CN116802214A

CN116802214A - Novel slow release prodrugs

Info

Publication number: CN116802214A
Application number: CN202180092124.5A
Authority: CN
Inventors: A·博斯哈特; J·阿勒斯卡格; B·施莱思; P·阿姆斯图茨; S·方泰纳
Original assignee: Molecular Partners AG
Current assignee: Molecular Partners AG
Priority date: 2020-12-16
Filing date: 2021-12-16
Publication date: 2023-09-22
Also published as: CN116802213A

Abstract

The present application relates to a composition comprising a binding moiety and a drug molecule, wherein the binding moiety reversibly binds to the drug molecule to form a prodrug complex which slowly releases the drug molecule in vivo after administration. The application also relates to methods of forming such compositions and methods of treatment using such compositions. Binding moieties, nucleic acids encoding the binding moieties, and methods of making these nucleic acids using host cells are also described.

Description

Novel slow release prodrugs

Cross Reference to Related Applications

The present application claims the priority of US 63/126356 submitted on month 12 and 16 of 2020, EP20216705 submitted on month 12 and 22 of 2020 and US 63/182,394 submitted on month 4 and 30 of 2021. The disclosures of these patent applications are incorporated by reference herein in their entirety for all purposes.

Sequence listing

The present application comprises a sequence listing submitted electronically in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy created at month 12 of 2021 was named 13081_0027-00304_sl.txt and was 54,310 bytes in size.

Technical Field

The present application relates to a composition comprising a binding moiety and a drug molecule, wherein the binding moiety reversibly binds the drug molecule to form a prodrug complex which slowly releases the drug molecule in vivo after administration. The application also relates to methods of forming such compositions and methods of treatment using such compositions. Binding moieties, nucleic acids encoding the binding moieties, and methods of making these nucleic acids using host cells are also described.

Background

Clearly, a viable drug candidate needs to meet certain efficacy criteria, but it is also important that the drug candidate have an acceptable safety profile. Many drug molecules have some undesirable off-target and/or on-target side effects, and some drug molecules may also cause undesirable on-target effects due to exaggerated and undesirable pharmacological effects on the intended target of the drug molecule. For certain drug classes, adverse effects cannot be avoided and therefore they must be alleviated. There are several options for this. For example, non-steroidal anti-inflammatory drugs (NSAIDs) can damage the inner lining of the stomach, so they are often co-administered with agents that protect the stomach, such as the proton pump inhibitor omeprazole (omeprazole). For other drug molecules, adverse effects or risks thereof may be reduced using specific dosage regimens, such that the drug is administered to the patient in multiple doses or continuously over a longer period of time. Examples of such situations may include taking the drug multiple times per day, or Intravenous (IV) infusion of the drug.

While divided doses and IV drug infusions are accepted and acceptable modes of administration, they are not without drawbacks. Heavy dosage regimens for patients, such as those requiring multiple administrations over a relatively short period of time, are associated with poor patient compliance and thus poorer treatment outcome. Although IV infusion methods are unlikely to suffer from poor patient compliance, the patient must be under medical care. This is destructive to the patient and results in an increased burden on the healthcare system. Thus, there is a need in the art for improved ways of alleviating the effects of undesirable drugs.

Particularly problematic adverse effects associated with the use of various drugs are Cytokine Release Syndrome (CRS) and hypercytokinemia, also known as "cytokine storm". For example, CRS or hypercytokinemia may occur after treatment with certain immunotherapies (such as monoclonal antibodies and CAR-T cells). Hypercytokine blood usually occurs rapidly after the first dose of drug, characterized by uncontrolled excessive release of cytokines in the body. Although cytokine release is a critical part of normal immune function, excessive cytokine release too fast into the blood can lead to symptoms such as hyperthermia, inflammation, severe fatigue, nausea and sometimes even multiple organ failure and death. After the participants developed severe hypercytokinemia, clinical trials of the drug sirailizumab (Theralizumab) intended for the treatment of B-cell chronic lymphocytic leukemia and rheumatoid arthritis had to be abandoned. The onset of symptoms occurred within one hour of administration and all participants in the trial required urgent hospital care. CRS is also caused by the massive and rapid release of cytokines into the blood by immune cells affected by immunotherapy. Symptoms of CRS include fever, nausea, headache, rash, rapid heart beat, hypotension, and dyspnea. Sometimes, CRS may be severe or life-threatening.

One class of drugs known to be associated with CRS is T cell cement (TCE). TCE is also associated with systemic endothelial activation and massive lymphocyte redistribution and neurotoxicity, especially after the first dose administration (Velasquez, blood,2018,131 (1), 30-38). These toxicities often affect clinical trial design and dose escalation strategies and have been shown to have dose limitations due to severity, especially in patients with high disease burden. Pre-medication and/or active intervention may also be required, ultimately leading to complex clinical trial designs.

Several strategies have been devised for clinical management of CRS associated with administration of drugs (e.g., T cell cement). These strategies include stepwise dosing (stepwise dose escalation), pretreatment with steroids, especially dexamethasone (dexamethasone), or treatment with tobalizumab (anti-IL 6 receptor antibody) (see e.g. Aldoss et al Current Oncology Reports,2019, 21:4). Pretreatment with steroid delays the onset of treatment, which is not recommended for invasive disease states, and the use of steroid in patients with high Body Mass Index (BMI) and/or blood pressure may be contraindicated. The FDA approved treatment with tolizumab in 2017 to avoid CRS. However, the immunosuppressive effects of such drugs can leave patients vulnerable to other infectious diseases.

In summary, there remains a need for new or improved methods to avoid, reduce or mitigate the adverse effects of, or the risks of, drug molecules used to treat diseases, including cancer.

Disclosure of Invention

The present application seeks to provide a new method of avoiding or reducing adverse effects or risks thereof following administration of a drug molecule. The present application provides a method for the slow release of active drug molecules into the body after administration of a drug product. This method uses a so-called "slow release" composition (also referred to herein as a prodrug complex) that releases the active drug molecule into the body for an extended period of time, thereby avoiding peaks in the concentration of the active drug molecule in the body shortly after administration. An example of a beneficial application of this method is the use of a slow release composition comprising TCE to reduce CRS risk after administration of TCE.

Prodrug strategies are often employed to alleviate problems associated with how a particular drug is absorbed, distributed, metabolized, and excreted. Many commonly available prodrugs contain small moieties (such as ester and amide groups) that hydrolyze in vivo, or groups that will be phosphorylated or dephosphorylated after administration.

In the present application we describe novel prodrug methods using binding moieties that bind reversibly to a drug molecule and when bound inhibit the biological activity of the drug molecule. Such biological activity of the drug molecule may be, for example, binding of the drug molecule to a biological target. The prodrug complexes of the application comprise such binding moieties that bind reversibly to the drug molecule. The binding properties of the binding moiety to the drug molecule allow for release of the drug molecule over time. Such release of the drug molecule over time may occur, for example, after administration of the prodrug complexes of the application to a subject (including a human). The binding moieties of the application have high affinity for and/or low rate of dissociation from the drug molecule. Binding moieties of the application include molecules having different structures, such as immunoglobulin molecules or non-immunoglobulin molecules. Binding moieties include antibodies, alternative scaffolds (such as engineered scaffolds), and polypeptides. An example of such an engineered scaffold is a designed ankyrin repeat domain.

In summary, the present invention provides slow release compositions comprising a binding moiety and a drug molecule, methods of making such compositions, and methods of treatment using such compositions. The invention also provides novel binding moieties, nucleic acids encoding the binding moieties, and methods of making these nucleic acids using host cells.

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

1. In a first embodiment, the present invention relates to a composition comprising (i) a binding moiety and (ii) a drug molecule; wherein the binding moiety reversibly binds to the drug molecule; and wherein the binding moiety, when bound, inhibits the biological activity of the drug molecule.

2. In a second embodiment, the present invention relates to a composition according to embodiment 1, wherein the binding moiety comprises an antibody, an alternative scaffold or a polypeptide.

3. In a third embodiment, the present invention relates to a composition according to embodiment 1 or 2, wherein the binding moiety comprises an immunoglobulin molecule or fragment thereof.

4. In a fourth embodiment, the present invention relates to a composition according to embodiment 1 or 2, wherein the binding moiety comprises a non-immunoglobulin molecule.

5. In a fifth embodiment, the present invention relates to a composition according to any one of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a chimeric antibody, a human antibody, a humanized antibody, a single domain antibody, a heavy chain variable domain (VH), a light chain variable domain (VL) or a variable domain (VHH).

6. In a sixth embodiment, the present invention relates to a composition according to any one of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from or associated with: addenptin (adnectin), a monoclonal antibody, an affibody (affibody), an African (affilin), an African (affimer), an aptamer, an African (affitin), an alpha antibody (alphabody), an anti-carrier (anti-calilin), a repeat protein domain, an armadin repeat domain, an Alzheimer (atrimer), an avermer (avimer), an ankyrin repeat domain, a fenomo (fynomer), a knottin (knottin), a Kunitzdomain (Kunitzdomain) or a T Cell Receptor (TCR).

In embodiment 6a, the present invention relates to a composition according to any one of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from or associated with adestine.

In embodiment 6b, the invention relates to a composition according to any of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from or associated with a monoclonal antibody.

In embodiment 6c, the present invention relates to a composition according to any one of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from or associated with an affibody.

In embodiment 6d, the present invention relates to a composition according to any one of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from or associated with agalin.

In embodiment 6e, the invention relates to a composition according to any one of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from or associated with alzheimer's disease.

In embodiment 6f, the invention relates to a composition according to any one of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from or associated with an aptamer.

In embodiment 6g, the invention relates to a composition according to any one of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from or associated with acitretin.

In embodiment 6h, the invention relates to a composition according to any one of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from or associated with an alpha antibody.

In embodiment 6i, the present invention relates to a composition according to any one of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from or associated with a repeat protein domain.

In embodiment 6j, the invention relates to a composition according to any one of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from or related to a adiadillo-repeat domain.

In embodiment 6k, the invention relates to a composition according to any one of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from or associated with alzheimer's disease.

In embodiment 6l, the invention relates to a composition according to any one of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from or associated with avermectin.

In embodiment 6m, the present invention relates to a method according to any one of embodiments 1 to 4

The composition, wherein the binding moiety comprises an antigen binding domain derived from or associated with an ankyrin repeat domain.

In embodiment 6n, the invention relates to a composition according to any one of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from or associated with fenomo.

In embodiment 6o, the present invention relates to a composition according to any one of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from or associated with a knottin.

In embodiment 6p, the present invention relates to a composition according to any one of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from or associated with a kunitz domain.

In embodiment 6q, the invention relates to a composition according to any one of embodiments 1 to 4, wherein the binding moiety comprises an antigen binding domain derived from or associated with a T Cell Receptor (TCR).

7. In a seventh embodiment, the present invention is directed to a composition according to any one of the preceding embodiments, wherein the biological activity of the drug molecule is binding of the drug molecule to a biological target.

8. In an eighth embodiment, the present invention relates to a composition according to any one of the preceding embodiments, wherein the biological activity of the drug molecule is an enzymatic activity.

9. In a ninth embodiment, the present invention is directed to a composition according to any one of the preceding embodiments, wherein the binding affinity of the binding moiety to the drug molecule allows release of the drug molecule over time after administration of the composition to a mammal.

10. In a tenth embodiment, the present invention relates to a composition according to embodiment 9, wherein the mammal is a human.

11. In an eleventh embodiment, the present disclosure is directed to a composition according to any preceding embodiment, wherein the binding moiety has a dissociation constant (K) of less than 10nM (such as less than 10nM, less than 1nM, less than 100pM, less than 10pM, or less than 1 pM) _D ) Binding to the drug molecule.

12. In a twelfth embodiment, the present disclosure is directed to a composition according to any one of the preceding embodiments, wherein the rate of dissociation (k _off ) Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-4 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-7 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-4 s ^-1 Or between about 1X 10 ^- ⁶ s ^-1 And about 1X 10 ^-5 s ^-1 Between them.

13. In a thirteenth embodiment, the present disclosure is directed to the composition of embodiment 11 or 12, wherein the dissociation constant (K _D ) Or dissociation rate (k) _off ) Is measured in Phosphate Buffered Saline (PBS).

14. In a fourteenth embodiment, the present disclosure is directed to a composition according to any one of the preceding embodiments, wherein the binding moiety has a blocking half-life (T _1/2 ) Wherein the blocking half-life is calculated according to the formula:

blocking

15. In a fifteenth embodiment, the present disclosure is directed to a composition according to embodiment 14, wherein the blocking half-life (T _1/2 ) At least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 15 hours, at least about 20 hours, such as at least about 20 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 50 hours, at least about 55 hours, or at least about 60 hours.

16. In a sixteenth embodiment, the present invention is directed to a composition according to any one of the preceding embodiments, wherein the binding moiety comprises a designed ankyrin repeat domain.

17. In a seventeenth embodiment, the present invention relates to a composition according to embodiment 16, wherein the engineered ankyrin repeat domain comprises an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of seq id nos: (1) 30 to 51, and (2) a sequence in which up to 9 amino acids in any one of SEQ ID NOs 30 to 51 are substituted with another amino acid.

18. In an eighteenth embodiment, the present invention relates to a composition according to embodiment 16, wherein the engineered ankyrin repeat domain comprises an amino acid sequence selected from the group consisting of seq id nos: (1) 1 to 10, and (2) a sequence having at least 85% amino acid sequence identity to any one of SEQ ID NOs 1 to 10.

19. In a nineteenth embodiment, the present invention is directed to a composition according to any one of the preceding embodiments, wherein the drug molecule comprises an antibody, alternative scaffold, or polypeptide.

20. In a twentieth embodiment, the present invention relates to a composition according to any one of the preceding embodiments, wherein the drug molecule comprises an immunoglobulin molecule or fragment thereof.

21. In a twenty-first embodiment, the present invention is directed to a composition according to any one of the preceding embodiments, wherein the drug molecule comprises a non-immunoglobulin molecule.

22. In a twenty-second embodiment, the present invention relates to a composition according to any one of the preceding embodiments, wherein the drug molecule comprises an antigen binding domain derived from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a chimeric antibody, a human antibody, a humanized antibody, a single domain antibody, a heavy chain variable domain (VH), a light chain variable domain (VL) or a variable domain (VHH).

23. In a twenty-third embodiment, the present invention is directed to a composition according to any one of the preceding embodiments, wherein the drug molecule comprises an antigen binding domain derived from or associated with: alder, monoclonal antibody, affibody, african, alzheimer's, aptamer, african, alpha antibody, anti-transporter, repeat domain, armadillo repeat domain, alzheimer's, avermectin, ankyrin repeat domain, fenomo, knotting element, kunitz domain or T Cell Receptor (TCR).

In embodiment 23a, the present invention relates to a composition according to any one of the preceding embodiments, wherein the binding moiety comprises an antigen binding domain derived from or associated with adestine.

In embodiment 23b, the invention relates to a composition according to any preceding embodiment, wherein the binding moiety comprises an antigen binding domain derived from or associated with a monoclonal antibody.

In embodiment 23c, the present invention relates to a group according to any one of the preceding embodiments

A compound, wherein the binding moiety comprises an antigen binding domain derived from or associated with an affibody.

23d in embodiment 23d, the present invention relates to a group according to any one of the preceding embodiments

A compound, wherein the binding moiety comprises an antigen binding domain derived from or associated with agalin.

In embodiment 23e, the present invention relates to a group according to any one of the preceding embodiments

A compound, wherein the binding moiety comprises an antigen binding domain derived from or associated with alzheimer's disease.

In embodiment 23f, the present invention relates to a group according to any one of the preceding embodiments

A compound, wherein the binding moiety comprises an antigen binding domain derived from or associated with an aptamer.

23g in embodiment 23g, the invention relates to a group according to any one of the preceding embodiments

A compound, wherein the binding moiety comprises an antigen binding domain derived from or associated with acitretin.

In embodiment 23h, the invention relates to a group according to any one of the preceding embodiments

A compound, wherein the binding moiety comprises an antigen binding domain derived from or associated with an alpha antibody.

In embodiment 23i, the present invention relates to a group according to any one of the preceding embodiments

A compound, wherein the binding moiety comprises an antigen binding domain derived from or associated with a repeat protein domain.

23j in embodiment 23j, the present invention relates to a group according to any one of the preceding embodiments

A compound, wherein the binding moiety comprises an antigen binding domain derived from or related to a adillo repeat domain.

In embodiment 23k, the present invention relates to a group according to any one of the preceding embodiments

In embodiment 23l, the invention relates to a group according to any one of the preceding embodiments

A compound, wherein the binding moiety comprises an antigen binding domain derived from or associated with avermectin.

In embodiment 23m, the invention relates to a composition according to any preceding embodiment, wherein the binding moiety comprises an antigen binding domain derived from or associated with an ankyrin repeat domain.

In embodiment 23n, the invention relates to a composition according to any preceding embodiment, wherein the binding moiety comprises an antigen binding domain derived from or associated with fenomo.

In embodiment 23o, the invention relates to a composition according to any preceding embodiment, wherein the binding moiety comprises an antigen binding domain derived from or associated with a knottin.

In embodiment 23p, the present invention relates to a composition according to any preceding embodiment, wherein the binding moiety comprises an antigen binding domain derived from or associated with a kunitz domain.

In embodiment 23q, the present invention relates to a composition according to any one of the preceding embodiments, wherein the binding moiety comprises an antigen binding domain derived from or associated with a T Cell Receptor (TCR).

24. In a twenty-fourth embodiment, the present invention is directed to a composition according to any one of the preceding embodiments, wherein the drug molecule has binding specificity for CD 3.

25. In a twenty-fifth embodiment, the present invention relates to a composition according to any of the preceding embodiments, wherein the drug molecule further comprises at least one binding domain having binding specificity for a Tumor Associated Antigen (TAA).

In embodiment 25a, the invention relates to a composition according to embodiment 25, wherein the Tumor Associated Antigen (TAA) is CD33.

In embodiment 25b, the invention relates to a composition according to embodiment 25, wherein the Tumor Associated Antigen (TAA) is CD123.

26. In a twenty-sixth embodiment, the present invention is directed to a composition according to any one of the preceding embodiments, wherein the drug molecule is a T cell cement drug molecule (TCE).

27. In a twenty-seventh embodiment, the present invention is directed to a composition according to any one of the preceding embodiments, wherein the drug molecule comprises a designed ankyrin repeat domain.

28. In a twenty-eighth embodiment, the present invention relates to a composition according to embodiment 27, wherein the engineered ankyrin repeat domain comprises an amino acid sequence selected from the group consisting of seq id no: (1) 12 to 15, and (2) a sequence having at least 85% amino acid sequence identity to any one of SEQ ID NOs 12 to 15.

29. In a twenty-ninth embodiment, the present invention is directed to a composition according to any one of the preceding embodiments, wherein the drug molecule comprises an antibody.

30. In a thirty-third embodiment, the present invention relates to a composition according to embodiment 29, wherein the drug molecule is an antibody that is a T-cell cement drug molecule (TCE).

31. In a thirty-first embodiment, the present invention relates to a composition according to embodiment 30, wherein the TCE comprises a binding domain that binds to CD3 and further comprises a binding domain that binds a Tumor Associated Antigen (TAA).

In embodiment 31a, the invention relates to a composition according to embodiment 31, wherein the Tumor Associated Antigen (TAA) is CD33.

In embodiment 31b, the invention relates to a composition according to embodiment 31, wherein the Tumor Associated Antigen (TAA) is CD123.

32. In a thirty-second embodiment, the present invention relates to a composition according to any one of embodiments 24 to 31b, wherein binding of the binding moiety to the TCE drug molecule inhibits binding of the TCE drug molecule to T cells and/or activation of T cells.

33. In a thirty-third embodiment, the present invention relates to a composition according to any one of embodiments 24 to 28 and 30 to 32, wherein the TCE is a bispecific antibody.

34. In a thirty-fourth embodiment, the present invention is directed to a composition according to any one of the preceding embodiments, wherein the binding moiety is an anti-idiotype conjugate of the drug molecule.

35. In a thirty-fifth embodiment, the present invention is directed to a composition according to any one of the preceding embodiments, further comprising a pharmaceutically acceptable carrier or excipient.

36. In a thirty-sixth embodiment, the present invention is directed to a composition according to any one of the preceding embodiments for use in therapy.

37. In a thirty-seventh embodiment, the present invention relates to a composition for use according to embodiment 36 for treating a proliferative disease, optionally wherein said proliferative disease is cancer.

38. In a thirty-eighth embodiment, the present invention relates to a method of treatment comprising the step of administering to a subject in need thereof a composition as defined according to any one of embodiments 1 to 35.

39. In a thirty-ninth embodiment, the present invention relates to a method according to embodiment 38, wherein the method is a method of treating a proliferative disease, optionally wherein the proliferative disease is cancer.

40. In a fortieth embodiment, the present invention is directed to a method of T cell activation in a subject in need thereof, comprising the step of administering to the subject the composition according to any one of embodiments 24-28 and 30-32, optionally wherein the composition further comprises a pharmaceutically acceptable carrier or excipient.

41. In a fortieth embodiment, the present invention is directed to a method of controlling the release of an active drug molecule in vivo, comprising administering to a subject in need thereof a composition according to any one of embodiments 1-35.

42. In a forty-second embodiment, the present invention relates to a method according to any one of embodiments 38-41, wherein said subject is a human.

43. In a forty-third embodiment, the present invention relates to a method of preparing a controlled release formulation, the method comprising the steps of:

(i) Providing a binding moiety as defined in any one of embodiments 1 to 6, 9 and 11 to 18;

(ii) Providing a drug molecule as defined in any of embodiments 19 to 23; and

(iii) The binding moiety and the active drug molecule are brought into equilibrium such that substantially all of the drug molecule is bound by the binding moiety.

44. In a forty-fourth embodiment, the present invention relates to a method of controlling the biological activity of a drug molecule, the method comprising combining a binding moiety as defined according to any one of embodiments 1 to 6, 9 and 11 to 18 with a drug molecule as defined according to any one of embodiments 19 to 23 to form a composition, and administering said composition to a patient in need thereof.

45. In a forty-fifth embodiment, the present invention relates to the method of embodiment 44, wherein said biological activity of said drug molecule is binding of said drug molecule to a biological target.

46. In a forty-sixth embodiment, the present invention relates to the method of embodiment 45, wherein said biological activity of said drug molecule is enzymatic activity.

47. In a forty-seventh embodiment, the present invention relates to a binding moiety having binding specificity for a drug molecule, wherein the binding moiety inhibits the biological activity of the drug molecule when bound thereto.

48. In a forty-eighth embodiment, the present invention relates to a binding moiety according to embodiment 47, wherein the binding moiety binds to the drug molecule to form a complex that reversibly inhibits the biological activity of the drug molecule.

49. In a forty-ninth embodiment, the present invention relates to a binding moiety according to any one of embodiments 47 or 48, wherein said biological activity of said drug molecule is binding of said drug molecule to a biological target.

50. In a fifty-first embodiment, the present disclosure is directed to the binding moiety of any one of embodiments 47 to 49, wherein the biological activity of the drug molecule is an enzymatic activity.

