated by the white-filled symbols. Human B cell activation was evaluated in terms of the upregulation of CD86 (measured as mean fluorescence intensity (MFI) and cell percentage (%)) in the absence (A) and presence (B) of HSA. Each value represents the mean of overlapping measurements. The data shown are representative of two independent experiments. Error bars indicate ±SEM. The EC50 values ​​and efficacy values ​​(nM) for all constructs in the presence of FAP-expressing CHO cells are shown in the table within the graph. [Figure 5-2] The bivalent nature of CD40 rescues the inhibitory effect induced by the HSA-binding domain. Human B cells were cultured in the presence of FAP-expressing CHO cells and treated with escalating concentrations of SMA014 (upward triangle), SMA104 (downward triangle), SMA091 (circle), SMA099 (diamond), AS579 (hexagon), and the agonist anti-CD40 mAb (square). As a control, B cells were co-cultured in the presence of FAP-negative CHO cells and treated with only the highest concentration of each construct, indicated by the white-filled symbols. Human B cell activation was evaluated in terms of the upregulation of CD86 (measured as mean fluorescence intensity (MFI) and cell percentage (%)) in the absence (A) and presence (B) of HSA. Each value represents the mean of overlapping measurements. The data shown are representative of two independent experiments. Error bars indicate ±SEM. The EC50 values ​​and efficacy values ​​(nM) for all constructs in the presence of FAP-expressing CHO cells are shown in the table within the graph. [Figure 6]Analysis of protein #5 (also known as SMA136) by size exclusion chromatography (SEC) and multiangle light scattering (MALS). The graph shows the SEC profile as molar mass over time. The determined molecular weight of protein #5 is shown. [Figure 7] Surface plasmon resonance (SPR) traces showing the binding of protein #5 (also known as SMA136) to human CD40 (A), human FAP (B), and human serum albumin (C). The determined KD values ​​are shown. [Figure 8] Surface plasmon resonance (SPR) traces showing the simultaneous binding of protein #5 to hCD40, hFAP, and HSA. The vertical lines (1, 2, and 3) indicate three injections: (1) binding of protein #5 to immobilized bio-hCD40; (2) binding of hFAP (diamond ◆, triangle ▲, and circle ●) or protein #5 (control; cross ×) to the bio-hCD40 / protein #5 complex; (3) binding of HSA (diamond ◆, triangle ▲) or hFAP (control; circle ●) to the bio-hCD40 / protein #5 / hFAP complex, followed by a 600-second dissociation period. The injection scheme is shown in Table 8 using the same symbols. Simultaneous binding of hCD40 / protein #5 / hFAP / HSA is measured dually in two different lanes (diamond and triangle symbols). [Figure 9-1] Protein #5 activates human B cells via CD40 in vitro. Human B cells were cultured in the presence of FAP-expressing CHO cells and treated with incrementally increasing concentrations of protein #5 (circle symbol) and anti-CD40 mAb (square symbol). Human B cell activation was evaluated in relation to the upcontrol of CD86 and CD69 (measured as mean fluorescence intensity (MFI) and cell percentage (%)). Each value represents the mean of overlapping measurements. The data shown represent 13 independent experiments. Error bars indicate ±SEM. The depicted table shows EC50 and efficacy values ​​for protein #5 (left column) and anti-CD40 mAb (right column). [Figure 9-2]Protein #5 activates human B cells via CD40 in vitro. Human B cells were cultured in the presence of FAP-expressing CHO cells and treated with incrementally increasing concentrations of protein #5 (circle symbol) and anti-CD40 mAb (square symbol). Human B cell activation was evaluated in relation to the upcontrol of CD86 and CD69 (measured as mean fluorescence intensity (MFI) and cell percentage (%)). Each value represents the mean of overlapping measurements. The data shown represent 13 independent experiments. Error bars indicate ±SEM. The depicted table shows EC50 and efficacy values ​​for protein #5 (left column) and anti-CD40 mAb (right column). [Figure 10-1] In vitro activation of human B cells by protein #5 is FAP-dependent. Data show that protein #5 does not induce upregulation of CD86 and CD69 in human B cells in the absence of FAP-expressing CHO cells in vitro. Experiments and data plots were performed using protein #5 (circles) and anti-CD40 mAb (squares), as shown in Figure 9, but in the presence of FAP-negative CHO cells. The data shown represent 13 independent experiments. Error bars indicate ±SEM. The table shows EC50 and efficacy values ​​for anti-CD40 mAb only. [Figure 10-2] In vitro activation of human B cells by protein #5 is FAP-dependent. Data show that protein #5 does not induce upregulation of CD86 and CD69 in human B cells in the absence of FAP-expressing CHO cells in vitro. Experiments and data plots were performed using protein #5 (circles) and anti-CD40 mAb (squares), as shown in Figure 9, but in the presence of FAP-negative CHO cells. The data shown represent 13 independent experiments. Error bars indicate ±SEM. The table shows EC50 and efficacy values ​​for anti-CD40 mAb only. [Figure 11]Schematic diagram of a human monocyte-derived dendritic cell (MDDC) activation assay using in vitro differentiated MDDCs and irradiated FAP-expressing (+FAP) CHO cells or non-FAP-expressing (-FAP) CHO cells. [Figure 12-1] Schematic diagram of the experimental design for in vivo antitumor efficacy studies. Mice were subcutaneously inoculated with MC38-FAP colon cancer cells on day 0. Mice were randomized to treatment groups based on tumor size and date, as shown in Figures 12A and 12B for each study. Four different studies were conducted using different schedules: (A) Early termination: Mice were sacrificed 4 days after the initial treatment, and tumors were analyzed by FACS (Studies PD1033 and PD1038); (B) Late termination: Mice were euthanized 10-11 days after the initial treatment, tumor size was measured over time to evaluate antitumor efficacy, and tumors were examined by FACS at the end of the study (Studies PD1032 and PD1035). Mice were treated with ibuprofen (ip) with AS598, AS608, or anti-CD40 antibody at the indicated time points. [Figure 12-2] Schematic diagram of the experimental design for in vivo antitumor efficacy studies. Mice were subcutaneously inoculated with MC38-FAP colon cancer cells on day 0. Mice were randomized to treatment groups based on tumor size and date, as shown in Figures 12A and 12B for each study. Four different studies were conducted using different schedules: (A) Early termination: Mice were sacrificed 4 days after the initial treatment, and tumors were analyzed by FACS (Studies PD1033 and PD1038); (B) Late termination: Mice were euthanized 10-11 days after the initial treatment, tumor size was measured over time to evaluate antitumor efficacy, and tumors were examined by FACS at the end of the study (Studies PD1032 and PD1035). Mice were treated with ibuprofen (ip) with AS598, AS608, or anti-CD40 antibody at the indicated time points. [Figure 13]Mouse body weight during antitumor efficacy studies. Mice were treated as shown in Figure 12. Mean body weight per treatment group (±SEM, n=10) is shown for studies PD1032(A) and PD1035(B). Dotted lines indicate the time of randomization and the start of treatment. Statistical analysis was performed using Kruskal-Wallis with multiple comparisons with the vehicle. Results were considered significant if *p<0.05, **p<0.01, and ***p<0.001. [Figure 14] Mean tumor volume during antitumor efficacy studies. Mice were treated as described in Figure 12, and tumor volume was measured every 3-4 days. Mean tumor volume per treatment group (±SEM, n=10) is shown for studies PD1032(A) and PD1035(B). Dotted lines indicate the time of randomization and the start of treatment. Arrows indicate the time of treatment. Statistical analysis was performed using Kruskall-Wallis with multiple comparisons to the vehicle and / or the negative control AS608 (in parentheses) at the end of the study. Results were considered statistically significant if **p<0.01***p<0.001****p<0.0001. [Figure 15] Average FAP activity in the presence of various FAP-specific recombinant binding proteins. The conversion of the substrate Z-GLY-PRO-AMC to a fluorescent product by recombinant human FAP was measured in the presence or absence of various recombinant proteins. FAP activity after 95 minutes of incubation is shown. Compared to FAP activity in the absence of the test molecule (first control: hFAP and substrate), all recombinant proteins tested (molecule 1-4) containing the FAP-binding domain showed no inhibitory effect on FAP enzyme activity. Partial inhibition of FAP activity was observed for molecule 5 (used as an assay control). Average FAP activity and standard deviation are shown from the quadret measurement. [Figure 16](A) Representative SPECT / CT images of MC38-FAP tumor-bearing mice 96 hours after injection of indium-111-labeled protein #7. Maximum intensity projection (MIP) produced using normalized intensity settings is shown. Labeled protein #7 localized to the tumor and preferentially accumulated. (B) Detection of protein #7 (top image) or control DARPin® protein (bottom image) by immunohistochemistry (IHC) in MC38-FAP tumors 24 hours after injection. Size bars are shown in the lower right corner of the image. (C) Time course of tissue distribution of indium-111-labeled DARPin® molecules. Tissue distribution of control DARPin® molecules (solid bar) and protein #7 (striped bar) was analyzed in tumor (left graph) and muscle (right graph) at the indicated time points. Data are obtained as the percentage of injected dose of DARPin® molecules per gram of tissue (%ID / g) and are expressed as mean ± SD. N=4 mice per time point [Figure 17] (A) Schematic diagram of the experimental design for the in vivo antitumor efficacy study. (B) Mean tumor growth volume during the antitumor efficacy study. Mice were treated and tumor volume was measured as described in Figure 17A and Example 9. Mean tumor volume per treatment group (±SEM, n=10) is shown for vehicle (triangle symbol), AS598 (circle symbol), and anti-CD40 antibody (square symbol). Arrows indicate the start of treatment. [Figure 18] Long-term efficacy in antitumor efficacy studies. Mice carrying MC38-FAP tumors were treated with the vehicle (triangle symbol), AS598 (square symbol), and AS608 (negative control) (circle symbol) as described in Example 10, and tumor volume was measured every 3-4 days. The mean tumor volume (±SEM) per treatment group is shown in (A), and the Kaplan-Meier survival curve is shown in (B). Arrows indicate the time of treatment. [Figure 19]Induction of antitumor immunological memory. (A) The experiment shown in Figure 18A was followed over a longer period and is accompanied by mean tumor growth curves shown for MC38-FAP tumor-bearing mice treated with vehicle (overfilled upward triangle), AS598 (overfilled circle), and AS608 (negative control) (overfilled downward triangle). Short arrows indicate the time of treatment. At approximately 120 days, tumor-free mice previously treated with AS598 were re-challenged with MC38-WT (downward solid triangle) or MC38-FAP (upward solid triangle) tumor cells and monitored until 200 days. Mean tumor growth curves for 8 mice per group are shown. (B) Naive control mice were challenged with MC38-WT (downward triangle) or MC38-FAP (upward triangle) tumor cells for approximately 120 days. Mean tumor growth curves for 5 mice per group are shown. [Figure 20] Toxicity evaluation. (A) Mice carrying MC38-FAP tumors were treated once with vehicle (n=10) (upward-pointing triangle), AS608 (negative control) (n=5) (downward-pointing triangle), AS598 (protein #7) (n=10) (circle), or anti-mCD40 antibody (n=10) (square), and serum cytokines were measured 24 hours later. Samples were collected from two independent experiments and analyzed together. (B) Mice carrying MC38-FAP tumors were treated once with vehicle (upward-pointing triangle), AS608 (negative control) (downward-pointing triangle), AS598 (protein #7) (circle), or anti-mCD40 antibody (square) (5 mice per group), and serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured 24 hours later. Samples were collected from two independent experiments and analyzed together. (C) Mice carrying MC38-FAP tumors were treated once with vehicle, AS608 (negative control) (not shown), AS598 (protein #7), or anti-mCD40 antibody (n=5-10). After 24 hours, the liver was collected and analyzed for tissue damage by immunohistochemistry (IHC). Representative photographs of the different treatments are shown. NEC, necrosis; ICI, immune cell infiltration. Size bars are shown in the lower right corner of the photographs. [Figure 21] Structural determination of human tumor necrosis factor receptor superfamily member 5 (hCD40) that forms a complex with the DARPin® protein, which has the amino acid sequence of SEQ ID NO: 3, by X-ray crystallography. [Modes for carrying out the invention]

[0010] 1. Overview Recombinant proteins comprising an engineered ankyrin repeat domain having binding specificity to FAP and CD40 are disclosed herein. Also disclosed are nucleic acids encoding the binding protein, pharmaceutical compositions comprising the binding protein or nucleic acid, and methods using the binding protein, nucleic acid, or pharmaceutical composition. In one embodiment, the materials and methods of the disclosure utilize the expression of FAP in tumor-associated stroma to enable, for example, specific targeting of CD40-expressing cells in tumors and selective activation of CD40 in those CD40-expressing cells.

[0011] CD40 agonist antibodies have demonstrated efficacy in preclinical mouse tumor models, and their clinical use has also shown some antitumor efficacy. However, the clinical development of agonist anti-CD40 antibodies is likely hindered by dose-limiting toxicity and the resulting low efficacy.

[0012] The multiselective recombinant proteins described herein promote the cancer targeting and tumor localization clustering of CD40, thereby addressing the challenges associated with previous therapeutic approaches. In nature, CD40 clustering is achieved by binding to a trimer CD40 ligand (CD40L, CD154) expressed as a membrane molecule on the surface of certain cells, such as activated CD4+ T cells. CD40 clustering on the cell membrane of cells targeted by CD40L, for example, is a prerequisite for activation of its signaling pathway. The multiselective recombinant proteins of the present invention disclosed herein utilize this clustering effect, and the activation of CD40 is associated with the expression of the tumor antigen FAP.

[0013] Fibroblast-activating protein αFAP (also known as seplase) is a type II membrane-bound glycoprotein that is abundantly expressed by cancer-associated fibroblasts in the stroma of many solid tumors. FAP is selectively expressed in reactive stromal fibroblasts in over 90% of epithelial malignancies (primary and metastatic), including lung, colorectal, bladder, ovarian, and breast cancers, as well as in malignant mesenchymal cells of bone and soft tissue sarcomas, but is generally absent in normal adult tissues (Brennen et al., "Mol Cancer Ther.", Vol. 11: pp. 257-266 (2012); Garin-Chesa et al., "Proc Natl Acad Sci USA", Vol. 87, pp. 7235-7239 (1990); Rettig et al., "Cancer Res.", Vol. 53: 3327-3335 (1993); Rettig et al., Proc Natl Acad Sci USA 85, 3110-3 114 (1988)). FAP is also expressed on certain malignant tumor cells.

[0014] While we do not wish to be bound by any particular theory, in the absence of the tumor antigen FAP (on normal, non-malignant, non-cancer-associated cells), minimal CD40 clustering will occur, and immune activation will be limited. In contrast, FAP is highly expressed in cancer-associated fibroblasts, and therefore, via FAP binding, the multiselective protein of this invention promotes CD40 clustering and activation in CD40-expressing immune cells such as B cells and antigen-presenting cells. The advantages of this strategy are twofold: systemic toxicity should be limited because activation is largely confined to tissues expressing FAP, and tumor-mediated CD40 clustering should drive potent agonist activity.

[0015] 2.Definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have meanings generally understood by those skilled in the art. Furthermore, unless specifically required by the context, singular terms shall include plural forms and plural terms shall include singular forms. Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization techniques described herein is well known and commonly used in the art.

[0016] The terms “comprising,” “having,” “including,” and “containing” should be interpreted as non-exclusive unless otherwise specified. Where a feature is described as “comprising” in an embodiment, it may also be “consisting of” or “essentially consisting of.” Any use of any example or illustrative wording provided herein (e.g., “etc.”) is intended solely to better illustrate the disclosure and, unless otherwise requested, does not limit the scope of the disclosure. Nothing in this specification should be interpreted as indicating any unclaimed element as essential to the practice of the disclosure. Except for the examples provided or unless otherwise indicated, all numbers representing quantities of components or reaction conditions used herein should be understood in all cases as modified by the term “about” in such a manner that it is interpreted by those skilled in the art.

[0017] The enumeration of value ranges in this specification is intended merely as a simplified way of referring individually to each distinct value and each endpoint that falls within that range, unless otherwise indicated herein, and each distinct value and endpoint is incorporated herein as if it were individually enumerated.

[0018] The term "polypeptide" refers to a molecule consisting of one or more chains of multiple amino acids, i.e., two or more amino acids, linked by peptide bonds. Preferably, a polypeptide consists of more than eight amino acids linked by peptide bonds. The term "polypeptide" also includes multiple chains of amino acids linked by cysteine ​​disulfide crosslinks. Polypeptides are well known to those skilled in the art.

[0019] The term "protein" refers to a molecule containing polypeptides in which at least a portion of the polypeptides have, or can have, a distinct 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 non-covalently or covalently linked, for example, by disulfide bonds between the two polypeptide chains. The portions of a protein that individually have, or can have, a distinct three-dimensional configuration by forming secondary and / or tertiary structures are called "protein domains." Such protein domains are well known to those skilled in the art.

[0020] Patent application No. 2002 / 020565 and Forrer et al., 2003 (Forrer, P., Stumpp, MT, Binz, HK, Pluckthun, A., 2003. FEBS Letters 539, 2-6) include a general description of the features, techniques, and applications of repeat proteins, repeat domains, and repeat modules.

[0021] The term "repeatable domain" refers to a protein domain that contains two or more consecutive repeating modules as structural units, where these repeating modules have structural and sequence homology. Preferably, the repeating domain also includes N-terminal and / or C-terminal capping modules. For clarity, capping modules can be repeating modules. Such repeating domains, repeating modules, and capping modules, sequence motifs, and their structural and sequence homologies are well known to those skilled in the art from examples such as the ankyrin repeating domain (Binz et al., J.Mol.Biol.332,489-503,2003, Binz et al.,2004,loc.cit, International Publication No. 2002 / 020565, International Publication No. 2012 / 069655), the high leucine repeating domain (International Publication No. 2002 / 020565), the tetratricopeptide repeating domain (Main,ER,Xiong,Y.,Cocco,MJ,D'Andrea,L.,Regan,L.,Structure 11(5),497-508,2003), and the armadillo repeating domain (International Publication No. 2009 / 040338). It is more well known to those skilled in the art that such repeating domains are different from proteins containing repeating amino acid sequences, and all repeating amino acid sequences can form individual domains (for example, the FN3 domain of fibronectin).

[0022] The term "ankyrin repeat domain" refers to a repeat domain that contains two or more consecutive ankyrin repeat modules as structural units, and these ankyrin repeat modules have structural homology and sequence homology.

[0023] The term "repeatable module" originally refers to the repeated amino acid sequence and structural unit of a designed repeatable domain, which is derived from the repeatable units of naturally occurring repeat proteins. Each repeatable module contained within a repeatable domain is derived from one or more repeatable units of a naturally occurring family or subfamily of repeat proteins, preferably the ankyrin repeat protein family. Therefore, the term "ankyrin repeatable module" originally refers to a repeatable module derived from the repeatable units of naturally occurring ankyrin repeat proteins. Ankyrin repeat proteins are well known to those skilled in the art. For example, see International Patent Publications WO2002 / 020565, WO2010 / 060748, WO2011 / 135067, WO2012 / 069654, WO2012 / 069655, WO2014 / 001442, WO2014 / 191574, WO2014 / 083208, WO2016 / 156596, and WO2018 / 054971.

[0024] The ankyrin repeat domains may be modularly assembled, optionally together with half-life extension domains, into the larger ankyrin repeat proteins according to this disclosure using standard recombinant DNA techniques (see, for example, Forrer, P., et al., FEBS letters 539, 2-6, 2003, International Patent Publications WO2012 / 069655 and WO2002 / 020565).

[0025] The term "designed" as used in terms such as designed repeat proteins, designed repeat domains, and designed ankyrin repeat domains refers to the characteristic that such repeat proteins and repeat domains are artificial and do not occur in nature.

[0026] The term "recombinant" as used in recombinant proteins, recombinant binding proteins, recombinant polypeptides, etc., means that the protein or polypeptide is produced by the use of recombinant DNA technology well 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, if such a recombinant bacterial expression plasmid is inserted into a suitable bacterium (e.g., E. coli), these bacteria can produce the polypeptide encoded by this recombinant DNA. The polypeptide or protein produced accordingly is called a recombinant polypeptide or recombinant protein.

[0027] In the context of this invention, the term "binding protein" refers to a protein containing a binding domain. The binding protein may also contain two, three, four, five or more binding domains. Preferably, the binding protein is a recombinant binding protein.

[0028] The term "binding domain" refers to a protein domain that exhibits binding specificity to a target. Preferably, this binding domain is a recombinant binding domain.

[0029] The term "target" refers to an individual molecule, such as a nucleic acid molecule, peptide, polypeptide or protein, carbohydrate, or any other naturally occurring molecule including any portion of such individual molecules, or a complex composed of two or more such molecules, or a sample of an entire cell or tissue sample, or any unnatural compound. Preferably, the target is a naturally occurring, or unnatural polypeptide or protein, or a polypeptide or protein comprising chemical modifications, such as being modified by naturally occurring or unnatural phosphorylation, acetylation, or methylation. For example, the target of each of the designed ankyrin repeat domains consisting of SEQ ID NOs: 39 to 42 is serum albumin.

[0030] Terms such as "having binding specificity for a target", "specifically binding to a target", "binding to a target with high specificity", "being specific for a target", or "target specificity" mean that a binding protein or binding domain reacts or binds with a particular target (e.g., a cell or substance) more frequently, more rapidly, for a longer period of time, and / or with a higher affinity than it reacts or binds with an alternative target (e.g., a cell or substance). For example, a binding domain that specifically binds to FAP may be defined as a binding domain that binds to FAP with a lower dissociation constant (i.e., binds with a higher affinity) than it binds to an irrelevant protein such as Escherichia coli maltose binding protein (MBP) in PBS. Preferably, the dissociation constant ("K D ") in PBS for the target is at least 10 2 times lower, more preferably at least 10 3 times lower, even more preferably at least 10 4 times lower, or most preferably at least 10 5 times lower than the corresponding dissociation constant for MBP. Methods for measuring the dissociation constant of protein-protein interactions, such as techniques based on surface plasmon resonance (SPR) (e.g., SPR equilibrium analysis) or isothermal titration calorimetry (ITC), are well known to those skilled in the art. The K of a specific protein-protein interactionD The measured values ​​may vary when measured under different conditions (e.g., salt concentration, pH). Therefore, K D The measurement of values ​​is preferably performed using a standardized protein solution and a standardized buffer such as PBS. Furthermore, by reading this definition, it is understood that, for example, an ankyrin repeat domain that specifically binds to a first target may or may not specifically or preferentially bind to a second target. Therefore, "specific binding" does not necessarily require (but may include) exclusive binding. Generally, an ankyrin repeat domain preferentially binds to a specific target molecule under the specified assay conditions and does not bind in significant amounts to other components present in the test sample.