51. In a fifty-first embodiment, the present disclosure is directed to a binding moiety according to any one of embodiments 47 to 50 having a binding affinity (K) for the drug molecule of less than 10nM, such as less than 10nM, less than 1nM, less than 100pM, less than 10pM, or less than 1pM _D )。

52. In a fifty-second embodiment, the present disclosure is directed to a binding moiety according to any one of embodiments 47 to 51, wherein the rate of dissociation (k) of the binding moiety from the drug molecule _off ) Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^- ⁴ s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-7 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1×

10 ^-4 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^- ⁶ s ^-1 Between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-4 s ^-1 Or between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-5 s ^-1 Between them.

53. In a fifty-third embodiment, the present disclosure is directed to the binding moiety of embodiment 51 or 52, wherein the dissociation constant (K _D ) Or dissociation rate (k) _off ) Is measured in Phosphate Buffered Saline (PBS).

54. In a fifty-fourth embodiment, the present disclosure is directed to the binding moiety of any one of embodiments 47 to 53, wherein the binding moiety has a blocking half-life (T _1/2 ) And wherein the blocking half-life is calculated according to the formula:

blocking

55. In a fifty-fifth embodiment, the present disclosure is directed to the binding moiety of embodiment 54, wherein the blocking half-life (T _1/2 ) At least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 15 hours, at least about 20 hours, such as at least about 20 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 50 hours, at least about 55 hours, or at least about 60 hours.

56. In a fifty-sixth embodiment, the present invention relates to a binding moiety according to any one of embodiments 47 to 55, wherein the binding moiety comprises an antibody, alternative scaffold or polypeptide.

57. In a fifty-seventh embodiment, the present invention is directed to a binding moiety according to any one of embodiments 47 to 56, wherein the binding moiety comprises an immunoglobulin molecule or fragment thereof.

58. In a fifty-eighth embodiment, the present disclosure is directed to the binding moiety according to any one of embodiments 47 to 56, wherein the binding moiety comprises a non-immunoglobulin molecule.

59. In a fifty-ninth embodiment, the present disclosure is directed to a binding moiety according to any one of embodiments 47 to 58, wherein the binding moiety comprises an antigen binding domain derived from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a chimeric antibody, a human antibody, a humanized antibody, a single domain antibody, a heavy chain variable domain (VH), a light chain variable domain (VL), or a variable domain (VHH).

60. In a sixtieth embodiment, the present disclosure is directed to a binding moiety according to any one of embodiments 47 to 59, wherein the binding moiety comprises an antigen binding domain derived from or associated with: alder, monoclonal antibody, affibody, african, alzheimer's, aptamer, african, alpha antibody, anti-transporter, repeat domain, armadillo repeat domain, alzheimer's, avermectin, ankyrin repeat domain, fenomo, knotting element, kunitz domain or T Cell Receptor (TCR).

60a in embodiment 60a, the present invention is directed to a method according to any one of embodiments 47 to 59

The composition, wherein the binding moiety comprises an antigen binding domain derived from or associated with adestine.

60b in embodiment 60b, the present invention is directed to a method according to any one of embodiments 47 to 59

The composition, wherein the binding moiety comprises an antigen binding domain derived from or associated with a monoclonal antibody.

60c in embodiment 60c, the present invention is directed to a method according to any one of embodiments 47 to 59

The composition, wherein the binding moiety comprises an antigen binding domain derived from or associated with an affibody.

60d in embodiment 60d, the present invention is directed to a method according to any one of embodiments 47 to 59

The composition, wherein the binding moiety comprises an antigen binding domain derived from or associated with agalin.

In embodiment 60e, the present invention is directed to any one of embodiments 47 to 59

The composition, wherein the binding moiety comprises an antigen binding domain derived from or associated with alzheimer's disease.

In embodiment 60f, the present invention is directed to any one of embodiments 47 to 59

The composition, wherein the binding moiety comprises an antigen binding domain derived from or associated with an aptamer.

60g in embodiment 60g, the present invention is directed to a method according to any one of embodiments 47 to 59

The composition, wherein the binding moiety comprises an antigen binding domain derived from or associated with acitretin.

60h in embodiment 60h, the present invention is directed to any one of embodiments 47 to 59

The composition, wherein the binding moiety comprises an antigen binding domain derived from or associated with an alpha antibody. .

In embodiment 60i, the present invention is directed to any one of embodiments 47 to 59

The composition, wherein the binding moiety comprises an antigen binding domain derived from or associated with a repeat protein domain.

In embodiment 60j, the present invention is directed to any one of embodiments 47 to 59

The composition, wherein the binding moiety comprises an antigen binding domain derived from or related to a armadillo repeat domain.

In embodiment 60k, the invention relates to a composition according to any one of embodiments 47 to 59, wherein the binding moiety comprises an antigen binding domain derived from or associated with alzheimer's disease.

In embodiment 60l, the invention relates to a composition according to any one of embodiments 47 to 59, wherein the binding moiety comprises an antigen binding domain derived from or associated with avermectin.

In embodiment 60m, the invention relates to a composition according to any one of embodiments 47 to 59, wherein the binding moiety comprises an antigen binding domain derived from or associated with an ankyrin repeat domain.

In embodiment 60n, the invention relates to a composition according to any one of embodiments 47 to 59, wherein the binding moiety comprises an antigen binding domain derived from or associated with fenomo.

In embodiment 60, the invention relates to a composition according to any one of embodiments 47 to 59, wherein the binding moiety comprises an antigen binding domain derived from or associated with a knottin.

In embodiment 60p, the present invention relates to a composition according to any one of embodiments 47 to 59, wherein the binding moiety comprises an antigen binding domain derived from or associated with a kunitz domain.

In embodiment 60q, the invention relates to a composition according to any one of embodiments 47 to 59, wherein the binding moiety comprises an antigen binding domain derived from or associated with a T Cell Receptor (TCR).

61. In a sixtieth embodiment, the present invention relates to a binding moiety according to any one of embodiments 47-60, wherein said binding moiety is a designed ankyrin repeat domain.

62. In a sixty-second embodiment, the present invention relates to the binding moiety according to embodiment 61, wherein the engineered ankyrin repeat domain comprises an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of seq id nos: (1) 30 to 51, and (2) a sequence in which up to 9 amino acids in any one of SEQ ID NOs 30 to 51 are substituted with another amino acid.

63. In a sixty-third embodiment, the present invention relates to a binding moiety according to any one of embodiments 61 or 62, wherein the designed ankyrin repeat domain comprises an amino acid sequence selected from the group consisting of seq id no: (1) 1 to 10, and (2) a sequence having at least 85% amino acid sequence identity to any one of SEQ ID NOs 1 to 10.

64. In a sixtieth embodiment, the present invention relates to a nucleic acid encoding a binding moiety according to any one of embodiments 47-60.

65. In a sixty-fifth embodiment, the present invention relates to a nucleic acid encoding the engineered ankyrin repeat domain of any one of embodiments 61-63.

66. In a sixty-sixth embodiment, the present invention relates to a host cell comprising a nucleic acid molecule according to embodiment 64 or 65.

67. In a sixtieth embodiment, the present invention is directed to a method of making a binding moiety according to any one of embodiments 47-63, comprising culturing a host cell according to embodiment 66 under conditions wherein the binding moiety is expressed.

68. In a sixty-eighth embodiment, the present invention relates to the method of embodiment 67, wherein the host cell is a prokaryotic host cell.

69. In a sixty-ninth embodiment, the present invention relates to a method according to embodiment 67, wherein said host cell is a eukaryotic host cell.

Drawings

Fig. 1: computer modeling of the concentration in plasma of total drug molecules ("total drug"), prodrug complexes ("co-drug"), unbound drug molecules ("free drug"), and unbound bound moieties ("free conjugate") after a single dose administration of a prodrug complex of the invention to a patient. Figure 1 illustrates the development of the concentration of different molecules and prodrug complexes over time (period of 10 weeks), starting with a substantially fully complexed drug molecule upon administration. The concentration of unbound (active) drug reached a higher concentration than the complexed (inactive) drug after about 10 days and accounted for the vast majority of the total drug after 5 weeks. Free conjugates are rapidly cleared from the system and therefore cannot be bound to a drug molecule after dissociation therefrom. As illustrated, the concentration of free (active) drug never reached the concentration of total drug at the time of administration. Thus, due to the slow release of the drug molecule from the prodrug complex, the same total amount of uncomplexed form of the drug molecule is administered followed by the C _max In contrast, the maximum concentration (C) of free (active) drug obtained after administration of the prodrug complexes of the invention _max ) Significantly reduced.

Fig. 2: computer simulation of the concentration of unbound drug molecules (free drug without conjugate, dotted line) or prodrug complex comprising drug molecules bound to the binding moiety (drug + conjugate, solid line) after repeated dose administration of unbound (active) drug molecules, wherein the same total amount of drug molecules is administered to the patient in each case. Display deviceThree different prodrug complexes were generated from binding moieties with different binding affinities for the drug molecules, as indicated. FIG. 2 shows that reduced C can be achieved with the prodrug complexes of the invention _max And a slow increase in exposure of the active drug molecule. In addition, C _max The extent of decrease and the slowing of the increase in exposure depends on the binding affinity of the binding moiety to the drug molecule. The higher the binding affinity, C _max The stronger the decrease in exposure, the slower the increase in exposure.

Fig. 3a: the binding moiety ("conjugate") is intimately associated with the drug molecule ("drug") prior to administration of the prodrug complex to a patient, thereby forming the prodrug complex. Since the concentration of both the binding moiety and the drug is high in the prodrug formulation, k _on The binding equilibrium is dominated and substantially all drug molecules are bound by the binding moiety. If bound by the binding moiety, the biological activity of the drug molecule is inhibited.

Fig. 3b: after administration to a patient, the concentration of the binding moiety and drug in the solution is greatly reduced and the equilibrium shifts in a direction that favors dissociation of the prodrug complex (i.e., k _off Dominant binding equilibrium). Thus, the drug molecules are decomplexed from the binding moiety over time, thereby slowly releasing the active drug molecules into the patient.

Fig. 4: surface Plasmon Resonance (SPR) data for the different binding moieties tested with the precursor of conjugate #9 (conjugate #1, conjugate #4 and conjugate # 5) for the parent conjugate as reference, showing that the parent conjugate has a blocking T of 1-2 hours _1/2 Whereas the binding moieties tested in example 2 showed far less dissociation after a 2 hour measurement period. This greatly reduced dissociation results in a much longer blocking T compared to the parental conjugate _1/2 。

Fig. 5a and 5b: standard tumor cell killing and T cell activation assays with TCE #1 and 3 different binding moieties (conjugate #4, conjugate #5 and conjugate # 9) and medium affinity parental conjugates. Sample preparation was as described in example 3. Tumor cell killing (OD as determined by Lactate Dehydrogenase (LDH) release ₄₉₂ -OD ₆₂₀ ) Shown in FIG. 5In a, T cell activation (cd25+ cells expressed as% cd8+ cells) is shown in fig. 5 b. IC50 values in nM are indicated.

Fig. 6a: by passing throughDetermination of tumor cells expressing TAA in the Presence of serial dilutions of TCE (TCE#1) (NucLight +.>Markers) T cell mediated growth inhibition (used as a surrogate for T cell mediated killing); see example 4. The total red object area (corresponding to tumor cells) was determined at regular intervals. Error bars show the standard error for each image (4 images per well).

Fig. 6b: from 4.5 days up to time pointGraph of AUC (area under the curve) data versus concentration of TCE #1 for the experiment. EC50 values are indicated as pM; see example 4.

Fig. 7a to 7e: "simple" for comparing the ability of ultra-high affinity and medium affinity binding moieties to block T cell mediated growth inhibition (used as a surrogate for T cell mediated killing) of TAA expressing tumor cells over time "Experiments were as described in example 4. The following conditions were tested: no TCE or binding moiety (background) was added; only 10nM binding moiety was added (conjugate #4, conjugate #5 and conjugate #9; FIG. 7 e); only TCE (tce#1) was added; adding pre-equilibrated TCE#1 and parent conjugate at a decreasing ratio of binding moiety TCE (TCE#1 + parent conjugate); the pre-equilibrated TCE and ultra high affinity binding moiety (binding moiety # 5) are added at a decreasing ratio of binding moiety to TCE (TCE #1+ binding moiety # 5). The TCE concentration was set to a final concentration of 10pM, approximately corresponding to EC of TCE #1 based on previous LDH experiments ₈₀ . The total red object area (corresponding to tumor cells) was determined at regular intervals. Error bar displayStandard error for each image (4 images per well).

Fig. 8a to 8d: a "simple" for comparing the ability of different ultra-high affinity binding moieties to block T cell mediated growth inhibition (used as a surrogate for T cell mediated killing) of TAA expressing tumor cells over time "And (5) experiment. Constant concentrations of TCE (TCE #1, 10pM final concentration) were mixed with titers of different binding moieties (conjugate #4, conjugate #5 and conjugate # 9). The mixture was concentrated at 100x (to allow>99.9% of TCE bound by the binding moiety) is pre-incubated for at least 24 hours, then at the beginning +.>Dilution was 100-fold immediately prior to the experiment. The negative control curves for only 10nM binding portion (conjugate #4, conjugate #5 and conjugate # 9) are shown in FIG. 8 d. The background curve is completed without the addition of TCE or binding moieties. The total red object area (corresponding to tumor cells; see example 4. Error bars show standard error for each image (4 images per well) was determined at regular intervals.

Fig. 9a to 9b: "complexity" for comparing the ability of ultra-high affinity binding moieties in the absence or presence of functional groove compartments to block T cell mediated inhibition of growth over time of tumor cells expressing TAA (used as a surrogate for T cell mediated killing) " Experiments (see example 6). Experiments were performed with an inlet containing uncoated beads (i.e. no functional groove compartment present) (fig. 9 a) or with an inlet containing beads coated with a CD3 binding domain capable of capturing dissociated binders (i.e. functional groove compartment present) (fig. 9 b). T cell mediated growth inhibition curves of TAA expressing tumor cells in the presence of T cells and prodrug complexes prepared with different molar ratios of TCE#1 to conjugate#9 are shown in FIG. 9a for the uncoated bead setup and in FIG. 9b for the coated bead setup. Such asAs indicated, the control was performed with unbound TCE #1 (instead of prodrug complex) and without any TCE or prodrug complex (background). The total red object area (corresponding to tumor cells) was determined at regular intervals. Error bars show the standard error for each image (36 images per well).

Fig. 10a to 10f: cytokine release as determined in the ex vivo assay was avoided by incorporating control or drug molecules into fresh human whole blood containing cd123+ and cd33+ target cells and incubating the samples on a rotating wheel to avoid blood clotting. The control and drug molecules tested included vehicle (star), 1nM α -CD123 x α -CD3 industrial control (filled triangles) and 1nM TCE#1 (open squares), or 1nM TCE#1+1.2nM conjugate#5 (open diamonds) compared thereto. (A) And (B) shows TCE drug molecules in unbound form (a) and in complex form (B) with conjugate # 5. Passing through the Meso Scale for 0 hours, 2 hours, 4 hours, 8 hours, 24 hours The techniques determine cytokine release of TNF- α (C), IFN- γ (D), IL-2 (E) and IL-6 (F). Data were from a single experiment performed in an immunoed AB, sweden with blood from one healthy human donor.

Fig. 11a to 11g: in vivo efficacy studies of the antitumor activity of a) unbound TCE with B) TCE complexed with two different conjugates in NOG mice bearing Molm-13 tumors, transplanted PBMCs were compared. C) Study design of in vivo efficacy study. PBMC from two human donors were transplanted on day 0 and Molm-13 tumor cells were injected on day 2 s.c. with a tumor size of about 70mm on day 6 ³ Treatment was started at that time. Unbound TCE#2 administered at two different doses of 200 μg/kg and 1000 μg/kg was compared to a complex of 1000 μg/kg TCE#2 and two different binders with different affinities for the CD3 binding domain. The complexes were pre-equilibrated with a 2-fold molar excess of conjugate to ensure 100% complexing rate of TCE at the beginning of treatment. Treatment was administered i.v. All treatment groups (including vehicle) except the non-half-life extended alpha-CD 33 x alpha-CD 3 industrial control (group 2 where treatment was given at 200 μg/kg daily) were 3 times per week. At a first therapeutic doseBlood samples were taken 4 hours before and after the first treatment dose to determine cytokine levels at the beginning of treatment with these relatively small tumors. D) Tumor growth curves for all six treatment groups of n=10 mice, humanized with PBMCs of donor a or donor B, were plotted as mean ± SEM, except for group 2 (industrial control), where two animals were excluded due to unsuccessful humanization of donor B. The treatment phase starting on day 6 and ending on day 16 is indicated. E) Tumor growth curves for all 6 treatment groups of n=5 mice humanized with PBMCs of donor a were plotted as mean ± SEM. F) Tumor growth curves for all six treatment groups of n=5 mice humanized with PBMCs of donor B were plotted as mean ± SEM, except group 2, where two animals were excluded due to unsuccessful humanization. G) Legend for all six treatment groups.

Fig. 12a to 12d: during in vivo efficacy studies, human cytokine levels were measured in serum of blood samples taken prior to (pre) or 4 hours after the first dose, where the tumor was relatively small at the beginning of treatment (about 70mm ³ ). For the six treatment groups introduced in fig. 11, cytokine levels of individual animals are plotted as filled symbols (donor a, n=5) or open symbols (donor B, n=5), with the mean shown as bars ± SD. As shown in fig. 11, two mice from treatment group 2 had to be excluded, since humanization of donor B was unsuccessful. A) TNF- α level in pg/ml B) IFN- γ level in pg/ml. C) IL-2 levels in pg/ml, and D) IL-6 levels in pg/ml.

Fig. 13a to 13d: comparison of in vivo safety studies of cytokine release of A) unbound TCE with B) TCE complexed with two different conjugates in PBMC-transplanted NOG mice carrying Molm-13 tumors. C) Study design of in vivo safety study. PBMCs from two human donors were transplanted on day 0, MOLM-13 tumor cells were injected subcutaneously (s.c.) into the posterior flank on day 2, and at tumor size of about 300mm on day 14 ³ -800mm ³ Single dose (challenge) is injected intravenously (i.v.). Unbound TCE #2 administered at 1000 μg/kg was compared to complexes of TCE #2 and three different conjugates Having a binding domain at K to CD3 _D Different affinities ranging from less than or equal to 1pM to two digits pM. The complexes were pre-equilibrated with a 2-fold molar excess of conjugate to ensure 100% complexing rate of TCE at the beginning of treatment. Blood samples were taken before dosing, 2 hours, 4 hours, 8 hours and 24 hours after the first therapeutic dose to determine cytokine levels with medium-sized tumors. D) In using established tumors (300 mm ³ -800mm ³ ) Human cytokine levels (TNF-. Alpha., IFN-. Gamma., IL-2 and IL-6) in serum from blood samples taken before or after single dose treatment. Cytokine levels were determined on undiluted serum samples by CBA human Th1/Th2/Th17 kit (BD Biosciences). Cytokine levels of individual animals are plotted as solid symbols (donor a, n=5) or open symbols (donor B, n=5), and mean values are shown as black and grey solid lines, respectively, for the unblocked TCE #2 and TCE #2+ conjugate treated groups. In vivo safety study 1 was performed using TCE #2±conjugate #1 or conjugate #4, while separate but comparable in vivo safety study 2 focused on TCE #2±conjugate #10.

Detailed Description

SUMMARY

The present invention relates to a composition comprising a binding moiety and a drug molecule, wherein the binding moiety reversibly binds to the drug molecule, and wherein when bound, the binding moiety inhibits the biological activity of the drug molecule. Such compositions of the invention are prodrug complexes that slowly release the drug molecule in vivo upon administration. The slow release of the active drug molecule after administration may minimize adverse effects or risks thereof that are otherwise associated with the drug molecule.

Without wishing to be bound by theory, it is understood that the binding moieties described herein bind tightly to the drug molecule in a non-covalent manner. Binding may occur at the active site of the drug molecule, or at another site on the drug molecule. The binding may be anti-idiotype. Regardless of the binding site, binding of the binding moiety inhibits the biological activity (i.e., mode of action) of the drug molecule. The drug molecules are formulated with an excess of binding moieties, resulting in complete or nearly complete complexing and inhibition of the drug molecules and formation of a composition (i.e., prodrug complex). When administered to a patient, the prodrug complex is strongly diluted, resulting in continuous release of the active drug molecule over time, as the re-association of the binding moiety becomes negligible due to i) the very low concentration of the binding moiety and drug in the body and ii) the rapid elimination of the binding moiety from the system.

Figures 1-3 provide further illustration and explanation of the slow release concept and nature of the binding moieties and compositions (prodrug complexes) of the present invention.

The invention also relates to methods of forming such compositions and methods of treatment using such compositions and the use of such compositions in therapy. For example, the present invention relates to the use of such compositions in the treatment of proliferative diseases, such as cancer.

Binding moieties having binding specificity for a drug molecule are also described, wherein binding of the binding moiety to the drug molecule forms a complex that reversibly inhibits the biological activity of the drug molecule. For example, such biological activity of the drug molecule is binding of the drug molecule to a biological target.

The invention also relates to nucleic acids encoding the binding moieties and methods of making these nucleic acids using host cells.

Definition of the definition

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein, and techniques thereof, are those well known and commonly employed in the art.

Unless otherwise indicated, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms. If aspects of the invention are described as "comprising" a feature, embodiments "consisting of" or "consisting essentially of" that feature are also contemplated. Any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure. Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as being modified in all instances by the term "about" as will be understood by those skilled in the relevant art. The term "about" as used herein is equivalent to ± 10% of a given value unless otherwise indicated.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value and each endpoint falling within the range, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

In the context of the present invention, the term "protein" refers to a molecule comprising a polypeptide, wherein at least a portion of the polypeptide has or is capable of achieving a defined three-dimensional arrangement by forming secondary, tertiary and/or quaternary structures within a single polypeptide chain and/or between multiple polypeptide chains. If a protein comprises two or more polypeptide chains, a single polypeptide chain may be non-covalently linked or covalently linked, for example, by disulfide bonds between the two polypeptides. Protein moieties that have or are able to acquire a defined three-dimensional arrangement by forming secondary and/or tertiary structures alone are referred to as "protein domains". Such protein domains are well known to those skilled in the art.

The term "drug molecule" (used interchangeably herein with the term "drug") refers to a therapeutic agent comprising a polypeptide or protein, wherein the polypeptide or protein contains a site capable of being bound by a binding moiety.

The term "recombinant" as used in the context of recombinant proteins, recombinant polypeptides, and the like means that the protein or polypeptide is produced using recombinant DNA techniques 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 DNA capable of in vitro expression. If, for example, such recombinant bacterial expression plasmids are inserted into an appropriate bacterium, for example, E.coli (Escherichia coli), these bacteria can produce the polypeptide encoded by the recombinant DNA. The corresponding polypeptide or protein produced is referred to as a recombinant polypeptide or recombinant protein.