[0031] Various assay formats can be used to select or characterize ankyrin repeat domains that specifically bind to the molecule of interest. For example, solid-phase ELISA immunoassays, immunoprecipitation, BIAcore® (GE Healthcare, Piscataway, NJ), fluorescence-activated cell sorting (FACS), Octet® (Menlo Park, California, ForteBio, Inc.), and Western blot analysis are some of the many assays that can be used to identify ankyrin repeat domains that specifically react with a target. Typically, a specific or selective reaction has at least 2x background signal or noise, more typically more than 10x background. More specifically, ankyrin repeat domains have an equilibrium dissociation constant (K). D If the value is <1 μM, for example, <100 nM, <10 nM, <100 pM, <10 pM, or <1 pM, it is said to "specifically bind" to the target.

[0032] K DThe value is often referred to as binding affinity. Binding affinity measures the strength of the sum of non-covalent interactions between a contact residue of one binding partner (e.g., the FAP or CD40 binding domain disclosed herein) and the contact residue of that binding partner (e.g., FAP or CD40). Unless otherwise indicated, as used herein, binding affinity refers to the binding affinity that reflects a 1:1 interaction between members of a binding pair or binding partner. For a binding protein containing two binding domains for one binding partner, binding affinity may refer to the binding affinity that reflects a 1:2 interaction between the binding protein and the binding partner.

[0033] Various methods for measuring binding affinity are well known in the art, and any of them can be used for the purposes of the present invention. For example, as illustrated herein, binding affinity refers to the dissociation rate of a particular ankyrin repeat domain and its binding target K D It can be represented as a value. K D is, "Offrate (K off Dissociation rate, also called "(K)", and binding rate, or are also referred to as "(K)". on This is the ratio of )」. 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.

[0034] 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). DThe values ​​may be measured by SPR using a biosensor system such as the BIACORE® system. BIAcore dynamic analysis involves analyzing the binding and dissociation of antigens from chips having immobilized molecules (e.g., molecules containing epitope-binding domains) on their surface. K of proteins D Another method for determining this is to use Bio-Layer Interferometry (e.g., Shah et al. J Vis Exp. 2014; (84): 51383). D The values ​​can be measured using OCTET® technology (Octet QKe system, ForteBio). Alternatively or additionally, the KinExA® (dynamic exclusion assay) assay, available from Sapidyne Instruments (Boise, Id.), can also be used. Any suitable method for evaluating the binding affinity between two binding partners is incorporated herein. Preferably, K D The value is determined in PBS by SPR, as described in Example 2, for example.

[0035] The term "polypeptide tag" refers to an amino acid sequence bound to a polypeptide / protein that is useful for the purification, detection, or targeting of the polypeptide / protein, or that improves the physicochemical behavior of the polypeptide / protein, or that has an effector function. Individual polypeptide tags, parts, and / or domains of a bound protein may be linked to each other directly or via polypeptide linkers. All of these polypeptide tags are well known in the art and readily available to those skilled in the art. Examples of polypeptide tags include small polypeptide sequences, e.g., His (e.g., the His tag consisting of SEQ ID NO: 57), myc tags, FLAG tags, or Strep tags, or enzymes (e.g., enzymes such as alkaline phosphatase) that can detect the polypeptide / protein, or parts that can be used for targeting (e.g., immunoglobulins or fragments thereof) and / or parts that can be used as effector molecules.

[0036] The term "polypeptide linker" refers to an amino acid sequence that can link, for example, two protein domains, a polypeptide tag and a protein domain, a protein domain and a non-polypeptide moiety such as polyethylene glycol, or two polypeptide tags. Such further domains, tags, non-polypeptide moieties, and linkers are known to those skilled in the art. An example of such a polypeptide linker is the linker consisting of SEQ ID NOs: 4 and 56.

[0037] The term "nucleic acid" or "nucleic acid molecule" refers to a polynucleotide molecule that may be a single-stranded or double-stranded ribonucleic acid (RNA) molecule or a deoxyribonucleic acid (DNA) molecule, including modified and artificial forms of DNA or RNA. Nucleic acid molecules may exist in isolated form or be contained in recombinant nucleic acid molecules or vectors.

[0038] In the context of this invention, the terms “medical condition,” “disease,” and “disorder” are used interchangeably and include, but are not limited to, autoimmune disorders, inflammatory disorders, retinopathy (particularly proliferative retinopathy), neurodegenerative disorders, infectious diseases, metabolic diseases, and neoplastic diseases. A “medical condition” may be characterized by inappropriate cell proliferation. A medical condition may be a state of hyperproliferation. A medical condition may be a neoplastic disease. The term “neoplastic disease,” as used herein, refers to an abnormal state or condition of cells or tissue characterized by rapidly growing cell proliferation or tumors. A medical condition may be a malignant neoplastic disease. A medical condition may be cancer. The terms “cancer” and “cancerous” are used herein to refer to or describe, typically a physiological condition in mammals characterized by unregulated cell proliferation. Cancer includes solid tumors and humoral tumors, as well as primary and metastatic tumors. A “tumor” includes one or more cancerous cells. A solid tumor typically also includes the tumor stroma. Examples of cancer include, but are not limited to, primary and metastatic cancers, lymphomas, blastomas, sarcomas, myelomas, melanomas, and leukemias, as well as any other epithelial malignancies and hematopoietic malignancies. More specific examples of such cancers include brain cancer, bladder cancer, breast cancer, ovarian cancer, kidney cancer, colorectal cancer, gastric cancer, head and neck cancer, lung cancer, pancreatic cancer, prostate cancer, malignant melanoma, osteosarcoma, soft tissue sarcoma, carcinoma, squamous cell carcinoma, clear cell renal cancer, head and neck squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer (NSCLC), renal cell carcinoma, small cell lung cancer (SCLC), triple-negative breast cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic lymphocytic leukemia (CL). L) Examples include chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), squamous cell carcinoma of the head and neck (SCCHN), chronic myeloid leukemia (CML), small lymphocytic lymphoma (SLL), malignant mesothelioma, liposarcoma, neuroblastoma, or synovial sarcoma.

[0039] The term “to treat” and related words do not necessarily imply a 100% or complete cure. Rather, the degree of treatment that a person skilled in the art would recognize as having potential benefits or therapeutic effects varies. In this regard, the methods for treating cancer of this disclosure may provide any amount or any level of treatment. Furthermore, the treatment provided by the methods of this disclosure may include treating (i.e., alleviating) one or more conditions or symptoms. Also, the treatment provided by the methods of this disclosure may include delaying the progression of cancer. For example, the methods may treat cancer by enhancing T cell activity or the immune response against cancer, reducing tumor or cancer growth or the appearance of new lesions, reducing metastasis of tumor cells, increasing cell death of tumor or cancer cells, or inhibiting the survival of tumor or cancer cells. In exemplary embodiments, the methods treat cancer by delaying the onset or recurrence of cancer by 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, 2 months, 4 months, 6 months, 1 year, 2 years, 4 years, or more. In exemplary embodiments, the method treats by increasing the survival of the subject. The term “treatment” also includes prophylactic treatment.

[0040] The therapeutic response in any given disease or condition can be determined by standardized response criteria specific to that disease or condition. Tumor responses can be evaluated using screening techniques such as magnetic resonance imaging (MRI) scans, radiographic contrast, computed tomography (CT) scans, positron emission tomography (PET) scans, bone scans, ultrasound, tumor biopsy sampling, counting of circulating tumor cells, and / or measurement of tumor antigens (e.g., prostate-specific antigen (PSA) and / or alpha-fetoprotein (AFP)). In addition to these therapeutic responses, subjects receiving treatment may experience beneficial effects such as improvement of disease-related symptoms.

[0041] The term "treatment" or "the act of treating" refers to both therapeutic measures and preventive or deterrent measures. Those requiring treatment include individuals already suffering from a disability and those whose disability should be prevented.

[0042] The term "therapeutic dose" refers to an amount sufficient to induce a desired biological, pharmacological, or therapeutic outcome in a subject. In the context of this invention, a therapeutic dose means a sufficient amount of binding protein to treat or prevent a disease or disorder with a reasonable benefit / risk ratio applicable to any medical treatment.

[0043] For the purposes of this procedure, the term “mammal” refers to any animal classified as a mammal, including humans, livestock and farm animals, non-human primates, and animals such as dogs, horses, cats, and cattle, including zoo animals, sports animals, or pet animals.

[0044] In one embodiment, the term "incubation" refers to incubation at pH 7.4. In one embodiment, this incubation at pH 7.4 refers to incubation in PBS.

[0045] 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.

[0046] The term "improved pharmacokinetic properties" refers to increased area under the curve, reduced clearance, or extended terminal phase half-life. The parameters of these pharmacokinetic properties and methods for measuring them are well known in the art (e.g., Mahmood, I., "Methods to determine pharmacokinetic profiles of therapeutic proteins," Drug Discov Today:Technol (2009), doi:10.1016 / j.ddtec.2008.12.001).

[0047] In the context of the present invention, the term "any amino acid" preferably means the 20 amino acids that are most abundant in nature, namely alanine (ala, A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0048] 3. Multiselective molecules targeting FAP and CD40 Multiselective molecules targeting FAP and CD40 are disclosed herein. These molecules are useful, for example, in the treatment of cancer. According to the present invention, the multiselective molecules provided herein that target FAP and CD40 are preferably designed repeat proteins, more preferably designed ankyrin repeat proteins.

[0049] 3.1. Ankyrin repeat domains and ankyrin repeat proteins 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, three, four, five, or more designed ankyrin repeat domains having the same or different target specificity (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 ankyrin repeat domain are small proteins (14 kDa) that can be selected to bind to a given target protein with high affinity and specificity. These characteristics, and the possibility of combining two, three, four, five, or more designed ankyrin repeat domains in a single protein, make designed ankyrin repeat proteins ideal candidates for agonists, antagonists, and / or inhibitors. Furthermore, such ankyrin repeat proteins can be engineered to carry various effector functions, such as cytotoxic agents or half-life extenders, enabling entirely new drug formats. In summary, engineered ankyrin repeat proteins represent a next-generation example of protein-based therapeutics with the potential to surpass existing antibody drugs.

[0050] The designed ankyrin repeat domains described herein generally comprise one or more designed repeat modules, preferably ankyrin repeat modules, as structural units (hereinafter also referred to as structural repeats or repeat units), wherein the repeat modules, preferably ankyrin repeat modules, have structural homology and sequence homology. The ankyrin repeat module generally consists of two antiparallel α-helices, followed by a beta-bulge and a beta-hairpin-containing loop connecting them to the next repeat unit, each having approximately 28 to 33 residues.

[0051] Recombinant proteins containing designed ankyrin repeat modules or their designed binding domains are also referred to herein as DARPin® proteins. See Stumpp et al., Curr Opin Drug Discov Devel., Vol. 10 (No. 2): pp. 153-159 (2007); and Binz et al., Nature Biotech., Vol. 22 (No. 5): 575-582 (2004). DARPin® proteins can be considered antibody mimetic molecules with high specificity and high binding affinity to target proteins. Generally, DARPin® proteins contain at least one ankyrin repeat module, e.g., at least two, three, or more ankyrin repeat modules. DARPin® is a trademark owned by Molecular Partners AG, Switzerland.

[0052] The ankyrin repeat domains described herein generally comprise a core scaffold providing structure and target-binding residues that bind to a target. The structural core comprises conserved amino acid residues, while the target-binding surface comprises different amino acid residues depending on the target. For example, an ankyrin repeat module may include the following sequence: xDxxGxTPLHLAxxxGxxxIVxVLLxxGADVNA (SEQ ID NO: 23), where "x" represents any amino acid, preferably not cysteine, glycine, or proline. As another example, an ankyrin repeat module may include any one of the amino acid sequences of SEQ ID NOs: 24-27.

[0053] Designed repeat protein libraries, including the designed ankyrin repeat protein library (International Publication No. 2002 / 020565, Binz et al., Nat. Biotechnol., Vol. 22, pp. 575-582, 2004; Stumpp et al., Drug Discov. Today, Vol. 13, pp. 695-701, 2008), can be used for the selection / screening of target-specific repeat domains that bind to targets with high affinity. Such target-specific repeat domains can then be used as valuable components of recombinant binding proteins for disease treatment. Methods for constructing such libraries are known to those skilled in the art (International Publication No. 2002 / 020565).

[0054] Multiple ankyrin repeat domains can be linked (either via covalent or non-covalent bonds) to form bispecific or multiselective specific molecules. Such multiselective specific molecules are disclosed herein, including molecules in which one FAP-binding domain and two CD40-binding domains are linked. Such molecules may also contain a half-life extension moiety at the N-terminus.

[0055] 3.2. N-terminus and C-terminus capping modules The repeat domains of recombinant proteins disclosed herein, preferably ankyrin repeat domains, preferably include an N-terminal and / or C-terminal capping module (hereinafter also referred to as a capping repeat or capping unit). The capping module is located at the N-terminus and / or C-terminus of the ankyrin repeat domain and typically forms a close tertiary interaction (i.e., a tertiary structural interaction) with the ankyrin repeat module(s), thereby providing a cap that laterally shields the hydrophobic core of the ankyrin repeat domain from exposure to solvents.

[0056] The N-terminal and / or C-terminal capping modules may originate from capping units or other structural units found in naturally occurring repeat proteins adjacent to repeat units. Examples of capping sequences are described in International Patent Publications WO2002 / 020565 and WO2012 / 069655, U.S. Patent Publication US20130296221, and Interlandi et al., J Mol Biol. 2008 Jan 18;375(3):837-54. Examples of N-terminal capping modules (i.e., N-terminal capping repeats) are Sequence IDs 11-16, and examples of C-terminal capping modules (i.e., C-terminal capping repeats) are Sequence IDs 18-21.

[0057] In exemplary embodiments, the N-terminal capping module comprises the amino acid sequence DLGKKLLEAARAGQDDEVRILLAAGADVNA (SEQ ID NO: 14) or DLGKKLLEAARAGQDDEVRELLKAGADVNA (SEQ ID NO: 15), wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in SEQ ID NO: 14 or SEQ ID NO: 15 may optionally be replaced by any amino acid, wherein SEQ ID NO: 14 or SEQ ID NO: 15 may optionally further comprise a "G", "S", or "GS" sequence at its N-terminus. In exemplary embodiments, the C-terminal capping module comprises the amino acid sequence QDIFGKTPADIAADAGHEDIAEVLQKAA (SEQ ID NO: 19) or QDKSGKTPADLAADAGHEDIAEVLQKAA (SEQ ID NO: 20), wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in SEQ ID NO: 19 or SEQ ID NO: 20 are optionally replaced by any amino acid.

[0058] Advantageously, in some embodiments, it has been found that by altering certain amino acid residues in the N-terminal capping module and / or C-terminal capping module of the designed ankyrin repeat domain herein, improvements in pharmacokinetic properties, including an extended terminal phase half-life of the designed ankyrin repeat domain and the recombinant binding protein containing the designed ankyrin repeat domain, can be obtained. The altered amino acid residues are mostly surface-exposed residues. Preferably, the altered amino acid residues are amino acid residues at positions 8 and 15 of the N-terminal capping module, where the position numbers correspond to the positions in SEQ ID NO: 11, and amino acid residues at positions 14 and 18 of the C-terminal capping module, where the position numbers correspond to the positions in SEQ ID NO: 18.

[0059] In one preferred embodiment, the designed ankyrin repeat domain provided herein comprises an N-terminal capping module having an amino acid sequence, where the amino acid at position 8 is Q and / or the amino acid at position 15 is L. Examples of such an N-terminal capping module are SEQ ID NOs: 11, 12, and 13. In one embodiment, the designed ankyrin repeat domain comprises an N-terminal capping module having an amino acid sequence, where the amino acid at position 4 is S, the amino acid at position 8 is Q, the amino acid at position 15 is L, the amino acid at position 17 is T, the amino acid at position 20 is T, and / or the amino acid at position 23 is Q. An example of such an N-terminal capping module is SEQ ID NOs: 16. In a preferred embodiment, the N-terminal capping module comprises an amino acid sequence of 30 amino acids. In a more preferred embodiment, the N-terminal capping module comprises an amino acid sequence consisting of 30 amino acids. Preferably, the position number of the N-terminal capping module is determined by aligning to SEQ ID NOs: 11 using the position number of SEQ ID NOs: 11. Preferably, the alignment does not contain an amino acid gap. Sequence alignment generation is a well-known procedure in the art. Any one of the N-terminal capping modules may optionally further contain a "G", "S", or "GS" sequence at its N-terminus.

[0060] For example, an N-terminal capping module having altered amino acid residues can include the following sequence: DLGxxLLQAAxxGQLDxVRxLxxxGADVNA (Sequence ID 17), where "x" represents any amino acid.

[0061] In exemplary embodiments, the N-terminal capping sequence comprises DLGKKLLQAARAGQLDEVRELLKAGADVNA (SEQ ID NO: 11), DLGKKLLQAARAGQLDEVRILLKAGADVNA (SEQ ID NO: 12), or DLGKKLLQAARAGQLDEVRILLAAGADVNA (SEQ ID NO: 13), wherein at positions other than 8 and 15, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acids in SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 may be optionally replaced by any amino acid, and SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 may optionally further contain a "G", "S", or "GS" sequence at its N-terminus. Accordingly, in one embodiment, the designed repeat domain of the present invention, preferably an ankyrin repeat domain, comprises an N-terminal capping module having the amino acid sequence DLGKKLLQAARAGQLDEVRELLKAGADVNA (SEQ ID NO: 11), wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 at positions other than 8 and 15 are optionally replaced by any amino acid, wherein SEQ ID NO: 11 may optionally further contain a "G", "S", or "GS" sequence at its N-terminus.

[0062] In another exemplary embodiment, the N-terminal capping sequence comprises DLGSKLLQAARAGQLDTVRTLLQAGADVNA (SEQ ID NO: 16), wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of SEQ ID NO: 16 at positions other than 4, 8, 15, 17, 20, and 23 are optionally replaced by any amino acid, wherein SEQ ID NO: 16 may optionally further comprise a "G", "S", or "GS" sequence at its N-terminus.

[0063] In a more preferred embodiment, the designed repeat domain provided herein, preferably an ankyrin repeat domain, comprises a C-terminal capping module having an amino acid sequence, where the amino acid at position 14 is R and / or the amino acid at position 18 is Q. Examples of such C-terminal capping modules are SEQ ID NOs: 18 and 19. In one embodiment, the designed ankyrin repeat domain comprises a C-terminal capping module having an amino acid sequence, where the amino acid at position 3 is T, the amino acid at position 4 is Q, the amino acid at position 6 is T, the amino acid at position 14 is R, the amino acid at position 18 is Q, the amino acid at position 19 is Q, the amino acid at position 22 is S, and / or the amino acid at position 26 is Q. An example of such a C-terminal capping module is SEQ ID NO: 21. In a preferred embodiment, the C-terminal capping module comprises an amino acid sequence of 28 amino acids. In a more preferred embodiment, the C-terminal capping module comprises an amino acid sequence consisting of 28 amino acids. Preferably, the position number of the C-terminal capping module is determined by aligning to SEQ ID NO: 18 using the position number of SEQ ID NO: 18. Preferably, the alignment does not include an amino acid gap.

[0064] For example, a C-terminal capping module with altered amino acid residues can include the following sequence: xDxxGxTPADxAARxGHQxIAxVLQxAA (SEQ ID NO: 22), where "x" represents any amino acid.

[0065] In an exemplary embodiment, the C-terminal capping sequence includes QDKSGKTPADLAARAGHQDIAEVLQKAA (SEQ ID NO: 18), where up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of SEQ ID NO: 18 at positions other than 14 and 18 are optionally replaced by any amino acid.

[0066] In another exemplary embodiment, the C-terminal capping sequence comprises QDTQGTTPADLAARAGHQQIASVLQQAA (SEQ ID NO: 21), where up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of SEQ ID NO: 21 at positions other than 3, 4, 6, 14, 18, 19, 22, and 26 are optionally replaced by any amino acid.

[0067] 3.3. FAP binding domain One intriguing stromal cell target is fibroblast-activating protein (FAP), a transmembrane serine protease highly expressed in cancer-associated stromal cells of virtually all epithelial cancers. FAP is also expressed during embryonic development, in wound tissue during healing, and in chronic inflammatory and fibrotic diseases such as cirrhosis and idiopathic pulmonary fibrosis. However, FAP has not been detected by immunohistochemistry in benign tumors or in most normal, quiescent adult stromal cells.

[0068] The recombinant proteins described herein include an ankyrin repeat domain, also referred to herein as the "FAP-binding domain," which specifically binds to FAP.

[0069] In some embodiments, the FAP-binding domain described herein includes an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 2. In exemplary embodiments, the FAP-binding domain described herein includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 2. In preferred embodiments, the FAP-binding domain described herein includes the amino acid sequence of SEQ ID NO: 2. In some embodiments, the FAP-binding domain described herein includes an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 8. In exemplary embodiments, the FAP-binding domain described herein includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 8. In preferred embodiments, the FAP-binding domain described herein includes the amino acid sequence of SEQ ID NO: 8. In some embodiments, the FAP-binding domain described herein comprises an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 9. In exemplary embodiments, the FAP-binding domain described herein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 9.In another exemplary embodiment, the FAP-binding domain described herein includes the amino acid sequence of SEQ ID NO: 9. In some embodiments, the FAP-binding domain described herein includes an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs: 28-38. In another exemplary embodiment, the FAP-binding domain described herein includes an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 28-38.