In the context of the present invention, the term "binding moiety" or "conjugate" refers to a conjugate comprising a polypeptide or protein, wherein the polypeptide or protein is capable of non-covalent binding to a drug molecule. The binding moiety need not necessarily bind to the active site of the drug molecule. However, the binding moiety must bind in a manner that inhibits the mode of action of the drug. This may be by binding to the active site of the drug, but may also be by binding to another site on the drug molecule to alter the conformation of the drug (i.e. allosteric inhibition) or to sterically hinder the active site of the drug molecule. The active site of the drug molecule is involved in the biological activity of the drug molecule. The biological activity may be an enzymatic activity or binding to a biological target. Binding of the binding moiety to the drug molecule inhibits the biological activity of the drug molecule. For example, binding of the binding moiety to the drug molecule inhibits enzymatic activity of the drug molecule or inhibits binding of the drug molecule to a biological target. The binding of the binding moiety to the drug molecule may be anti-idiotype. Thus, the binding moiety may be an anti-idiotype conjugate of a drug molecule.

Binding moieties useful in the present invention include antibodies, alternative scaffolds, and polypeptides. As used herein, the term "antibody" refers not only to intact antibody molecules, such as those typically produced by the immune system when the immune system detects an exogenous antigen, but also to any fragments, variants, and synthetic or engineered analogs of the antibody molecule that retain antigen binding capacity. Such fragments, variants and analogs are also well known in the art and are used periodically in vitro and in vivo. Thus, the term "antibody" includes intact immunoglobulin molecules, antibody fragments such as, for example, fab ', F (ab') 2, and single chain V region fragments (scFv), bispecific or multispecific antibodies, chimeric antibodies, humanized antibodies, antibody fusion proteins, non-conventional antibodies, and proteins comprising an antigen-binding domain derived from an immunoglobulin molecule. As used herein, the term "alternative scaffold" refers to any molecule comprising or consisting of a protein but not an antibody.

Any of these different structural types of binding moieties can bind tightly to the drug molecule, thereby blocking the mode of action of the drug molecule and forming a prodrug complex. The prodrug complex slowly uncomplexes in the body, releasing the drug molecule into the body. In a preferred embodiment, the binding moiety comprises an ankyrin repeat domain having binding specificity for a drug molecule. In another preferred embodiment, the binding moiety comprises an antibody having binding specificity for a drug molecule. In another preferred embodiment, the binding moiety comprises an alternative scaffold having binding specificity for a drug molecule, wherein the alternative scaffold does not comprise an ankyrin repeat domain. In a preferred embodiment, the drug molecule comprises an antibody and the binding moiety is an anti-idiotype antibody having binding specificity for said antibody comprised in said drug molecule. In another preferred embodiment, the drug molecule comprises an antibody and the binding moiety is an anti-idiotype alternative scaffold having binding specificity for said antibody comprised in said drug molecule, such as for example an ankyrin repeat domain. In another preferred embodiment, the drug molecule comprises an alternative scaffold, such as for example an ankyrin repeat domain, and the binding moiety is an anti-idiotype antibody having binding specificity for said alternative scaffold comprised in said drug molecule. In another preferred embodiment, the drug molecule comprises an alternative scaffold, such as for example an ankyrin repeat domain, and the binding moiety is an anti-idiotype alternative scaffold having binding specificity for said alternative scaffold comprised in said drug molecule, such as for example an ankyrin repeat domain.

The term "nucleic acid" or "nucleic acid molecule" refers to a polynucleotide molecule, which may be a single-or double-stranded ribonucleic acid (RNA) molecule or a deoxyribonucleic acid (DNA) molecule, and includes modified and artificial forms of DNA or RNA. The nucleic acid molecule may be present in isolated form or comprised in a recombinant nucleic acid molecule or vector.

The term "target" refers to a single molecule, such as a nucleic acid molecule, polypeptide or protein, carbohydrate or any other naturally occurring molecule, including any portion of such a single molecule, or a complex of two or more such molecules, or an entire cell or tissue sample, or any unnatural compound. Preferably, the target is a naturally occurring or non-natural polypeptide or protein, or a polypeptide or protein that contains a chemical modification (e.g., natural or non-natural phosphorylation, acetylation, or methylation). In some embodiments, the biological target is an immune cell, such as a T cell, a B cell, a Natural Killer (NK) cell, or another type of immune cell. In some other embodiments, the biological target is a tumor cell.

In the context of the present invention, the term "polypeptide" relates to a molecule consisting of a chain of a plurality (i.e. two or more) amino acids linked via peptide bonds. Preferably, the polypeptide consists of more than eight amino acids linked via peptide bonds. The term "polypeptide" also includes multiple chains of amino acids linked together by S-S bridges of cysteines. Polypeptides are well known to those skilled in the art.

Patent applications WO2002/020565 and Forrer et al, 2003 (Forrer, p., stumpp, m.t., binz, h.k., pluckthun, a.,2003.FEBS Letters 539,2-6) contain general descriptions of repeat protein features and repeat domain features, techniques and applications. The term "repeat protein" refers to a protein comprising one or more repeat domains. Preferably, the repeat protein comprises one, two, three, four, five or six repeat domains. In addition, the repeat protein may comprise additional non-repeat protein domains, polypeptide tags, and/or peptide linkers. The repeat domain may be a binding domain.

The term "repeat domain" refers to a protein domain comprising two or more consecutive repeat modules as structural units, wherein the repeat modules have structural and sequence homology. Preferably, the repeat domain further comprises an N-terminal and/or C-terminal capping module. For clarity, the end-capping module may be a repeating module. Such repeat domains, repeat modules and end-capping modules, sequence motifs and structural and sequence homologies are well known to practitioners in the art from the following examples: ankyrin repeat domains (Binz et al, J.mol. Biol.332,489-503,2003; binz et al, nature Biotech.22 (5): 575-582 (2004); WO2002/020565; WO 2012/069655), leucine rich repeat domains (WO 2002/020565), triangular tetrapeptide repeat domains (Main, E.R., xiong, Y., cocco, M.J., D' Andrea, L., regan, L., structure 11 (5), 497-508, 2003) and so repeat domains (WO 2009/040338). It is also well known to those skilled in the art that such repeat domains differ from proteins comprising repeat amino acid sequences, wherein each of the repeat amino acid sequences is capable of forming a single domain (e.g., the FN3 domain of fibronectin).

The term "ankyrin repeat domain" refers to a repeat domain comprising two or more consecutive ankyrin repeat modules as structural units. The ankyrin repeat domains may be assembled modularly into larger ankyrin repeat proteins, optionally with half-life extending domains, using standard recombinant DNA techniques (see e.g. Forrer, p. Et al, FEBS letters, volume 539, pages 2-6, 2003, WO2002/020565, WO2016/156596, WO 2018/054971).

As used in the context of engineered ankyrin repeat proteins and engineered repeat domains, etc., the term "engineered" refers to the property of such repeat proteins and repeat domains, respectively, being artificial and not present in nature. The engineered repeat proteins described herein comprise at least one engineered repeat domain. Preferably, the designed repeat domain is a designed ankyrin repeat domain.

The term "target interaction residue" refers to an amino acid residue of a binding moiety that facilitates direct interaction with a drug molecule. For example, if the binding moiety is a designed ankyrin repeat domain, the term "target interaction residue" refers to an amino acid residue of the designed ankyrin repeat domain that facilitates direct interaction with a drug molecule.

The term "framework residue" or "framework position" refers to an amino acid residue of a repeat module that contributes to the folding topology, i.e., contributes to the folding of the repeat module or contributes to the interaction with an adjacent module. Such contributions may be interactions with other residues in the repeat module, or effects on the conformation of the polypeptide backbone as present in the alpha-helix or beta-sheet, or amino acid extensions involved in the formation of linear polypeptides or loops.

Such framework and target interaction residues can be identified by analysis of structural data obtained by physicochemical methods such as X-ray crystallography, NMR and/or CD spectroscopy, or by comparison with known and relevant structural information well known to practitioners in the field of structural biology and/or bioinformatics.

The term "repeat module" refers to the repeat amino acid sequence and structural units of a designed repeat domain that originally originated from the repeat unit of a naturally occurring repeat protein. Each repeat module comprised in the repeat domain is derived from one or more repeat units of a naturally occurring family or subfamily of repeat proteins, preferably the ankyrin family of repeat proteins. Furthermore, each repeat module comprised in a repeat domain may comprise a "repeat sequence motif" derived from a homologous repeat module obtained from a repeat domain selected on the target and having the same target specificity.

Thus, the term "ankyrin repeat module" refers to a repeat module that is originally derived from a repeat unit of a naturally occurring ankyrin repeat protein. Ankyrin repeat proteins are well known to those skilled in the art. Designed ankyrin repeat proteins have been previously described; see, for example, international patent publication nos. WO2002/020565, WO2010/060748, WO2011/135067, WO2012/069654, WO2012/069655, WO2014/001442, WO2014/191574, WO2014/083208, WO2016/156596, and WO2018/054971, all of which are incorporated by reference in their entirety. Typically, an ankyrin repeat module comprises about 31 to 33 amino acid residues that form two alpha helices separated by a loop.

The repeat module may comprise a position having an amino acid residue that has not been randomized in the library for selection of a target-specific repeat domain ("non-randomized position" or "fixed position" are used interchangeably herein) and a position having an amino acid residue that has been randomized in the library for selection of a target-specific repeat domain ("randomized position"). The non-randomized positions comprise framework residues. The randomized positions comprise target interaction residues. By "randomized" is meant that two or more amino acids are allowed at the amino acid positions of the repeat module, e.g., wherein any of the typically twenty naturally occurring amino acids are allowed, or wherein a majority of the twenty naturally occurring amino acids are allowed, such as amino acids other than cysteine, or amino acids other than glycine, cysteine, and proline.

The term "repeat motif" refers to an amino acid sequence derived from one or more repeat modules. Preferably, the repeat module is from a repeat domain having binding specificity for the same target. Such repeat sequence motifs comprise framework residue positions and target interaction residue positions. The framework residue positions correspond to framework residue positions of the repeat module. Likewise, the target interaction residue position corresponds to the position of the target interaction residue of the repeat module. The repeat motif comprises non-randomized positions and randomized positions.

The term "repeat unit" refers to an amino acid sequence comprising the sequence motif of one or more naturally occurring proteins, wherein the "repeat unit" is present in multiple copies and exhibits a defined folding topology that is common to all of the motifs that determine protein folding. Examples of such repeating units include leucine-rich repeating units, ankyrin repeating units, armadillo repeating units, thirty-tetrapeptide repeating units, HEAT repeating units, and leucine-rich variant repeating units.

A binding moiety "specifically binds" or "preferentially binds" (used interchangeably herein) a particular drug molecule if the binding moiety reacts or associates more frequently, more rapidly, longer in duration, with higher affinity, and/or with higher avidity than with an alternative target (e.g., a cell or substance). By comparing binding to the appropriate drug molecule with binding to the alternative drug molecule under a given set of conditions, the binding specificity of the binding moiety can be tested. In some embodiments, a binding molecule is considered specific if it binds to an appropriate drug molecule with an affinity that is at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, or at least 1000-fold higher than the affinity to bind to the alternative drug molecule. It will also be appreciated by reading this definition that, for example, a binding moiety that specifically or preferentially binds to a first drug molecule may or may not specifically or preferentially bind to a second drug molecule. Thus, "specific binding" does not necessarily require (although it may include) exclusive binding. Generally, the anchor binding moiety preferentially binds to a particular drug molecule and does not bind in significant amounts to other components present in the test sample under the indicated assay conditions.

The binding of any molecule to another molecule is controlled by two forces, the binding rate (k _on ) Dissociation rate (k) _off ). Any combination [ B ]]Targets [ T ]]The affinity of (2) can be determined by the equilibrium dissociation constant K _D Is represented as k _off /k _on Is a quotient of (2).

k _on Is the second order rate constant of the binding reaction, in M ^-1 s ^-1 Whereas dissociation reaction k _off Is a first order rate constant with the unit of s ^-1 . It is clear from this that the association reaction depends on the concentration of the reactants, whereas the dissociation is independent of the concentration, following a simple exponential decay function. Thus, the formation of prodrug complexes is affected by k _on And k _off And (5) controlling.

The half-life of the prodrug complex is referred to herein as the "blocking half-life" or "blocking T _1/2 ", because the prodrug complex (or binding portion thereof)" blocks "or" inhibits "the mode of action of the drug. Blocking T _1/2 Can be calculated according to the following formula:

blockingWhere "ln (2)" is the natural logarithm of the number 2 (natural logarithm), approximately 0.693. It can be similarly said that the binding moiety has a blocking half-life (or blocks T) when complexed with a drug molecule in the compositions of the invention _1/2 ). A variety of assay formats may be used to select or characterize binding moieties that specifically bind to a drug molecule of interest. For example, solid phase ELISA immunoassays, immunoprecipitation, BIAcore ^TM (GE Healthcare, piscataway, NJ), fluorescence Activated Cell Sorting (FACS), octet ^TM (ForteBio Inc., menlo Park, calif.) and Western blot analysis are a number of assays that can be used to identify binding moieties that specifically bind to target drug molecules. Typically, the specific or selective binding will be at least twice the background signal or noise, and more typically more than 10 times the background signal. Even particularly when the equilibrium dissociation constant (K _D ) Value of<1 mu m, such as<1μΜ、<100nM、<10nM、<1nM、<100pM、<10pM or<At 1pM, the binding moiety is considered to "specifically bind" to the target.

Various methods of measuring binding affinity are known in the art, any of which may be used for the purposes of the present invention. For example, as exemplified herein, the binding affinity of a particular binding moiety for a drug molecular target can be expressed as K _D Values, which refer to dissociation constants of the binding moiety and the drug molecular target. K (K) _D Is the dissociation rate (also called "dissociation rate (k) _off ) ") with association rate or" binding rate (k) _on ) "ratio. Thus, K is _D Equal to k _off /k _on And is expressed as molar concentration (M), and K _D The smaller the binding affinity the stronger.

K _D The value may be determined using any suitable method. For measuring K _D One exemplary method of (a) is surface plasmon Daughter resonance (SPR) (see, e.g., nguyen et al, sensors (Basel), 5.5, 5; 15 (5): 10481-510). K (K) _D The values may be used by SPR using a biosensor system such asThe system measures. BIAcore kinetic analysis involves, for example, analysis of binding and dissociation of antigens to a chip having immobilized molecules (e.g., molecules comprising epitope binding domains) on its surface. K for determining protein _D Another method of (a) is by using biological layer interferometry (see, e.g., shah et al, J Vis Exp.2014 (84): 51383). K (K) _D The value can be used +.>Technology (Octet QKe system, forteBio) measurements. Alternatively or additionally, +.>(kinetic exclusion assay) assay, purchased from Sapidyne Instruments (Boise, supra). Any method suitable for assessing the binding affinity between two binding partners is contemplated herein. Surface Plasmon Resonance (SPR) is particularly preferred. Most preferably, K is determined in PBS and by SPR _D Values.

The term "PBS" means a phosphate buffered saline solution containing 137mM NaCl, 10mM phosphate, and 2.7mM KCl and having a pH of 7.4.

As used herein, the term "substantially" means at least 95%. Thus, as used herein, the phrase "substantially all of the drug molecules are bound by the binding moiety" and the like means that at least 95% of all of the drug molecules are bound by the binding moiety. In some embodiments, "substantially" means at least 96%, or at least 97%, or at least 98%, or at least 99%. In some embodiments, "substantially" means at least 99.9%.

The term "treating" and words related thereto do not necessarily imply 100% or complete cure. Rather, there are treatments that one of ordinary skill in the art would consider to have potential beneficial effects or varying degrees of therapeutic effects. In this regard, the methods of treatment and medical uses described herein may provide any amount or level of treatment. Further, the treatment provided by the methods of the present disclosure may include treating (i.e., alleviating) one or more disorders or symptoms. In an exemplary aspect, the invention provides a method of treatment with a drug molecule, the method comprising administering a prodrug complex to a patient, wherein the adverse effect or risk thereof experienced by the patient administered the prodrug complex is reduced compared to the adverse effect or risk thereof that would be experienced by the patient if the same amount of the drug molecule were administered without the prodrug complex (i.e., in the form of a complex having a binding moiety of the invention). Thus, the use of the binding moieties of the invention to form prodrug complexes allows therapeutic methods with reduced adverse effects or reduced risk thereof, and/or therapeutic methods using higher doses of drug molecules or administering drug molecules within a shortened period of time.

The therapeutic response in any given disease or disorder can be determined by standardized response criteria specific to that disease or disorder. Subjects undergoing therapy may experience an improvement in symptoms associated with the disease, or a beneficial effect of reduced risk of adverse effects associated with administration of the therapeutic agent.

As used herein, the term "proliferative disease" refers to a disease characterized by overproduction of cells. Examples of proliferative diseases include, but are not limited to, cancer, atherosclerosis, rheumatoid arthritis, psoriasis, idiopathic pulmonary fibrosis, scleroderma, and cirrhosis. In a preferred embodiment, the proliferative disease is cancer.

Binding portion

The binding moieties described herein are polypeptides or proteins having a variety of different structures that can bind to a drug molecule with high affinity. Examples of binding moieties for use in the present invention include antibodies, alternative scaffolds, and polypeptides.

Antibodies include any polypeptide or protein comprising an antigen binding domain derived from an antibody or immunoglobulin molecule. The antigen binding domains may be derived from, for example, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and single domain antibodies, for example, heavy chain variable domains (VH), light chain variable domains (VL), and variable domains (VHH) from, for example, human or camelid sources. In some cases, it is advantageous that the antigen binding domain is derived from the same species in which the binding moiety will ultimately be used. For example, for use in humans, it may be beneficial for the antigen binding domains of the binding moieties described herein to comprise human or humanized antigen binding domains. Antibodies can be obtained using techniques well known in the art.

In one embodiment, the binding moiety is a camelid nanobody. Camelid nanobodies (also known as camelid single domain antibodies or VHHs) are derived from the camelid family of mammals, such as llamas, camels and alpacas. Unlike other antibodies, camelid antibodies lack light chains and consist of two identical heavy chains. Camelid antibodies typically have a relatively low molecular weight of about 15 kDa.

In one embodiment, the binding moiety is a shark antibody domain. Shark antibody domains, such as camelid nanobodies, also lack light chains.

Alternative scaffolds include any polypeptide or protein comprising a binding domain that is capable of binding an antigen (such as a drug molecule) and is not derived from an antibody or immunoglobulin molecule. The binding domain of the alternative scaffold may comprise or be derivable from a variety of different polypeptide or protein structures. Alternative scaffolds include, but are not limited to, adestin (monoclonal antibodies), affibodies, aphorins, alzheimer's and aptamers, alfa, alpha antibodies, anti-caligenes, armadine-based scaffolds, alfa, avermer, ankyrin-based scaffolds (such as Proteins), fenomo, knotting element, and kunitz domain peptides. Alternative stents are described in the following: for example Yu et al Annu Rev Anal Chem (Palo Alto Calif.) 2017, month 6, 12; 10 (1) 293-320. Doi:10.1146/annurevacchem-061516-045205.

Adinatine was originally derived from the tenth extracellular domain of human fibronectin type III protein (10 Fn 3). The fibronectin type III domain has 7 or 8 β -strands distributed between two β -sheets that stack upon themselves to form the core of the protein, and also contains loops (similar to CDRs) that link the β -strands to each other and are exposed to solvents. At least three such loops are present at each edge of the beta sheet sandwich, where an edge is the boundary of the protein perpendicular to the beta chain direction (see U.S. patent No. 6,818,418). Because of this structure, this non-antibody scaffold mimics antigen binding properties that are similar in nature and affinity to those of antibodies. These scaffolds can be used in vitro loop randomization and shuffling strategies, which are similar to the affinity maturation process of antibodies in vivo.

The affibody affinity ligand consists of a triple helix bundle based on a scaffold of one of the IgG binding domains of protein a, a surface protein from the bacterium staphylococcus aureus (Staphylococcus aureus). The scaffold domain consists of 58 amino acids, 13 of which are randomized to generate an affinity library with a large number of ligand variants (see, e.g., U.S. patent No. 5,831,012). The affibody molecule mimics an antibody, but is quite small, having a molecular weight of about 6kDa, compared to the molecular weight of about 150kDa of the antibody. The binding sites of the affibody molecules are similar to those of antibodies, although of different sizes.

Kefir is a synthetic antibody mimetic structurally derived from human ubiquitin (historically also derived from gamma-B crystallin). The agafilm consists of two identical domains, mainly with beta-sheet structure, with a total molecular mass of about 20kDa. They contain several surface exposed amino acids suitable for modification. Kefir is similar to antibodies in terms of its affinity and specificity for antigens, but is structurally dissimilar.

Alzheimer's disease is a peptide aptamer with a structure known as SQT (cystatin A quadruple mutant-Tracy). Aptamers and alzheimer's are short peptides responsible for affinity binding to inert and rigid protein scaffolds for structural constraints, where both the N-terminus and the C-terminus of the binding peptide are embedded in the inert scaffold.

The acitretin is a variant of the DNA binding protein Sac7d, which is engineered to obtain specific binding affinity. Sac7d was originally derived from the archaebacteria hyperthermophiles sulfolobus acidocaldarius (Sulfolobus acidocaldarius) and bound to DNA to prevent its thermal denaturation. The alfa is commercially known as nano-feitin (Nanofitin).

Alpha antibodies are small (about 10 kDa) proteins that are engineered to bind a variety of antigens and are therefore antibody mimics. Alpha antibody scaffolds were designed based on coiled coil structure calculations. Standard alpha antibody scaffolds contain three alpha-helices, each consisting of four heptad repeats (7 residue fragments), connected by glycine/serine rich linkers. The standard heptapeptide sequence is "IAAIQKQ". The ability of alpha antibodies to target extracellular and intracellular proteins and their high binding affinity may allow them to bind targets that antibodies cannot reach.

Anti-cargo proteins are a group of binding proteins with a robust and conserved β -barrel structure found in lipocalins. Lipocalins are a class of extracellular proteins comprising a peptide chain (150-190 amino acids) that are responsible for the recognition, storage and transport of various biomolecules, such as signaling molecules.

Armadia-based, repeated protein scaffolds are abundant in eukaryotes and are involved in a wide range of biological processes, particularly those associated with nuclear transport. A scaffold based on armadine, repeated proteins is generally composed of three to five internal repeats and two end-capping elements. They also have tandem elongated supercoiled structures that enable binding to their corresponding peptide ligands in an extended conformation.

Alzheimer's disease is a scaffold derived from trimeric plasma proteins called tetranectins, which belong to the family of C-type lectins consisting of three identical units. The structure of the C-type lectin domain (CTLD) within tetranectin has five flexible loops that mediate interactions with targeting molecules.

Avermectin is derived from a protein containing a native a domain, such as HER3, and consists of many different "a domain" monomers (2-10) linked by amino acid linkers. The methods described in, for example, U.S. patent application publication nos. 2004/0175756, 2005/0053973, 2005/0048512, and 2006/0008844 can be used to produce avermectin that can bind to a target antigen.

In one embodiment, the binding moiety is an ankyrin repeat protein. Engineered ankyrin repeat proteins (such asProteins) can function like antibody mimetic proteins, typically exhibiting highly specific and high affinity target binding. The engineered ankyrin repeat protein comprises one or more engineered ankyrin repeat domains. The engineered ankyrin repeat domains are derived from natural ankyrin repeat proteins, and each engineered ankyrin repeat domain typically binds to a target protein with high specificity and affinity. Due to their high specificity, stability, potency and affinity, and due to their flexibility in forming monospecific, bispecific or multispecific proteins, engineered ankyrin repeat proteins are particularly suitable for use as high affinity binding moieties. Designed ankyrin repeat protein drug candidates also exhibit advantageous development properties including rapid, low cost and high yield manufacturing and shelf life of up to several years at 4 ℃. Designed ankyrin repeat proteins are preferred embodiments of the binding moiety of the invention. />Is a registered trademark owned by Molecular Partners AG.