[0070] In some embodiments, the sequence of SEQ ID NO: 2 is subjected to 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 or fewer substitutions. In some embodiments, the sequence of SEQ ID NO: 2 is subjected to 5 or fewer substitutions. In some embodiments, the sequence of SEQ ID NO: 2 is subjected to 4 or fewer substitutions. In some embodiments, the sequence of SEQ ID NO: 2 is subjected to 3 or fewer substitutions. In some embodiments, the sequence of SEQ ID NO: 2 is subjected to 2 or fewer substitutions. In some embodiments, the sequence of SEQ ID NO: 2 is subjected to 1 or fewer substitutions. In some embodiments, the substitution(s) are K of the protein containing the sequence of SEQ ID NO: 2. D Compared to the value, K DThe value will not be changed by more than 1000 times, more than 100 times, or more than 10 times. In some embodiments, the sequence of SEQ ID NO: 8 may have 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 or fewer substitutions. In some embodiments, the sequence of SEQ ID NO: 8 may have 5 or fewer substitutions. In some embodiments, the sequence of SEQ ID NO: 8 may have 4 or fewer substitutions. In some embodiments, the sequence of SEQ ID NO: 8 may have 3 or fewer substitutions. In some embodiments, the sequence of SEQ ID NO: 8 may have 2 or fewer substitutions. In some embodiments, the sequence of SEQ ID NO: 8 may have 1 or fewer substitutions. In some embodiments, the substitution(s) may be K of the protein containing the sequence of SEQ ID NO: 8. D Compared to the value, K D The values ​​should not be changed by more than 1000 times, more than 100 times, or more than 10 times. In certain embodiments, substitutions are conservative substitutions as shown in Table 1. In certain embodiments, substitutions are made outside the structural core residues of the ankyrin repeat domain, for example, within the beta loop connecting the alpha helix. In certain embodiments, substitutions are made within the structural core residues of the ankyrin repeat domain. For example, the ankyrin domain may include the consensus sequence: xDxxGxTPLHLAxxxGxxxIVxVLLxxGADVNA (SEQ ID NO: 23), where "x" preferably represents any amino acid other than cysteine, glycine, or proline, or it may include xDxxGxTPLHLAxxxGHLEIVEVLLKzGADVNA (SEQ ID NO: 24), where "x" preferably represents any amino acid other than cysteine, glycine, or proline, and "z" is selected from the group consisting of asparagine, histidine, or tyrosine. In one embodiment, the substitution is performed on the residue designated as "x". In another embodiment, the substitution is performed outside the residue designated as "x".

[0071] In addition, the second-to-last position may be "A" (see, for example, sequence numbers 2, 8, 9, 28-31, and 38) or "L" (see, for example, sequence numbers 32-37), and / or the last position may be "A" (see, for example, sequence numbers 2, 8, 9, 28-31, and 38) or "N" (see, for example, sequence numbers 32-37). Therefore, in some embodiments, the FAP-binding domain comprises an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs: 2, 8, 9, 28-31, and 38, and optionally, the second-to-last A is substituted with L, and / or the last A is substituted with N. In an exemplary embodiment, the FAP-binding domain comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 2, 8, 9, 28-31, and 38, and optionally, the second-to-last A is substituted with L, and / or the last A is substituted with N. In some embodiments, the FAP-binding domain comprises an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 2, with the second-to-last A being substituted with L and / or the last A being substituted with N. In exemplary embodiments, the FAP-binding domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, with the second-to-last A being substituted with L and / or the last A being substituted with N.In preferred embodiments, the FAP-binding domain described herein comprises the amino acid sequence of SEQ ID NO: 2, optionally with the second-to-last A replaced by L and / or the last A replaced by N. In some embodiments, the FAP-binding domain comprises an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs: 9, 28-31, and 38, optionally with the second-to-last A replaced by L and / or the last A replaced by N. In an exemplary embodiment, the FAP-binding domain comprises an amino acid sequence that is at least 90% identical to one of sequence numbers 9, 28-31, and 38, with the second-to-last A being substituted with L and / or the last A being substituted with N. In another exemplary embodiment, the FAP-binding domain comprises an amino acid sequence that is one of sequence numbers 9, 28-31, and 38, with the second-to-last A being substituted with L and / or the last A being substituted with N. In some embodiments, the FAP-binding domain comprises an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs.In an exemplary embodiment, the FAP-binding domain comprises an amino acid sequence that is at least 90% identical to any one of sequence numbers 32-37, with the second-to-last L being substituted with A and / or the last N being substituted with A. In another exemplary embodiment, the FAP-binding domain comprises an amino acid sequence that is any one of sequence numbers 32-37, with the second-to-last L being substituted with A and / or the last N being substituted with A. The sequence may optionally contain G, S, or GS at its N-terminus (see below).

[0072] In addition, the FAP-binding domain may optionally further include a "G", "S", or "GS" sequence at its N-terminus (see, for example, SEQ ID NO: 38 compared to SEQ ID NO: 2). Thus, in some embodiments, the FAP-binding domains provided herein include (i) an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs: 2, 8, 9, and 28-37, and (ii) further including G, S, or GS at its N-terminus. In exemplary embodiments, the FAP-binding domain comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 2, 8, 9, and 28-37, and further comprises G, S, or GS at its N-terminus. In exemplary embodiments, the FAP-binding domain comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 2, 8, 9, and 28-37, and further comprises G, S, or GS at its N-terminus. In exemplary embodiments, the FAP-binding domain comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 2, 8, 9, and 28-37, and further comprises G, S, or GS at its N-terminus. Therefore, in some embodiments, the FAP-binding domains provided herein include an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 38, wherein the G at position 1 and / or the S at position 2 of SEQ ID NO: 38 are optionally omitted.

[0073] Therefore, in one particularly preferred embodiment, the FAP-binding domain described herein comprises the amino acid sequence of SEQ ID NO: 2, wherein its N-terminus optionally further comprises G, S, or GS, and optionally the second-to-last A is substituted with L, and / or the last A is substituted with N.

[0074] In certain embodiments, the affinity between the FAP-binding domain or a recombinant protein containing an FAP-binding domain and its target (i.e., FAP) is K D This is described in relation to. In an exemplary embodiment, K D It is about 10 -1 M or less, about 10 -2 M or less, about 10 -3 M or less, about 10 -4 M or less, about 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, about 10 -9 M or less, about 10 -10 M or less, about 10 -11 M or less, about 10 -12 M or less, about 10 -13 M or less, about 10 -14 M or less, about 10 -5 M~about 10 -15 M, about 10 -6 M~about 10 -15 M, about 10 -7 M~about 10 -15 M, about 10 -8 M~about 10 -15 M, about 10 -9 M~about 10 -15 M, about 10 -10 M~about 10 -15 M, about 10 -5 M~about 10 -14 M, about 10 -6 M~about 10 -14 M, about 10 -7 M~about 10 -14 M, about 10 -8 M~about 10 -14 M, about 10 -9 M~about 10 -14 M, about 10 -10 M~about 10-14 M, about 10 -5 M to about 10 -13 M, about 10 -6 M to about 10 -13 M, about 10 -7 M to about 10 -13 M, about 10 -8 M to about 10 -13 M, about 10 -9 M to about 10 -13 M, or about 10 -10 M to about 10 -13 M.

[0075] In an exemplary embodiment, the FAP binding domain binds to FAP with a K value of about 100 nM, about 90 nM, about 80 nM, about 75 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 250 pM, about 200 pM, about 150 pM, about 100 pM, about 50 pM, about 40 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, or about 1 pM or less, and preferably binds to human FAP with a KD value of 100 nM, 90 nM, 80 nM, 75 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 250 pM, 200 pM, 150 pM, 140 pM, 130 pM, or 120 pM or less. In an exemplary embodiment, the FAP binding domain binds to FAP with a K D value. In another exemplary embodiment, the FAP binding domain binds to FAP with a K D value. In another exemplary embodiment, the FAP binding domain binds to FAP with a K D value. In another exemplary embodiment, the FAP binding domain binds to FAP with a K D value. In a preferred embodiment, the FAP binding domain binds to FAP with a K D value. Preferably, the FAP binding domain has the amino acid sequence of SEQ ID NO: 2.

[0076] In exemplary embodiments, recombinant proteins containing the FAP-binding domain have K levels of approximately 100 nM, 90 nM, 80 nM, 75 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, or less than 1 pM. D Binds to FAP at a KD value of 100 nM, 90 nM, 80 nM, 75 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, or 300 pM or less. In one exemplary embodiment, the recombinant protein binds to FAP at a KD value of approximately 100 nM or less. D It binds to FAP by value. In another exemplary embodiment, the FAP-binding domain is Kn about 10nM or less. D It binds to FAP at a value of approximately 1 nM or less. In another exemplary embodiment, the recombinant protein has a K value of approximately 1 nM or less. D It binds to FAP at a value of approximately 500 pM or less. In another exemplary embodiment, the recombinant protein has a K value of approximately 500 pM or less. D It binds to FAP at a certain value. In one preferred embodiment, the recombinant protein has a K value of about 300 pM or less. D It binds to FAP by value. Preferably, the recombinant protein has the amino acid sequence of SEQ ID NO: 5.

[0077] In certain embodiments, the FAP is human FAP (SEQ ID NO: 52).

[0078] [Table 1]

[0079] 3.4. CD40 binding domain The recombinant proteins disclosed herein also utilize CD40-induced immune cell costimulatory activity.

[0080] The recombinant proteins described herein include an ankyrin repeat domain that specifically binds to CD40, also referred to herein as the “CD40-binding domain.” Similar to CD40 agonist antibodies, the CD40-binding domain activates the CD40 / CD40L signaling pathway. The recombinant proteins described herein may also include two or more CD40-binding domains, for example, two or three or more CD40-binding domains. Thus, the recombinant proteins described herein may include a first and a second CD40-binding domain, or a first, second, and a third CD40-binding domain. The embodiments provided below describe such a first CD40-binding domain, a second CD40-binding domain, and / or a third CD40-binding domain. Preferably, the recombinant proteins described herein include two CD40-binding domains, namely a first CD40-binding domain and a second CD40-binding domain.

[0081] In some embodiments, the CD40 binding domain or each of the CD40 binding domains independently contains an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 3. In exemplary embodiments, the CD40 binding domain or each of the CD40 binding domains independently contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 3. In preferred embodiments, the CD40 binding domain or each of the CD40 binding domains contains the amino acid sequence of SEQ ID NO: 3. In some embodiments, the CD40 binding domain or each of the CD40 binding domains independently contains an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 10. In an exemplary embodiment, the CD40 binding domain or each of the CD40 binding domains independently contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 10. In another exemplary embodiment, the CD40 binding domain or each of the CD40 binding domains contains the amino acid sequence of SEQ ID NO: 10.In some embodiments, the CD40 binding domain or each of the CD40 binding domains independently comprises an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs: 43 - 50. In an exemplary embodiment, the CD40 binding domain or each of the CD40 binding domains independently comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 43 - 50. In another exemplary embodiment, the CD40 binding domain or each of the CD40 binding domains independently comprises an amino acid sequence of any one of SEQ ID NOs: 43 - 50.

[0082] In some embodiments, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer substitutions are made to the sequence of SEQ ID NO: 3. In some embodiments, 5 or fewer substitutions are made to the sequence of SEQ ID NO: 3. In some embodiments, 4 or fewer substitutions are made to the sequence of SEQ ID NO: 3. In some embodiments, 3 or fewer substitutions are made to the sequence of SEQ ID NO: 3. In some embodiments, 2 or fewer substitutions are made to the sequence of SEQ ID NO: 3. In some embodiments, 1 or fewer substitutions are made to the sequence of SEQ ID NO: 3. In some embodiments, the substitution(s) is / are the K of the protein comprising the sequence of SEQ ID NO: 3 D value, compared to the K DThe values ​​should not be changed by more than 1000 times, more than 100 times, or more than 10 times. In certain embodiments, substitutions are conservative substitutions as shown in Table 1. In certain embodiments, substitutions are made outside the structural core residues of the ankyrin repeat domain, for example, within the beta loop connecting the alpha helix. In certain embodiments, substitutions are made within the structural core residues of the ankyrin repeat domain. For example, each of the ankyrin domain or ankyrin-binding domain may contain the consensus sequence: xDxxGxTPLHLAxxxGxxxIVxVLLxxGADVNA (SEQ ID NO: 23), where "x" preferably represents any amino acid other than cysteine, glycine, or proline, or it may contain xDxxGxTPLHLAxxxGHLEIVEVLLKzGADVNA (SEQ ID NO: 24), where "x" preferably represents any amino acid other than cysteine, glycine, or proline, and "z" is selected from the group consisting of asparagine, histidine, or tyrosine. In one embodiment, the substitution is performed on the residue designated as "x". In another embodiment, the substitution is performed outside the residue designated as "x".

[0083] In addition, the second-to-last position may be "A" (see, for example, SEQ ID NOs: 3, 10, 43, 44, and 48-50) or "L" (see, for example, SEQ ID NOs: 45-47), and / or the last position may be "A" (see, for example, SEQ ID NOs: 3, 10, 43, 44, and 48-50) or "N" (see, for example, SEQ ID NOs: 45-47). Therefore, in some embodiments, the CD40 binding domain or each of the CD40 binding domains independently comprises an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs: 3, 10, 43, 44, and 48-50, wherein the second-to-last A is substituted with L and / or the last A is substituted with N. In exemplary embodiments, the CD40-binding domain or each of the CD40-binding domains independently comprises an amino acid sequence that is at least 90% identical to one of sequence numbers 3, 10, 43, 44, and 48-50, wherein the second-to-last A is substituted with L and / or the last A is substituted with N. In some embodiments, the CD40 binding domain or each of the CD40 binding domains independently comprises an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 3, wherein the second-to-last A is substituted with L and / or the last A is substituted with N.In exemplary embodiments, the CD40-binding domain or each of the CD40-binding domains independently comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, optionally having the second-to-last A replaced by L and / or the last A replaced by N. In preferred embodiments, the CD40-binding domain or each of the CD40-binding domains described herein comprises the amino acid sequence of SEQ ID NO: 3, optionally having the second-to-last A replaced by L and / or the last A replaced by N. In some embodiments, the CD40 binding domain or each of the CD40 binding domains independently comprises an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs: 10, 43, 44, and 48-50, wherein the second-to-last A is substituted with L and / or the last A is substituted with N. In exemplary embodiments, each CD40-binding domain, or each of such CD40-binding domains, independently comprises an amino acid sequence that is at least 90% identical to one of sequence numbers 10, 43, 44, and 48-50, optionally having the second-to-last A replaced by L and / or the last A replaced by N. In another exemplary embodiment, each CD40-binding domain, or each of such CD40-binding domains, independently comprises an amino acid sequence that is one of sequence numbers 10, 43, 44, and 48-50, optionally having the second-to-last A replaced by L and / or the last A replaced by N. The sequence may optionally contain G, S, or GS at its N-terminus (see below).

[0084] In addition, each of the CD40-binding domains provided herein may optionally further contain a "G", "S", or "GS" sequence at its N-terminus (see, for example, SEQ ID NO: 50 compared to SEQ ID NO: 3). Thus, in some embodiments, each of the CD40-binding domains provided herein independently contains an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs: 3, 10, and 43-49, and further contains G, S, or GS at its N-terminus. In exemplary embodiments, the CD40-binding domain or each of the CD40-binding domains independently comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 3, 10, and 43-49, and further comprises G, S, or GS at its N-terminus. In exemplary embodiments, the CD40-binding domain or each of the CD40-binding domains independently comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 3, 10, and 43-49, and further comprises G, S, or GS at its N-terminus. In exemplary embodiments, the CD40-binding domain or each of the CD40-binding domains independently comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 3, 10, and 43-49, and further comprises G, S, or GS at its N-terminus.Therefore, in some embodiments, the CD40 binding domain provided herein or each of said CD40 binding domains comprises an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 50, wherein the G at position 1 and / or the S at position 2 of SEQ ID NO: 50 are optionally omitted.

[0085] Therefore, in one particularly preferred embodiment, the CD40 binding domain described herein or each of said CD40 binding domains comprises the amino acid sequence of SEQ ID NO: 3, wherein its N-terminus optionally further comprises G, S, or GS, and optionally the second-to-last A is substituted with L, and / or the last A is substituted with N.

[0086] In some preferred embodiments, any one of the CD40 binding domains described herein includes Q at position 8, L at position 15, R at position 143, and / or Q at position 147, where the position numbers correspond to the positions of sequence number 3. Thus, in some embodiments, the CD40 binding domains described herein or each of said CD40 binding domains includes (1) Q at position 8, and (2) R at position 143, and / or Q at position 147, where the position numbers correspond to the positions of sequence number 3. In some embodiments, the CD40 binding domains described herein or each of said CD40 binding domains includes (1) L at position 15, and (2) R at position 143, and / or Q at position 147, where the position numbers correspond to the positions of sequence number 3. In some embodiments, the CD40 binding domains described herein or each of the CD40 binding domains include (1) Q at position 8 and L at position 15, and (2) R at position 143 and / or Q at position 147, where the position numbers correspond to the positions of sequence number 3. Thus, in some embodiments, the CD40 binding domains described herein or each of the CD40 binding domains include (1) Q at position 8 and / or L at position 15, and (2) R at position 143, where the position numbers correspond to the positions of sequence number 3. In some embodiments, the CD40 binding domains described herein or each of the CD40 binding domains include (1) Q at position 8 and / or L at position 15, and (2) Q at position 147, where the position numbers correspond to the positions of sequence number 3. In some embodiments, the CD40 binding domain described herein or each of said CD40 binding domains includes (1) Q at position 8 and / or L at position 15, and (2) R at position 143 and Q at position 147, where the position numbers correspond to the positions of SEQ ID NO: 3. In some more preferred embodiments, the CD40 binding domain described herein or each of said CD40 binding domains includes Q at position 8, L at position 15, R at position 143, and Q at position 147, where the position numbers correspond to the positions of SEQ ID NO: 3.

[0087] Furthermore, in a more preferred embodiment, each of the CD40-binding domains described herein includes Q at position 8, L at position 15, R at position 143, and Q at position 147, where the position numbers correspond to the positions of SEQ ID NO: 3, resulting in improved pharmacokinetic properties of the CD40-binding domain compared to a CD40-binding domain having the same amino acid sequence except for the amino acids at positions 8, 15, 143, and 147, which are different from Q, L, R, and Q, respectively, where the position numbers correspond to the positions of SEQ ID NO: 3.

[0088] In certain embodiments, the affinity between a recombinant protein containing a CD40-binding domain or each of said CD40-binding domains, or multiple CD40-binding domains, and its target (i.e., CD40) is K D This is described in relation to. In an exemplary embodiment, K D It is about 10 -1 M or less, about 10 -2 M or less, about 10 -3 M or less, about 10 -4 M or less, about 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, about 10 -9 M or less, about 10 -10 M or less, about 10 -11 M or less, about 10 -12 M or less, about 10 -13 M or less, about 10 -14 M or less, about 10 -5 M~about 10 -15 M, about 10 -6 M~about 10 -15 M, about 10 -7 M~about 10 -15 M, about 10 -8 M~about 10 -15 M, about 10 -9 M~about 10 -15 M, about 10 -10 M~about 10 -15 M, about 10 -5 M~about 10 -14 M, about 10 -6 M~about 10 -14 M, about 10 -7M~about 10 -14 M, about 10 -8 M~about 10 -14 M, about 10 -9 M~about 10 -14 M, about 10 -10 M~about 10 -14 M, about 10 -5 M~about 10 -13 M, about 10 -6 M~about 10 -13 M, about 10 -7 M~about 10 -13 M, about 10 -8 M~about 10 -13 M, about 10 -9 M~about 10 -13 M, or about 10 -10 M~about 10 -13 It is M.

[0089] In exemplary embodiments, the CD40 binding domain or each of the CD40 binding domains independently has a K content of approximately 100 nM, approximately 90 nM, approximately 80 nM, approximately 75 nM, approximately 60 nM, approximately 50 nM, approximately 40 nM, approximately 30 nM, approximately 20 nM, approximately 10 nM, approximately 5 nM, approximately 2 nM, approximately 1 nM, approximately 900 pM, approximately 800 pM, approximately 700 pM, approximately 600 pM, approximately 500 pM, approximately 400 pM, approximately 300 pM, approximately 250 pM, approximately 200 pM, approximately 150 pM, approximately 100 pM, approximately 50 pM, approximately 40 pM, approximately 30 pM, approximately 25 pM, approximately 20 pM, approximately 15 pM, approximately 10 pM, approximately 5 pM, or approximately 1 pM or less. D Binding to CD40 at a KD value, preferably 100 nM, 90 nM, 80 nM, or 75 nM or less. In an exemplary embodiment, the CD40 binding domain or each of said CD40 binding domains independently binds to CD40 at a KD value of about 100 nM or less. D It binds to CD40 by value. In a preferred embodiment, the CD40 binding domain or each of the CD40 binding domains independently has a K of about 75 nM or less. D It binds to CD40 by value. Preferably, the CD40 binding domain or each of the CD40 binding domains has the amino acid sequence of SEQ ID NO: 3.

[0090] In exemplary embodiments, recombinant proteins containing two CD40-binding domains have K levels of approximately 100 nM, 90 nM, 80 nM, 75 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, or less than 1 pM. D Binding to CD40 at a KD value of approximately 100 nM, 90 nM, 80 nM, 75 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 250 pM, 200 pM, 150 pM, 140 pM, 130 pM, 120 pM, 115 pM, 110 pM, 105 pM, or 100 pM or less. In an exemplary embodiment, the recombinant protein binds to CD40 at a KD value of approximately 100 nM or less. D It binds to CD40 at a value of . In another exemplary embodiment, the recombinant protein has a K value of approximately 1 nM or less. D It binds to CD40 at a value of 500 pM or less. In another exemplary embodiment, the recombinant protein has a K value of 500 pM or less. D It binds to CD40 at a value of . In a preferred embodiment, the recombinant protein has a K value of about 100 pM or less. D It binds to CD40 at a specific value. Preferably, the recombinant protein has the amino acid sequence of SEQ ID NO: 5.

[0091] In some embodiments, two or more CD40 binding domains are preferred to further promote CD40 clustering and immune cell co-stimulation. CD40 ligand has been reported to bind to CD40 on immune cells as a trimer. However, trimerization alone is not sufficient to activate the CD40 signaling pathway. Higher-order clustering of CD40 is required for its activation. As described herein, through FAP binding, the multi-selective molecule already promotes CD40 clustering in the tumor environment. To further promote CD40 clustering, two or more CD40 binding domains can be used to produce a "cross-linking" effect on the cell surface. For example, as shown in Figure 4, a monovalent CD40 binder (FC) was sufficient to activate the CD40 pathway. Higher potency can be achieved by using two CD40 binding domains (FCC), or three CD40 binding domains (FCCC). Figure 4 also shows that two CD40 binding domains are sufficient to activate the CD40 pathway with high potency and that it is not necessary to have three CD40 binding domains for efficient CD40 clustering.

[0092] In certain embodiments, CD40 is human CD40 (SEQ ID NO: 51).

[0093] 3.5. Half-life extension moiety A "half-life extension moiety" extends the in vivo serum half-life of the recombinant proteins described herein as compared to the same protein without a half-life extension moiety. Examples of half-life extension moieties 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).

[0094] In some embodiments, the recombinant multiselective proteins described herein include an ankyrin repeat domain, also referred herein as the “serum albumin-binding domain,” which specifically binds to serum albumin. The recombinant proteins described herein may also include two or more serum albumin-binding domains, for example, two or three or more serum albumin-binding domains. Thus, the recombinant proteins described herein may include a first and a second serum albumin-binding domain, or a first, second, and a third serum albumin-binding domain. The embodiments provided below describe such a first serum albumin-binding domain, a second serum albumin-binding domain, and / or a third serum albumin-binding domain. Preferably, the recombinant proteins described herein include only one serum albumin-binding domain.

[0095] In some embodiments, the serum albumin-binding domain described herein comprises an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 1. In exemplary embodiments, the serum albumin-binding domain described herein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1. In preferred embodiments, the serum albumin-binding domain described herein comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the serum albumin-binding domain described herein comprises an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs: 39-42. In an exemplary embodiment, the serum albumin-binding domain described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 39-42. In another exemplary embodiment, the serum albumin-binding domain described herein comprises an amino acid sequence of any one of SEQ ID NOs: 39-42.

[0096] In some embodiments, the sequence of SEQ ID NO: 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer substitutions are made to the sequence of SEQ ID NO: 1. In some embodiments, the sequence of SEQ ID NO: 1 is subjected to 5 or fewer substitutions. In some embodiments, the sequence of SEQ ID NO: 1 is subjected to 4 or fewer substitutions. In some embodiments, the sequence of SEQ ID NO: 1 is subjected to 3 or fewer substitutions. In some embodiments, the sequence of SEQ ID NO: 1 is subjected to 2 or fewer substitutions. In some embodiments, the sequence of SEQ ID NO: 1 is subjected to 1 or fewer substitutions. In some embodiments, the substitution(s) are K of the protein containing the sequence of SEQ ID NO: 1. D Compared to the value, K D The values ​​should not be changed by more than 1000 times, more than 100 times, or more than 10 times. In certain embodiments, substitutions are conservative substitutions as shown in Table 1. In certain embodiments, substitutions are made outside the structural core residues of the ankyrin repeat domain, for example, within the beta loop connecting the alpha helix. In certain embodiments, substitutions are made within the structural core residues of the ankyrin repeat domain. For example, the ankyrin domain may include the consensus sequence: xDxxGxTPLHLAxxxGxxxIVxVLLxxGADVNA (SEQ ID NO: 23), where "x" preferably represents any amino acid other than cysteine, glycine, or proline, or it may include xDxxGxTPLHLAxxxGHLEIVEVLLKzGADVNA (SEQ ID NO: 24), where "x" preferably represents any amino acid other than cysteine, glycine, or proline, and "z" is selected from the group consisting of asparagine, histidine, or tyrosine. In one embodiment, the substitution is performed on the residue designated as "x". In another embodiment, the substitution is performed outside the residue designated as "x".

[0097] In addition, the second-to-last position may be "A" (see, for example, SEQ ID NOs: 1, 39, 40, and 42) or "L" (see, for example, SEQ ID NOs: 41), and / or the last position may be "A" (see, for example, SEQ ID NOs: 1, 39, 40, and 42) or "N" (see, for example, SEQ ID NOs: 1). Therefore, in some embodiments, the serum albumin-binding domain comprises an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs: 1, 39, 40, and 42, with the second-to-last A being substituted with L and / or the last A being substituted with N. In exemplary embodiments, the serum albumin-binding domain comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 1, 39, 40, and 42, with the second-to-last A being substituted with L and / or the last A being substituted with N. In some embodiments, the serum albumin-binding domain comprises an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 1, with the second-to-last A being substituted with L and / or the last A being substituted with N. In exemplary embodiments, the serum albumin-binding domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, with the second-to-last A being substituted with L and / or the last A being substituted with N.In preferred embodiments, the serum albumin-binding domain described herein comprises the amino acid sequence of SEQ ID NO: 1, optionally having the second-to-last A replaced by L and / or the last A replaced by N. In some embodiments, the serum albumin-binding domain comprises an amino acid sequence that is at least 80%, at least 81%, 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 to any one of SEQ ID NOs: 39, 40, and 42, optionally having the second-to-last A replaced by L and / or the last A replaced by N. In an exemplary embodiment, the serum albumin-binding domain comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NOs: 39, 40, and 42, with the second-to-last A being substituted with L and / or the last A being substituted with N. In another exemplary embodiment, the serum albumin-binding domain comprises an amino acid sequence of one of SEQ ID NOs: 39, 40, and 42, with the second-to-last A being substituted with L and / or the last A being substituted with N. The sequence may optionally contain G, S, or GS at its N-terminus (see below).

[0098] In addition, the serum albumin-binding domain may optionally further include a "G", "S", or "GS" sequence at its N-terminus (see, for example, SEQ ID NO: 1 compared to SEQ ID NO: 42). Thus, in some embodiments, the serum albumin-binding domain provided herein comprises (i) an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 42, and further comprises G, S, or GS at its N-terminus. In an exemplary embodiment, the serum albumin-binding domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 42, and further comprises G, S, or GS at its N-terminus. In an exemplary embodiment, the serum albumin-binding domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 42, and further comprises G, S, or GS at its N-terminus. Therefore, in some embodiments, the serum albumin-binding domains provided herein include an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 1 and one of SEQ ID NO: 39-41, wherein the G at position 1 and / or the S at position 2 of SEQ ID NO: 1 and one of SEQ ID NO: 39-41 are optionally omitted.In exemplary embodiments, the serum albumin-binding domain comprises an amino acid sequence that is at least 90% identical to one of sequence numbers 1, 30, and 31, wherein the G at position 1 and / or the S at position 2 of sequence numbers 1 and one of sequence numbers 39-41 are optionally omitted.

[0099] Therefore, in one particularly preferred embodiment, the serum albumin-binding domain described herein comprises the amino acid sequence of SEQ ID NO: 1, wherein the G at position 1 and / or the S at position 2 are optionally omitted, the second-to-last A is optionally replaced with L, and / or the last A is replaced with N.

[0100] In certain embodiments, the affinity between a serum albumin-binding domain or a recombinant protein containing a serum albumin-binding domain and its target (i.e., serum albumin) is K D This is described in relation to. In an exemplary embodiment, K D It is about 10 -1 M or less, about 10 -2 M or less, about 10 -3 M or less, about 10 -4 M or less, about 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, about 10 -9 M or less, about 10 -10 M or less, about 10 -11 M or less, about 10 -12 M or less, about 10 -13 M or less, about 10 -14 M or less, about 10 -5 M~about 10 -15 M, about 10 -6 M~about 10 -15 M, about 10 -7 M~about 10 -15 M, about 10 -8 M~about 10 -15 M, about 10 -9 M~about 10 -15 M, about 10 -10 M~about 10 -15 M, about 10 -5 M~about 10 -14M, about 10 -6 M~about 10 -14 M, about 10 -7 M~about 10 -14 M, about 10 -8 M~about 10 -14 M, about 10 -9 M~about 10 -14 M, about 10 -10 M~about 10 -14 M, about 10 -5 M~about 10 -13 M, about 10 -6 M~about 10 -13 M, about 10 -7 M~about 10 -13 M, about 10 -8 M~about 10 -13 M, about 10 -9 M~about 10 -13 M, or about 10 -10 M~about 10 -13 It is M.

[0101] In exemplary embodiments, the serum albumin-binding domain has a K content of approximately 100 nM, 90 nM, 80 nM, 75 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, or less than 1 pM. D Binds to serum albumin at a KD value of 100 nM, 90 nM, 80 nM, 75 nM, 70 nM, 60 nM, 50 nM, 40 nM, or 35 nM or less. In an exemplary embodiment, the serum albumin-binding domain binds to serum albumin at a KD value of approximately 100 nM or less. D It binds to serum albumin at a value of approximately 50 nM or less. In another exemplary embodiment, the serum albumin-binding domain has a K value of approximately 50 nM or less. D It binds to serum albumin at a certain value. In a preferred embodiment, the serum albumin-binding domain has a K content of about 35 pM or less. DIt binds to serum albumin. Preferably, the serum albumin-binding domain has the amino acid sequence of SEQ ID NO: 1.

[0102] In exemplary embodiments, the recombinant protein containing the serum albumin domain has a K content of approximately 100 nM, 90 nM, 80 nM, 75 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, or less than 1 pM. D The recombinant protein binds to serum albumin at a KD value of approximately 100 nM, 90 nM, 80 nM, 75 nM, 70 nM, 60 nM, or 50 nM or less. In an exemplary embodiment, the recombinant protein binds to serum albumin at a KD value of approximately 100 nM or less. D It binds to serum albumin at a certain value. In another exemplary embodiment, the recombinant protein has a K content of approximately 75 nM or less. D It binds to serum albumin at a certain value. In one preferred embodiment, the recombinant protein has a K content of about 50 nM or less. D It binds to serum albumin at a specific value. Preferably, the recombinant protein has the amino acid sequence of SEQ ID NO: 5.

[0103] In certain embodiments, serum albumin is human serum albumin (SEQ ID NO: 53).

[0104] 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.

[0105] In some embodiments, the Fc domain includes an uninterrupted native sequence of the Fc domain (i.e., the wild-type sequence). In some embodiments, the immunoglobulin Fc domain includes a mutant 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 WO2005 / 063815) and / or to increase homogeneity during recombinant protein production. 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, Eu-numbered). The absence of lysine may increase homogeneity during recombinant protein production. In some embodiments, the Fc domain includes lysine located at the C-terminal position (K447, Eu-numbered).

[0106] 3.6. Linker The recombinant proteins described herein may include linkers. A “linker” is a molecule or group of molecules that can bind to two distinct entities (e.g., an FAP-binding domain and a CD40-binding domain) and provide spacing and flexibility between the two entities so that they can achieve conformations that specifically bind to their respective targets (e.g., FAP and CD40). Protein linkers are particularly preferred and can be expressed as components of recombinant proteins using standard recombinant DNA techniques well known in the art.

[0107] Ankyrin repeat domains can be linked, for example, by disulfide bonds, polypeptide bonds, or covalent or non-covalent bonds via crosslinking agents to generate heterodimeric proteins. Recombinant proteins may contain linkers between FAP and any binding domain including the CD40 binding domain, and between any binding domain and any half-life extension portion (which may itself be a binding domain).

[0108] In some embodiments, the linker is a peptidyl linker. In some embodiments, the peptidyl linker contains about 1 to 30 amino acid residues. Exemplary linkers include, for example, glycine-rich peptides; peptides containing glycine and serine; and sequences such as [Gly-Gly-Ser]. n A peptide having the formula n = 1, 2, 3, 4, 5 or 6; or the sequence [Gly-Gly-Gly-Gly-Ser] n Examples of peptides include those having (SEQ ID NO: 56), where n is 1, 2, 3, 4, 5, or 6. Glycine-rich peptide linkers contain a peptide linker, and at least 25% of the residues are glycine. Glycine-rich peptide linkers are well known in the art (e.g., Chichili et al. Protein Sci. February 2013; 22(2): pp. 153-167).

[0109] In some embodiments, the peptidyl linker is a proline-threonine-rich peptide linker. In an exemplary embodiment, the linker is the proline-threonine-rich peptide linker of SEQ ID NO: 4.

[0110] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 4.

[0111] 3.7. FAP / CD40 dual-targeting, bispecific, or multi-selective molecules The multiselective molecules of the present invention include any combination of binding domains and linkers as described herein, optionally selected from among them. That is, any one of the domains and linkers described in sections 3.3 to 3.6 above can be combined in the multiselective molecules of the present invention. Furthermore, the binding domain of the multiselective molecules of the present invention may include either the N-terminal capping module and / or the C-terminal capping module described in section 3.2 above.

[0112] In some embodiments, the recombinant protein of the present invention comprises, from the N-terminus to the C-terminus, (i)) a first ankyrin repeat domain that specifically binds to serum albumin, (ii) a second ankyrin repeat domain that specifically binds to FAP, (iii) a third ankyrin repeat domain that specifically binds to CD40, and (iv) a fourth ankyrin repeat domain that specifically binds to CD40. The first ankyrin repeat domain may be any one of the serum albumin-binding domains described in Section 3.5, the second ankyrin repeat domain may be any one of the FAP-binding domains described in Section 3.3, and the third and fourth ankyrin repeat domains may be any one of the CD40-binding domains described in Section 3.4. The third and fourth ankyrin repeat domains may have the same sequence or may have different sequences.

[0113] In some preferred embodiments, the multi-selective recombinant protein of the present invention comprises, from the N-terminus to the C-terminus, (serum albumin binding domain)-(linker)-(FAP binding domain)-(linker)-(CD40 binding domain)-(linker)-(CD40 binding domain), wherein the linker preferably comprises the amino acid sequence of SEQ ID NO: 4.

[0114] In certain embodiments, the recombinant protein of the present invention comprises an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 5. In one preferred embodiment, the recombinant protein of the present invention comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5. In a more preferred embodiment, the recombinant protein of the present invention comprises the amino acid sequence of SEQ ID NO: 5.

[0115] In certain embodiments, one of the four binding domains of SEQ ID NO: 5 is subjected to 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 or fewer substitutions. In some embodiments, one of the four binding domains of SEQ ID NO: 5 is subjected to 10 or fewer substitutions. In some embodiments, one of the four binding domains of SEQ ID NO: 5 is subjected to 5 or fewer substitutions. In some embodiments, one of the four binding domains of SEQ ID NO: 5 is subjected to 4 or fewer substitutions. In some embodiments, one of the four binding domains of SEQ ID NO: 5 is subjected to 3 or fewer substitutions. In some embodiments, one of the four binding domains of SEQ ID NO: 5 is subjected to 2 or fewer substitutions. In some embodiments, one of the four binding domains of SEQ ID NO: 5 is subjected to 1 or fewer substitutions. In some embodiments, the substitution(s) are K of the protein containing the sequence of SEQ ID NO: 5. D Compared to the value, K is FAP-bound, CD40-bound, or serum albumin-bound. D The value should not be changed by more than 1000 times, more than 100 times, or more than 10 times. In certain embodiments, the substitution is a conservative substitution as shown in Table 1.

[0116] In certain embodiments, the recombinant protein of the present invention comprises an amino acid sequence that is at least 80%, at least 81%, 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 to SEQ ID NO: 6.

[0117] In a specific embodiment, the multiselective recombinant protein of the present invention is added to human CD40 in PBS. -8 Less than M, 10 -9 Less than M, 5 x 10 -10 Less than M, 3 x 10 -10Less than M, or 2 × 10 -10 Dissociation constant less than M (K D ) binds. In a preferred embodiment, the recombinant protein of the present invention is 10 -9 Dissociation constant less than M (K D It binds to human CD40 in PBS.

[0118] In a particular embodiment, the multiselective recombinant protein of the present invention is added to human FAP in PBS. -8 Less than M, 10 -9 Less than M, 5 x 10 -10 Less than M, or 3 × 10 -10 Dissociation constant less than M (K D ) binds. In a preferred embodiment, the recombinant protein of the present invention is bonded to human FAP in PBS by 10 -9 Dissociation constant less than M (K D They are joined together using ).

[0119] In a specific embodiment, the multiselective recombinant protein of the present invention is mixed with human serum albumin in PBS, 10 -7 Less than M, 7 x 10 -8 Less than M, or 5 × 10 -8 Dissociation constant less than M (K D ) binds to human serum albumin in PBS. In a preferred embodiment, the recombinant protein of the present invention is bonded to human serum albumin in PBS. -7 Dissociation constant less than M (K D They are joined together using ).

[0120] In certain embodiments, the multiselective recombinant protein of the present invention is 10 -8 Less than M, 10 -9 Less than M, 5 x 10 -10 Less than M, 3 x 10 -10 M, or 2×10 -10 The dissociation constant (K D The recombinant protein binds to human CD40 in PBS at a value of 10 -8 Less than M, 10 -9 Less than M, 5 x 10 -10 Less than M, or 3 x 10 -10 Dissociation constant less than M (K DIt binds to human FAP in PBS at a value of ). In a preferred embodiment, the recombinant protein of the present invention binds to human CD40 in PBS at a value of 10 -9 Dissociation constant less than M (K D The recombinant protein is bound to human FAP in PBS, and the recombinant protein is 10 -9 Dissociation constant less than M (K D They are joined together using ).

[0121] In a particular embodiment, the recombinant protein of the present invention is 10 -8 Less than M, 10 -9 Less than M, 5 x 10 -10 Less than M, 3 x 10 -10 M, or 2×10 -10 The dissociation constant (K D The recombinant protein binds to human CD40 in PBS at a value of 10 -8 Less than M, 10 -9 Less than M, 5 x 10 -10 Less than M, or 3 x 10 -10 Dissociation constant less than M (K D The recombinant protein binds to human FAP in PBS at a value of 10 -7 Less than M, 7x10 -8 Less than M, or 5x10 -8 Dissociation constant less than M (K D It binds to human serum albumin in PBS at a value of 10. In a preferred embodiment, the recombinant protein of the present invention binds to human CD40 in PBS at a value of 10. -9 Dissociation constant less than M (K D The recombinant protein is bound to human FAP in PBS, and the recombinant protein is 10 -9 Dissociation constant less than M (K D ) binds. In a more preferred embodiment, the recombinant protein of the present invention is bonded to human CD40 in PBS. -9 Dissociation constant less than M (K D The recombinant protein is bound to human FAP in PBS at 10 -9 Dissociation constant less than M (K D The recombinant protein binds to human serum albumin in PBS at 10 -7 Dissociation constant less than M (K D They are joined together using ).

[0122] In certain preferred embodiments, the recombinant binding protein of the present invention simultaneously binds to binding FAP, CD40, and serum albumin, and preferably, this simultaneous binding is measured by surface plasmon resonance (SPR), and more preferably, as described in Example 3.

[0123] In certain embodiments, the multiselective recombinant protein of the present invention induces B cell activation upon binding to FAP and CD40. In certain embodiments, the B cells are human B cells. In certain embodiments, the biological activity of the multiselective recombinant protein is evaluated by an in vitro B cell activation assay that measures the expression of costimulatory molecules such as CD86 and CD69. Increased expression of these costimulatory molecules (CD86 and CD69) in B cells has been reported to indicate CD40 activation.

[0124] In certain embodiments, the multiselective recombinant protein of the present invention inhibits human CD40 in CD40-expressing B cells in the presence of FAP-expressing CHO cells by approximately 10 -8 M or less, or about 10 -9 Activation occurs at EC50 values ​​below M.

[0125] In certain embodiments, the multiselective recombinant protein is evaluated by an in vitro B cell activation assay, with a molecular weight of approximately ≤100 nM, ≤75 nM, ≤65 nM, ≤55 nM, ≤45 nM, ≤35 nM, ≤25 nM, ≤15 nM, ≤10 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, or ≤0.1 nM, ≤0.01 nM to ≤50 nM, ≤0.01 nM to ≤25 nM, ≤0.01 nM to ≤10 nM, ≤0.01 nM to ≤5 nM, ≤0.01 nM to ≤1 nM, ≤0.01 nM ~ about 0.1nM, about 0.01nM - about 0.07nM, about 0.04nM - about 50nM, about 0.04nM - about 25nM, about 0.04n M ~ about 10nM, about 0.04nM - about 5nM, about 0.04nM - about 1nM, about 0.04nM - about 0.1nM, about 0.04nM - about 0.0 7nM, about 0.1nM to about 50nM, about 0.1nM to about 25nM, about 0.1nM to about 10nM, about 0.1nM to about 5nM, about 0.1n Half effective concentration (EC 50 ) has.

[0126] In an exemplary embodiment, the multiselective recombinant protein has an EC50 of about 10 nM or less, as assessed by an in vitro B cell activation assay. In another exemplary embodiment, the multiselective recombinant protein has an EC50 of about 1 nM or less, as assessed by an in vitro B cell activation assay. In yet another exemplary embodiment, the multiselective recombinant protein has an EC50 of about 0.01 nM to about 10 nM, preferably about 0.1 nM to about 1.0 nM, or about 0.18 nM to about 0.85 nM, as assessed by an in vitro B cell activation assay. In yet another exemplary embodiment, the multiselective recombinant protein has an EC50 of about 0.01 nM to about 0.1 nM, preferably about 0.04 nM to about 0.07 nM, as assessed by an in vitro B cell activation assay.

[0127] In a particular embodiment, the B cell activation assay is a human B cell activation assay. In an exemplary embodiment, EC 50This is measured using GraphPad Prism (version 8.1.2) according to the manufacturer's instructions. In exemplary embodiments, EC 50 The values ​​are determined by fitting the data with a 4-parameter logistic fit model using Graphpad Prism software. In an exemplary embodiment, EC 50 The value is determined using the method described in the examples.