Fenomo is a small globular protein (approximately 7 kDa) evolved from amino acids 83-145 of Src homology domain 3 (SH 3) of human Fyn tyrosine kinase. Fenomo are attractive binding molecules due to their high thermal stability, cysteine-free scaffold and human origin, which reduces potential immunogenicity.

Knottins, also known as cysteine knot miniproteins, are typically proteins 30 amino acids in length, which contain three antiparallel β -sheets and a restriction ring tethered by a disulfide bond that creates a cysteine knot. This disulfide bond imparts high thermostability, making knottin an attractive antibody mimetic.

Kunitz domain peptides or kunitz domain inhibitors are a class of protease inhibitors having an irregular secondary structure containing about 60 amino acids with three disulfide bonds and three loops that can be mutated without destabilizing the structural framework.

In one embodiment, the binding moiety is a polypeptide or protein comprising an antigen binding domain derived from a T Cell Receptor (TCR).

Examples of binding portions

Examples of ankyrin repeat domains for use in the present invention are provided by SEQ ID NOS: 1 to 10 (see examples).

The ankyrin repeat domains of SEQ ID NOS 1 to 10 bind specifically with high affinity to a CD3 specific binding molecule having an amino acid sequence selected from SEQ ID NOS 12 to 15. For example, the ankyrin repeat domains of SEQ ID NOS.1 to 10 bind specifically with high affinity to a CD3 specific binding molecule having the amino acid sequence of SEQ ID NO. 13 or the amino acid sequence of SEQ ID NO. 14. In one embodiment, the ankyrin repeat domains of SEQ ID NOS.1 to 10 specifically bind to a CD3 specific binding molecule having the amino acid sequence of SEQ ID NO. 13. In another embodiment, the ankyrin repeat domains of SEQ ID NOS: 1 to 10 specifically bind to a CD3 specific binding molecule having the amino acid sequence of SEQ ID NO: 14. A CD3 specific binding molecule having an amino acid sequence selected from SEQ ID NOs 12 to 15 may be genetically fused with another binding molecule having specificity for a Tumor Associated Antigen (TAA) to form a T cell cement (TCE) drug molecule. Binding of the ankyrin repeat domain of any one of SEQ ID NOs 1 to 10 to such a T-cell cement drug molecule inhibits binding of the T-cell cement drug molecule to CD3 and thus inhibits the biological activity of the T-cell cement drug molecule.

Thus, in one embodiment, the binding moiety of the invention is an ankyrin repeat domain comprising an amino acid sequence having at least about 85% sequence identity to an ankyrin repeat domain selected from the group consisting of SEQ ID NOs 1 to 10.

In one embodiment, the binding moiety is an ankyrin repeat domain comprising an amino acid sequence having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to an ankyrin repeat domain selected from the group consisting of SEQ ID NOs 1 to 10.

In one embodiment, the binding moiety is an ankyrin repeat domain, wherein said ankyrin repeat domain is selected from the group consisting of SEQ ID NOs 1 to 10.

In one embodiment, the binding moiety is a designed ankyrin repeat domain comprising an amino acid sequence selected from the group consisting of: (1) 1 to 10, and (2) a sequence having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to any one of SEQ ID NOs 1 to 10.

In one embodiment, the binding moiety is a designed ankyrin repeat protein comprising an amino acid sequence selected from the group consisting of seq id no: (1) 30 to 51, and (2) a sequence in which up to 9 amino acids in any one of SEQ ID NOs 30 to 51 are substituted with another amino acid. In one embodiment, the binding moiety is a engineered ankyrin repeat protein comprising an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of seq id no: (1) 30 to 51, and (2) a sequence in which up to 8 amino acids in any one of SEQ ID NOs 30 to 51 are substituted with another amino acid. In one embodiment, the binding moiety is a engineered ankyrin repeat protein comprising an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of seq id no: (1) 30 to 51, and (2) a sequence in which up to 7 amino acids in any one of SEQ ID NOs 30 to 51 are substituted with another amino acid. In one embodiment, the binding moiety is a designed ankyrin repeat protein comprising an amino acid sequence selected from the group consisting of seq id no: (1) 30 to 51, and (2) a sequence in which up to 6 amino acids in any one of SEQ ID NOs 30 to 51 are substituted with another amino acid. In one embodiment, the binding moiety is a engineered ankyrin repeat protein comprising an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of seq id no: (1) 30 to 51, and (2) a sequence in which up to 5 amino acids in any one of SEQ ID NOs 30 to 51 are substituted with another amino acid. In one embodiment, the binding moiety is a engineered ankyrin repeat protein comprising an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of seq id no: (1) 30 to 51, and (2) a sequence in which up to 4 amino acids in any one of SEQ ID NOs 30 to 51 are substituted with another amino acid. In one embodiment, the binding moiety is a engineered ankyrin repeat protein comprising an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of seq id no: (1) 30 to 51, and (2) a sequence in which up to 3 amino acids in any one of SEQ ID NOs 30 to 51 are substituted with another amino acid. In one embodiment, the binding moiety is a engineered ankyrin repeat protein comprising an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of seq id no: (1) 30 to 51, and (2) a sequence in which up to 2 amino acids in any one of SEQ ID NOs 30 to 51 are substituted with another amino acid. In one embodiment, the binding moiety is a engineered ankyrin repeat protein comprising an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of seq id no: (1) 30 to 51, and (2) a sequence in which at most 1 amino acid in any one of SEQ ID NO 30 to 51 is substituted with another amino acid.

In all binding moieties described herein, the amino acid sequence of the binding moiety may be substituted with one or more amino acids. In some embodiments, up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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 substitutions are made in any of the binding moieties described herein.

In some embodiments, up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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 substitutions are made in any ankyrin repeat domain relative to any of the sequences of SEQ ID NOs 1 to 10. In some embodiments, up to 15 substitutions are made relative to any of the sequences of SEQ ID NOs 1 to 10. In some embodiments, up to 14 substitutions are made relative to any of the sequences of SEQ ID NOs 1 to 10. In some embodiments, up to 13 substitutions are made relative to any of the sequences of SEQ ID NOs 1 to 10. In some embodiments, up to 12 substitutions are made relative to any of the sequences of SEQ ID NOs 1 to 10. In some embodiments, up to 11 substitutions are made relative to any of the sequences of SEQ ID NOs 1 to 10. In some embodiments, up to 10 substitutions are made relative to any of the sequences of SEQ ID NOs 1 to 10. In some embodiments, up to 9 substitutions are made relative to any of the sequences of SEQ ID NOs 1 to 10. In some embodiments, up to 8 substitutions are made relative to any of the sequences of SEQ ID NOs 1 to 10. In some embodiments, up to 7 substitutions are made relative to any of the sequences of SEQ ID NOs 1 to 10. In some embodiments, up to 6 substitutions are made relative to any of the sequences of SEQ ID NOs 1 to 10. In some embodiments, up to 5 substitutions are made relative to any of the sequences of SEQ ID NOs 1 to 10. In some embodiments, up to 4 substitutions are made relative to any of the sequences of SEQ ID NOs 1 to 10. In some embodiments, up to 3 substitutions are made relative to any of the sequences of SEQ ID NOs 1 to 10. In some embodiments, up to 2 substitutions are made relative to any of the sequences of SEQ ID NOs 1 to 10. In some embodiments, up to 1 substitution is made with respect to any of the sequences of SEQ ID NOs 1 to 10.

In some embodiments, amino acid substitutions are all made at framework positions. In some embodiments, amino acid substitutions are all made at non-random positions. The localization of random positions in the designed ankyrin repeat domain is disclosed, for example, in Binz et al, nature Biotech.22 (5): 575-582 (2004).

In some embodiments, K is associated with an unobtainable binding moiety _D Amino acid substitutions result in K compared to the values _D The value changes by no more than about 1000 times, no more than about 100 times, or no more than about 10 times. For example, in some embodiments, the binding moiety to any one of the sequences comprising SEQ ID NOS: 1 to 10 binds K to a drug molecule comprising any one of the sequences comprising SEQ ID NOS: 12 to 15 _D Amino acid substitutions result in K compared to the values _D The value changes by no more than about 1000-fold, no more than about 300-fold, no more than about 100-fold, no more than about 50-fold, no more than about 25-fold, no more than about 10-fold, or no more than about 5-fold.

In certain embodiments, the amino acid substitutions in the binding moiety are conservative substitutions according to table 1 below.

TABLE 1 amino acid substitutions

When the binding moiety is an ankyrin repeat domain, in some embodiments, substitution may be made outside of the structural core residues of the ankyrin repeat domain, for example in the β loop joining the α -helices. In other embodiments, substitutions may be made within the structural core residues of the ankyrin repeat domain. For example, the ankyrin domain may comprise a consensus sequence: xDxGxTPLHxxxGxxxLVxVLLxxGADVNA (SEQ ID NO: 52), wherein "x" represents any amino acid (preferably not cysteine, glycine or proline); or xxxGxTPLHLAxGHLEIVLLKzGADVNA (SEQ ID NO: 53), wherein "x" represents any amino acid (preferably not cysteine, glycine or proline) and "z" is selected from the group consisting of asparagine, histidine or tyrosine. In one embodiment, a residue designated "x" is substituted. In another embodiment, the substitution is made outside of the residue designated "x".

In addition, the penultimate position of any ankyrin repeat domain of the binding moiety may be "a" or "L", and/or the last position may be "a" or "N". Thus, in some embodiments, the ankyrin repeat domain of the binding moiety comprises an amino acid sequence that is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to any one of SEQ ID NOs 1 to 10, and wherein optionally a at the penultimate position is substituted with L, and/or a at the last position is substituted with N. In an exemplary embodiment, the ankyrin repeat domain of the binding moiety comprises an amino acid sequence at least about 90% identical to any one of SEQ ID NOs 1 to 10, and wherein optionally a at the penultimate position is substituted with L and/or a at the last position is substituted with N. Furthermore, the sequence of any ankyrin repeat domain of the binding moiety may optionally comprise G, S or GS (see below) at its N-terminus.

Furthermore, each ankyrin repeat domain of the binding moiety may optionally comprise a "G", "S" or "GS" sequence at its N-terminus. Thus, in some embodiments, the ankyrin repeat domain of the binding moiety comprises an amino acid sequence that is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to any one of SEQ ID NOs 1 to 10, and further optionally comprises G, S or GS at its N-terminus. In an exemplary embodiment, the ankyrin repeat domain of the binding moiety comprises an amino acid sequence at least about 90% identical to any one of SEQ ID NOs 1 to 10, and wherein said ankyrin repeat domain further optionally comprises G, S or GS at its N-terminus. Furthermore, the sequence of any ankyrin repeat domain of the binding moiety may optionally have an a substituted with L in the penultimate position and/or an a substituted with N in the last position (see above).

N-terminal and C-terminal capping sequences

When the binding moiety described herein comprises an ankyrin repeat domain, the ankyrin repeat domain may comprise an N-terminal or C-terminal capping sequence. A capping sequence refers to an additional polypeptide sequence fused to the N-terminal or C-terminal end of an ankyrin repeat motif or module, wherein the capping sequence forms a tight tertiary interaction (i.e., tertiary structural interaction) with an adjacent ankyrin repeat motif or module of an ankyrin repeat domain, thereby providing a cover protecting the hydrophobic core of one side of the ankyrin repeat domain from exposure to solvent.

The N-terminal and/or C-terminal capping sequences may be derived from capping units or other structural units present in naturally occurring repeat proteins adjacent to the repeat unit. Examples of capping sequences are described in the following: international patent publication nos. WO2002/020565 and WO2012/069655, U.S. patent publication No. US2013/0296221, and inter et al, J Mol biol.2008, 1 month, 18 days; 375 (3):837-54. Examples of N-terminal ankyrin capping modules (i.e., N-terminal capping repeats) are SEQ ID NOS: 21 to 23, and examples of C-terminal capping modules (i.e., C-terminal capping repeats) include SEQ ID NO:28.

Drug molecules

In the context of the present invention, a drug molecule is a therapeutic agent comprising a polypeptide or protein, wherein the polypeptide or protein contains a site capable of being bound by a binding moiety. The drug molecules used in the present invention are not particularly limited as long as they can be bound by the binding moiety. This means, for example, that the drug molecule may belong to the same "class" as the binding moiety or to a different "class" than the binding moiety, such that, for example, both the drug molecule and the binding moiety may be antibodies, or both the drug molecule and the binding moiety may be alternative scaffolds (e.g., ankyrin repeat proteins), or the drug molecule may be antibodies and the binding moiety may be alternative scaffolds (e.g., ankyrin repeat proteins), or the drug molecule may be alternative scaffolds (e.g., ankyrin repeat proteins) and the binding moiety may be antibodies. This further means that, for example, the drug molecule itself may comprise different structural parts, e.g. a combination of an antibody part and an alternative scaffold part, or a combination of parts of different alternative scaffold structures. Where both the drug molecule and the binding moiety are antibodies, it will be clear to one skilled in the art that antibodies will differ from one another, including with respect to binding specificity. Similarly, where both the drug molecule and the binding moiety are alternative scaffolds, it will be apparent to those skilled in the art that alternative scaffolds will differ from one another, including with respect to binding specificity.

In addition, the drug molecules used in the present invention may contain half-life extending moieties. The half-life extending moiety extends the serum half-life of the drug molecule in vivo, as compared to the same molecule without the half-life extending moiety. Examples of half-life extending 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). In some cases, the half-life extending moiety can comprise an ankyrin repeat domain having binding specificity for HSA. In other cases, the half-life extending moiety may comprise an immunoglobulin domain, such as an Fc domain, e.g., a human IgG ₁ Or a variant or derivative thereof.

In some embodiments, the drug molecules for use in the present invention comprise an alternative scaffold, wherein the alternative scaffold is selected from the group consisting of adestin (monoclonal antibody), affibodies, aphilin, alzheimer's and aptamer, aphilin, alpha antibody, anti-carrier, armadin-based scaffold, alzheimer's, avermectin, ankyrin-based scaffold (such as Proteins), fenomo, knotting element, and kunitz domain peptides. . Alternative stents are described in the following: for example Yu et al Annu Rev Anal Chem (Palo Alto Calif.) 2017, month 6, 12; 10 (1) 293-320. Doi:10.1146/annurevacchem-061516-045205.

Drug molecules for use in the present invention also include, but are not limited to, different classes of drugs currently approved for clinical use, such as:

(1) Immune Checkpoint Inhibitors (ICI);

(2) A bispecific antibody; and

(3) Genetically modified immune cells, such as T cells, in particular immune cells expressing Chimeric Antigen Receptors (CARs), such as CAR-T cells.

Drug molecules for use in the present invention also include, but are not limited to, drug molecules that up-regulate or down-regulate immune checkpoint activity, referred to herein as "immune checkpoint modulators". An immune checkpoint is a molecule in the immune system that increases (co-stimulatory molecules) or decreases (inhibitory molecules) immune signals. In cancer patients, tumors can use these immune checkpoints to protect themselves from immune system attacks, particularly T cell attacks. Drug molecules for immune checkpoint therapy may block inhibitory immune checkpoint molecules or activate stimulatory immune checkpoint molecules, thereby restoring immune system function. In recent years, immune checkpoint drugs have become an important new cancer treatment option. Immune checkpoint molecules include, but are not limited to, CD27, CD137L, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, OX40L, DNAM-1, PD-L1, PD-L2, CTLA-4, CD8, CD40, CEACAMI, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, C5AR1, CCR8, CD226, CD28, CD33, CD38, CD3e, CD47, CD94, ETAR, NKG2A, SIRP α, TLR8, TNFRSF18, IDO1, IDO2, TDO, KIR, LAG-3, TIM-4, and VISTA. In one embodiment, the drug molecule is an immune checkpoint modulator.

In one embodiment, the pharmaceutical molecules used in the present invention comprise antibodies. In another embodiment, the drug molecule comprises a bispecific antibody. In another embodiment, the drug molecule comprises a multispecific antibody. In another embodiment, the drug molecule comprises an antibody that is a T cell cement drug molecule (TCE).

Bispecific antibodies include TCE. An example of a bispecific antibody as TCE is what is known as BiTe ^TM Molecules of the molecule. These are anticancer drugs consisting of two single-chain variable fragments (scFvs) on a single peptide chain. This TCE binds to CD3 (cluster of differentiation 3) molecules on the surface of T cells via one of scFvs, while the other scFv binds to a Tumor Associated Antigen (TAA) on the surface of tumor cells. By binding to CD3 and linking T cells to tumor cells, T cells are "activated" and can exert cytotoxic activity on tumor cells. One example of a bispecific antibody as TCE is bolafirumab (blinatumomab), which binds to CD3 on the surface of T cells and CD19 on the surface of B cells. The bordetention is approved for the treatment of acute lymphoblastic leukemia.

In one embodiment, the drug molecules used in the present invention comprise an alternative scaffold. In another embodiment, the drug molecule comprises a bispecific alternative scaffold. In another embodiment, the drug molecule comprises a multispecific alternative scaffold. In another embodiment, the drug molecule comprises an alternative scaffold molecule that is a T cell cement drug molecule (TCE). In a preferred embodiment, the alternative scaffold is an ankyrin repeat domain. In a further preferred embodiment, the drug molecule for use in the present invention comprises an alternative scaffold, wherein the alternative scaffold is an ankyrin repeat domain having binding specificity for CD3, and wherein the ankyrin repeat domain comprises an amino acid sequence selected from SEQ ID NOs 12 to 15. In a further preferred embodiment, the drug molecule comprises an ankyrin repeat domain having binding specificity for CD3, wherein said ankyrin repeat domain comprises an amino acid sequence selected from SEQ ID NOs 12 to 15. In one embodiment, the drug molecule comprises an ankyrin repeat domain having binding specificity for CD3, wherein said ankyrin repeat domain comprises an amino acid sequence selected from SEQ ID NOs 13 to 14. In one embodiment, the drug molecule comprises an ankyrin repeat domain having binding specificity for CD3, wherein said ankyrin repeat domain comprises SEQ ID No. 13. In one embodiment, the drug molecule comprises an ankyrin repeat domain having binding specificity for CD3, wherein said ankyrin repeat domain comprises SEQ ID No. 14.

Bispecific or multispecific alternative scaffold molecules include TCE. In one embodiment, the drug molecule is a bispecific or multispecific alternative scaffold molecule, wherein the alternative scaffold molecule is a TCE comprising (i) a CD3 specific binding domain as an ankyrin repeat domain, and (ii) a TAA specific binding domain as an ankyrin repeat domain. Similar to that called BiTe ^TM TCE of the molecule, which comprises an alternative scaffold, is an anticancer drug that binds to CD3 molecules on the surface of T cells via one of the binding domains, while the other binding domain binds to a Tumor Associated Antigen (TAA) on the surface of tumor cells. By binding to CD3 and linking T cells to tumor cells, T cells are "activated" and can exert cytotoxic activity on tumor cells.

When the drug molecule is TCE, the preferred binding site of the binding moiety of the invention is the CD3 specific binding domain of TCE. By binding to the CD3 specific binding domain of TCE, the binding moiety blocks the mode of action of TCE by preventing TCE from binding to T cells. In one embodiment, the binding of the binding moiety to the CD3 specific binding domain of TCE is an anti-idiotype.

A number of bispecific antibodies have been described as TCEs, including those listed below, with their respective binding targets provided in brackets. For example, as described above, bonauzumab (CD 19xCD3; amgen) binds to the CD3 antigen on T cells and to the CD19 antigen on tumor cells produced by the B cell lineage. Other bispecific antibodies as TCEs include, but are not limited to AMG330 (CD 33xCD3; amgen); fu Tuozhu mab (flotatuzumab) (CD 123xCD3; macrogenetics); MCLA117 (Clec 12AXCD3; merus); AMG160 (HLE PSMAxCD3, amgen); AMG427 (HLE FLT3xCD3, amgen); AMG562 (HLE CD19xCD3, amgen); AMG596 (HLE EGFRvIIIxCD3, amgen); AMG673 (HLE CD33xCD3, amgen); AMG701 (HLE BCMAxCD3, amgen); AMG757 (HLE DLL3xCD3, amgen); AMG910 (HLE Claudin18.2xCD3, amgen); ornitumumab (CD 20xCD3, regeneron); mo Tuozhu mab (mosuteuzumab) (CD 20xCD3, roche); lattice Luo Feituo mab (glofithamab) (CD 20xCD3, roche); and elcatuzumab (epcoritamab) (CD 20xCD3, genmab). Any such TCE may be used as a pharmaceutical molecule in the compositions of the present invention.

In one embodiment, the drug molecules used in the present invention comprise antibodies and alternative scaffolds. In another embodiment, the drug molecule comprises two different alternative scaffolds. In another embodiment, the drug molecule comprises a T Cell Receptor (TCR) -derived antigen recognition domain.

In one embodiment, the drug molecules used in the present invention comprise genetically modified immune cells. In a preferred embodiment, the genetically modified immune cell expresses a Chimeric Antigen Receptor (CAR). In one embodiment, the genetically modified immune cell is a genetically modified T cell, such as a CAR-expressing T cell (CAR-T cell). In another embodiment, the genetically modified immune cell is a genetically modified Natural Killer (NK) cell, such as a CAR-expressing NK cell (CAR-NK cell).

Binding affinity

The binding moieties and drug molecules described herein bind non-covalently to form a prodrug complex. The binding of any molecule to another molecule is controlled by two forces, the binding rate (k _on ) Dissociation rate (k) _off ). Any combination [ B ]]Targets [ T ]]Affinity of (2) can be determined byEquilibrium dissociation constant K _D Is represented as k _off /k _on Is a quotient of (2).

In certain embodiments, the binding affinity of the binding moiety to the drug molecule is K _D Description. In an exemplary embodiment, K _D Is 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 ^-7 M to about 10 ^-15 M, about 10 ^-8 M to about 10 ^- ¹⁵ M, about 10 ^-9 M to about 10 ^-15 M, about 10 ^-10 M to about 10 ^-15 M, about 10 ^-11 M to about 10 ^-15 M, about 10 ^-12 M to about 10 ^-15 M, about 10 ^-7 M to about 10 ^-14 M, about 10 ^-8 M to about 10 ^-14 M, about 10 ^-9 M to about 10 ^-14 M, about 10 ^-10 M to about 10 ^-14 M, about 10 ^-11 M to about 10 ^-14 M, about 10 ^-12 M to about 10 ^-14 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, about 10 ^-10 M to about 10 ^-13 M, about 10 ^-11 M to about 10 ^-13 M or about 10 ^-12 M to about 10 ^-13 M。

In exemplary embodiments, the binding moiety is K below or less than _D Value binding drug molecules: about 100nM, about 50nM, about 25nM, about 10nM, about 5nM, about 2nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 100pM, about 50pM, about 25pM, about 10pM, about 5pM, about 2pM, about 1pM, about 500fM, about 250fM, about 100fM, about 50fM, about 25fM, about 10fM, about 5fM, about 2fM or about 1fM. In one exemplary embodiment, the binding moiety is smallK at or equal to about 1nM _D The values bind to the drug molecules. In another exemplary embodiment, the binding moiety is at a K of less than or equal to about 100pM _D The values bind to the drug molecules. In another exemplary embodiment, the binding moiety is at a K of less than or equal to about 10pM _D The values bind to the drug molecules. In another exemplary embodiment, the binding moiety is at a K of less than or equal to about 1pM _D The values bind to the drug molecules.