[0128] In certain embodiments, the multiselective recombinant protein has a terminal phase half-life of at least 10 hours, at least 20 hours, at least 30 hours, at least 40 hours, or about 44 hours in a mouse model. In certain embodiments, the multiselective recombinant protein has a terminal phase half-life of at least 1 day, at least 2 days, at least 3 days, at least 4 days, or about 2.8 days, or about 4.5 days in a cynomolgus monkey model.

[0129] In certain embodiments, the multiselective recombinant protein of the present invention can inhibit tumor growth in an FAP-expressing MC38 colon cancer mouse model. In one embodiment, the recombinant protein of the present invention can inhibit tumor growth in an FAP-expressing MC38 colon cancer mouse model under treatment conditions as described in Example 6.

[0130] In certain embodiments, the multiselective recombinant protein does not inhibit FAP protease activity. In certain embodiments, in the presence of the multiselective recombinant protein, FAP protease activity is reduced by 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% compared to the control (the control may be FAP protease activity in the absence of the multiselective recombinant protein). In exemplary embodiments, FAP activity is measured using the method illustrated in Example 7.

[0131] In a particular embodiment, the multiselective recombinant protein of the present invention comprises two CD40-binding domains, where each of the CD40-binding domains independently contains Q at position 8, L at position 15, R at position 143, and / or Q at position 147, where the position numbers correspond to the positions of SEQ ID NO: 3. In a preferred embodiment, the multiselective recombinant protein of the present invention comprises two CD40-binding domains, where each of the CD40-binding domains contains Q at position 8, L at position 15, R at position 143, and Q at position 147, where the position numbers correspond to the positions of SEQ ID NO: 3.

[0132] In one embodiment, the multiselective recombinant protein of the present invention comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5, and further comprises one or any combination of the following properties: (i) The recombinant protein is mixed with human CD40 in PBS, and -8 Less than M, 10 -9 Less than M, 5 x 10 -10 Less than M, 3 x 10 -10 Less than M, or 2 × 10 -10 Dissociation constant less than M (K D (ii) The recombinant protein is bound to human FAP in PBS by 10 -8 Less than M, 10 -9 Less than M, 5 x 10 -10 Less than M, or 3 × 10 -10 Dissociation constant less than M (K D (iii) The recombinant protein is bound to human serum albumin in PBS by 10 -7 Less than M, 7 x 10 -8 Less than M, or 5 × 10 -8 Dissociation constant less than M (K D (iv) The recombinant protein binds to human CD40 in CD40-expressing B cells in the presence of FAP-expressing CHO cells by approximately 10 -8 M or less or about 10 -9(v) The recombinant binding protein is activated at EC50 values ​​of M or less; (v) The recombinant binding protein can bind simultaneously with FAP, CD40, and serum albumin; (vi) The recombinant protein does not inhibit FAP protease activity, or the reduction in FAP protease activity in the presence of the recombinant protein is ≤25%, ≤20%, ≤15%, or ≤10%; (vii) The recombinant protein has a terminal phase half-life of at least 10 hours, at least 20 hours, at least 30 hours, at least 40 hours, or about 44 hours in the mouse model; (viii) The recombinant protein has a terminal phase half-life of at least 1 day, or at least 44 hours in the cynomolgus monkey model. (ix) The recombinant protein has a terminal phase half-life of 2 days, at least 3 days, at least 4 days, or about 2.8 days, or about 4.5 days; (x) The recombinant protein can inhibit tumor growth in a FAP-expressing MC38 colon cancer mouse model; (x) One of the two CD40-binding domains or each of the two CD40-binding domains independently contains Q at position 8, L at position 15, R at position 143, and / or Q at position 147, where the position numbers correspond to the positions of SEQ ID NO: 3, or more preferably, each of the two CD40-binding domains independently contains Q at position 8, L at position 15, R at position 143, and Q at position 147, where the position numbers correspond to the positions of SEQ ID NO: 3.

[0133] In certain embodiments, the multiselective recombinant protein of the present invention comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, and the recombinant protein is added to human CD40 in PBS. -9 Dissociation constant less than M (K D ) is bound to and / or to human FAP in PBS, 10 -9 Dissociation constant less than M (K D They are joined together using ).

[0134] In certain embodiments, the multiselective recombinant protein of the present invention comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, and the recombinant protein is mixed with human CD40 in PBS. -9 Dissociation constant less than M (K D ) is bound to human FAP in PBS and 10 -9Dissociation constant less than M (K D ) is bound to human serum albumin in PBS and 10 -7 Dissociation constant less than M (K D They are joined together using ).

[0135] In certain embodiments, the multiselective recombinant protein of the present invention comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, and in the presence of FAP-expressing CHO cells, it can express human CD40 in CD40-expressing B cells for about 10 -8 M or less, or about 10 -9 Activation occurs at EC50 values ​​below M.

[0136] In certain embodiments, the multiselective recombinant protein of the present invention comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, and co-bounds to human CD40, human FAP, and human serum albumin, preferably by surface plasmon resonance (SPR), and more preferably as described in Example 3.

[0137] In certain embodiments, the multiselective recombinant protein of the present invention comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, and the recombinant protein does not inhibit FAP protease activity, or the reduction in FAP protease activity in the presence of the recombinant protein is 25% or less, 20% or less, 15% or less, or 10% or less.

[0138] In certain embodiments, the multiselective recombinant protein of the present invention comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5 and has a terminal phase half-life of at least 10 hours, at least 20 hours, at least 30 hours, at least 40 hours, or about 44 hours in a mouse model.

[0139] In certain embodiments, the multiselective recombinant protein of the present invention comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5 and has a terminal phase half-life of at least 1 day, at least 2 days, at least 3 days, at least 4 days, or about 2.8 days, or about 4.5 days, in a cynomolgus monkey model.

[0140] In certain embodiments, the multiselective recombinant protein of the present invention comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, and the recombinant protein can inhibit tumor growth in a FAP-expressing MC38 colon cancer mouse model, preferably such tumor inhibition is measured as described in Example 6.

[0141] In certain embodiments, the multiselective recombinant protein of the present invention comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, and one of the two CD40-binding domains or each of the two CD40-binding domains independently contains Q at position 8, L at position 15, R at position 143, and / or Q at position 147, where the position numbers correspond to the positions of SEQ ID NO: 3. In certain preferred embodiments, the multiselective recombinant protein of the present invention comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, and each of the two CD40-binding domains independently contains Q at position 8, L at position 15, R at position 143, and Q at position 147, where the position numbers correspond to the positions of SEQ ID NO: 3.

[0142] In one embodiment, the recombinant protein of the present invention comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, and the recombinant protein has a K content of 100 nM or less. D The recombinant protein binds to human FAP, human CD40, and human serum albumin at a specific value, and has a terminal phase half-life in a cynomolgus monkey model of at least 1 day, at least 2 days, at least 3 days, at least 4 days, or about 2.8 days, or about 4.5 days.

[0143] In one embodiment, the recombinant protein of the present invention comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, and the recombinant protein has a K content of 100 nM or less. DThe values ​​bind to human FAP, human CD40, and human serum albumin, and in the presence of the recombinant protein, the FAP protease activity is reduced by 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less compared to the control, and typically and preferably, the control is the FAP protease activity in the absence of the recombinant protein, and more typically and preferably, the FAP protease activity is measured as described in Example 7.

[0144] In one embodiment, the recombinant protein of the present invention comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, and the recombinant protein has a K content of 100 nM or less. D The recombinant protein binds to human FAP, human CD40, and human serum albumin at a value, and has a terminal phase half-life in a cynomolgus monkey model of at least 1 day, at least 2 days, at least 3 days, at least 4 days, or about 2.8 days, or about 4.5 days, and in the presence of the recombinant protein, the FAP protease activity is reduced by 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less compared to the control, and typically and preferably, the control is the FAP protease activity in the absence of the recombinant protein, and more typically and preferably, the FAP protease activity is measured as described in Example 7.

[0145] In certain embodiments, the multiselective recombinant protein of the present invention comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, and the protein is mixed with human CD40 in PBS. -9 Dissociation constant less than M (K D ) binds to human FAP in PBS, and the protein then binds to human FAP in PBS. -9 Dissociation constant less than M (K D The protein binds to human CD40 in CD40-expressing B cells in the presence of FAP-expressing CHO cells, and this protein binds to human CD40 in CD40-expressing B cells in the presence of FAP-expressing CHO cells in approximately 10 minutes. -8The protein is activated at EC50 values ​​of M or less, can simultaneously bind to FAP and CD40, does not inhibit FAP protease activity, or the reduction in FAP protease activity in the presence of the recombinant protein is 25% or less, and has a terminal phase half-life of at least 10 hours in a mouse model.

[0146] In certain embodiments, the recombinant protein described herein comprises a first ankyrin repeat domain that specifically binds to serum albumin, a second ankyrin repeat domain that specifically binds to fibroblast-activating protein (FAP), a third ankyrin repeat domain that specifically binds to CD40, and a fourth ankyrin repeat domain that specifically binds to CD40, wherein the ankyrin repeat domains are arranged from the N-terminus to the C-terminus according to the following formula: (serum albumin-binding domain)-(linker)-(FAP-binding domain)-(linker)-(CD40-binding domain)-(linker)-(CD40-binding domain), and the recombinant protein is fused to human FAP in PBS. -9 Dissociation constant less than M (K D ) specifically binds to human CD40 and 10 in PBS, and the recombinant protein binds to human CD40 and 10 -9 Dissociation constant less than M (K D ) specifically binds to human serum albumin in PBS and 10 -7 Dissociation constant less than M (K D) specifically binds, the FAP-binding domain being one of the FAP-binding domains described in Section 3.3, each of the two CD40-binding domains being independently one of the CD40-binding domains described in Section 3.4, the serum albumin-binding domain being one of the serum albumin-binding domains described in Section 3.5, and the linker being one of the linkers described in Section 3.6. In certain embodiments, the multiselective recombinant protein co-binds to human CD40, human FAP, and human serum albumin, preferably by surface plasmon resonance (SPR), more preferably as described in Example 3. In certain embodiments, the recombinant protein binds human CD40 in CD40-expressing B cells in the presence of FAP-expressing CHO cells for about 10 -8It is activated at EC50 values ​​of M or less. In certain embodiments, the multiselective recombinant protein does not inhibit FAP protease activity, or the reduction in FAP protease activity in the presence of the recombinant protein is 25% or less. In certain embodiments, the multiselective recombinant protein has a terminal phase half-life of at least 20 hours in a mouse model. In certain embodiments, the multiselective recombinant protein has a terminal phase half-life of at least 3 days in a cynomolgus monkey model. In certain embodiments, the multiselective recombinant protein can inhibit tumor growth in an FAP-expressing MC38 colon cancer mouse model, and this tumor growth inhibition is preferably measured as described in Example 6. In certain embodiments, one of the two CD40-binding domains or each of the two CD40-binding domains of the multiselective recombinant protein independently contains Q at position 8, L at position 15, R at position 143, and / or Q at position 147, where the position numbers correspond to the positions in Sequence ID No. 3. In certain embodiments, each of the two CD40-binding domains of the multiselective recombinant protein contains Q at position 8, L at position 15, R at position 143, and Q at position 147, where the position numbers correspond to the positions of SEQ ID NO: 3. In certain embodiments, the multiselective recombinant protein contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5. In certain embodiments, the multiselective recombinant protein contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 5. In certain embodiments, the multiselective recombinant protein comprises a polypeptide having an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5.

[0147] 3.8 Methods for generating nucleic acids and multiselective proteins This disclosure also provides polynucleotides encoding recombinant proteins described herein. This disclosure also provides methods for producing any of the polynucleotides described herein. This disclosure also provides recombinant proteins obtained by such methods. Polynucleotides can be produced and expressed by procedures well known in the art.

[0148] In one embodiment, the Disclosure provides a polynucleotide or a composition comprising a polynucleotide encoding a recombinant multiselective protein, wherein the dull protein comprises a first ankyrin repeat domain that specifically binds to fibroblast-activating protein (FAP), a second ankyrin repeat domain that specifically binds to CD40, and optionally a half-life extension moiety.

[0149] In one embodiment, the Disclosure provides a polynucleotide or a polynucleotide-containing composition comprising a nucleic acid sequence encoding a recombinant protein including SEQ ID NOs. 1, 2, 3, and / or 4. In one embodiment, the Disclosure provides a polynucleotide or a polynucleotide-containing composition comprising a nucleic acid sequence encoding a recombinant protein including SEQ ID NOs. 5 and / or 6. In one embodiment, the Disclosure provides a nucleic acid comprising the nucleic acid sequence SEQ ID NO. 58.

[0150] In another aspect, the Disclosure provides polynucleotides encoding recombinant proteins and their variants, such variant polynucleotides sharing at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, 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%, or at least 99% sequence identity with any nucleic acid disclosed herein, such as the nucleic acid sequence of SEQ ID NO: 58.

[0151] In another aspect, the disclosure provides polynucleotides encoding recombinant proteins and their variants, such variant polynucleotides can be hybridized to the sequence of SEQ ID NO: 58 under highly stringent conditions. "Highly stringent conditions" include (1) using low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C, (2) using a denaturing agent such as formamide during hybridization, e.g., 50% (v / v) formamide and 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer, pH 6.5 and 750 mM sodium chloride, 75 mM sodium citrate at 42°C, or (3) 50% formamide, 5 × SSC (0.75 M The process involves using NaCl (0.075M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 pg / mL), 0.1% SDS, and 10% dextran sulfate at 42°C, washing in 0.2× SSC (sodium chloride / sodium citrate) at 42°C and 50% formamide at 55°C, followed by a high-stringency wash with 0.1× SSC containing EDTA at 55°C.

[0152] Polynucleotides complementary to any such sequence are also included in this disclosure. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (recombinant, cDNA, or synthetic) or RNA molecules. RNA molecules may include hnRNA molecules containing introns and corresponding to DNA molecules in a one-to-one manner, and mRNA molecules without introns. Additional coding or non-coding sequences may, but are not required, be present within the polynucleotides of this disclosure, and polynucleotides may, but are not required, be ligated to other molecules and / or supporting materials.

[0153] Those skilled in the art will understand that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode recombinant proteins (or their individual domains), including the amino acid sequences described herein. Some of these polynucleotides have minimal homology to the nucleotide sequences of any native gene. Polynucleotides that vary due to differences in codon usage are specifically contemplated in this disclosure.

[0154] This disclosure also includes codon-optimized polynucleotides, in which the nucleic acid sequence is optimized to maximize expression in a particular cell. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a target host cell by replacing at least one codon in the original sequence (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) with a codon that is more or most frequently used in the gene of that host cell, while maintaining the original amino acid sequence. Different species exhibit specific biases for specific codons of specific amino acids. Codon bias (differences in codon use between organisms) often correlates with the translation efficiency of messenger RNA (mRNA), which is thought to depend, among other things, on the characteristics of the codon being translated and the availability of a particular transfer RNA (tRNA) molecule. The dominance of selected tRNAs in a cell generally reflects the codon most frequently used in peptide synthesis. Thus, genes can be tuned for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, and these tables can be adapted in many ways (e.g., Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000)). Computer algorithms for codons that optimize specific sequences for expression in specific host cells are also available, such as Gene Forge (Aptagen; Jacobus, Pa.). In some embodiments, one or more codons in a sequence encoding a recombinant protein (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) correspond to the codon most frequently used for a particular amino acid.

[0155] A suitable cloning vector may be constructed according to standard techniques or selected from a large number of cloning vectors available in the art. The selected cloning vector may be modified according to the host cell to which it is intended to be used, but a useful cloning vector generally possesses self-renewal capability, may have a single target on a specific restriction endonuclease, and / or may contain a marker gene that can be used when selecting a clone containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, BlueScript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.

[0156] Expression vectors are further provided. Expression vectors are generally replicable polynucleotide constructs containing the polynucleotides described herein. It is suggested that expression vectors must be replicable in host cells, either as an episome or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, adenoviruses, adeno-associated viruses, viral vectors including retroviruses, cosmids, and expression vectors disclosed in PCT Publication WO87 / 04462. Vector components may generally include, but are not limited to, a signal sequence, an origin of replication, one or more marker genes, and one or more suitable transcriptional regulatory elements (e.g., promoters, enhancers, and terminators). For expression (i.e., translation), one or more translational regulatory elements such as ribosome binding sites, translation initiation sites, and stop codons are also usually required.

[0157] Vectors containing the target polynucleotide and / or the polynucleotide itself can be introduced into host cells by any of several suitable means, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances, particulate guns, lipofection, and infection (e.g., the vector is an infectious agent such as vaccinia virus). The selection of the introduction vector or polynucleotide often depends on the characteristics of the host cell.

[0158] Examples of host cells include Escherichia coli cells, yeast cells, insect cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells. Preferred host cells include many known cells in the art, such as Escherichia coli cells, CHO cells, human embryonic kidney (HEK) 293 cells, or Sp2.0 cells.

[0159] 4.Treatment method The recombinant proteins described herein can be used, for example, to treat subjects with medical conditions such as cancer.

[0160] This disclosure provides a method for treating a medical condition, comprising administering a therapeutically effective amount of a recombinant protein, nucleic acid, or pharmaceutical composition described herein to a subject in need of treatment. In certain embodiments, the subject is human. In preferred embodiments, the medical condition is cancer. In certain embodiments, the cancer is a solid tumor. In certain embodiments, cancer cells express FAP. In certain embodiments, tumor stromal cells express FAP.

[0161] In some embodiments, cancer is brain cancer, bladder cancer, breast cancer, clear cell renal cancer, cervical cancer, colon and rectal cancer, endometrial cancer, gastric cancer, head and neck cancer, head and neck squamous cell carcinoma, lip cancer, oral cancer, liver cancer, cervical cancer, lung squamous cell carcinoma, melanoma, mesothelioma, non-small cell lung cancer (NSCLC), non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, urothelial carcinoma, sarcoma, small cell lung cancer (SCLC), head and neck squamous cell carcinoma (SCCHN), triple-negative breast cancer, or thyroid cancer.

[0162] In some embodiments, the cancer is an adrenocortical tumor, hydatidiform soft part sarcoma, cell tumor, chondrosarcoma, colorectal cancer, tendonoid tumor, fibroplastic round cell tumor, endocrine tumor, yolk sac tumor, epithelioid hemangioendothelioma, Ewing's sarcoma, germ cell tumor, hepatoblastoma, hepatocellular carcinoma, melanoma, renal tumor, neuroblastoma, non-rhabdomyosarcoma soft part sarcoma (NRSTS), osteosarcoma, paravertebral sarcoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, synovial sarcoma, or Wilms' tumor.

[0163] In some embodiments, the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or chronic myeloid leukemia (CML).

[0164] In some embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), or small lymphocytic lymphoma (SLL).

[0165] In fact, cancers that can be treated include, but are not limited to, alveolar rhabdomyosarcoma, bone cancer, anal cancer, anal canal cancer, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, or pleural cancer, nose, nasal cavity, or middle ear cancer, oral cancer, vulvar cancer, esophageal cancer, gastrointestinal carcinoid tumor, hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer, peritoneal, retinal and mesentery cancer, pharyngeal cancer, small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, ureteral cancer, and bladder cancer.

[0166] In certain embodiments, cancer is selected from the group consisting of cancers of the head and neck, ovaries, cervix, bladder, and esophagus, pancreatic cancer, gastrointestinal cancer, stomach cancer, breast cancer, endometrial cancer, and colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancer, and bronchioloalveolar carcinoma.

[0167] In certain embodiments, the cancer is non-small cell lung cancer (NSCLC), head and neck cancer, kidney cancer, trinecologically negative breast cancer, or gastric cancer. In certain embodiments, the cancer is non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, kidney cancer, breast cancer, melanoma, ovarian cancer, liver cancer, pancreatic cancer, colon cancer, prostate cancer, gastric cancer, lymphoma, or leukemia. In certain embodiments, the cancer is brain cancer.

[0168] The recombinant proteins described herein may be used before or after surgery to remove a tumor, or before, during, or after radiotherapy. Recombinant proteins may be used to treat tumors that are large enough to be found by palpation or by imaging techniques known in the art, such as MRI, ultrasound, or CAT scans. In some embodiments, the recombinant proteins are at least about 200 mm 3 , 300mm 3 , 400mm 3 , 500mm 3 , 750mm 3 , or up to 1000mm 3 It is used to treat advanced tumors having the following dimensions.

[0169] 5. Pharmaceutical composition and administration In another aspect, the disclosure also provides pharmaceutical compositions comprising recombinant multiselective proteins described herein.

[0170] The pharmaceutical composition may contain pharmaceutically acceptable carriers, diluents, or excipients. Typical pharmaceutical carriers include phosphate-buffered saline, water, emulsions such as oil / water or water / oil emulsions, and various types of wetting agents.

[0171] Pharmaceutical compositions include, for example, acidifying agents, additives, adsorbents, aerosol propellants, air replacement agents, alkalizing agents, anti-hardening agents, anticoagulants, antimicrobial preservatives, antioxidants, disinfectants, base materials, binders, buffers, chelating agents, coating agents, colorants, drying agents, detergents, diluents, disinfectants, disintegrants, dispersants, dissolution accelerators, dyes, emollients, emulsifiers, emulsifying stabilizers, fillers, film-forming agents, seasonings, flavorings, flow enhancers, and gels. It may contain any pharmaceutically acceptable ingredients, including fermenting agents, granulating agents, humectants, lubricants, mucosal adhesives, ointment bases, ointments, oily vehicles, organic bases, pastel bases, pigments, plasticizers, abrasives, preservatives, chelating agents, skin penetration agents, solubilizers, solvents, stabilizers, suppository bases, surfactants, surfactants, suspending agents, sweeteners, therapeutic agents, thickeners, tonic agents, toxic agents, thickeners, water absorbents, water-miscible cosolvents, water softeners, or wetting agents. See, for example, "Handbook of Pharmaceutical Excipients," 3rd edition, AH Kibbe (Pharmaceutical Press, London, UK, 2000). See also "Remington's Pharmaceutical Sciences," 16th edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980).

[0172] Pharmaceutical compositions can be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition may be, for example, about 4 or about 5 to about 8.0, or about 4.5 to about 7.5, or about 5.0 to about 7.5. In exemplary embodiments, the pH of the pharmaceutical composition is 5.5 to 7.5.

[0173] The recombinant multiselective proteins described herein can be administered to subjects via any preferred route of administration, including parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal administration. Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. For further details, see Pharmaceuticals and Pharmacy Practice, JBLippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986).

[0174] The dose of the activator of this disclosure administered throughout the course of a treatment regimen should be sufficient to treat the cancer within a clinically acceptable time frame (e.g., 1 to 4 weeks or longer (e.g., 5 to 20 weeks or longer)) from the time of administration. In certain embodiments, the period may be longer. The dose is determined by the efficacy of the particular activator and the condition of the animal (e.g., human), and sometimes by the body weight of the animal being treated (e.g., human). The extent to which the cancer is treated at the time of administration of a particular dose can be expressed, for example, by the cytotoxicity of the activator or the degree of tumor regression achieved by the activator. Methods for measuring the cytotoxicity of recombinant multiselective proteins and methods for assaying tumor regression are well known in the art. For example, and not intended to limit this disclosure, the dose of the activator of this disclosure may be about 0.0001 to about 1 g / kg body weight / day of the subject being treated, about 0.0001 to 0.001 g / kg body weight / day, or about 0.01 mg to about 1 g / kg body weight / day. The dosage unit also refers to the amount in milligrams per square meter of body surface area, i.e., rag / m². 2 It may also be represented as follows.

[0175] The recombinant multiselective proteins described herein can be used in combination with other therapeutic agents, such as other anticancer agents. Each therapeutic agent may be administered simultaneously (e.g., with the same agent or at the same time), in parallel (i.e., in separate agents administered immediately after the other in any order), or sequentially in any order. Sequential administration is effective when the therapeutic agents in combination therapy are in different dosage forms (e.g., one agent is a tablet or capsule and the other is a sterile liquid) and / or on different dosing schedules, for example, a chemotherapeutic agent administered at least daily and a biotherapeutic agent administered less frequently, such as once a week, once every two weeks, or once every three weeks. [Examples]

[0176] Example 1 - Design of a highly selective binding protein We produced multiselective binding proteins in various formats and determined their FAP specificity, efficacy, and CD40 activation effect. All of these multiselective proteins contained FAP-specific binding domains and CD40-specific binding domains. We evaluated the effects of (i) adding human serum albumin (HSA) binding domains(s), (ii) increasing the binding titer by adding further CD40 binding domains(s), and (iii) altering the order of binding domains within the protein.

[0177] To compare different formats, we designed an in vitro assay to measure the upregulation of the costimulatory receptor CD86, which is expressed on human B cells upon CD40 induction. This cell assay used primary human B cells in the presence or absence of FAP-expressing cells. Upregulation of the CD86 costimulatory molecule was evaluated as a marker of B cell activation. An anti-CD40 monoclonal antibody whose mechanism of action is independent of FAP-mediated crosslinking was used as a reference material.

[0178] Multiple selectivity proteins in different formats. As outlined in Table 2, several multiple selectivity protein formats were produced by initiation by the parent molecule (SEQ ID NO: 59; SMA014), which included one FAP-specific binding domain and one CD40-specific binding domain, as well as several other multiple selectivity protein formats.

[0179] [Table 2]

[0180] In Table 2, "C," "F," and "H" represent ankyrin repeat domains that specifically bind to CD40, FAP, and HSA, respectively. The order of the different domains shown in Table 2 reflects the actual sequence of the domains from the N-terminus to the C-terminus in the molecular structure of the protein. All proteins also have a His tag (SEQ ID NO: 57) at the N-terminus to facilitate purification.

[0181] Materials and methods For reference, a CD40 monoclonal antibody was used. Binding of this CD40 mAb (IgG2 mAb) to CD40 results in activation of antigen-presenting cells independently of FAP. The anti-CD40 mAb corresponds to sequence 21.4.1 of U.S. Patent Application No. 7,338,660B2.

[0182] CHO cells were cultured at 37°C in DMEM medium containing 10% FBS with 5% CO2, and the cells were separated by dividing them every 2-3 days using acutase.

[0183] The FAP-expressing CHO cell line is a stably transfected clonal cell line expressing human FAP on its cell surface. A plasmid containing a GFP fusion of the human FAP ORF was obtained from OriGene Technologies (#RG204692). The cDNA encoding human FAP (without GFP) was subcloned using standard molecular biology techniques. This plasmid was then transfected into CHO cells to generate a stable transfectant overexpressing human FAP using lipofectamine. Selective pressure was applied using different concentrations of Geneticin G-418 (Promega, V8091). FAP expression was analyzed by flow cytometry using an anti-FAP antibody corresponding to ESC11 (International Publication No. 2011 / 040972). The FAP-CHO transfectant population from G-418 under condition 1.9 mg / mL (FAP-CHO-1.9) showed lower FAP expression levels, while the population from condition 1.7 mg / mL (FAP-CHO-1.7) showed higher FAP expression levels. The data in this example were produced using FAP-CHO-1.7.

[0184] In vitro B cell activation assay. The design of the in vitro B cell activation assay is schematically shown in Figure 1. Buffy coat was obtained from the Zurich Blood Center and diluted with PBS. Peripheral blood mononuclear cells (PBMCs) were then isolated by density centrifugation using Leucosep tubes. After several washing steps, positive selection (Human CD19 MicroBeads Kit) was used according to the manufacturer's recommendations to isolate CD19. + B cells were concentrated from PBMCs. 1 × 10 5 / Well CD19 + B cells and 5 × 10 4 FAP-expressing CHO cells or CHO wild-type (WT-CHO) cells were seeded together in RPMI 1640 medium with or without 600 μM HSA + 10% FBS into a 96-well plate, along with dose titrations of the indicated molecules (400, 200, 40, 8, 5, 1.6, 0.3, 0 nM). The cultures were incubated at 37°C in 5% CO2 for 24 hours, and CD20 was performed.+ The upregulation of CD86 and CD69 on B cells was evaluated by flow cytometry using AttuneNxT.

[0185] FACS staining, flow cytometer setup, and antibody dilution. Cells were first washed with 150 μL of PBS and then incubated with 100 μL of BD human Fc-Block diluted in PBS (1:100) for 20 minutes at room temperature (RT). After Fc-blocking incubation, cells were incubated with 100 μL of directly labeled antibody diluted in FACS buffer (see Table 3 below for dilution factors) and incubated in the dark at 4°C for a further 20 minutes. Cells were washed with PBS, resuspended in 100 μL of Live / Dead stain (1:1000) diluted in PBS, and incubated in the dark at 4°C for 20 minutes. 100 μL of FACS buffer containing the FBS reaction was added to stop the Live / Dead staining reaction. Cells were washed again with PBS and fixed with BD cell fixation solution (1:10) diluted in water according to the manufacturer's recommendations. Antibody dilution and FACS setup are outlined in Table 3 below. FACS machine compensation was performed using compensation beads as recommended by the manufacturer (ThermoFisher; AbC® Total Antibody Compensation Bead Kit). Raw_fcs files were analyzed using FlowJo software (version 10.0.3). Cells were gated with live cells using Live-Dead discrimination dye, and then gated with CD20-positive cells for CD86 as shown in Figure 2. MFI and the percentage of positive cells for CD86 were exported and plotted using GraphPad Prism software, version 8.1.2.

[0186] [Table 3]

[0187] EC50 determination. The EC50 value was determined using GraphPad Prism version 7.02 by transforming the x-value (concentration) in log mode and fitting it to the nonlinear mode log (agonist) versus response with a variable gradient (3-parameter) equation for determining the EC50 value.

[0188] Efficacy determination. The efficacy value was determined by calculating the average of overlapping MFI values ​​at the highest concentration (400 nM) using GraphPad Prism version 7.02.

[0189] result HSA-binding domains(s) impair the potency and efficacy of the bispecific FAPxCD40 ankyrin repeat-binding protein (FC format). SMA014, a bispecific FAPxCD40 DARPin® molecule in FC format, was cloned in different formats with one (HFC, SMA087) or two (HFCH, SMA095) HSA-binding ankyrin repeat-binding domains and tested in in vitro B-cell activation assays. As shown in Figure 3, HSA-binding domains(s) impaired both the potency and efficacy of the original bispecific binding protein in FC format, and the level of impairment correlated with the number of HSA-binding domains attached to the binding protein. Importantly, the inhibition was more pronounced in the presence of 600 μM albumin, mimicking the physiological concentration of albumin in human serum. It is reasonable to assume that the HSA-binding complex / albumin may sterically hinder the binding of the CD40 domain and / or FAP domain(s), and therefore its activity. As expected, the DARPin® protein and anti-CD40 mAb upregulated CD86 in a dose-dependent manner, and the DARPin® protein was active only in the presence of FAP-expressing CHO cells (Figure 3). In the absence or presence of HSA, the HSA-binding domain did not affect the FAP-specific mode of action. As expected, the agonist anti-CD40 mAb induced activation of human B cells independently of FAP expression and activated B cells in the presence of either FAP-CHO cells or WT-CHO cells. The mean EC50 (potency) and maximum MFI (efficacy) values ​​for two independent experiments are summarized in Tables 4 and 5 for FAP-CHO and Table 6 for WT-CHO, respectively.

[0190] In summary, the addition of one or more half-life-extending HSA-binding domains impaired the functionality of the bispecific FAPxCD40-binding protein (FC format), the inhibition increased with the number of added HSA-binding domains, and the inhibition was more pronounced in the presence of physiological albumin concentrations.

[0191] The bivalent nature of CD40 increases potency and efficacy and rescues the inhibitory effect induced by the HSA-binding domain. The applicant then investigated how CD40 titer affects the performance of the bispecific FAPxCD40 DARPin® molecule. The FC-format bispecific FAPxCD40 DARPin® molecule, SMA014, was cloned in different formats with one (FCC, SMA104) or two (FCCC, SMA105) CD40-binding DARPin domains and tested in an in vitro B-cell activation assay. As shown in Figure 4, the bivalent and trivalent formats induced stronger upregulation of CD86, indicating that the binding titer favorably contributes to the molecular performance. Specifically, the bivalent nature of CD40 (SMA104) strongly increased the molecular potency (20-fold) and efficacy (2-fold) in the presence of FAP-expressing CHO cells. Trivalent CD40 (SMA105) only slightly increased the molecular potency compared to divalent CD40 and did not have any further effect on potency. In the absence of FAP, the divalent CD40 format (SMA104) did not induce upregulation of CD86 on human B cells, but the trivalent CD40 format (SMA105) showed slight activation at the highest concentration even in the absence of FAP, suggesting the possibility of FAP-independent activation induced by the trivalent CD40 conjugate. Considering these results, the divalent CD40 format was selected for further characterization. In particular, the applicant investigated whether divalent CD40 could rescue the inhibitory effect of the HSA-binding domain. To address this issue, the divalent CD40 construct SMA104 was cloned with additional HSA-binding domains at different locations (clones SMA091, SMA099, and AS579; see Table 2 above for information on these domain formats). All tested formats showed improved potency and efficacy compared to SMA014 (Figure 5A). Importantly, under more physiological conditions in the presence of HSA, the activity of all formats with an HSA-binding domain decreased, but SMA091 still showed improved activity compared to SMA014 (Figure 5B).None of the ankyrin repeat-binding proteins enhanced CD86 expression on B cells, even in the absence of the highest concentration of FAP (Figures 5A and 5B). As expected, agonist anti-CD40 mAbs induced activation of human B cells independently of FAP expression, and activated B cells via either FAP-CHO or WT-CHO. The mean EC50 (potency) and maximum MFI (efficacy) values ​​from two independent experiments are summarized in Tables 4 and 5 for FAP-CHO and in Table 6 for WT-CHO, respectively.

[0192] [Table 4]

[0193] [Table 5]

[0194] [Table 6]

[0195] Conclusion: The FC format bispecific FAPxCD40 ankyrin repeat protein exhibited good biological activity and favorable physical properties in functional cell assays. However, this binding protein may require half-life extension domains to enable its clinical development. Therefore, different formats were analyzed to determine whether and how the number and location of half-life extension HSA binding domains affect the molecular activity. Half-life extension domains had a detrimental effect on the molecular activity, and this effect was observed to increase with the number of half-life extension domains and the presence of HSA. Furthermore, surprisingly, CD40 bivalent (due to having two CD40 binding domains) strongly increased the potency (20-fold) and efficacy (2-fold) of the binding protein while maintaining a stringent FAP-specific mechanism of action, whereas CD40 trivalent (due to having three CD40 binding domains) only slightly increased potency compared to CD40 bivalent and showed no further effect on efficacy. In addition, the trivalent CD40-binding protein showed slight activation at the highest concentration even in the absence of FAP, suggesting a partial loss of FAP-specific mode of action. It was further found that the bivalent CD40 could rescue the inhibitory effect of one half-life extension domain by increasing the potency and efficacy of the binding protein. Specifically, at physiological concentrations of HSA, the HFCC-formatted binding protein retained activity and FAP specificity comparable to the FC-formatted binding protein. In conclusion, by adding a second CD40-binding domain, we were able to produce a molecule with a half-life extension domain that prevents the adverse effects of the HSA-binding half-life extension domain and possesses similar functional properties to the parent binding protein in FC format, but with features that facilitate its clinical development. For all these reasons, the HFCC domain format was selected for further investigation.This format is used in the binding proteins of the present invention, which include the amino acid sequences of SEQ ID NO: 5 (DARPin® Protein #5 or simply "Protein #5"), SEQ ID NO: 6 (DARPin® Protein #6 or simply "Protein #6"), and SEQ ID NO: 7 (DARPin® Protein #7 or simply "Protein #7"), as described in the following examples.

[0196] Example 2 - Biophysical properties, binding affinity, and binding specificity of the multiselective binding protein of the present invention This example describes experiments conducted to determine (1) the biophysical properties of the multiselective binding protein of the present invention, such as aggregate formation, and (2) its binding affinity and species cross-reactivity to various target proteins (i.e., FAP, CD40, and serum albumin).

[0197] Aggregate formation Protein #5 (sometimes also referred to as SMA136 herein) was analyzed by size exclusion chromatography (SEC) and multi-angle light scattering (MALS). Figure 6 shows the results of this analysis. This SEC profile demonstrates that protein #5 is monomeric and monodisperse in solution and does not form aggregates.

[0198] Binding affinity to target protein Summary. The binding of DARPin® protein #5 ("protein #5") to human, cynomolgus monkey, and mouse-derived CD40, FAP, and serum albumin was analyzed by surface plasmon resonance (SPR). The results showed that protein #5 specifically binds to (i) human and cynomolgus monkey-derived CD40, (ii) human, mouse, and cynomolgus monkey-derived serum albumin, and (iii) human and cynomolgus monkey-derived FAP. Specific binding to mouse CD40 and mouse FAP was not detected. The dynamic parameters of protein #5 are outlined in Table 7. SPR traces of protein #5 binding to human CD40, human FAP, and human serum albumin are shown in Figure 7.

[0199] [Table 7] * The value represents the average of two measurements. ** The value represents the average of three consecutive measurements. †Chi 2 / Rmax > 10% is defined as an inaccurate fit. !K of MSA binding on protein #5 D This is overestimated due to the strong nonspecific binding of MSA on the chip. Not detected: Standard deviation that could not be determined because replication did not occur.

[0200] Materials and methods All SPR measurements were performed using a ProteOn XPR36 instrument (BioRad) and electrophoresis buffer containing 0.005% Tween 20 (PBST) in PBS pH 7.4. A 1:1 Langmuir model was used to fit the SPR traces.

[0201] ProteOn setup of protein #5 binding to CD40 in different species: CD40 proteins from different species (human, cynomolgus monkey, mouse) (AcroBio system) were biotinylated using standard methods well known in this field to obtain bio-hCD40, bio-cCD40, and bio-mCD40 proteins. The bio-hCD40, bio-cCD40, and bio-mCD40 proteins were immobilized on NLC chips (BioRad) to levels of 300 RU, 250 RU, and 300 RU, respectively. The interaction of protein #5 with CD40 was measured by injecting protein #5 at serial dilutions of 3, 1.5, 0.75, 0.38, and 0.19 nM with a constant flow of 100 μL / min, with association for 120 seconds and dissociation for 900 seconds. The measurement was repeated two or three times, and the target was regenerated between each measurement using 10 mM glycine pH 2 at a flow rate of 100 μL / min for 18 seconds. The signal was double-referenced against a control lane treated with electrophoresis buffer (PBST).

[0202] ProteOn setup of protein #5 that binds to serum albumin from different species. First, human FAP (hFAP) was coated to a level of 700 RU on a GLC chip (BioRad), and then 50 nM protein #5 was immobilized as the analyte at a constant flow rate of 100 μL / min to a level of 120 RU for 120 seconds (0 seconds of dissociation). Serum albumin binding to protein #5 was measured by injecting human serum albumin (HSA), cynomolgus monkey serum albumin (CSA), and mouse serum albumin (MSA) at serial dilutions of 100, 33, 11, and 3.7 nM at a constant flow rate of 100 μL / min with 120 seconds of association and 600 seconds of dissociation. HSA, CSA, and MSA binding were measured continuously, and the hFAP / protein #5 complex was regenerated each time with 10 mM glycine pH 2 at a flow rate of 100 μL / min for 18 seconds. Therefore, protein #5 had to be re-immobilized on hFAP after each regeneration step. The signal was double-referenced against the control lane ((a) PBST electrophoresis buffer; (b) coated with an unrelated target to which protein #5 did not bind). Dynamics were calculated during the first 300 seconds of dissociation.

[0203] ProteOn setup for protein #5 that binds to FAPs of different species. hFAP, cynomolgus monkey FAP (cFAP), and mouse FAP (mFAP) were immobilized on GLC tips (BioRad) in 10 mM sodium acetate buffer pH 5.3 to levels of 1200 RU, 800 RU, and 2000 RU, respectively. The interaction between FAP and protein #5 was measured by applying protein #5 at serial dilutions of 50, 25, 12, 6.5, and 3.13 nM with a constant flow of 100 μL / min, with association for 120 seconds and dissociation for 1800 seconds. The target was regenerated using 10 mM glycine pH 2 and 124 mM H3P04. The signal was double-referenced against a PBST-treated control lane.

[0204] Results and Conclusions Surface plasmon resonance measurements showed that protein #5 had essentially identical binding affinities (K) of 10³±12 pM and 10¹±11 pM, respectively. D Protein #5 demonstrated strong binding to human and cynomolgus monkey CD40. Protein #5 is not cross-reactive to mouse CD40 due to its potentially low sequence identity of the extracellular domain, which is only 57.5%. Protein #5 has a binding affinity of 50 nM (K D Protein #5 showed binding to human serum albumin at a similar binding affinity (K). Furthermore, protein #5 showed binding to human FAP and cynomolgus monkey FAP, and showed similar binding affinity (K). D The concentrations were 0.307 nM and 0.339 nM, respectively, but no cross-reactivity was detected for mouse FAP.

[0205] Example 3 - Simultaneous binding of CD40, FAP, and protein #5 to serum albumin as analyzed by surface plasmon resonance. The following experiments describe surface plasmon resonance experiments performed to analyze the simultaneous binding of multiselective specific proteins, including SEQ ID NO: 5, to human CD40, human FAP, and human serum albumin.

[0206] Materials and Methods. SPR measurements were performed using a ProteOn XPR36 instrument (BioRad). The electrophoresis buffer was PBS pH 7.4 (PBST) containing 0.005% Tween20®. Biotinylated human CD40 (bio-hCD40-Fc) was immobilized to a level of 550 RU on a NeutrAvidin-coated NLC sensor tip. In the first analyte step (analyte 1), 25 nM protein #5 was immobilized on bio-hCD40 with association for 120 seconds and dissociation for 0 seconds. Immediately after this first step, the second analyte step (analyte 2) was performed by injecting either PBST, 25 nM protein #5, or 50 nM hFAP with association for 120 seconds and dissociation for 0 seconds. In the third step (analyte 3), either PBST, 50nMhFAP, or 50nMHSA was applied with 120 seconds of association and 600 seconds of dissociation (injection scheme: see Table 8). The entire experiment was performed at a constant flow rate of 100 μL / min. This setting allowed for the binding of hFAP and HSA only if protein #5 (analyte 1) was already bound to CD40 (immobilized on the chip). The signals were double-referenced against the PBST-treated control lanes of L6 and A6.

[0207] [Table 8]

[0208] Results. SPR traces of the simultaneous binding of protein #5 to hCD40, hFAP, and HSA are shown in Figure 8. Briefly, 550 RU of biotinylated human CD40 was immobilized on a neutraavidin chip. In the first association step, protein #5 bound saturally to hCD40, reaching a total signal of 200 RU as shown in injection 1 in Figure 8. In the second association step (Figure 8, injection 2), hFAP bound to the complex protein #5 / CD40, resulting in an increase of 200 RU. It should be noted that protein #5 had already begun to dissociate from hCD40 during the time interval between injection 1 and injection 2 (loss of 75 RU). The third association step (Figure 8, injection 3) resulted in the binding of human serum albumin to the complex hCD40 / protein #5 / hFAP (increase of 80 RU), indicating that simultaneous binding of protein #5 to all three targets had occurred.

[0209] In conclusion, surface plasmon resonance studies demonstrate that protein #5 can simultaneously bind to CD40, FAP, and serum albumin.

[0210] Example 4 - Activation of human B cells via CD40 The objective of this study was to evaluate the biological activity and FAP-specific mechanism of action of multiselective binding proteins, protein #5 (containing SEQ ID NO: 5) and protein #6 (containing SEQ ID NO: 6). Proteins #5 and #6 were tested in cell assays using primary human B cells, in or out of the presence of FAP-expressing cells. Upregulation of co-stimulatory molecules, particularly CD86 and CD69, was evaluated as a marker of CD40 signaling-mediated B cell activation. Anti-CD40 monoclonal antibodies with mechanisms of action independent of FAP-mediated crosslinking were used as reference materials. The potency, efficacy, and FAP specificity of protein #5 or protein #6 were evaluated in comparison to the reference materials.

[0211] In vitro data obtained from the human B cell activation assay showed that proteins #5 and #6 can activate B cells via CD40 activation, as reflected by the upregulation of CD86 and CD69, and that proteins #5 and #6 can activate B cells only in the presence of FAP-positive cells, but not in the presence of FAP-negative cells, thus confirming a mechanism of action that is strictly dependent on FAP-mediated crosslinking.