In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 1, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein the binding moiety is at K _D Values bind to the drug molecules: less than about 100nM, such as less than about 50nM, about 25nM, about 10nM, about 5nM, about 2nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 100pM, about 50pM, about 25pM, about 10pM, about 5pM, about 2pM, about 1pM, about 500fM, about 250fM, about 100fM, about 50fM, about 25fM, about 10fM, about 5fM, about 2fM, or about 1fM. In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 1, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein the binding moiety comprises a K in the range of about 1pM to about 100pM _D The value binds to the drug molecule.

In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 2, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein the binding moiety is at K _D Values bind to the drug molecules: less than about 100nM, such as less than about 50nM, about 25nM, about 10nM, about 5nM, about 2nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 100pM, about 50pM, about 25pM, about 10pM, about 5pM, about 2pM, about 1pM, about 500fM, about 250fM, about 100fM, about 50fM, about 25fM, about 10fM, about 5fM, about 2fM, or about 1fM. In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 2, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein the binding moiety comprises a K in the range of about 1pM to about 100pM _D The value binds to the drug molecule.

In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 3, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein the binding moiety is at K _D Values bind to the drug molecules: less than about 100nM, such as less than about 50nM, about 25nM, about 10nM, about 5nM, about 2nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 100pM, about 50pM, about 25pM, about 10pM, about 5pM, about 2pM, about 1pM, about 500fM, about 250fM, about 100fM, about 50fM, about 25fM, about 10fM, about 5fM, about 2fM, or about 1fM. In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 3, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein the binding moiety comprises a K in the range of about 1pM to about 100pM _D The value binds to the drug molecule.

In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID NO. 4, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NO. 12 to 15,wherein the binding moiety is at least K _D Values bind to the drug molecules: less than about 100nM, such as less than about 50nM, about 25nM, about 10nM, about 5nM, about 2nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 100pM, about 50pM, about 25pM, about 10pM, about 5pM, about 2pM, about 1pM, about 500fM, about 250fM, about 100fM, about 50fM, about 25fM, about 10fM, about 5fM, about 2fM, or about 1fM. In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 4, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein the binding moiety comprises a K in the range of about 1pM to about 100pM _D The value binds to the drug molecule.

In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 5, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein the binding moiety is at K _D Value binding the drug molecule binding: less than about 100nM, such as less than about 50nM, about 25nM, about 10nM, about 5nM, about 2nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 100pM, about 50pM, about 25pM, about 10pM, about 5pM, about 2pM, about 1pM, about 500fM, about 250fM, about 100fM, about 50fM, about 25fM, about 10fM, about 5fM, about 2fM, or about 1fM. In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 5, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein the binding moiety comprises a K in the range of about 1pM to about 100pM _D The value binds to the drug molecule.

In some embodiments, the binding moiety comprises a polypeptide having at least about 85% sequence identity to SEQ ID NO. 6An ankyrin repeat domain of the amino acid sequence of SEQ ID NO 12 to 15, and a pharmaceutical molecule comprising an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein said binding moiety is represented by K _D Values bind to the drug molecules: less than about 100nM, such as less than about 50nM, about 25nM, about 10nM, about 5nM, about 2nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 100pM, about 50pM, about 25pM, about 10pM, about 5pM, about 2pM, about 1pM, about 500fM, about 250fM, about 100fM, about 50fM, about 25fM, about 10fM, about 5fM, about 2fM, or about 1fM. In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 6, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein the binding moiety comprises a K in the range of about 1pM to about 100pM _D The value binds to the drug molecule.

In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 7, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein the binding moiety is at K _D Values bind to the drug molecules: less than about 100nM, such as less than about 50nM, about 25nM, about 10nM, about 5nM, about 2nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 100pM, about 50pM, about 25pM, about 10pM, about 5pM, about 2pM, about 1pM, about 500fM, about 250fM, about 100fM, about 50fM, about 25fM, about 10fM, about 5fM, about 2fM, or about 1fM. In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 7, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein the binding moiety is at about 1pM to about 100p K in M range _D The value binds to the drug molecule.

In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 8, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein the binding moiety is at K _D Values bind to the drug molecules: less than about 100nM, such as less than about 50nM, about 25nM, about 10nM, about 5nM, about 2nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 100pM, about 50pM, about 25pM, about 10pM, about 5pM, about 2pM, about 1pM, about 500fM, about 250fM, about 100fM, about 50fM, about 25fM, about 10fM, about 5fM, about 2fM, or about 1fM. In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 8, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein the binding moiety comprises a K in the range of about 1pM to about 100pM _D The value binds to the drug molecule.

In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 9, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein the binding moiety is at K _D Values bind to the drug molecules: less than about 100nM, such as less than about 50nM, about 25nM, about 10nM, about 5nM, about 2nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 100pM, about 50pM, about 25pM, about 10pM, about 5pM, about 2pM, about 1pM, about 500fM, about 250fM, about 100fM, about 50fM, about 25fM, about 10fM, about 5fM, about 2fM, or about 1fM. In some embodiments, the binding moiety comprises an ankyrin heavy having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 9A multiple domain, and a drug molecule comprising an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein said binding moiety is at a K in the range of about 1pM to about 100pM _D The value binds to the drug molecule.

In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 10, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein the binding moiety is at K _D Values bind to the drug molecules: less than about 100nM, such as less than about 50nM, about 25nM, about 10nM, about 5nM, about 2nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 100pM, about 50pM, about 25pM, about 10pM, about 5pM, about 2pM, about 1pM, about 500fM, about 250fM, about 100fM, about 50fM, about 25fM, about 10fM, about 5fM, about 2fM, or about 1fM. In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 10, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein the binding moiety comprises a K in the range of about 1pM to about 100pM _D The value binds to the drug molecule.

In an exemplary embodiment, the binding moiety is selected from the group consisting of K _off Value binding drug molecules: between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-4 s ^-1 Such as between about 1 x 10 ^-8 s ^-1 And about 1X 10 ^-7 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^- ⁶ s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-5 s ^-1 Between or about1×10 ^-7 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-4 s ^-1 Between, or between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-5 s ^-1 Between them.

In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 1, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein the binding moiety is represented by k _off Values bind to the drug molecules: between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-4 s ^-1 Such as between about 1 x 10 ^-8 s ^-1 And about 1X 10 ^-7 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-4 s ^-1 Between, or between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-5 s ^-1 Between them. In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 1, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein the binding moiety is present at about 1x 10 ^-7 s ^-1 And about 1x 10 ^-4 s ^-1 K between _off The value binds to the drug molecule.

In some embodiments, the binding moiety comprises a polypeptide having at least about 85% sequence identity to SEQ ID NO. 2An ankyrin repeat domain of an amino acid sequence, and a drug molecule comprising an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein said binding moiety binds said drug molecule with the following koff value: between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-4 s ^-1 Such as between about 1x 10 ^-8 s ^-1 And about 1X 10 ^-7 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^- ⁵ s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-4 s ^-1 Between, or between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-5 s ^-1 Between them. In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 2, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein the binding moiety is present in an amount of between about 1 x 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 The koff value between them binds the drug molecule.

In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 3, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein the binding moiety is represented by k _off Values bind to the drug molecules: between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-4 s ^-1 Such as between about 1 x 10 ^-8 s ^-1 And about 1X 10 ^-7 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-4 s ^-1 Between, or between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-5 s ^-1 Between them. In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 3, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein the binding moiety is present in an amount of between about 1 x 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 K between _off The value binds to the drug molecule.

In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 4, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein the binding moiety is represented by k _off Values bind to the drug molecules: between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-4 s ^-1 Such as between about 1 x 10 ^-8 s ^-1 And about 1X 10 ^-7 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-5 s ^-1 Between which,Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-4 s ^-1 Between, or between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-5 s ^-1 Between them. In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 4, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein the binding moiety is present at about 1x 10 ^-7 s ^-1 And about 1x 10 ^-4 s ^-1 K between _off The value binds to the drug molecule.

In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 5, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein the binding moiety is represented by k _off Values bind to the drug molecules: between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-4 s ^-1 Such as between about 1x 10 ^-8 s ^-1 And about 1X 10 ^-7 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-4 s ^-1 Between, or between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-5 s ^-1 Between them. In some embodiments, the binding moiety comprises an ankyrin having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 5A repeat domain and a drug molecule comprising an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein said binding moiety is present at about 1x 10 ^-7 s ^-1 And about 1x 10 ^-4 s ^-1 K between _off The value binds to the drug molecule.

In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 6, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein the binding moiety is represented by k _off Values bind to the drug molecules: between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-4 s ^-1 Such as between about 1x 10 ^-8 s ^-1 And about 1X 10 ^-7 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-4 s ^-1 Between, or between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-5 s ^-1 Between them. In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 6, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein the binding moiety is present at about 1x 10 ^-7 s ^-1 And about 1x 10 ^-4 s ^-1 K between _off The value binds to the drug molecule.

In some embodiments, the binding moiety comprises a polypeptide havingAn ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 7, and a pharmaceutical molecule comprising an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein the binding moiety is represented by k _off Values bind to the drug molecules: between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-4 s ^-1 Such as between about 1 x 10 ^-8 s ^-1 And about 1X 10 ^-7 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-4 s ^-1 Between, or between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-5 s ^-1 Between them. In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 7, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein said binding moiety is present in an amount of between about 1 x 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 K between _off The value binds to the drug molecule.

In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 8, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein the binding moiety is represented by k _off Values bind to the drug molecules: between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-4 s ^-1 Such as between about 1 x 10 ^-8 s ^-1 And about 1X 10 ^-7 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-4 s ^-1 Between, or between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-5 s ^-1 Between them. In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 8, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein the binding moiety is present in an amount of between about 1 x 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 K between _off The value binds to the drug molecule.

In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 9, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein the binding moiety is represented by k _off Values bind to the drug molecules: between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-4 s ^-1 Such as between about 1 x 10 ^-8 s ^-1 And about 1X 10 ^-7 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-4 s ^-1 Between, or between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-5 s ^-1 Between them. In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 9, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein said binding moiety is present in an amount of between about 1 x 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 K between _off The value binds to the drug molecule.

In some embodiments, the binding moiety comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 10, and the drug molecule comprises an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to any one of SEQ ID NOs 12 to 15, wherein the binding moiety is represented by k _off Values bind to the drug molecules: between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-4 s ^-1 Such as between about 1 x 10 ^-8 s ^-1 And about 1X 10 ^-7 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-5 s ^-1 Between about 1X 10 ^-7 s ^-1 And about 1X 10 ^-6 s ^-1 Between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-4 s ^-1 Between, or between about 1X 10 ^-6 s ^-1 And about 1X 10 ^-5 s ^-1 Between them. In some embodiments, the binding moiety comprises a polypeptide havingAn ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 10 and a pharmaceutical molecule comprising an ankyrin repeat domain having an amino acid sequence with at least about 85% sequence identity to SEQ ID No. 13 or 14, wherein said binding moiety is present in an amount of between about 1 x 10 ^-7 s ^-1 And about 1X 10 ^-4 s ^-1 K between _off The value binds to the drug molecule.

When the binding moiety and the drug molecule are bound together, the drug molecule is unable to exert biological activity, such as, for example, binding to a biological target molecule. Thus, the activity of the drug is "blocked" and the binding moiety binds to it. Half-life of blockade (hereinafter referred to as "blockade T _1/2 ") is the time it takes half of the prodrug complex to dissociate. Thus T _1/2 Is the "half-life" of the prodrug complex. Blocking T _1/2 Can be calculated according to the following formula:

blocking

To achieve slow release of the drug into the subject, the blocking half-life should be at least about 2 hours, at least about 5 hours, at least about 10 hours, at least about 20 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 50 hours, at least about 60 hours, at least about 70 hours, at least about 80 hours, at least about 90 hours, at least about 100 hours, at least about 150 hours, or at least about 200 hours. The blocking half-life may fall between any of the above values. For example, the blocking half-life may range from about 10 hours to about 250 hours, from about 20 hours to about 250 hours, from about 40 hours to about 200 hours, or from about 50 hours to about 100 hours.

In some embodiments, the compositions of the present invention comprise, consist essentially of, or consist of: a binding moiety comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to SEQ ID No. 1, and a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to any of SEQ ID nos. 12 to 15, wherein the prodrug complex exhibits a blocking half-life of at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 50 hours, at least about 60 hours, at least about 70 hours, at least about 80 hours, at least about 90 hours, at least about 100 hours, at least about 150 hours, or at least about 200 hours.

In some embodiments, the compositions of the present invention comprise, consist essentially of, or consist of: a binding moiety comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to SEQ ID No. 2, and a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to any of SEQ ID nos. 12 to 15, wherein the prodrug complex exhibits a blocking half-life of at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 50 hours, at least about 60 hours, at least about 70 hours, at least about 80 hours, at least about 90 hours, at least about 100 hours, at least about 150 hours, or at least about 200 hours.

In some embodiments, the compositions of the present invention comprise, consist essentially of, or consist of: a binding moiety comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to SEQ ID No. 3, and a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to any of SEQ ID nos. 12 to 15, wherein the prodrug complex exhibits a blocking half-life of at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 50 hours, at least about 60 hours, at least about 70 hours, at least about 80 hours, at least about 90 hours, at least about 100 hours, at least about 150 hours, or at least about 200 hours.

In some embodiments, the compositions of the present invention comprise, consist essentially of, or consist of: a binding moiety comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to SEQ ID No. 4, and a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to any of SEQ ID nos. 12 to 15, wherein the prodrug complex exhibits a blocking half-life of at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 50 hours, at least about 60 hours, at least about 70 hours, at least about 80 hours, at least about 90 hours, at least about 100 hours, at least about 150 hours, or at least about 200 hours.

In some embodiments, the compositions of the present invention comprise, consist essentially of, or consist of: a binding moiety comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to SEQ ID No. 5, and a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to any of SEQ ID nos. 12 to 15, wherein the prodrug complex exhibits a blocking half-life of at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 50 hours, at least about 60 hours, at least about 70 hours, at least about 80 hours, at least about 90 hours, at least about 100 hours, at least about 150 hours, or at least about 200 hours.

In some embodiments, the compositions of the present invention comprise, consist essentially of, or consist of: a binding moiety comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to SEQ ID No. 6, and a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to any of SEQ ID nos. 12 to 15, wherein the prodrug complex exhibits a blocking half-life of at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 50 hours, at least about 60 hours, at least about 70 hours, at least about 80 hours, at least about 90 hours, at least about 100 hours, at least about 150 hours, or at least about 200 hours.

In some embodiments, the compositions of the present invention comprise, consist essentially of, or consist of: a binding moiety comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to SEQ ID No. 7, and a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to any of SEQ ID nos. 12 to 15, wherein the prodrug complex exhibits a blocking half-life of at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 50 hours, at least about 60 hours, at least about 70 hours, at least about 80 hours, at least about 90 hours, at least about 100 hours, at least about 150 hours, or at least about 200 hours.

In some embodiments, the compositions of the present invention comprise, consist essentially of, or consist of: a binding moiety comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to SEQ ID No. 8, and a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to any of SEQ ID nos. 12 to 15, wherein the prodrug complex exhibits a blocking half-life of at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 50 hours, at least about 60 hours, at least about 70 hours, at least about 80 hours, at least about 90 hours, at least about 100 hours, at least about 150 hours, or at least about 200 hours.

In some embodiments, the compositions of the present invention comprise, consist essentially of, or consist of: a binding moiety comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to SEQ ID No. 9, and a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to any of SEQ ID nos. 12 to 15, wherein the prodrug complex exhibits a blocking half-life of at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 50 hours, at least about 60 hours, at least about 70 hours, at least about 80 hours, at least about 90 hours, at least about 100 hours, at least about 150 hours, or at least about 200 hours.

In some embodiments, the compositions of the present invention comprise, consist essentially of, or consist of: a binding moiety comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to SEQ ID No. 10, and a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85% amino acid sequence identity to any of SEQ ID nos. 12 to 15, wherein the prodrug complex exhibits a blocking half-life of at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 50 hours, at least about 60 hours, at least about 70 hours, at least about 80 hours, at least about 90 hours, at least about 100 hours, at least about 150 hours, or at least about 200 hours.

There is no particular upper limit on the blocking half-life. However, for certain disease indications and related drug molecules, those skilled in the art will appreciate that if the blocking half-life of the prodrug complex is too long, the drug may be released into the body too slowly to have the desired therapeutic benefit. Thus, the blocking half-life should be such that the desired therapeutic benefit of a given drug molecule is still observable.

Composition and method for producing the same

In one embodiment, the present invention relates to a composition comprising a binding moiety as defined herein; a drug molecule as defined herein; wherein the binding moiety binds reversibly to a drug molecule; and wherein the binding moiety, when bound, inhibits the biological activity of the drug molecule. In one embodiment, the biological activity is binding of the drug molecule to a biological target. In one embodiment, the binding affinity of the binding moiety to the drug molecule allows for release of the drug molecule over time following in vivo administration. As used herein, the term "composition" is used interchangeably with "prodrug complex".

Any of the protein binding moieties listed above may be combined with any of the drug molecules listed above to form a composition (i.e., prodrug complex), provided that the binding moiety has the desired binding affinity and specificity for the drug molecule.

The composition may comprise any of the binding moieties and drug molecule combinations described above,in particular with binding coefficients such as K _D And k _off ) Or any specifically disclosed combination of blocking half-lives.

As mentioned above, engineered ankyrin repeat domains are preferred binding moieties for use in the present invention. Thus, in one embodiment, the composition comprises a binding moiety comprising a designed ankyrin repeat domain. In another embodiment, the composition comprises a binding moiety comprising a designed ankyrin repeat domain and a drug molecule comprising a designed ankyrin repeat domain. In another embodiment, the composition comprises a drug molecule comprising a designed ankyrin repeat domain binding moiety and comprising two, three, four, five or more designed ankyrin repeat domains. In another embodiment, the composition comprises a binding moiety comprising a designed ankyrin repeat domain and a drug molecule comprising an alternative scaffold. In another embodiment, the composition comprises a binding moiety comprising a designed ankyrin repeat domain and a drug molecule comprising an antibody. In another embodiment, the composition comprises a binding moiety of a designed ankyrin repeat domain and a drug molecule comprising an antigen binding domain derived from a T Cell Receptor (TCR). In one embodiment, the composition comprises a binding moiety comprising a designed ankyrin repeat domain and a drug molecule as a T cell cement drug molecule. In one embodiment, the composition comprises a binding moiety comprising a designed ankyrin repeat domain and a drug molecule that is an immune checkpoint modulator drug molecule. In one embodiment, the composition comprises a drug molecule comprising a binding moiety comprising a engineered ankyrin repeat protein and a drug molecule that is a bispecific antibody drug molecule. In one embodiment, the composition comprises a binding portion of a designed ankyrin repeat domain and a drug molecule that is a CAR-expressing immune cell (such as a CAR-T cell or CAR-NK cell).

In one embodiment, the composition comprises (i) a binding moiety comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of seq id no: (1) SEQ ID NO. 1 and (2) a sequence having at least about 85% amino acid sequence identity to SEQ ID NO. 1; and (ii) a drug molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15 and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In one embodiment, a composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID NO:1, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to any of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 1, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 1, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 13 and 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID NO:1, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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:1 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 1, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 1 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 1, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 1 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 13 to 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 1, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 1, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (i) a binding moiety comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (1) 2, and (2) a sequence having at least about 85% amino acid sequence identity to SEQ ID No. 2; and (ii) a drug molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In one embodiment, a composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID NO:2, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to any of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 2, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 2, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 13 and 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 2, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 2 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 2, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 2 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 2, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 2 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 13 to 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 2, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 2, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (i) a binding moiety comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of seq id no: (1) 3, and (2) a sequence having at least about 85% amino acid sequence identity to SEQ ID No. 3; and (ii) a drug molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In one embodiment, a composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID NO:3, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to any of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 3, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 3, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 13 and 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID NO:3, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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:3 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 3, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 3 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID NOs 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 3, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 3 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 13 to 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 3, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 3, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (i) a binding moiety comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of seq id no: (1) 4, and (2) a sequence having at least about 85% amino acid sequence identity to SEQ ID No. 4; and (ii) a drug molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In one embodiment, a composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID NO:4, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to any of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 4, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 4, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 13 and 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 4, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 4 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 4, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 4 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 4, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 4 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 13 to 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 4, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 4, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (i) a binding moiety comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of seq id no: (1) 5, and (2) a sequence having at least about 85% amino acid sequence identity to SEQ ID No. 5; and (ii) a drug molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In one embodiment, a composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID NO:5, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to any of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 5, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 5, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 13 and 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 5, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 5 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 5, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 5 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 5, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 5 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 13 to 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 5, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 5, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (i) a binding moiety comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of seq id no: (1) SEQ ID NO. 6, and (2) a sequence having at least about 85% amino acid sequence identity to SEQ ID NO. 6; and (ii) a drug molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In one embodiment, a composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID NO:6, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to any of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 6, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 6, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 13 and 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 6, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 6 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 6, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 6 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID NOs 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 6, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 6 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 13 to 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 6, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 6, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (i) a binding moiety comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of seq id no: (1) 7, and (2) a sequence having at least about 85% amino acid sequence identity to SEQ ID No. 7; and (ii) a drug molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In one embodiment, a composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID NO:7, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to any of SEQ ID NOs.

In one embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 7, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 7, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 13 and 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 7, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 7 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 7, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 7 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 7, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 7 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 13 to 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 7, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 7, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (i) a binding moiety comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of seq id no: (1) 8, and (2) a sequence having at least about 85% amino acid sequence identity to SEQ ID NO 8; and (ii) a drug molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In one embodiment, a composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID NO:8, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to any of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 8, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 8, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 13 and 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 8, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 8 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 8, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 8 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 8, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 8 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 13 to 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 8, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 8, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (i) a binding moiety comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of seq id no: (1) SEQ ID NO. 9, and (2) a sequence having at least about 85% amino acid sequence identity to SEQ ID NO. 9; and (ii) a drug molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In one embodiment, a composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID NO 9, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to any of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 9, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 9, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 13 and 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 9, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 9 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 9, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 9 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 9, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 9 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 13 to 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 9, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 9, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (i) a binding moiety comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of seq id no: (1) 10, and (2) a sequence having at least about 85% amino acid sequence identity to SEQ ID No. 10; and (ii) a drug molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In one embodiment, a composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID NO 10, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to any of SEQ ID nos. 13 and 14.

In one embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 10, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% amino acid sequence identity to SEQ ID No. 10, and (2) a pharmaceutical molecule comprising any one of SEQ ID NOs 13 and 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 10, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 10 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain having an amino acid sequence selected from the group consisting of: (a) SEQ ID NOS 12 to 15, and (2) a sequence having at least about 85% amino acid sequence identity with SEQ ID NOS 12 to 15.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 10, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 10 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having the amino acid sequence of SEQ ID No. 10, wherein optionally up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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. 10 have been substituted with other amino acids, and (2) a pharmaceutical molecule comprising any of SEQ ID nos. 13 to 14.

In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 10, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 12 to 15. In another embodiment, the composition comprises (1) a binding moiety comprising an ankyrin repeat domain having SEQ ID No. 10, and (2) a pharmaceutical molecule comprising an ankyrin repeat domain comprising any one of SEQ ID nos. 13 and 14.

In one embodiment, the drug molecule comprised in any of the compositions described above is a bispecific or multispecific drug molecule. In another embodiment, the drug molecule comprised in any of the compositions described above is a T cell cement drug molecule.

Antibodies and alternative scaffolds, as described above, are also binding moieties for use in the invention. Thus, in one embodiment, the composition comprises a binding moiety comprising an antibody or alternative scaffold. In another embodiment, the composition comprises (1) a binding moiety comprising an antibody or alternative scaffold, and (2) a drug molecule comprising a designed ankyrin repeat domain. In another embodiment, the composition comprises (1) a binding moiety comprising an antibody or alternative scaffold, (2) and a drug molecule comprising an antibody. In one embodiment, the composition comprises (1) a binding moiety comprising an antibody or alternative scaffold, and (2) a drug molecule that is a T cell cement drug molecule. In one embodiment, the composition comprises (1) a binding moiety comprising an antibody or alternative scaffold, and (2) a drug molecule that is an immune checkpoint inhibitor drug molecule. In one embodiment, the composition comprises (1) a binding moiety comprising an antibody or alternative scaffold, and (2) a drug molecule that is a bispecific antibody drug molecule. In one embodiment, the composition comprises (1) a binding moiety comprising an antibody or alternative scaffold, and (2) a drug molecule that is an immune cell activating drug molecule. In one embodiment, the composition comprises (1) a binding moiety comprising an antibody or alternative scaffold, and (2) a drug molecule that is a genetically modified immune cell (such as a CAR-expressing immune cell, such as a CAR-T cell or CAR-NK cell).

Since the compositions of the present invention require that substantially all of the drug molecules bind to the binding moiety, the pharmaceutical composition is preferably administered for a time sufficient for such binding to occur and reach equilibrium. The time required to establish equilibrium will depend on the binding constant (k _on And k _off )。

Accordingly, in one embodiment, the present invention relates to a method of preparing a controlled release formulation comprising the steps of:

(i) Providing a binding moiety as defined herein;

(ii) Providing a drug molecule as defined herein; and

(iii) The binding moiety and the active drug molecule are brought into equilibrium such that substantially all of the drug molecule is bound to the binding moiety.

In one embodiment, the drug molecule and the binding moiety are allowed to equilibrate for at least about 1 hour, such as at least about 2 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, or at least about 72 hours.

The concentration of the binding moiety in the composition should be relatively high to ensure k _on The equilibrium constant is significantly higher than k _off Equilibrium constant, which means that substantially all of the drug molecules are bound by the binding moiety and that the mode of action of the drug molecules is substantially inhibited. In one embodiment, the binding moiety and the drug molecule are provided in a 1:1 molar ratio. In some embodiments, the binding moiety should be present in molar excess compared to the amount of drug molecule. Thus, in these embodiments, the binding moiety is provided in a molar ratio to the drug molecule of at least 1.2:1, at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 20:1, at least 50:1, at least 100:1, at least 200:1, at least 400:1, or at least 1000:1.

The invention also relates to pharmaceutical compositions comprising the compositions described herein and a pharmaceutically acceptable carrier or excipient. Also described herein are uses and methods of treatment using the pharmaceutical compositions. The methods and uses encompassed by the present invention are described in more detail below. It should be noted that the pharmaceutical compositions, methods and uses treat indications of diseases treated by the pharmaceutical molecules used to prepare the pharmaceutical compositions.

The primary function of the binding moiety is to bind to the drug molecule to achieve slow release of the drug molecule. Thus, in some embodiments, the binding moiety does not substantially alter the in vivo biological effect of the drug molecule, except that the drug molecule is released into the body at a slower rate than direct administration without prior complexing with the protein binding moiety. However, the binding moiety may comprise additional functional moieties. Such additional functional moieties may provide particular advantages, such as bioactivity, or may be used to label/tag the binding moiety for detection purposes, for example.

The pharmaceutical compositions described herein may be prepared using methods known in the art.

The pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient. Standard pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions such as oil/water or water/oil emulsions, and various types of wetting agents.

The pharmaceutical composition may comprise any pharmaceutically acceptable ingredient, including, for example, acidulants, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anti-caking agents, anticoagulants, antimicrobial preservatives, antioxidants, antimicrobial agents, binders, adhesives, buffers, chelating agents, coating agents, colorants, desiccants, detergents, diluents, disinfectants, disintegrants, dispersing agents, dissolution enhancers, dyes, emollients, emulsifiers, emulsion stabilizers, fillers, film formers, flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesive agents, ointment bases, ointments, oleaginous vehicles, organic bases, lozenge bases, pigments, plasticizers, polishing agents, preservatives, chelating agents, skin penetrating agents, solubilizing agents, solvents, stabilizers, suppository bases, surfactants, suspending agents, sweeteners, therapeutic agents, thickening agents, tonicity agents, toxicity agents, water-absorbing agents, water-miscible co-solvents, water softeners, or wetting agents. See, e.g., third edition Handbook of Pharmaceutical Excipients, a.h. kibbe (Pharmaceutical Press, london, UK, 2000), incorporated by reference in its entirety. Remington's Pharmaceutical Sciences, sixteenth edition, e.w. martin (Mack Publishing co., easton, pa., 1980), incorporated by reference in its entirety.

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

In one embodiment, the invention relates to a method of immune cell activation, such as T cell activation or NK cell activation, in a subject in need thereof, comprising the step of administering to said subject a pharmaceutical composition as described herein.

In another embodiment, the invention relates to a method of controlling the release of an active drug molecule in vivo, the method comprising administering to a subject in need thereof a pharmaceutical composition as described herein.

In another embodiment, the present invention relates to a method of treating a subject, the method comprising the step of administering to a subject in need thereof an effective amount of a pharmaceutical composition as defined herein. In some embodiments, the method is a method of treating a proliferative disease. In some embodiments, the method is a method of treating cancer.

In another embodiment, the invention relates to a composition as defined herein for use in therapy. In another embodiment, the invention relates to a pharmaceutical composition as defined herein for use in therapy. Preferably, a pharmaceutical composition as defined herein is provided for use in the treatment of a proliferative disease. In a more preferred embodiment, the proliferative disease is cancer.

The pharmaceutical compositions of the invention are typically administered to subjects who have been identified as having a high risk of specific side effects typically associated with drug molecules and/or who require large doses of drug molecules such that problematic side effects have an increased likelihood. In some embodiments, the subject is a mammal. In a preferred embodiment, the subject is a human.

In some embodiments, a single administration of the pharmaceutical composition may be sufficient. In other embodiments, repeated administration may be necessary. Various factors will influence the number and frequency of administration, such as the age and general health of the subject, as well as the nature of the drug molecule and typical dosage regimen.

The pharmaceutical compositions described herein may be administered to a subject via any suitable route of administration, such as parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal administration. Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. See Pharmaceutics and Pharmacy Practice, J.B.Lippincott Company, philadelphia, pa., banker and Chalmers editions, pages 238-250, 1982, and ASHP Handbook on Injectable Drugs, toissel, 4 th edition, pages 622-630, 1986 for additional details.

The pharmaceutical compositions described herein may be used in combination with another therapeutic agent. Each therapeutic agent may be administered simultaneously (e.g., in the same drug or simultaneously), concurrently (i.e., administered one after the other in any order in separate drugs), or sequentially in any order. Sequential administration may be useful when the therapeutic agents in combination therapy are in different dosage forms (e.g., one agent is a tablet or capsule and the other agent is a sterile liquid) and/or are administered with different dosing regimens, e.g., an analgesic administered at least daily and a less frequently administered biologic therapeutic, such as once weekly or once every two weeks.

Additional therapeutic agents include, but are not limited to, analgesics, steroids, anticancer agents, antibiotics, penicillins, antihypertensives, antidiabetic agents, anticonvulsants, antiemetics, antidepressants, checkpoint inhibitors (e.g., anti-PD-1, PD-L1, tim-3, LAG-3, etc.), immune agonists (e.g., 41-BB, CD28, CD40, etc.), cytokines (IL-15, IL-12, etc. or anti-TGFb, anti-IL-10, etc.), immunostimulants, T cell stimulators, immunooncologic modes (e.g., CAR-T and cell therapies, antibody Drug Conjugates (ADC), oncolytic viruses, etc.), chemotherapeutics, radioimmunoconjugates, and the like.

Nucleic acids and methods

The invention also relates to nucleic acids encoding binding moieties as described herein. In one embodiment, the nucleic acid encodes a binding moiety comprising a designed ankyrin repeat domain as defined herein. Examples of such nucleic acids are provided by SEQ ID NOS.16 to 19. The invention also relates to host cells comprising said nucleic acids.

The invention also relates to a method of preparing a binding moiety as defined herein, comprising culturing a host cell as defined herein under conditions in which said recombinant binding protein is expressed. In some embodiments, the host cell is a eukaryotic host cell. In other embodiments, the host cell is a prokaryotic host cell. In one embodiment, a method of making a binding moiety comprises culturing a host cell under conditions that express the recombinant binding protein, wherein the binding moiety comprises a designed ankyrin repeat domain, and wherein the host cell is a prokaryotic host cell, such as, for example, e. In another embodiment, a method of making a binding moiety comprises culturing a host cell under conditions that express the recombinant binding protein, wherein the binding moiety comprises an antibody, and wherein the host cell is a eukaryotic host cell, such as, for example, a CHO cell.

Examples

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

Material

Chemicals were purchased from Sigma-Aldrich (USA). The oligonucleotides were from Microsynth (Switzerland). Unless otherwise indicated, DNA polymerase, restriction enzyme and buffer were from New England Biolabs (USA) or Fermentas/Thermo Fisher Scientific (USA). Inducible E.coli expression strains are used for cloning and protein production, for example E.coli XL1-blue (Stratagene, USA) or BL21 (Novagen, USA). NLC chips for SPR measurement were from BioRad (BioRad, USA). HTRF reagent is from Cisbio (Cisbio, france). The pan-T cell isolation kit was from Miltenyi Biotec (Germany). The polyester insert for the "complex" Incucyte of 24-well format was from Corning (USA) and the neutravidin beads were from Thermo Fischer (USA). The nucleic Red lentivirus is from Sartorius (Germany). The cytotoxicity detection (by LDH release) kit was from Roche.

Molecular biology

Unless otherwise indicated, methods were performed according to known protocols (see, e.g., sambrook j., fritsch e.f. and manitis t. Molecular Cloning: A Laboratory Manual, cold Spring Harbor Laboratory 1989, new York).

Engineered ankyrin repeat libraries

Methods for generating a designed ankyrin repeat protein library have been described in the following: for example, U.S. patent No. 7,417,130; binz et al, 2003, supra; binz et al, 2004, supra. By such methods, a library of engineered ankyrin repeat proteins with randomized ankyrin repeat modules and/or randomized end capping modules can be constructed. For example, such libraries can thus be assembled based on an immobilized N-terminal capping module (e.g., the N-terminal capping module of SEQ ID NO:21, 22, or 23) or a randomized N-terminal capping module according to SEQ ID NO:24, one or more randomized repeat modules according to the sequence motif of SEQ ID NO:25, 26, or 27, and an immobilized C-terminal capping module (e.g., the C-terminal capping module of SEQ ID NO: 28) or a randomized C-terminal capping module according to SEQ ID NO: 29. Preferably, such libraries are assembled without any of amino acids C, G, M, N (preceding the G residue) and P at randomized positions of the repeat or end-capping module.

Furthermore, such randomized modules in such libraries may comprise additional polypeptide loop insertions with randomized amino acid positions. Examples of such polypeptide loop insertions are Complementarity Determining Region (CDR) loop libraries of antibodies or de novo generated peptide libraries. For example, such loop insertions can be designed using the structure of the N-terminal ankyrin repeat domain of human ribonuclease L (Tanaka, N., nakanishi, M, kusakabe, Y, goto, Y., kitade, Y, nakamura, K.T., EMBO J.23 (30), 3929-3938, 2004) as a guide. Similar to such ankyrin repeat domains in which ten amino acids are inserted in β -turns that exist near the boundary of two ankyrin repeats, ankyrin repeat libraries may contain randomized loops (with fixed and randomized positions) of variable length (e.g., 1 to 20 amino acids) inserted in one or more β -turns of the ankyrin repeat domain.

The N-terminal end capping module of the ankyrin repeat library preferably has a RILLAA, RILLKA or RELLKA motif, and any such C-terminal end capping module of the ankyrin repeat library preferably has a KLN, KLA or KAA motif.

The design of such ankyrin repeat libraries can be guided by the known structure of ankyrin repeat domains that interact with the target. Examples of such structures identified by their Protein Database (PDB) unique accession number or identification code (PDB-ID) are 1WDY, 3V31, 3V30, 3V2X, 3V2O, 3UXG, 3TWQ-3TWX, 1N11, 1S70 and 2ZGD.

Examples of designed ankyrin repeat libraries, such as N2C and N3C designed ankyrin repeat libraries, have been described (U.S. Pat. No. 7,417,130; binz et al, 2003, supra; binz et al, 2004, supra). The numbers in N2C and N3C describe the number of randomized repeat modules that exist between the N-terminal and C-terminal end capping modules.

The nomenclature used to define the repeat units and the positions within the modules is based on Binz et al, 2004 (supra), modified in that the boundaries of the ankyrin repeat modules and ankyrin repeat units are shifted by one amino acid position. For example, position 1 of the ankyrin repeat module of Binz et al 2004 (supra) corresponds to position 2 of the ankyrin repeat module of the disclosure, and thus position 33 of the ankyrin repeat module of Binz et al 2004 (supra) corresponds to position 1 of the following ankyrin repeat module of the disclosure.

Example 1: selection of ankyrin repeat domains with binding specificity for the CD3 specific binding domain

SUMMARY

Use of coresSugar displays (Hanes, J. And Pluckthun, A., PNAS 94,4937-42,1997), in a manner similar to that described by Binz et al, 2004 (supra), from the standpoint of specific conditions and additional counter-selection stepsA plurality of ankyrin repeat domains with binding specificity for the CD3 specific binding domain of the T cell cement molecule (TCE) are selected in the library. The binding and specificity of selected clones for the CD3 specific binding domain was assessed by e.coli crude extract Homogeneous Time Resolved Fluorescence (HTRF), indicating successful selection of multiple binding proteins that specifically bind to the CD3 specific binding domain. These initially identified binding proteins were further developed to obtain binding proteins with even higher affinity and/or even lower dissociation rates for the CD3 specific binding domain of TCE. For example, the ankyrin repeat domains of SEQ ID NOs 1 to 10 constitute the amino acid sequence of a binding protein comprising ankyrin repeat domains with binding specificity and high binding affinity and/or low dissociation rate with the CD3 specific binding domain of the bispecific T cell cement molecule.

CD3 specific binding domains as targets and selection materials

The CD3 specific binding domain of bispecific TCE is used as a target and selection material. Such a target domain is a polypeptide selected from the group consisting of SEQ ID NOS.11-15. The target protein is biotinylated using standard methods.

Selection of target-specific ankyrin repeat proteins by ribosome display

The designed ankyrin repeat protein libraries (N2C and N3C) were used in ribosome display selection against the CD3 specific binding domain (SEQ ID NO: 11) used as target (see Binz et al, nat Biotechnol22, 575-582 (2004); zahnd et al, nat Methods 4,269-279 (2007); hanes et al, proc Natl Acad Sci USA 95,14130-14135 (1998)).

Four rounds of selection were performed for each target and library. Four rounds of selection employed standard ribosome display selection, using decreasing target concentration and increasing wash stringency to increase selection pressure from round 1 to round 4 (Binz et al, 2004, supra). After each round of selection, the number of Reverse Transcription (RT) -PCR cycles was continuously reduced, thereby adjusting the yield due to enrichment of the conjugate. The resulting 3 pools were then subjected to conjugate screening.

As shown by crude extract HTRF, selected clones specifically bind to the CD3 specific binding domain of TCE

Using standard protocols, a crude extract of e.coli cells expressing ankyrin repeat proteins was used to identify individually selected ankyrin repeat proteins in solution that specifically bind to the CD3 specific binding domain of TCE by Homogeneous Time Resolved Fluorescence (HTRF) assay. The ankyrin repeat clones selected by ribosome display were cloned into a derivative of the pQE30 (Qiagen) expression vector in a format in which the clones were covalently linked to the Human Serum Albumin (HSA) binding domain and the CD3 binding domain (SEQ ID NO: 11), transformed into E.coli XL1-Blue (Stratagene), plated onto LB-agar (containing 1% glucose and 50. Mu.g/ml ampicillin) and then incubated overnight at 37 ℃. Individual colonies were picked into 96-well plates (each clone in a single well) containing 165 μl of growth medium (LB containing 1% glucose and 50 μg/ml ampicillin) and incubated overnight at 37 ℃ with shaking at 800 rpm. 150 μl of fresh LB medium containing 50 μg/ml ampicillin was inoculated with 8.5 μl of overnight culture in a fresh 96-deep well plate. After 120 min incubation at 37℃and 850rpm, expression was induced with IPTG (0.5 mM final concentration) for 6 hours. Cells were harvested by centrifugation of the plates, the supernatant was discarded and the pellet was frozen overnight at-20℃and then resuspended in 10 μl μ l B-PERII (Thermo Scientific) and incubated for one hour at room temperature with shaking (600 rpm). Then 160 μl PBS was added and cell debris was removed by centrifugation (3220 g for 10 min).

The extract of each lysed clone was used as PBSTB (supplemented with 0.1% TweenAnd 0.1% (w/v) BSA in PBS, pH 7.4) at 1:800 dilution (final concentrateDegree) was applied to the wells of 384 well plates with 12.5nM (final concentration) biotinylated CD3 binding domain, 1:300 (final concentration) anti-FLAG-D2 HTRF antibody-FRET acceptor conjugate (Cisbio) and 1:300 (final concentration) anti-strep-Tb antibody FRET donor conjugate (Cisbio, france) and incubated for 120 min at 4 ℃. HTRF was read out on Tecan M1000 using 340nm excitation wavelength and a 620±10nm emission filter for background fluorescence detection and a 665±10nm emission filter for detection of specifically bound fluorescent signals.

The same lysate was mixed with 12.5nM (final concentration) biotinylated HSA, 1:300 (final concentration) anti-FLAG-D2 HTRF antibody-FRET acceptor conjugate (Cisbio) and 1:300 (final concentration) anti-strep-Tb antibody FRET donor conjugate (Cisbio, france) into wells of 384-well plates and incubated at 4℃for 120 min. HTRF was read out on Tecan M1000 using 340nm excitation wavelength and a 620±10nm emission filter for background fluorescence detection and a 665±10nm emission filter for detection of specifically bound fluorescent signals.

The extracts of each of the lytic clones were tested for binding to the biotinylated CD3 binding target domain and for inhibition of unhindered binding to biotinylated HSA in order to assess specific binding to the CD3 binding domain.

Further analysis and selection of binding proteins with very high affinity for target proteins

A total of 744 binding proteins were initially identified. Based on the binding profile, 172 candidates were selected for expression in 96-well format and purified to homogeneity in parallel with DNA sequencing. Candidates were characterized biophysically by size exclusion chromatography, sypro-Orange thermal stability assessment (see Niesen et al, nat Protoc2, 2212-2221, (2007)), proteon Surface Plasmon Resonance (SPR) target affinity assessment, ELISA, target protein competition HTRF experiments, and/or SDS-PAGE.

In the further development of the binding proteins initially identified, two different methods were used to produce conjugates with very high affinity for the target protein and/or very low dissociation rates from the target protein. In the first approach, one of the binding proteins identified initially ("parent" binding proteins) is selected as the appropriate starting point for affinity maturation. The affinity maturation procedure involves saturation mutagenesis of each random position of the ankyrin repeat domain that serves as a starting point. Sequences generated by the affinity maturation procedure were screened for lower dissociation rates by competing HTRF. The beneficial mutations thus identified are incorporated into the binding protein by protein engineering. Binding properties of affinity matured and engineered binding proteins were verified by Surface Plasmon Resonance (SPR). Conjugates #1 to #4 (SEQ ID NOS: 1 to 4) derived from the parent conjugate were produced by this method.

In the second method, the selection pool obtained after four rounds of ribosome display selection against the target protein (SEQ ID NO: 11) was used as starting point for additional dissociation rate selection. The off-rate selection from the pool was performed consecutively for the other two biotinylated target proteins (SEQ ID NO:13 and SEQ ID NO: 14), each in the presence of an excess of the respective non-biotinylated target proteins. In some cases, the off-rate selection process is combined with error-prone PCR. The binding properties of hundreds of binding proteins produced by this procedure were then assessed by SPR.

Based on these second methods, conjugates #5 to #10 (SEQ ID NOS: 5 to 10) that bind to the CD3 specific binding target domain with high affinity and/or low dissociation rate were selected for further analysis.

In summary, the 10 binding proteins (SEQ ID NOS: 1 to 10) derived from these two different methods constitute the binding moiety of the present invention.

These selected binding proteins with binding specificity for the TCE CD3 binding domain were cloned into pQE (QIAgen, germany) based expression vectors as described below, providing an N-terminal His-tag (SEQ ID NO: 20) to facilitate simple protein purification. Constructing an expression vector encoding a combination of:

Conjugate #1 (SEQ ID NO:1, having His-tag fused to its N-terminus (SEQ ID NO: 20));

conjugate #2 (SEQ ID NO:2, having His-tag fused to its N-terminus (SEQ ID NO: 20));

conjugate #3 (SEQ ID NO:3, having His-tag fused to its N-terminus (SEQ ID NO: 20)).

Conjugate #4 (SEQ ID NO:4, having a His-tag fused to its N-terminus (SEQ ID NO: 20)).

Conjugate #5 (SEQ ID NO:5, having His-tag fused to its N-terminus (SEQ ID NO: 20)).

Conjugate #6 (SEQ ID NO:6, having His-tag fused to its N-terminus (SEQ ID NO: 20)).

Conjugate #7 (SEQ ID NO:7, having His-tag fused to its N-terminus (SEQ ID NO: 20));

conjugate #8 (SEQ ID NO:8, having His-tag fused to its N-terminus (SEQ ID NO: 20));

conjugate #9 (SEQ ID NO:9, having His-tag fused to its N-terminus (SEQ ID NO: 20)); and

conjugate #10 (SEQ ID NO:10, having a His-tag fused to its N-terminus (SEQ ID NO: 20)).