[0212] Materials and methods In vitro B cell activation assays, FACS staining, flow cytometer setup and antibody dilution, EC50 determination and efficacy determination were performed essentially as described in Example 1.

[0213] The anti-CD40 antibody, FAP-expressing CHO cells, and FAP-non-expressing CHO cells used as references were those described in Example 1.

[0214] result Proteins #5 and #6 induce upregulation of costimulatory molecules in human B cells through an FAP-dependent mechanism of action. We evaluated the ability of proteins #5 and #6 to activate human B cells via CD40 in the presence of FAP-expressing CHO cells. Protein #5 induced upregulation of costimulatory molecules CD86 and CD69 in human B cells co-cultured with FAP-expressing CHO cells, with EC50 values ​​ranging from 0.04 nM to 0.07 nM (Figure 9). Conversely, protein #5 did not activate human B cells in the presence of non-FAP-expressing CHO cells or wild-type (WT)-CHO cells (Figure 10). As expected, agonist anti-CD40 mAbs induced activation of human B cells independently of FAP expression, activating B cells via either FAP-CHO or WT-CHO. Similar results were obtained for protein #6 as for protein #5.

[0215] conclusion Proteins #5 and #6 induced upregulation of two different costimulatory molecules, CD86 and CD69, in primary human B cells only in the presence of FAP-positive CHO cells, but not in the presence of FAP-negative CHO cells, confirming a mechanism of action strictly dependent on FAP-mediated crosslinking. In the presence of FAP, protein #5 showed similar potency and efficacy to the comparative anti-CD40 monoclonal antibody. Protein #5 induced upregulation of costimulatory molecules in a dose-dependent manner at EC50s of 0.04 nM to 0.07 nM in the presence of FAP-expressing CHO cells. Similar results were obtained for protein #6.

[0216] Example 5 - Activation of human dendritic cells via CD40 We performed a study using human monocyte-derived dendritic cells (MDDCs) that is conceptually similar to the one described for B cells in Example 4 (see schematic diagram in Figure 11).

[0217] The objective of this study was to evaluate the biological activity and FAP-specific mechanism of action of multiselective binding proteins, protein #5 (containing SEQ ID NO: 5) and protein #6 (containing SEQ ID NO: 6). Proteins #5 and #6 were tested in cell assays using human MDDCs in the presence or absence of FAP-expressing cells. Upregulation of the secretion of co-stimulatory molecules, particularly CD86, CD83, and CD80, as well as IL-12, was evaluated as a marker of MDDC activation mediated by CD40 signaling. Anti-CD40 monoclonal antibodies with mechanisms of action independent of FAP-mediated crosslinking were used as reference materials. The potency, efficacy, and FAP specificity of protein #5 or protein #6 were evaluated in comparison to the reference materials.

[0218] In vitro data obtained from human MDDC activation assays showed that proteins #5 and #6 can activate MDDC via CD40 activation, as reflected by the upregulation of costimulatory molecules and IL-12 secretion. Furthermore, proteins #5 and #6 can activate MDDC only in the presence of FAP-positive cells, but not in the presence of FAP-negative cells, confirming a mechanism of action strictly dependent on FAP-mediated crosslinking. Protein #5 could induce dose-dependent upregulation of costimulatory molecules and IL-12 secretion in the presence of FAP-expressing CHO cells at EC50 levels of 0.03 nM to 7.67 nM and 0.83 nM to 7.63 nM, respectively, but could not induce these effects in the presence of FAP-negative CHO cells. Similar results were obtained for protein #6.

[0219] In conclusion, this study demonstrated that proteins #5 and #6 can activate human MDDC via CD40 in vitro in a FAP-dependent manner.

[0220] Example 6 - Protein #7 demonstrated antitumor activity in vivo. In the following examples, the dose-dependent in vivo efficacy of repeated doses of the multiselective binding protein, DARPin® protein #7, was evaluated in a mouse MC38 colon cancer model. Protein #7 is a mouse substitute for protein #5 or protein #6, containing an FAP-binding domain that binds to mouse FAP and a CD40-binding domain that binds to mouse CD40. The MC38 carcinoma model has been previously shown to be susceptible to CD40 agonist treatment. Since syngeneic mouse tumors such as MC38 have generally been found to express very low levels of stromal FAP compared to human tumor stroma, the MC38 cell line was transfected to express FAP, thereby better mimicking the FAP expression observed in human tumors.

[0221] In the described studies, protein #7 (N-terminal His-tagged (SEQ ID NO: 57); also referred to as AS598) was tested at a dose of 2.5 mg / kg in studies PD1032, PD1033, PD1035, and PD1038, and also at a dose of 12.5 mg / kg in studies PD1032 and PD1033. A commercially available anti-CD40 antibody (FGK45; BioXell) that binds to mouse CD40 was used as a positive control. A non-FAP-binding mutant of protein #7 (referred to as AS608; SEQ ID NO: 67 (SEQ ID NO: 57) with an N-terminal His-tagged gene, in which the FAP-binding domain is replaced by an unbound ankyrin repeat domain) was used as a negative control molecule to demonstrate the dependence of protein #7's pharmacological activity on binding to mFAP.

[0222] The multiselective binding proteins of the present invention, such as protein #7, are intended to locally activate CD40 in tumor tissue to reduce systemic toxicity. Therefore, to evaluate the safety profile of protein #7, several parameters of systemic toxicity, including weight loss, elevated serum cytokines and aminotransferases, and liver tissue damage, were determined in addition to tumor growth and inhibition, compared with anti-mCD40 antibodies known to induce hepatotoxicity.

[0223] Materials and methods: Tumor Experiments: Tumor experiments were performed schematically as shown in Figure 12. MC38-mFAP polyclonal tumor cells were subcutaneously inoculated into the right flank region of syngeneic mice (C57BL / 6JRj) (day 0). Mice were randomized to treatment groups and treated on the same day (PD1032 on day 25, PD1033 on day 39, PD1035 on day 26, and PD1038 on day 37). Subsequently, test substances (AS598, AS608, FGK45) were administered to tumor-bearing mice according to the prescribed regimens as shown in Table 9 (see also Figure 12). Tumor growth was monitored every 3-4 days by caliper measurement until days 36, 43, 36, and 40, respectively, after inoculation. On day 36 of the experiment (PD1032 and PD1035), the mice were sacrificed, the tumors were removed, and immunophenotyping was performed by flow cytometry. On days 43 (PD1033) and 40 (PD1038) of the experiment, the mice were sacrificed, the tumors were removed, and immunophenotyping was performed by flow cytometry.

[0224] PD1032 and PD1035 were two independent primary studies of antitumor efficacy, while PD1033 and PD1038 were two adjacent studies for tumor environment analysis using FACS. In the primary studies, AS598 was administered three times every four days at doses of 2.5 mg / kg (studies PD1032 and PD1035) and 12.5 mg / kg (study PD1032). The non-FAP-targeted control AS608 was administered three times every four days at 2.5 mg / kg (study PD1035). In the early-termination study for FACS analysis, AS598 was administered twice with a three-day interval at doses of 2.5 mg / kg (studies PD1033 and PD1038) and 12.5 mg / kg (study PD1033). The non-FAP-targeted control AS608 was similarly administered twice at a dose of 2.5 mg / kg with a 3-day interval (study PD1038). The anti-CD40 antibody FGK45 was used as a positive control in all studies and administered at 5 mg / kg (equivalent molar dose to AS598 at 2.5 mg / kg) on ​​the same schedule as the DARPin® protein.

[0225] [Table 9] Note: N: Number of animals; the number of mice / group in PD10033 is different from that in PD1038 (N / N).

[0226] Tumor inoculation: Under standard isoflurane anesthesia, MC38-mFAP polyclonal tumor cells (9 × 10) in 0.2 mL of PBS were administered to the right posterior ventral / dorsal region of 90 (PD1032 + PD1033) or 105 (PD1035 + PD1038) female C57BL6 mice for tumor development. 6 ) was administered subcutaneously.

[0227] Tumor Measurement: Tumor measurements were performed twice a week starting 15 days after tumor inoculation (PD1032 and PD1033) or 14 days after tumor inoculation (PD1035 and PD1038). The length and width of the tumor were measured using calipers. Tumor volume was calculated using the following formula: (length x width) 2 xπ) / 6

[0228] Randomization: Group randomization based on tumor volume was performed on days 25, 39, 26, and 37 after tumor inoculation. From the original 90 mice that received tumor transplants (PD1032 and PD1033), 40 mice were randomized to four subgroups of 10 animals each 25 days after tumor inoculation (PD1032). The remaining 50 mice were randomized to four subgroups of 5 animals each 39 days after tumor inoculation (PD1033). From the original 105 mice that received tumor transplants (PD1035 and PD1038), 40 mice were randomized to four subgroups of 10 animals each 26 days after tumor inoculation (PD1035). The remaining 65 mice were randomized to four subgroups of 6 animals each 37 days after tumor inoculation (PD1038).

[0229] Observation and Data Collection: After randomization of the groups, animals were checked twice a week, with body weight and tumor measurements taken in between. Animals were also checked for tumor growth and any effects of treatment on normal behavior, including motility, visual estimation of food and water consumption, weight gain / loss, decrease in eye / coat luster, and any other abnormal effects. Deaths and observed clinical signs were recorded based on the number of animals in each subset.

[0230] Sampling: Tumors were removed, weighed, and used for FACS, while the remaining material was fixed in formalin. The spleen was removed, half was used for FACS, and the other half was fixed in formalin. The liver was removed and fixed in formalin. Blood samples were collected in Multivette 600Z Gel (Sarstedt #15.1674) on days 24, 26, and 36 for PD1032, day 43 for PD1033, days 27 and 36 for PD1035, and day 41 for PD1038, and centrifuged at 15,000 rpm for 5 minutes. Serum was collected and stored at -80°C for possible later analysis.

[0231] Statistical Analysis: Statistical analysis was performed using Prism 8.2.0 software (GraphPad Software). All statistics involving multiple comparisons were performed using the Kruskal-Wallis nonparametric test, followed by Dunn's multiple comparison test for all groups compared to the vehicle. Nonparametric Mann-Whitney two-tailed analysis was performed to compare the differences between the two groups.

[0232] Liver enzymes. In mouse models and clinical trials in humans, agonist antibodies against CD40 have been shown to significantly, but transiently, increase liver enzymes such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT). In humans, AST is found in various tissues, including the liver, brain, pancreas, heart, kidneys, lungs, and skeletal muscle. When any of these tissues are damaged, AST is released into the bloodstream. Elevated AST levels indicate tissue damage, but are not specific to the liver itself. In contrast, ALT is found primarily in the liver. Any elevation of ALT is a direct indicator of liver damage.

[0233] Therefore, AST and ALT levels were determined in these experiments as measures of hepatotoxicity. Measurements were taken 24 hours after treatment. The 24-hour post-treatment timeframe was deemed most appropriate based on literature and in-house time titration experiments. ALT and AST analysis was performed using kits MAK052 and MAK055 (Sigma-Aldrich), respectively, in accordance with the manufacturer's guidelines.

[0234] Cytokine levels. Blood was collected from mice carrying MC38-FAP colon carcinoma tumors treated according to Figure 12, 24 hours after the first infusion (main study = PD1032 and PD1035), 24 hours after the second infusion (adjacent study = PD1033 and PD1038), and at the end of the study (main study = PD1032 and PD1035). Eleven different cytokines (TNF-alpha, IL-6, IFN-gamma, IL12p70, MCP-1, MIP-1-alpha, MIP-1-beta, IP-10, IL-10, IL-2, and IL-1-beta) were analyzed using the Luminex assay (R&D Systems) as recommended by the manufacturer.

[0235] Immunohistochemical (IHC) analysis of liver tissue. Liver tissue was collected 24 hours after the initial infusion, washed with PBS, immediately fixed with formalin, and embedded in paraffin blocks. Hematoxylin / eosin staining was performed on paraffin-embedded liver sections, and the tissue was analyzed by a pathologist in a blinded manner.

[0236] result: Overall health and weight. During the study period, no adverse health effects or signs of weight loss were observed in mice treated with AS598 (Figures 13A and 13B). In contrast, consistent with previous reports, FGK45 treatment resulted in significant but transient weight loss after the initial injection (Figures 13A and 13B).

[0237] Tumor growth: Tumor growth was measured every 3-4 days during the study period. Treatment in studies PD1032 and PD1035 resulted in an average tumor volume of 300 mm³. 3If the threshold was exceeded, treatment was initiated on days 25 and 26, respectively. The mean tumor growth curves for the different treatment groups in both studies are shown in Figures 14A and 14B.

[0238] AS598 demonstrated statistically significant antitumor efficacy compared to the vehicle group at both tested doses (Figures 14A and 14B). The antitumor efficacy was similar to that observed with the positive control anti-CD40 antibody FGK45. The non-FAP-bound control AS608 used in study PD1035 showed no antitumor efficacy, demonstrating that the antitumor efficacy observed in this tumor model was FAP-dependent.

[0239] In addition to tumor volume measurement, tumors were dissected from mice at the end of the study to determine tumor weight. The results and conclusions from tumor weight measurement were consistent with the results and conclusions from tumor volume measurement (data not shown).

[0240] Furthermore, the results obtained in studies PD1033 and PD1038 were consistent with the results obtained in studies PD1032 and PD1035 (data not shown).

[0241] Blood cytokine levels. FGK45 significantly increased the blood levels of eight of the measured cytokines: TNF-alpha, IL-6, IFN-gamma, IL12p70, MCP-1, MIP-1-alpha, MIP-1-beta, and IP-10 (Figure 20A, and data not shown). In contrast, AS598 (2.5 mg / kg or 12.5 mg / kg), AS608, or the vehicle did not increase the blood levels of any of the measured cytokines (Figure 20A, and data not shown).

[0242] Liver enzymes and damage. As expected, FGK45 induced a significant increase in ALT levels, which was detected 24 hours after the first infusion but not after subsequent infusions (Figure 20B). This was consistent with the literature and previous in-house studies. In contrast to FGK45, AS598 did not induce any increase in ALT levels (Figure 20B). In addition to ALT, AST also increased 24 hours after the first infusion in the FGK45-treated group, but not in AS598-treated or control animals (Figure 20B).

[0243] IHC of liver tissue. Histological analysis revealed extensive tissue damage characterized by centripetal multifocal inflammation with mononuclear leukocyte agglutination, thrombosis, and necrosis in mice treated with anti-mCD40 antibody (Figure 20C). In contrast, IHC analysis of livers from mice treated with protein #7 showed no evidence of hepatotoxicity and had a similar histological profile to that observed in livers treated with vehicle (Figure 20C).

[0244] In summary, in contrast to anti-mCD40 antibodies, protein #7 did not show signs of systemic toxicity with respect to weight loss, elevated pro-inflammatory cytokines (e.g., IL-6, TNFα, IFNγ, and IL-12p70), elevated aminotransferase levels (AST and ALT), or liver tissue damage.

[0245] Conclusion: In addition to serum albumin, we produced protein #7, a surrogate mouse-specific binding protein with binding specificity to mouse FAP and mouse CD40. Protein #7, tested in mouse cell-based in vitro assays, showed comparable results to proteins #5 and #6 in human cell-based in vitro assays (see, e.g., Example 4), demonstrating strictly FAP-dependent activation of CD40 (data not shown). As shown in this example, protein #7 (with an N-terminal His-tag; AS598) was also active in vivo and substantially inhibited the progression of FAP-positive tumors. Furthermore, in contrast to the anti-mouse CD40 antibody (FGK45), the antitumor activity of protein #7 was not associated with elevated serum cytokine levels or tested indicators of hepatotoxicity. Elevated serum cytokine levels and hepatotoxicity manifest as dose-limiting toxicities for some clinical CD40-activating antibodies. The presented data support a mechanism of action dependent on FAP-mediated crosslinking of the CD40 receptor, both in vitro and in vivo, resulting in tumor-localized CD40 activation without peripheral or non-tumor organ toxicity.

[0246] In conclusion, a tumor-targeted CD40 agonist-specific DARPin® protein was produced that locally activates the CD40 receptor in FAP-positive tumors and induces substantial antitumor activity in the absence of systemic toxicity.

[0247] Example 7: Protease activity of FAP in the presence or absence of the multiselective binding protein of the present invention This example describes FAP activity assays performed in the presence or absence of various multiselective binding proteins of the present invention to determine whether endogenous FAP enzyme activity is inhibited upon binding of multiselective recombinant proteins.

[0248] FAP is a type II single transmembrane serine protease whose expression is highly upregulated in tumor-like tissue remodeling sites (e.g., expressed on the surface of stromal fibroblasts in >90% of epithelial carcinomas), wound healing, embryonic tissues, and inflammatory sites (e.g., atherosclerosis / arthritis), although FAP expression is difficult to detect in non-affected adult organs. This atypical serine protease possesses both dipeptidyl peptidase (exopeptidase) and endopeptidase activity and cleaves substrates after proline binding. Structurally, FAP consists of a short cytoplasmic N-terminal sequence (4aa), a single transmembrane helix (21aa), and an extracellular domain (735aa) that forms an eight-bladed β-propeller and an α / β-hydrolase domain. FAP is active as a homodimer. The catalytic triplicate structure essential for FAP protease activity consists of residues Ser624, Asp702, and His734. The active site is accessible either through the central pore of the beta-propeller or through the cavity at the interface between the beta-propeller and the hydrolase domain.

[0249] The protease activity of FAP results in the cleavage of various substrates, including neuropeptide Y, type I collagen, and α2-antiplasmin. It also produces the substrate Z-GLY-PRO-AMC, which can be cleaved by either exopeptidase or endopeptidase activity, resulting in a product that can be measured with a fluorescence reader.

[0250] Table 10 outlines the molecules tested in the FAP activity assay.

[0251] [Table 10]

[0252] FAP activity assay. Human FAP (rhFAP) target was diluted to 0.67 μg / mL in assay buffer (50 mM Tris, 1 M NaCl, 1 mg / ml BSA, pH 7.5), and 45 μL per well was added to a 96-well plate (resulting in a final hFAP concentration of 0.3 nM in the activity assay). As shown in Table 10, molecules 1-5 were applied in a 500-fold molar excess by adding 5 μL of 3 μM molecule to the target sample (final concentration 150 nM). Finally, 50 μl of 100 μM Z-GLY-PRO-AMC substrate (final concentration 50 μM) was added to obtain a total volume of 100 μL in each well. Molecular number 5 was used as a control to demonstrate partial inhibition of FAP activity.

[0253] Before measurement, the plate was centrifuged at 4000 rpm for 2 minutes to remove any air bubbles that might interfere with the assay. Fluorescence was measured every 5 minutes for 95 minutes with excitation at 380 nm and emission at 460 nm using a fluorescence reader with manual gain set to 105%. Quadruple measurements were performed and the results are shown as mean and standard deviation.

[0254] Results. In the first step, dose-response curves were measured using FAP concentrations ranging from 0.01 nM to a maximum of 1.2 nM with a fixed substrate concentration of 50 μM. A linear time-dependent signal increase was observed over 95 minutes at a target rhFAP concentration of 0.3 nM (R0.999). 2 (Measurements were taken every 5 minutes). To determine the effect of protein binding on the enzymatic activity of FAP, the assay was performed under FAP saturation conditions by adding an FAP-binding molecule (e.g., molecular number 1 (sequence number 5)) at an excess of 500 times the molar ratio (150 nM) of FAP (0.3 nM). This concentration of molecular number 1 (sequence number 5) was 500 times the binding affinity of molecular number 1 (sequence number 5) to human FAP (Kd = 0.3 nM).

[0255] As outlined in Figure 15, molecules 1–4 did not inhibit endogenous dipeptidyl FAP enzyme activity. Partial inhibition of FAP activity was observed with an alternative FAP-binding molecule (molecule 5), which was used as an assay control.

[0256] Conclusion. The multi-selective binding protein of the present invention, for example, molecular number 1 (HFCC) or molecular number 2 (HFCC) * C * C * ) or their FAP-binding domains (F or F * Binding to FAP, such as ), did not affect the protease activity of FAP, as measured by its ability to cleave the fluorescence-generating substrate Z-GLY-PRO-AMC.

[0257] Example 8: Preferential localization and accumulation of the multi-selective binding protein of the present invention in tumor tissue. This example describes experiments conducted to investigate whether the multiselective binding protein of the present invention can preferentially localize and accumulate in tumor tissue, possibly via binding to FAP expressed in tumor tissue. For this purpose, the syngeneic MC38-FAP mouse model described in Example 6 was used as a preferred experimental system. Protein #7, also described in Example 6, was used as a representative multiselective binding protein of the present invention.

[0258] We investigated the localization and accumulation of protein #7 in tumor tissue using three different methodologies: SPECT / CT imaging, immunohistochemistry (IHC), and in vivo tissue distribution analysis.

[0259] Materials and methods Tumor inoculation and treatment: As described above, 9 × 10 6 Individual MC38-FAP mouse colon cancer cells were subcutaneously inoculated. The tumor was 500 mm. 3 When the size was reached, approximately 150 KBq of radioconjugated molecules (protein #7 or control DARPin® protein, which has four corresponding ankyrin repeat domain structures and binds to HSA but not to FAP or CD40) corresponding to 2.5 mg / kg = 50 μg / mouse were injected into the tail vein of the mouse.

[0260] Indium-111 Labeling: The DARPin® molecule was conjugated with radioactive labeling on the free SH group of the cysteine ​​attached to the C-terminus of the DARPin® molecule using a bifunctional chelating agent that has specific reactivity to sulfhydryl (SH) groups, in order to label indium-111 and maleimide-DTPA (Chematech, catalog number: C107). The molecules were stirred at room temperature (RT) for 1 hour in metal-free PBS (pH 7.4, PSI grade) and 0.05 mM EDTA. Maleimide-DTPA in DMSO (Sigma-Aldrich) in a 10-fold molar excess exceeding the amount of protein was added, and the mixture was incubated at room temperature for 1 hour. The reaction solution was transferred to an ultrafiltration tube (Amicon Amicon Ultra 15, 10 kDa molecular weight cutoff), and 4 mL of metal-free PBS was added. The tube was centrifuged at 4000 × g for 6 minutes at room temperature. After spinning, the flow-through was discarded, and 4 mL of metal-free PBS was added. The tube was then centrifuged again as described above. The filtered supernatant was transferred to a low-protein-bound Eppendorf reaction tube, and the concentration was determined. Site-specific conjugation was determined using electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). The final conjugate product was stored at 4°C.