Selection of high level and soluble expression of binding proteins for analysis

For further analysis, the conjugates were expressed in E.coli cells and purified according to standard protocols using their His-tag. 1000ml of the culture (TB, 50mg/l ampicillin, 37 ℃) was inoculated with 50ml of the fixed overnight culture (TB, 1% glucose, 50mg/l ampicillin; 37 ℃). Cultures were induced with 0.5mM IPTG at an absorbance of 1.0 to 1.5 at 600nm and incubated at 37℃for 4-5 hours with shaking. The culture was centrifuged and the resulting pellet was resuspended in 25ml TBS ₅₀₀ (50 mM Tris-HCl,500mM NaCl,pH8) and lysed (sonication or French press). After lysis, the samples were mixed with 50KU DNase/ml, incubated for 15 minutes, then heat treated at 62.5℃for 30 minutes, centrifuged and the supernatant collected and filtered. Triton X100 (1% (v/v) final concentration) and imidazole (20 mM final concentration) were added to the homogenate. The protein was purified on a nickel-nitrilotriacetic acid (Ni-NTA) column according to standard protocols and resins known to those skilled in the art, followed bySize exclusion chromatography was performed on the system. Purification of highly soluble ankyrin repeat proteins with binding specificity for the TCE CD3 binding domain from E.coli cultures (up to 200mg ankyrin repeat protein per liter of culture), purity as estimated from 4% -12% SDS-PAGE>95％。

Example 2: SPR binding assay

An important feature of the binding moiety of the present invention is its affinity for drug molecules. Related aspects include the rate of dissociation of the binding moiety from the drug molecule and the resulting blocking half-life. High affinity, low off-rate and significant blocking half-life are required to achieve slow release of the drug molecule from the complex, wherein the drug molecule is reversibly bound by the binding moiety. See fig. 1-3 for further description and explanation of the function and nature of the prodrug complexes of the invention.

Surface Plasmon Resonance (SPR) assays were used to determine the binding affinity and dissociation rate of the ankyrin repeat binding domain to the CD3 binding domain of the TCE drug molecule.

All SPR data were generated using a Bio-Rad Proteon XPR36 instrument with PBS-T (0.005% Tween 20) as running buffer. The new neutravidin sensor chip (NLC) was air initialized and conditioned according to the Bio-Rad manual.

SPR data were generated for the precursors of conjugates #1, #4, #5 and conjugate #9 (as listed in example 1 above) bound to biotinylated CD3 specific binding domains with SEQ ID NOs 13 and 14, respectively, on the chip for the parental conjugates used in the affinity maturation process (see example 1). Data were generated at 25 ℃ and the dissociation rate over 2 hours was measured using a binding agent concentration (i.e., single trace) of 100 nM. SPR traces obtained with SEQ ID NO. 13 as target protein are shown in FIG. 4. The precursors of conjugates #1, #4, #5 and #9 all exhibited significantly reduced dissociation rates compared to the parental conjugate. Similar results were obtained with SEQ ID NO. 14 as target protein.

Generation of a junction to biotinylated CD3 specific binding domains with SEQ ID NOs 13 and 14, respectively, on chip Further SPR data for compounds #1 to conjugate #9 (as listed in example 1 above). Data were generated at 33℃and the binding rate (k) over 2 hours was measured using several binder concentrations (26.7 nM, 3nM, 1 nM) (i.e., multiple traces) _on ) Dissociation rate (k) _off ) And deriving equilibrium dissociation constant (K _D ). The blocking half-life (T) _1/2 ). The results are provided in table 2 below:

TABLE 2

Table 2 provides k for some ultra-high affinity binders (analytes) for two different CD3 binding proteins (SEQ ID NO:13 and SEQ ID NO: 14) _on 、k _off And K _D Values, wherein formula T is used _1/2 ＝ln2/k _off ＝0.693/k _off From the dissociation rate (k _off ) Calculation of blocking T _1/2 . The dissociation rates of the conjugates were all lower than 1X 10 ^-4 s ^-1 。K _D Values are all below 1X 10 ^-10 M. SPR measurements for conjugates #5 to #9 (SEQ ID NOS: 5 to 9) did not yield more accurate values than those shown in Table 2 due to the lack of at least a 10% signal drop (k) during the dissociation phase _off Based on which the calculation is performed).

All conjugates exhibited a blocking half-life of at least 3 hours (T _1/2 ) Some of which exhibit a blocking half-life of at least 10 hours. Thus, these experiments showed that conjugates #1 to #9 have very high binding affinity for the CD3 specific binding domain used in TCE drug molecules. In addition, the dissociation rates of conjugate #1 to conjugate #9 from these target proteins are very low, and therefore have very significant blocking half-lives. Conjugate #10 exhibited similar properties as conjugate #9 when tested in a comparable SPR assay.

Example 3: t cell activation/killing assay

In this example, three binding moieties and parent conjugates of the invention were tested for their potential to inhibit T cell activation by T cell cement (referred to herein as TCE # 1) and killing of tumor cells by activated T cells. TCE#1 comprises a CD3 specific binding domain (SEQ ID NO: 13) covalently linked to a Tumor Associated Antigen (TAA) specific binding domain (TAA binding domain). TAA is expressed on tumor cells used in the assay.

A sample of the prodrug complex was prepared at a concentration 100 times the final measured concentration as indicated below to ensure that substantially all TCE was bound by the binding moiety.

Preparation of prodrug complex samples and addition to T cell activation/killing assays

1. Preparation of constant TCE of final 10pM 200x concentrate (2 nM)

2. Binding portion titers were prepared, starting finally with 10nM,200 Xconcentrated (starting 2000nM,1:3 serial dilutions: 2000nM, 666nM, 222nM, 74nM, 25nM, 8.2nM, 2.7nM, 0.91nM, 0.30 nM).

3.1:1 mixing TCE with binding moiety titrant (either TCE alone or binding moiety alone with assay medium) →100×

4. Equilibration at 37℃for at least 24 hours in a pH controlled incubator

5. 1:100 (2. Mu.l in 200. Mu.l volume) dilution to T cell activation/killing experiments → 1x before assay start

6. Incubation was carried out for 48 hours

For the T cell activation assay, target tumor cells and effector T cells (pan T cells from healthy blood donors) were combined at 5:1 effector to target cell ratio, prodrug complex samples were added, and the mixture was incubated at 37 ℃ for 48 hours. The supernatant was analyzed for LDH release from killed tumor cells and was analyzed by FACS (using CD25 monoclonal antibody (BC 96), perCP-cyanine5.5, eBioscience ^TM ) Determination of CD8 ⁺ Level of activation marker (CD 25) on T cells. As indicated in fig. 5a and 5b, various controls were included (i.e., T cell only, tumor cell only, triton control, binding moiety only).

The results are provided in fig. 5a and 5 b. It can be seen that the three binding moieties of the present invention (conjugate #4 (SEQ ID NO: 4)); conjugate #5 (SEQ ID NO: 5); and conjugate #9 (SEQ ID NO: 9)) inhibited T cell activation and tumor cell killing at much lower concentrations than the parent conjugate, consistent with the much higher binding affinity of the binding moiety of the invention. IC50 values (in nM) are provided in fig. 5a and 5 b.

Example 4: "simple" Measurement

Standard T cell activation assays (such as those in example 3 above) provide a single readout after a defined time point. For the purposes of the present invention, multiple readouts at different time points are required to obtain time resolved views of T cell mediated tumor cell growth inhibition (used as a surrogate for T cell mediated tumor cell killing). Analysis (see https:// www.essenbioscience.com/en/products/incucyte /) may provide such information. The Incucyte assay principle is to co-incubate T cells (or pan T cells that are hpbmcs) with tumor cells that have been transduced with NucLight Red lentivirus that reds the cells and are therefore detectable with an Incucyte camera, and by determining the Red object (i.e., by Incucyte The area of (a nuclear restricted mKate2 protein (red fluorescent protein)) labelled tumor cells can be monitored in a time-resolved manner by means of an Incucyte camera for tumor cell growth in the presence of T cells.

In the initial experiment, T cell cement (TCE # 1) was prepared, concentrated 100-fold, and serially diluted 2-fold (20 pM final concentration in the assay) starting from a concentration of 2 nM. TCE#1 was added to TAA expressing pan T cells and tumor cells and labeled with NucLight Red for passageAnd (5) detecting. During 4.5 days, in +.>The growth of red-labeled tumor cells was observed in the device. FIG. 6a shows that at higher concentrations of TCE#1 (starting from 20 pM), TCE#1 effectively inhibited tumor cell growth, and as the concentration was decreased (in 2-fold steps), the inhibition of tumor cell growth gradually decreased, transitioning between 0.16pM and 0.64pM (EC) ₅₀ About 0.27 pM). Thus, below 0.1pM, TCE has no significant effect on tumor cell growth, while between 0.1pM and 1pM, TCE is in the dynamic range of T cell mediated tumor cell growth inhibition, while in>At 1pM, TCE results in complete T cell mediated tumor cell growth inhibition (used as a surrogate for T cell mediated tumor cell killing). FIG. 6b illustrates that these findings and show an EC50 of about 0.27pM for TCE activity. / >

Titration of prodrug complexes "simple" Measurement setup

To investigate the effect of the binding moieties of the invention on TCE-mediated inhibition of TAA-expressing tumor cell growth, further samples of various drug complexes were takenAnd (5) measuring. Drug complex samples were prepared by titrating the binding moiety to a constant concentration of TCE (TCE # 1). The mixture was prepared as a 100x concentrated stock solution to ensure that substantially all drug molecules were complexed by the binding moiety at the beginning of the experiment (i.e., the prodrug complex was added to T cells and tumor cells). The equilibrated 100x concentrated stock was diluted 100-fold by addition to the cells and the change in growth of red-labeled tumor cells was followed over a 4.5 day duration as an alternative to T cell mediated tumor cell killing (see fig. 7 and 8).

Preparation and addition of prodrug complex samplesIn the experiment:

1. preparation of constant TCE of final 10pM 200x concentrate (2 nM)

2. Preparation of binding portion titres with final initial concentration of 10nM,200 Xconcentrated (initial 2. Mu.M)

3.1:1 mixing TCE with binding moiety titrant (either TCE alone or binding moiety alone with assay medium) →100x concentrated stock solution sample

4. Equilibrated at 37℃for at least 24 hours in a pH controlled incubator

5. Dilution to experiment (T cells+tumor cells labeled with NucLight Red) 1:100 before the start of the assay (2. Mu.l in 200. Mu.l volume)

6. At the position ofTumor cell proliferation was determined for up to 5 days

In the first experiment, a simple one was studiedDetermination of the parent Binder (K) _D About 200 pM) and an ultra-high affinity binding moiety (K) having SEQ ID NO. 5 _D About 6pM or less) (conjugate # 5). Figures 7a to 7d clearly show that the parent conjugate shows a blocking loss in the sample between 330-fold and 110-fold molar excess of the parent conjugate relative to the drug molecule, whereas the ultra-high affinity binding moiety (conjugate # 5) completely blocks the activity of the drug molecule, even though the binding moiety is much lower by a 4-fold molar excess relative to the drug molecule in the prodrug complex sample.

In a second experiment, different ultra-high affinity binding moieties were studied in a similar setting (fig. 8 a-8 c). As the molar excess of binding moiety relative to the drug molecule (tce#1) in the drug complex sample was reduced, loss of inhibition of the antitumor activity of the drug molecule was observed. This inhibition is a loss that is shown to be related to the binding affinity of the binding moiety to the drug molecule. Conjugate #4 has shown a loss of inhibition of TCE activity at a 4-fold molar excess resulting in increased inhibition of tumor cell growth, while the other two conjugate portions conjugate #5 and conjugate #9 still effectively inhibited TCE activity at a 4-fold molar excess, resulting in a normal growth curve without any T cell mediated inhibition. Only when the molar ratio was reduced to 1:1, conjugate #5 and conjugate #9 showed a loss of inhibition of TCE activity, resulting in an increase in T cell mediated inhibition of tumor cell growth. Even at a 1:1 molar ratio, conjugate #9 can inhibit TCE activity to some extent, resulting in weak inhibition of tumor cell growth.

The data confirm that the data set of the data set,the assay is a sensitive method to study TCE blocking of the ultra-high affinity binding moiety and the binding moiety exhibits blocking activity as expected by the ultra-high affinity exhibited by SPR in example 2.

Taken together, these experiments indicate that the binding moieties of the present invention can effectively inhibit the biological activity of a drug molecule, depending on the binding affinity of the binding moiety to the drug molecule, as demonstrated herein in the exemplary manner of TCE #1 as a drug molecule and the conjugates described in example 1 as binding moieties.

Example 5: "Complex" Measurement />

In the in vivo case, the binding moiety, due to its short half-life, is rapidly eliminated once dissociated from the drug molecule. In contrast, if a drug molecule is designed to have a long half-life, the drug molecule can circulate in the body for a much longer period of time, whether or not it is complexed. Methods for producing drug molecules with long half-lives are well known in the art and include, for example, fusion with half-life extending moieties. To simulate rapid renal elimination of conjugates, "complications"Provided with an additional so-called "sump" chamber. In this chamber, a large excess of bead immobilized target protein of the binding moiety tested (relative to the amount of binding moiety contained in the prodrug complex added to the main chamber >10,000-fold molar excess). Such immobilized target proteins, once dissociated from the prodrug complex and diffused into the cell, scavenge the free binding moiety.

In the experiments described herein, TCE (tce#1) was used as a drug molecule. Thus, the immobilized scavenger protein added to the chamber is the CD3 specific binding domain (SEQ ID NO: 11) bound by the binding moiety being tested with high affinity.

Experimental setup

As in example 4, a TAA expressing tumor cell line was used as the target cell line, which had been transduced with Incucyte Nuclight Red lentivirus for passageAnd (5) detecting by a camera. Pan T cells were added together with different prodrug complexes. Prodrug complexes were generated by pre-equilibrating 10pM tce#1 (final concentration) with different amounts of conjugate #9 at 123pM (12.3-fold molar excess), 41pM (4.1-fold molar excess) and 13.7pM (1.37-fold molar excess). Each of these prodrug complexes is then used for "complex"/-for>In the setup, it had either uncoated beads that did not sequester the free binding moiety (fig. 9 a) as negative control, or beads coated with CD3 specific binding domain, thus having functional channels (fig. 9 b). In addition, unbound TCE #1 (10 pM) and background (i.e. without prodrug complex or TCE, but with beads) served as controls. By determining the basis- >The area of the red object detected by the camera was used to measure the growth of tumor cells for 5 daysSegments.

Figure 9a shows that in wells with a chamber containing uncoated beads, the binding moiety tested completely inhibited TCE activity, so no T cell mediated inhibition of tumor cell growth was observed, except for the prodrug complex produced with the lowest binding to TCE ratio (1.37:1). This is consistent with the results shown in example 4. In contrast, in wells with functional compartments containing beads coated with a CD3 specific binding domain (fig. 9 b), the binding moiety tested only partially inhibited TCE activity, so T cell mediated inhibition of tumor cell growth was observed even at the highest binder: TCE ratio (12.3:1). Inhibition of T cell-mediated tumor cell growth (as a surrogate for T cell-mediated tumor cell killing) in wells with prodrug complexes began with significant delay compared to controls with unbound TCE, only about 1.5-2 days after the start of the experiment. Inhibition of tumor cell growth in wells with unbound TCE has begun about 1 day after the start of the experiment. The delayed activity of TCE drug molecules reflected in the delayed inhibition of tumor cell growth reveals that the binding moiety can delay the biological activity of the drug molecule when complexed with the drug molecule to form a prodrug complex. These data support the concept that the binding moiety of the present invention can form a prodrug complex with a drug molecule, resulting in a slow release of the drug molecule and thus a down-regulation of the activity of the drug molecule when the prodrug complex is administered to a patient compared to administration of an unbound drug molecule.

Example 6: in an ex vivo human whole blood assay, transient blocking of CD3 effects of TCE drug molecules by complexing with conjugates Cytokine release should be reduced in part。

To investigate the effect of the conjugate on the cytokine release profile of the TCE drug molecule (in this example, TCE#1 (SEQ ID NO: 54), α -CD123 x α -CD33 x α -CD 3T cell cement), an ex vivo human whole blood assay was performed at Immuneed AB, sweden.

The whole blood circuit system used may be used to study interactions between blood and drug samples, including effects on cytokine release. The blood circuit system uniquely includes immune cells, immunoglobulins, and the complete complement and coagulation cascade system in the blood (Fletcher, E.A.K. et al, int Immunopharmacol,2018.54: pages 1-11). Cytokine release was determined by incorporating the test article into fresh human whole blood from one healthy human donor containing cd123+ and cd33+ target cells, followed by incubation of the sample on a rotating wheel to avoid blood clotting and to mimic blood circulation.

As a control, vehicle or 1nM of an anti-CD 123x anti-CD 3 industrial control test preparation was incorporated into human whole blood. Tce#1 molecules were incorporated at a concentration of 1nM and compared to 1nM tce#1+1.2nM conjugate # 5. The complexed TCE was pre-equilibrated with a 1.2-fold molar excess of conjugate to ensure 100% complexing of TCE at the start of the experiment. The test article is shown in fig. 10A and 10B.

By Meso ScaleThe techniques determine cytokine release of human TNF- α, IFN- γ, IL-2 and IL-6 at time points of 0 hours, 2 hours, 4 hours, 8 hours, 24 hours.

As shown in fig. 10C-10F, during the 24 hours of measurement, an increase in the level of all cytokines relative to vehicle and a lesser degree of unbound TCE #1 was observed in response to the a-CD 123 x a-CD 3 industrial control. However, at all time points, the complex TCE consisting of TCE#1 complexed with conjugate #5 greatly reduced cytokine release of TNF- α, IFN- γ, IL-2 and IL-6 compared to unbound TCE#1.

In summary, TCE prodrug complexes comprising complexes of TCE (such as TCE # 1) and conjugates (such as conjugate # 5) were shown to be effective in suppressing cytokine release in ex vivo human whole blood assays.

Example 7: in vivo efficacy studies, TCE prodrug complexes maintain optimal antitumor activity

The objective of the in vivo efficacy study was to compare unbound TCE drug molecules (in this example, TCE#2 (SEQ ID NO: 55), an α -HSA xα -CD123 xα -CD 3T cell cement comprising half-life extending moietiesA complex of TCE #2 with one of two different binders, which have an anti-tumor activity in the two-digit pM (binder # 4) or even 1pM or lower range (binder # 10), and a complexed TCE drug molecule (TCE prodrug complex) (in this example). NOG mice were humanized on day 0 with PBMCs from two human donors (n=5 donor a and n=5 donor B). Subcutaneous injection 1x10 on day 2 ⁶ Molm-13 tumor cells, treatment was started on day 6 after randomization, tumor size was about 70mm ³ . Quality control included analysis of PBMC subpopulations and viability by FC 1 day after isolation, and successful humanization of mouse blood by FC on day 19. The survival and CD33 positivity of the Molm-13 cells were tested 1 day prior to injection.

Test articles for in vivo efficacy studies are shown in fig. 11A and 11B. Study design and treatment groups are summarized in fig. 11C. TCE#2 was administered at a dose of 200. Mu.g/kg or 1000. Mu.g/kg, and combined with 1000. Mu.g/kg TCE#2 and conjugate#4 (K _D About 20 pM) or conjugate #10 (K) _D Less than or equal to 1 pM). The complex was pre-equilibrated with a 2x molar excess of conjugate to ensure a 100% rate of complex at the beginning of treatment. Treatment was administered i.v. All treatment groups (including vehicle) except anti-CD 33x anti-CD 3 industrial control group 2 (where treatment was given at 200 μg/kg daily) were 3 times per week from day 6 to day 16. Blood samples were taken before and 4 hours after the first therapeutic dose to determine cytokine levels at the beginning of treatment with these relatively small tumors. Tumor growth was monitored and the width and length measured 3 times per week with calipers, and tumor volume was calculated using length x width x height x pi/6.

Fig. 11D shows tumor growth curves for all 6 treatment groups of n=10 mice humanized with PBMCs of donor a or donor B, plotted as mean ± SEM. For group 2 (α -CD33x α -CD3 industrial control), two animals humanized with PBMCs of donor B were excluded from analysis due to unsuccessful humanization.

For the combined donor a and donor B, strong tumor growth inhibition was observed for the non-half-life extended anti-CD 33x anti-CD 3 industrial control group given daily at 200 μg/kg, and tumor eradication was observed for TCE #2 with a half-life extension given 3 times per week at 1000 μg/kg. Less significant tumor growth inhibition was observed for TCE #2 administered 3 times per week at 200 μg/kg, and for TCE prodrug complex containing a 1000 μg/kg TCE #2+2x molar excess of conjugate #4 at 3 doses per week. Under these experimental conditions, no similar tumor growth inhibition was observed for the TCE prodrug complex comprising a 1000 μg/kg TCE #2+2x molar excess of conjugate # 10.

Computer modeling predicts a similar AUC between 200 μg/kg free TCE and 1000 μg/kg TCE-conjugate complex derived from conjugate #4 (K _D About 20 pM) of TCE #2, transient blocking. In contrast, the very high affinity of PK of tce#2 to conjugate #10 (K _D Less than 1 pM) resulted in lower exposure of active TCE because a greater proportion of TCE#2+ conjugate#10 complex could be eliminated from circulation before the conjugate could release active TCE#2. Thus, the "sweet spot" is the interaction of binding affinity with the serum half-life of TCE in terms of optimally reducing cytokine release while maintaining full antitumor efficacy. The slow release TCE prodrug kit provided allows for matching of sufficient conjugate to TCE.

Fig. 11E shows the tumor growth curves of all six treatment groups of n=5 mice humanized with PBMCs of donor a plotted as mean ± SEM, while fig. 11F shows the tumor growth curves of all six treatment groups of n=5 mice humanized with PBMCs of donor B, except group 2, where two animals were excluded due to unsuccessful humanization. Significant donor-donor variability was observed in this experiment, with PBMCs from donor a showing stronger inhibition of growth of Molm-13 cells than PBMCs from donor B. Fig. 11G shows the legends of fig. 11D to 11F.

Figure 12 shows the cytokine levels determined in the serum of blood samples taken 4 hours before and after the first therapeutic dose of the in vivo efficacy study, while the tumor is still relatively small. Human cytokine levels were determined on undiluted serum samples by CBA human Th1/Th2/Th17 kit (BD Biosciences).

For the α -CD33X1-CD 3 industrial control, a significant increase in cytokines from 0 to 4 hours could be observed for all measured cytokines (TNF- α, IFN- γ, IL-2 and IL-6), while for TCE #2 there was a lesser increase at both doses administered. However, this increase in cytokines was prevented by TCE prodrug complexes comprising tce#2+ conjugate #4 or conjugate #10, supporting the following findings: complexing of the TCE drug molecule with the high affinity binder results in a slow release of active TCE and thus changes the exposure profile.

In summary, a slow release TCE prodrug complex (complex comprising tce#2 and conjugate#4 (K to the CD3 effector moiety) _D About 20 pM)) showed an antitumor efficacy equal to unbound TCE at the lower dose and decreased mild cytokine levels of TCE #2 4 hours after the first therapeutic dose.

Example 8: slow release TCE prodrug complex in vivo safety studies to reduce cytokine release

The purpose of two independent in vivo safety studies was to compare the extent of cytokine release triggered by unbound TCE drug molecules (TCE #2, an α -HSA x α -CD123 x α -CD33 x α -CD 3T cell cement in this example) and slow release TCE prodrug complexes (complexes of TCE #2 with one of three different binders, in this example, with a double bit pM (binder #1 and binder #4, study 1) or even the less than or equal to 1pM range (binder #10, study 2).