[0261] SPECT / CT Imaging Study: Single-photon emission computed tomography (SPECT) and X-ray computed tomography (CT) images were acquired using a NanoSPECT / CTPlus camera (version 1.2, Bioscan). SPECT and CT images were acquired using the Nucline software (version 1.02). While CT images were reconstructed using the Nucline software, SPECT images were reconstructed using the HISPECT software (version 1.4.3049, Scivis GmbH). Fusions of SPECT and CT data were analyzed using VivoQuant® post-processing software (version 3.5, Invicro, USA). Whole-body activity was measured using a gamma-ray counter tube before image acquisition. SPECT / CT in vivo images were acquired from anesthetized mice at 4, 24, 48, 72, and 96 hours after injection of 111In-DARPin® molecules (by inhalation of a 2% isofluorane / oxygen mixture). The image acquired 96 hours after injection is shown in Figure 16A. All SPECT projections took 20 to 60 seconds per frame, resulting in a scan time of 15 to 45 minutes per image. CT scans were performed with a tube voltage of 55 kVp and a tube current of 145 μA, and an exposure time of 1000 milliseconds per projection.

[0262] IHC Study: Formalin-fixed paraffin-embedded (FFPE) tumor tissue slides were deparaffinized for antigen recovery by first deparaffinizing at 70°C for 8 minutes for 3 cycles, followed by deparaffinizing at 95°C for 48 minutes in pH 7.4 (EDTA-based solution, CC1 conditions on a Ventana automated staining system). The slides were then incubated with rabbit anti-DARPin® antibody conjugate (proprietary, working concentration: 2.0 μg / mL) at 37°C for 2 hours. The anti-DARPin® antibody conjugate was detected by an HRP system using an Omni Rabbit HRP autodispenser (Roche Diagnostics) at 37°C for 20 minutes. Finally, the slides were stained with hematoxylin, dehydrated with an ethanol gradient (70% > 90% > 100% and 100%, 1 minute each step), washed with xylene for 2 minutes, and coverslips were applied to the slides with cytoplasmic mounting medium. The slides were scanned with Vectra Polaris, and DARPin® protein accumulation was qualitatively evaluated.

[0263] In vivo tissue distribution study: MC38-FAP tumor-bearing mice were treated as described above (tumor inoculation and treatment), and euthanized 4, 24, 48, 72, or 96 hours after injection. The target organs were dissected, weighed, and radioactivity was determined as counts per minute (CPM) using a gamma counter (Packard Cobra II Gamma D5010). The CPM values ​​per organ analyzed were then converted to μg of DARPin® protein per organ (DARPin® protein / μg of organ), using the total amount of DARPin® protein per mouse as a reference value. The μg of DARPin® protein / μg of organ was converted to μg of DARPin® protein per gram of tissue (DARPin® protein / μg of tissue), and plotted on a graph as the percentage of injected dose per gram of tissue (ID / g%) using 4 mice / time point, expressed as mean ± SD.

[0264] result SPECT / CT imaging experiments revealed the preferential localization and accumulation of protein #7, the multiselective binding protein of the present invention, in tumor tissue (Figure 16A). Some off-tumor uptake was observed, mainly in the spleen, likely due to on-target distribution induced by the CD40-specific domain of protein #7.

[0265] MC38-FAP tumor-bearing mice were sacrificed, and tumors were analyzed for the presence of DARPin® molecules by IHC. IHC analysis showed a strong presence of protein #7, the multiselective binding protein of the present invention, in tumor tissue (Figure 16B, upper panel). In contrast, negative control DARPin® molecules that bind to HSA rather than FAP or CD40 were not detected at significant levels in tumor tissue (Figure 16B, lower panel). These data confirm that the tumor localization and accumulation of protein #7 are mediated by the FAP-specific binding domain and / or CD40-specific binding domain. Any faint signals observed in tumors treated with the negative control were likely caused by the presence of albumin in the tumor microenvironment and the binding of negative control DARPin® molecules to it, or simply by tissue permeability resulting in molecular diffusion.

[0266] In in vivo tissue distribution studies in which radiolabeled protein #7 or control DARPin® protein was injected into MC38-FAP tumor-bearing mice, accumulation of protein #7, the multiselective binding protein of the present invention, was specifically shown in tumor tissue (Figure 16C, left panel), but not in other organs, such as muscle (Figure 16C, right panel), bone marrow, liver, and kidney (data not shown). Consistent with SPECT / CT studies, some extratumor accumulation was observed only in the spleen, which was likely due to CD40 expression in this lymphoid organ (data not shown). Importantly, the radiolabeled-negative control DARPin® protein, which binds to HSA but not to FAP or CD40, did not accumulate in tumors or any other tissues. This confirms that tumor localization and accumulation of protein #7 occur via a mechanism mediated by FAP and / or CD40-specific binding.

[0267] conclusion The data presented in this embodiment provide experimental evidence that the multiselective binding protein of the present invention can preferentially localize to and accumulate in tumor tissue in a FAP and / or CD40-dependent manner. These results are consistent with the finding that the FAP-specific ankyrin repeat domain can effectively mediate tumor localization and accumulation of the multiselective binding protein through binding to tumor-expressed FAP.

[0268] Example 9: Tumor inhibition by the multiselective binding protein of the present invention is dependent on FAP expression in the tumor. Example 6 demonstrated that protein #7 (with an N-terminal His-tag; AS598) was active in vivo and substantially inhibited the progression of FAP-positive tumors. This example provided further evidence for the FAP-dependent mechanism of action of protein #7 using a syngeneic mouse model with low FAP expression. Specifically, a non-transfected MC38-WT cell line producing tumors with very low FAP expression levels in vivo was used to conduct efficacy studies.

[0269] Materials and methods: Tumor Experiment: The tumor experiment was performed as schematically shown in Figure 17A. MC38-WT colon cancer tumor cells were subcutaneously inoculated into the flank region of female syngeneic mice (C57BL / 6J) (Day 0). The mice were raised to approximately 77 mm on Day 7. 3 Mice were randomized to treatment groups based on tumor size. The test substances (protein #7 (with an N-terminal His-tag; AS598) and anti-CD40 antibody) were administered intraperitoneally (ip) to tumor-bearing mice according to the prescribed regimens shown in Table 11. The test substances were administered every 4 days at equimolar concentrations. Tumor growth was monitored every 3-4 days by caliper measurement until individual mice died due to ethical tumor volume limits.

[0270] [Table 11] N: Animal number

[0271] Tumor inoculation: Under standard isoflurane anesthesia, 1 × 10¹⁶ cells in 0.1 mL of PBS were inoculated into the flank of the mouse. 6 Individual MC38-WT tumor cells were subcutaneously inoculated.

[0272] Tumor Measurement: Tumor measurements were performed twice a week after tumor inoculation. The length and width of the tumor were measured using calipers. Tumor volume was calculated using the following formula: V = (L × W × W) / 2

[0273] Randomization: On day 7, randomization was performed using a "consistently matched distribution" randomization method (StudyDirect® software).

[0274] Observation and Data Collection: After tumor cell inoculation, animals were monitored daily for morbidity and mortality. At the time of routine monitoring, animals were examined for tumor growth and any effects of the treatment on behavior, including motility, food and water consumption, weight gain / loss, and any other abnormalities. Mortality and observed clinical signs were recorded for each individual animal.

[0275] Statistical analysis was performed using the Kruskal-Wallis nonparametric test, followed by Dunn's multiple comparison test for all groups compared to the vehicle. * p<0.05 ** p<0.01 *** If p < 0.001, the result was considered statistically significant.

[0276] Results and conclusions: As shown in Figure 17B, protein #7 is this FAP LOW In tumor models, it showed no statistically significant antitumor efficacy compared to the vehicle, thus providing further evidence for the FAP-dependent mechanism of action of protein #7. In contrast, an anti-CD40 antibody whose in vivo activity is not dependent on the presence of FAP in the tumor microenvironment inhibited tumor progression as expected, thus confirming the sensitivity of the MC38-WT tumor cell line to CD40 agonists.

[0277] Example 10: Long-term antitumor effect of the multi-selective binding protein of the present invention This example describes a study in which MC38-FAP tumor-bearing mice were treated and then followed over a longer period to further investigate the full therapeutic potential of the multiselective binding protein of the present invention.

[0278] Materials and methods Tumor Experiment: The tumor experiment was essentially carried out as described above in Study PD1035 and Example 6 in Figure 14B, but in this case, the mice were monitored over time and if they showed signs of distress as defined in the government-approved animal protocol, or if the tumor reached a predetermined tumor size of 2000 mm 3Mice were sacrificed only if the endpoints defined by were exceeded. Test substances (protein #7 (with N-terminal His-tag; AS598) and a non-FAP-binding mutant of protein #7 (AS608; as a negative control molecule, SEQ ID NO: 67 (SEQ ID NO: 57) with N-terminal His-tag) were administered to tumor-bearing mice according to the prescribed regimens as shown in Table 12. Tumor growth was monitored every 3-4 days by caliper measurement until individual mice died due to ethical tumor volume limits.

[0279] [Table 12] N: Animal number

[0280] Tumor inoculation and randomization: Under standard isoflurane anesthesia, 9 × 10¹⁴ oz in 0.2 mL of PBS were administered to the flanks of mice. 6 Individual MC38-FAP tumor cells were subcutaneously inoculated. The tumor was approximately 300 mm. 3 Once the mice reached their average size, they were randomized as described above.

[0281] Tumor Measurement: Tumor measurements were performed twice a week after tumor inoculation. The length and width of the tumor were measured using calipers. The tumor volume was calculated using the following formula: V = (L × W × W × π) / 6

[0282] Observation and Data Collection: At routine monitoring points, animals were examined for tumor growth and any effects of treatment on behavior, including motility, food and water consumption, weight gain / loss, and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals.

[0283] Results and Conclusions The results provide evidence that protein #7 can inhibit and eliminate MC38-FAP tumors and confer long-term protection against tumor recurrence. MC38-FAP tumors were no longer detectable approximately 20 days after the last treatment with protein #7 (Figure 18A). Furthermore, mice treated with protein #7 survived for at least 200 days after inoculation (i.e., the entire measurement period), demonstrating the sustained and long-lasting antitumor effect of protein #7 (Figure 18B). In contrast, MC38-FAP tumors treated with a negative control that binds to the CD40 target but not to the FAP target progressed similarly to the vehicle group (Figure 18A), and the mice either died or had to be sacrificed at a similar rate to the vehicle group (Figure 18B). These data confirm the FAP-dependent mechanism of action of protein #7 in vivo. Overall, these data provide evidence that the multiselective binding protein of the present invention can induce potent and persistent antitumor activity in a FAP-dependent manner in the MC38-FAP tumor mouse model.

[0284] Example 11: Induction of protective antitumor immunological memory by the multiselective binding protein of the present invention Mice carrying well-established MC38-FAP tumors were able to eliminate the tumors to an undetectable size after treatment with protein #7, as shown in Figure 18. To study the potential of protein #7-induced anti-tumor immunomemory, MC38-FAP tumor cells were re-challenged in the same tumor-free mice.

[0285] Materials and methods Tumor Experiments: Tumor experiments and treatments were carried out essentially as described in Study PD1035, Example 6 and Figure 14B, and Example 10 in Figure 18B. Mice treated with protein #7 were monitored for approximately 120 days before being rechallenged with MC38-WT or MC38-FAP tumor cells (1 or 9 × 10 in 0 or 2 mL of PBS, respectively). 6(Cells / mouse). In parallel, naive mice were challenged with the exact same tumor cells. Tumor growth was monitored every 3-4 days by caliper measurements until individual mice died due to ethical tumor volume limits.

[0286] Results and Conclusions After an initial weak tumor growth, all mice previously treated with protein #7 completely eliminated the rechallenged MC38-FAP tumors (Figure 19A), suggesting the presence of an immunological antitumor memory response. Interestingly, the same phenomenon was observed in mice rechallenged with MC38-WT tumor cells (Figure 19A), suggesting that protein #7 contributed to the establishment of immunological memory, directed more broadly to tumor antigens than to FAP-related antigens. Furthermore, naive control groups inoculated with either MC38-WT or MC38-FAP tumor cells clearly demonstrated the tumor-forming potential of the tumor cells used (Figure 19B), thus confirming that the absence of tumor growth in the rechallenged mice shown in Figure 19A was due to an immune-mediated antitumor response. In conclusion, experimental evidence was obtained suggesting that the multiselective binding protein of the present invention has the ability to induce protective antitumor immunological memory, and furthermore, that this immunological memory is not limited to FAP-related antigens in the MC38-FAP tumor model.

[0287] Example 12: X-ray structural analysis of a complex of human tumor necrosis factor receptor superfamily member 5 (hCD40) bound to a CD40-specific binding protein. The objective of this study was to produce and analyze a recombinant hCD40 complex bound by the CD40-specific binding protein of the present invention using X-ray crystallography. The CD40-specific binding protein used in this structural analysis was DARPin®, which has the amino acid sequence of SEQ ID NO: 3.

[0288] Materials and methods Protein synthesis. hCD40 was expressed in Hi5 cells in the presence of tunicamycin to inhibit glycosylation. Protein from the culture supernatant was purified by HIS-Trap, THB digest, negative HIS-Trap, and SEC. The purified hCD40 was mixed with CD40-specific DARPin® protein (SEQ ID NO: 3) in a 1:1.2 ratio. Excess DARPin® protein was removed from the DARPin® protein complex via SEC in hCD40:10 mM HEPES / NaOH pH 7, 150 mM NaCl. The sample was concentrated to obtain 36.7 mg / mL. This procedure yielded a homogeneous protein with a purity exceeding 95%, as determined by Coomassi staining SDS-PAGE.

[0289] Crystallization. The purified proteins were used in crystallization tests employing both standard screening with approximately 1200 different conditions and crystallization conditions identified using literature data. The initially obtained conditions were optimized using standard strategies, systemically changing parameters that significantly affect crystallization, such as temperature, protein concentration, and titration ratio. These conditions were also refined by systematically changing pH or precipitant concentration.

[0290] Final crystallization conditions: 30w / v% PEG 4K 0.24M LiSO4 0.1M Tris pH = 8.50 0.35M NaBr

[0291] Data Collection and Processing. Crystals were rapidly frozen and measured at a temperature of 100K. Using cryogenic conditions, X-ray diffraction data was collected from a composite crystal of hCD40 bound to ligand DARPin® protein (SEQ ID NO: 3) using SWISS LIGHT SOURCE (SLS, Villigen, Switzerland). The crystals belong to space group C2. The data were processed using the programs autoPROC, XDS, and autoPROC, AIMLESS. Data collection and processing statistics for the DARPin® protein are listed in Table 13 below.

[0292] [Table 13]

[0293] Structural modeling and refinement. Phase information necessary for structural determination and analysis was obtained by molecular substitution. A previously elucidated structure of hCD40 was used as a search model. Subsequent model construction and refinement were performed according to standard protocols using COOT and the software package CCP4, respectively. Approximately 4.9% of the measured reflectances were excluded from the refinement procedure for calculating the free R factor, a measure for cross-validating the accuracy of the final model (see Table 14 below). TLS refinement (using REFMAC5 and CCp4) yielded lower R factors and higher quality electron density maps. Automatically generated local NCS constraints were applied (new REFMAC5 version keyword "ncsr local"). Ligand parameterization and corresponding library file generation were performed using GRADE (Global Phasing Limited). F 3.0 was matched using REFMAC5. o -F c A water model was constructed using COOT's "Find waters" algorithm by placing water molecules at the peaks of the map and verifying all water using COOT's validation tool. The criteria for the list of suspected water were as follows: 80 Å 2 Larger B factor, 2F less than 1.2σ o -Fc Maps were used to determine the nearest contact distance (less than 2.3 Å) or the nearest contact distance (greater than 3.5 Å). Suspected water molecules and water molecules within ligand-binding sites (less than 10 Å from ligand) were manually identified. The Ramachandran plot of the final model showed 92.2% of all residues in the most preferred region, 7.8% in the additionally acceptable region, and 0.0% in the acceptable region. No residues were found in the unacceptable region (Table 14). Statistics for the final structure and refinement process are listed in Table 14 below.

[0294] [Table 14]

[0295] result The structure was elucidated and refined to a final resolution of 2.29 Å. Structural analysis using X-ray crystallography revealed that the DARPin® protein (SEQ ID NO: 3) binds to the cysteine-rich domain (CRD) 1 (amino acids 23-59) of the CD40 receptor (SEQ ID NO: 51), and binds to the CD40 receptor on one side opposite to the binding site of the CD40 ligand (CD40L). This indicates that there is no direct binding site competition between the DARPin protein and CD40L (Figures 21A and 21B). The CRD1 domain of the CD40 receptor is located away from the cell membrane.

[0296] It has been reported that potent CD40 agonist antibodies bind to the distal epitope of the CD40 receptor membrane (Yu et al., "Cancer Cell" Vol. 33, pp. 664-675, e664 (2018)). Similarly, the X-ray crystallographic study described in this example showed that the CD40-specific binding protein (SEQ ID NO: 3) interacts with CRD1 of the CD40 receptor, which is far from the cell membrane. As has already been suggested for CD40 agonist antibodies, epitopes further from the cell membrane experience less steric hindrance, thus improving access to the CD40-specific binding protein that constitutes the localization molecule (e.g., the recombinant protein consisting of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6), potentially leading to more efficient clustering of the CD40 receptor and, consequently, more efficient activation of the CD40 receptor. Furthermore, the interaction region between the CD40-specific binding protein (SEQ ID NO: 3) and CRD1 was shown to be opposite to the binding site of CD40L, suggesting that there is no direct binding competition between the binding protein of the present invention and CD40L. Compounds that do not compete with CD40L may have additive or synergistic effects with the ligand, potentially leading to better receptor activation (e.g., Yu et al., ibid.; Challa et al., "Allergy" Vol. 54, pp. 576-583 (1999); Pound et al., "Int Immunol" Vol. 11, pp. 11-20 (1999)).

[0297] This specification is best understood in light of the teachings of the references cited herein. The embodiments herein are provided as examples 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 incorporated into the invention. All publications, patents, and GenBank sequences cited herein are incorporated in their entirety by reference. Unless the material incorporated by reference is inconsistent with or contradicts this specification, this specification takes precedence over any such material. The references herein do not constitute an admission that such references are prior art to the invention.

[0298] 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 by the following embodiments.

Claims

1. Recombinant protein, It comprises a first ankyrin repeat domain that specifically binds to serum albumin, a second ankyrin repeat domain that specifically binds to fibroblast-activating protein (FAP), a third ankyrin repeat domain that specifically binds to CD40, and a fourth ankyrin repeat domain that specifically binds to CD40. The first to fourth ankyrin repeat domains are arranged from the N-terminus to the C-terminus in the formula: (First ankyrin repeat domain) - (Second ankyrin repeat domain) - (Third ankyrin repeat domain) - (Fourth ankyrin repeat domain) Recombinant proteins arranged according to the following rules.

2. The recombinant protein according to claim 1, wherein the second ankyrin repeat domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

2.

3. The recombinant protein according to claim 1 or 2, wherein each of the third and fourth ankyrin repeat domains independently comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

3.

4. The recombinant protein according to claim 3, wherein each of the third and fourth ankyrin repeat domains independently contains Q at position 8, L at position 15, R at position 143, and Q at position 147, where positions 8, 15, 143, and 147 correspond to positions from the N-terminus of the amino acid sequence described in SEQ ID NO:

3.

5. The recombinant protein according to any one of claims 1 to 4, wherein the first ankyrin repeat domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

1.

6. From the N-terminus to the C-terminus, the following equation: A recombinant protein according to any one of claims 1 to 5, comprising (first ankyrin repeat domain) - (linker) - (second ankyrin repeat domain) - (linker) - (third ankyrin repeat domain) - (linker) - (fourth ankyrin repeat domain), wherein each linker comprises the amino acid sequence of SEQ ID NO:

4.

7. The second ankyrin repeat domain has a K of 100 nM or less. Ｄ It binds to human FAP at a value, and / or each of the third and fourth ankyrin repeat domains has a K value of 100 nM or less. Ｄ It binds to human CD40 at a certain value, and / or the first ankyrin repeat domain has a K content of 100 nM or less. Ｄ A recombinant protein according to any one of claims 1 to 6, which binds to human serum albumin in a specific value.

8. A recombinant protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, wherein the recombinant protein has a K content of 100 nM or less. Ｄ Recombinant proteins that bind to human FAP, human CD40, and human serum albumin.

9. A recombinant protein containing the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO:

6.

10. When the recombinant protein was evaluated by flow cytometry in an in vitro human B cell activation assay, it exhibited a half-effect concentration (EC) of 0.01 nM to 10 nM. ５０ A recombinant protein according to any one of claims 1 to 9, having )

11. The recombinant protein according to any one of claims 1 to 10, wherein the binding of the recombinant protein to FAP is evaluated by cleavage of the fluorescent substrate Z-GLY-PRO-AMC, and does not inhibit the prolyl endopeptidase activity of FAP by more than 25%.

12. A nucleic acid encoding a recombinant protein according to any one of claims 1 to 11.

13. A pharmaceutical composition comprising a recombinant protein according to any one of claims 1 to 11 or a nucleic acid according to claim 12, and a pharmaceutically acceptable carrier or excipient.

14. A host cell comprising the nucleic acid described in claim 12.

15. A method for producing a recombinant protein according to any one of claims 1 to 11, comprising the step of culturing the host cells according to claim 14 under conditions in which the recombinant protein is expressed.

16. A pharmaceutical composition according to claim 13 for treating a medical condition, wherein the medical condition is cancer.

17. The pharmaceutical composition according to claim 16, wherein the cancer is a solid tumor.

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