Test articles for in vivo safety studies are shown in fig. 13A and 13B. Study design and treatment groups are summarized in fig. 13C. The reason for separating in vivo safety studies from in vivo efficacy studies is that a tumor of moderate size is required to have a sufficient amount of target cells and thus elicit a measurable cytokine response after the first administration of TCE.

NOG mice were humanized on day 0 with PBMCs from two human donors (n=5 donor a and n=5 donor B). Subcutaneous injection 1x10 on day 2 ⁶ Molm-13 tumor cells and tumor size was about 300mm on day 14 ³ -800mm ³ Single doses (challenge) were given intravenously. Quality control included analysis of PBMC subpopulations and viability by FC 1 day after isolation, and successful humanization of mouse blood by FC on day 15 (study 1) or day 19 (study 2). Unbound TCE #2 with a half-life extension administered at 1000 μg/kg was compared to TCE prodrug complexes (complexes of TCE #2 with conjugate #1, conjugate #4 or conjugate # 10). The complexes were pre-equilibrated with a 2-fold molar excess of conjugate to ensure a 100% rate of complexing at the beginning of treatment. Blood samples were taken before dosing, 2 hours, 4 hours, 8 hours and 24 hours after the first therapeutic dose to determine cytokine levels with medium-sized tumors. FIG. 13D shows human cytokine levels (TNF-. Alpha., IFN-. Gamma., IL-2 and IL-6) in serum of blood samples taken at indicated time points before or after single dose treatment. Cytokine levels were determined on undiluted serum samples by CBA human Th1/Th2/Th17 kit (BD Biosciences).

All cytokines were elevated after a single dose of unbound TCE #2, while a reduced cytokine release was observed for the three different TCE prodrug complexes tested. The decrease in cytokine release did correlate with binding affinity and was followed by the top row (K _D About 40pM conjugate # 1) to middle row (K _D About 20pM conjugate # 4) to bottom row (K _D Conjugate #10 +.1 pM) increased to become more pronounced. Conjugate #10 almost completely inhibited cytokine release.

In summary, slow release TCE prodrug complexes are able to reduce cytokine release in vivo compared to unbound TCE. Furthermore, binding affinity for the CD3 effector moiety in TCE correlates with a decrease in cytokine release.

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

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

Sequence(s)

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Sequence listing

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<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "designed ankyrin repeat domain"

<400> 3

Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Phe Lys Gly Leu Thr Pro Leu His Leu Ala Ala Ala His Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Val Tyr Gly Trp Thr Pro Leu His Trp Ala Ala Ala Lys Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Lys Asp Trp Leu Gly Ile Thr Pro Leu His Leu Ala Ala Ser His Gly

100 105 110

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

115 120 125

Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala

130 135 140

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

145 150 155

<210> 4

<211> 157

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "designed ankyrin repeat domain"

<400> 4

Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Phe Lys Gly Leu Thr Pro Leu His Leu Ala Ala Glu His Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Val Tyr Gly Trp Thr Pro Leu His Trp Ala Ala Ala Lys Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Lys Asp Trp Leu Gly Ile Thr Pro Leu His Leu Ala Ala Ser His Gly

100 105 110

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

115 120 125

Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala

130 135 140

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

145 150 155

<210> 5

<211> 157

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "designed ankyrin repeat domain"

<400> 5

Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

His Tyr Gly Trp Thr Pro Leu His Ile Ala Ala Gln Ile Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Trp Ile Gly Tyr Thr Pro Leu His Leu Ala Ala Ser His Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Lys Asp Val Ser Gly Lys Thr Pro Leu His Val Ala Ala Ala His Gly

100 105 110

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

115 120 125

Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala

130 135 140

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

145 150 155

<210> 6

<211> 157

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "designed ankyrin repeat domain"

<400> 6

Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Gln Tyr Gly Trp Thr Pro Leu His Leu Ala Ala Tyr Ser Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Trp Val Gly Trp Thr Pro Leu His Leu Ala Ala Ser His Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Lys Asp Glu Ala Gly Arg Thr Pro Leu His Ile Ala Ala Lys Gln Gly

100 105 110

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

115 120 125

Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala

130 135 140

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

145 150 155

<210> 7

<211> 157

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "designed ankyrin repeat domain"

<400> 7

Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

His Tyr Gly Trp Thr Pro Leu His Leu Ala Ala Ala Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Trp Ile Gly Tyr Thr Pro Leu His Ile Ala Ala Ser His Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Lys Asp Ser Ser Gly Lys Thr Pro Leu His Ile Ala Ala Gln His Gly

100 105 110

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

115 120 125

Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala

130 135 140

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

145 150 155

<210> 8

<211> 157

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "designed ankyrin repeat domain"

<400> 8

Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

His Tyr Gly Trp Thr Pro Leu His Ile Ala Ala Gln Arg Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Trp Leu Gly Trp Thr Pro Leu His Val Ala Ala Ser His Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Lys Asp Leu Ser Gly Arg Thr Pro Leu His Ile Ala Ala Arg Gln Gly

100 105 110

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

115 120 125

Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala

130 135 140

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

145 150 155

<210> 9

<211> 159

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "designed ankyrin repeat domain"

<400> 9

Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

His Tyr Gly Trp Thr Pro Leu His Leu Ala Ala Ser Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Trp Ile Gly Trp Thr Pro Leu His Leu Ala Ala Ser Phe Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Lys Asp Val Ser Gly Lys Thr Pro Leu His Ile Ala Ala Arg Gln Gly

100 105 110

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

115 120 125

Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala

130 135 140

Gly His Gln Asp Ile Ala Glu Val Leu Gln Leu Gln Lys Ala Ala

145 150 155

<210> 10

<211> 157

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "designed ankyrin repeat domain"

<400> 10

Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

His Tyr Gly Trp Thr Pro Leu His Leu Ala Ala Ser Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Trp Ile Gly Trp Thr Pro Leu His Leu Ala Ala Ser Phe Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Lys Asp Val Ser Gly Lys Thr Pro Leu His Ile Ala Ala Arg Gln Gly

100 105 110

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

115 120 125

Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala

130 135 140

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

145 150 155

<210> 11

<211> 124

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "CD3 specific binding domain"

<400> 11

Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala Lys Asn

20 25 30

Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu

35 40 45

Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asn Asp Lys Arg Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

100 105 110

His Gly Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 12

<211> 124

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "CD3 specific binding domain"

<400> 12

Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Ser Gln Gly Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu

35 40 45

Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Asp Lys Gly Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 13

<211> 124

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "CD3 specific binding domain"

<400> 13

Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn

20 25 30

Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu

35 40 45

Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Asp Lys Gly Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 14

<211> 124

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "CD3 specific binding domain"

<400> 14

Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn

20 25 30

Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu

35 40 45

Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asn Asp Lys Arg Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 15

<211> 124

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "CD3 specific binding domain"

<400> 15

Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn

20 25 30

Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu

35 40 45

Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Thr Asn Lys Arg Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Arg Asp Thr Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

100 105 110

His Arg Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 16

<211> 471

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic Polynucleotide'

<220>

<221> Source

<223 >/annotation = "nucleic acid encoding a designed ankyrin repeat domain"

<400> 16

gacttaggaa agaaattgct gcaagccgca cgcgccggtc aacttgatga ggtgcgcgaa 60

ttattgaagg caggtgcaga cgtgaacgct aaagacttta agggacttac tcctttacac 120

ttagcggccg cacatggtca tttggaaatt gtggaggtcc tgttgaaggc tggcgccgac 180

gtgaacgcca aagatgttta cggttggacc ccattacaca ttgctgccgc ctcgggacat 240

ctggaaattg ttgaggttct gcttaaagct ggcgcagacg ttaatgccaa ggactggttg 300

gggattacgc ccttacacct ggccgcgtca catggacatt tagagattgt agaagtcctg 360

ttaaaggcgg gcgcggacgt taatgcccaa gacaaaagtg gcaaaacacc agcggatctg 420

gccgctcgtg ctggacacca ggacattgct gaagtgctgc agaaggcagc g 471

<210> 17

<211> 471

<212> DNA

<213> artificial sequence

<220>

<221> Source

<220>

<221> Source

<223 >/annotation = "nucleic acid encoding a designed ankyrin repeat domain"

<400> 17

gacttaggaa agaaattgct gcaagccgca cgcgccggtc aacttgatga ggtgcgcgaa 60

ttattgaagg caggtgcaga cgtgaacgct aaagacttta agggacttac tcctttacac 120

ttagcggccg agcatggtca tttggaaatt gtggaggtcc tgttgaaggc tggcgccgac 180

gtgaacgcca aagatgttta cggttggacc ccattacact gggctgccgc caagggacat 240

ctggaaattg ttgaggttct gcttaaagct ggcgcagacg ttaatgccaa ggactggttg 300

gggattacgc ccttacacct ggccgcgtca catggacatt tagagattgt agaagtcctg 360

ttaaaggcgg gcgcggacgt taatgcccaa gacaaaagtg gcaaaacacc agcggatctg 420

gccgctcgtg ctggacacca ggacattgct gaagtgctgc agaaggcagc g 471

<210> 18

<211> 477

<212> DNA

<213> artificial sequence

<220>

<221> Source

<220>

<221> Source

<223 >/annotation = "nucleic acid encoding a designed ankyrin repeat domain"

<400> 18

gacttgggga aaaaactgct tcaggctgca cgcgctggtc agttagatga ggtgcgtgag 60

ttgttaaaag ctggagcgga cgtaaatgct aaagatcatt acggatggac acccctgcat 120

cttgcagcgt cagaaggtca ccttgaaatc gtcgaggtcc ttttgaaagc aggggcagat 180

gtcaacgcca aggactggat cggttggacc cctcttcatt tagctgcttc gttcggtcat 240

ctggagattg tagaagtttt attaaaagcc ggtgcggatg tgaatgcaaa agatgtctca 300

gggaaaaccc cgttacacat cgccgctcgt caagggcatt tagagatcgt cgaggtactg 360

ttgaaagcgg gcgcagatgt caatgcacag gacaagtccg gcaaaactcc agcggattta 420

gctgcgcgcg caggacacca agacattgcg gaagtcttac aactgcagaa ggcagcg 477

<210> 19

<211> 471

<212> DNA

<213> artificial sequence

<220>

<221> Source

<220>

<221> Source

<223 >/annotation = "nucleic acid encoding a designed ankyrin repeat domain"

<400> 19

gacttgggga aaaaactgct tcaggctgca cgcgctggtc agttagatga ggtgcgtgag 60

ttgttaaaag ctggagcgga cgtaaatgct aaagatcatt acggatggac acccctgcat 120

cttgcagcgt cagaaggtca ccttgaaatc gtcgaggtcc ttttgaaagc aggggcagat 180

gtcaacgcca aggactggat cggttggacc cctcttcatt tagctgcttc gttcggtcat 240

ctggagattg tagaagtttt attaaaagcc ggtgcggatg tgaatgcaaa agatgtctca 300

gggaaaaccc cgttacacat cgccgctcgt caagggcatt tagagatcgt cgaggtactg 360

ttgaaagcgg gcgcagatgt caatgcacag gacaagtccg gcaaaactcc agcggattta 420

gctgcgcgcg caggacacca agacattgcg gaagtcctgc agaaggcagc g 471

<210> 20

<211> 12

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic peptides'

<220>

<221> Source

<223 >/comment= "His-tag"

<400> 20

Met Arg Gly Ser His His His His His His Gly Ser

1 5 10

<210> 21

<211> 30

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/comment = "N-terminal end capping Module"

<400> 21

Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

20 25 30

<210> 22

<211> 30

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/comment = "N-terminal end capping Module"

<400> 22

Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

20 25 30

<210> 23

<211> 30

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/comment = "N-terminal end capping Module"

<400> 23

Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala

20 25 30

<210> 24

<211> 30

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/comment = "N-terminal end capping Module"

<220>

<221> MOD_RES

<222> (4)..(4)

<223> any amino acid

<220>

<221> MOD_RES

<222> (11)..(12)

<223> any amino acid

<400> 24

Asp Leu Gly Xaa Lys Leu Leu Gln Ala Ala Xaa Xaa Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala

20 25 30

<210> 25

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<220>

<221> MOD_RES

<222> (1)..(1)

<223> any amino acid

<220>

<221> MOD_RES

<222> (3)..(4)

<223> any amino acid

<220>

<221> MOD_RES

<222> (6)..(6)

<223> any amino acid

<220>

<221> MOD_RES

<222> (14)..(15)

<223> any amino acid

<220>

<221> MOD_RES

<222> (27)..(27)

<223> any amino acid

<400> 25

Xaa Asp Xaa Xaa Gly Xaa Thr Pro Leu His Leu Ala Ala Xaa Xaa Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Xaa Gly Ala Asp Val Asn

20 25 30

Ala

<210> 26

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<220>

<221> MOD_RES

<222> (3)..(4)

<223> any amino acid

<220>

<221> MOD_RES

<222> (6)..(6)

<223> any amino acid

<220>

<221> MOD_RES

<222> (14)..(15)

<223> any amino acid

<400> 26

Lys Asp Xaa Xaa Gly Xaa Thr Pro Leu His Leu Ala Ala Xaa Xaa Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 27

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<220>

<221> MOD_RES

<222> (3)..(4)

<223> any amino acid

<220>

<221> MOD_RES

<222> (6)..(6)

<223> any amino acid

<220>

<221> MOD_RES

<222> (11)..(11)

<223> any amino acid

<220>

<221> MOD_RES

<222> (14)..(15)

<223> any amino acid

<400> 27

Lys Asp Xaa Xaa Gly Xaa Thr Pro Leu His Xaa Ala Ala Xaa Xaa Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 28

<211> 28

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic peptides'

<220>

<221> Source

<223 >/comment = "C-terminal end capping Module"

<400> 28

Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly

1 5 10 15

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

20 25

<210> 29

<211> 28

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic peptides'

<220>

<221> Source

<223 >/comment = "C-terminal end capping Module"

<220>

<221> MOD_RES

<222> (1)..(1)

<223> any amino acid

<220>

<221> MOD_RES

<222> (3)..(4)

<223> any amino acid

<220>

<221> MOD_RES

<222> (6)..(6)

<223> any amino acid

<220>

<221> MOD_RES

<222> (11)..(11)

<223> any amino acid

<220>

<221> MOD_RES

<222> (15)..(15)

<223> any amino acid

<400> 29

Xaa Asp Xaa Xaa Gly Xaa Thr Pro Ala Asp Xaa Ala Ala Arg Xaa Gly

1 5 10 15

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

20 25

<210> 30

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 30

Lys Asp Phe Lys Gly Leu Thr Pro Leu His Leu Ala Ala Ala His Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 31

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 31

Lys Asp Val Tyr Gly Trp Thr Pro Leu His Ile Ala Ala Ala Ser Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 32

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 32

Lys Asp Trp Leu Gly Ile Thr Pro Leu His Leu Ala Ala Ser His Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 33

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 33

Lys Asp Val Tyr Gly Trp Thr Pro Leu His Trp Ala Ala Ala Ser Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 34

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 34

Lys Asp Val Tyr Gly Trp Thr Pro Leu His Trp Ala Ala Ala Lys Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 35

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 35

Lys Asp Phe Lys Gly Leu Thr Pro Leu His Leu Ala Ala Glu His Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 36

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 36

Lys Asp Val Tyr Gly Trp Thr Pro Leu His Trp Ala Ala Ala Lys Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 37

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 37

Lys Asp His Tyr Gly Trp Thr Pro Leu His Ile Ala Ala Gln Ile Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 38

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 38

Lys Asp Trp Ile Gly Tyr Thr Pro Leu His Leu Ala Ala Ser His Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 39

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 39

Lys Asp Val Ser Gly Lys Thr Pro Leu His Val Ala Ala Ala His Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 40

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 40

Lys Asp Gln Tyr Gly Trp Thr Pro Leu His Leu Ala Ala Tyr Ser Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 41

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 41

Lys Asp Trp Val Gly Trp Thr Pro Leu His Leu Ala Ala Ser His Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 42

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 42

Lys Asp Glu Ala Gly Arg Thr Pro Leu His Ile Ala Ala Lys Gln Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 43

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 43

Lys Asp His Tyr Gly Trp Thr Pro Leu His Leu Ala Ala Ala Glu Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 44

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 44

Lys Asp Trp Ile Gly Tyr Thr Pro Leu His Ile Ala Ala Ser His Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 45

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 45

Lys Asp Ser Ser Gly Lys Thr Pro Leu His Ile Ala Ala Gln His Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 46

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 46

Lys Asp His Tyr Gly Trp Thr Pro Leu His Ile Ala Ala Gln Arg Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 47

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 47

Lys Asp Trp Leu Gly Trp Thr Pro Leu His Val Ala Ala Ser His Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 48

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 48

Lys Asp Leu Ser Gly Arg Thr Pro Leu His Ile Ala Ala Arg Gln Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 49

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 49

Lys Asp His Tyr Gly Trp Thr Pro Leu His Leu Ala Ala Ser Glu Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 50

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 50

Lys Asp Trp Ile Gly Trp Thr Pro Leu His Leu Ala Ala Ser Phe Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 51

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<400> 51

Lys Asp Val Ser Gly Lys Thr Pro Leu His Ile Ala Ala Arg Gln Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

20 25 30

Ala

<210> 52

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<220>

<221> MOD_RES

<222> (1)..(1)

<223> any amino acid

<220>

<221> MOD_RES

<222> (3)..(4)

<223> any amino acid

<220>

<221> MOD_RES

<222> (6)..(6)

<223> any amino acid

<220>

<221> MOD_RES

<222> (13)..(15)

<223> any amino acid

<220>

<221> MOD_RES

<222> (17)..(19)

<223> any amino acid

<220>

<221> MOD_RES

<222> (22)..(22)

<223> any amino acid

<220>

<221> MOD_RES

<222> (26)..(27)

<223> any amino acid

<400> 52

Xaa Asp Xaa Xaa Gly Xaa Thr Pro Leu His Leu Ala Xaa Xaa Xaa Gly

1 5 10 15

Xaa Xaa Xaa Leu Val Xaa Val Leu Leu Xaa Xaa Gly Ala Asp Val Asn

20 25 30

Ala

<210> 53

<211> 33

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "ankyrin repeat Module"

<220>

<221> MOD_RES

<222> (1)..(1)

<223> any amino acid

<220>

<221> MOD_RES

<222> (3)..(4)

<223> any amino acid

<220>

<221> MOD_RES

<222> (6)..(6)

<223> any amino acid

<220>

<221> MOD_RES

<222> (14)..(15)

<223> any amino acid

<400> 53

Xaa Asp Xaa Xaa Gly Xaa Thr Pro Leu His Leu Ala Ala Xaa Xaa Gly

1 5 10 15

His Leu Glu Ile Val Glu Val Leu Leu Lys Glx Gly Ala Asp Val Asn

20 25 30

Ala

<210> 54

<211> 421

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "TCE (without half-life extending moiety)".

<400> 54

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Leu Asp

20 25 30

Trp Leu Gly His Thr Pro Leu His Leu Ala Ala Tyr Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Ile

50 55 60

Asp Asp Asn Asn Gly Phe Thr Pro Leu His Leu Ala Ala Ile Asp Gly

65 70 75 80

His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn

85 90 95

Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn

100 105 110

Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala Gly Ser Pro

115 120 125

Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

130 135 140

Thr Pro Thr Gly Ser Asp Leu Gly Asp Lys Leu Leu Leu Ala Ala Thr

145 150 155 160

Ser Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp

165 170 175

Val Asn Ala Lys Asp Tyr Asp Gly Asp Thr Pro Leu His Leu Ala Ala

180 185 190

Asp Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

195 200 205

Asp Val Asn Ala Lys Asp Tyr Ser Gly Ser Thr Pro Leu His Ala Ala

210 215 220

Ala Ala Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Val Phe Gly Tyr Thr Pro Ala Asp Leu

245 250 255

Ala Ala Tyr Val Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu

290 295 300

Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu

325 330 335

His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly Val Thr Pro

355 360 365

Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp Gly Thr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala

420

<210> 55

<211> 569

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/annotation = "description of artificial sequence: synthetic polypeptide'

<220>

<221> Source

<223 >/annotation = "TCE (with half-life extending moiety)".

<400> 55

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val

165 170 175

Asn Ala Leu Asp Trp Leu Gly His Thr Pro Leu His Leu Ala Ala Tyr

180 185 190

Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp

195 200 205

Val Asn Ala Ile Asp Asp Asn Asn Gly Phe Thr Pro Leu His Leu Ala

210 215 220

Ala Ile Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile

245 250 255

Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Asp Lys Leu Leu

290 295 300

Leu Ala Ala Thr Ser Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Asp Gly Asp Thr Pro Leu

325 330 335

His Leu Ala Ala Asp Glu Gly His Leu Glu Ile Val Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Ser Gly Ser Thr Pro

355 360 365

Leu His Ala Ala Ala Ala Tyr Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Val Phe Gly Tyr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Tyr Val Gly His Glu Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr

420 425 430

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly

435 440 445

Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg

450 455 460

Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly

465 470 475 480

Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe

485 490 495

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn Asp Lys

500 505 510

Arg Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile

515 520 525

Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Arg Asp Ser

530 535 540

Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Gln Asp

545 550 555 560

Ile Ala Glu Val Leu Gln Lys Ala Ala

565

Claims

1. A composition comprising (i) a binding moiety and (ii) a drug molecule;

wherein the binding moiety reversibly binds to the drug molecule; and is also provided with

Wherein the binding moiety, when bound, inhibits the biological activity of the drug molecule.

2. The composition of claim 1, wherein the binding moiety comprises an antibody, alternative scaffold, or polypeptide.

3. The composition of any preceding claim, wherein the biological activity of the drug molecule is binding of the drug molecule to a biological target.

4. The composition of any preceding claim, wherein the binding affinity of the binding moiety to the drug molecule allows release of the drug molecule over time following administration of the composition to a mammal.

5. The composition of any preceding claim, wherein the binding moiety has a dissociation constant (K _D ) Binding to the drug molecule.

6. The composition of any preceding claim, wherein the rate of dissociation (k) of the binding moiety from the drug molecule _off ) Between about 1X 10 ^-8 s ^-1 And about 1X 10 ^-4 s ^-1 Between them.

7. The composition of any preceding claim, wherein the binding moiety has a blocking half-life (T _1/2 ) Wherein the blocking half-life is calculated according to the formula:

8. the composition of claim 7, wherein the blocking half-life (T _1/2 ) For at least about 2 hours.

9. The composition of any preceding claim, wherein the binding moiety comprises a designed ankyrin repeat domain.

10. The composition of claim 9, wherein the engineered ankyrin repeat domain comprises an ankyrin repeat module comprising an amino acid sequence selected from the group consisting of seq id nos: (1) 30 to 51, and (2) a sequence in which up to 9 amino acids in any one of SEQ ID NOs 30 to 51 are substituted with other amino acids.

11. The composition of claim 9, wherein the engineered ankyrin repeat domain comprises an amino acid sequence selected from the group consisting of: (1) 1 to 10, and (2) a sequence having at least 85% amino acid sequence identity to any one of SEQ ID NOs 1 to 10.

12. The composition of any preceding claim, wherein the drug molecule has binding specificity for CD 3.

13. The composition of any preceding claim, wherein the drug molecule is a T cell cement drug molecule (TCE).

14. The composition of any one of claims 12 to 13, wherein binding of the binding moiety to the TCE drug molecule inhibits binding of the TCE drug molecule to T cells and/or activation of T cells.

15. A composition according to any preceding claim for use in therapy.